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Stem Cells and Regeneration in the Digestive Organs (X6)

Background

The cytokine TRAIL (tumor necrotic factor-related apoptosis-inducing ligand) selectively induces apoptosis in cancer cells, but cancer stem cells (CSCs) that contribute to cancer-recurrence are frequently TRAIL-resistant. Here we examined hitherto unknown effects of the dietary anti-carcinogenic compound phenethyl isothiocyanate (PEITC) on attenuation of proliferation and tumorigenicity and on up regulation of death receptors and apoptosis in human cervical CSC.

Cancer stem-like cells were enriched from human cervical HeLa cell line by sphere-culture method and were characterized by CSC-specific markers analyses (flow cytometry) and Hoechst staining. Cell proliferation assays, immunoblotting, and flow cytometry were used to assess anti-proliferative as well as pro-apoptotic effects of PEITC exposure in HeLa CSCs (hCSCs). Xenotransplantation study in a non-obese diabetic, severe combined immunodeficient (NOD/SCID) mouse model, histopathology, and ELISA techniques were further utilized to validate our results in vivo.

PEITC attenuated proliferation of CD44high/+/CD24low/, stem-like, sphere-forming subpopulations of hCSCs in a concentration- and time-dependent manner that was comparable to the CSC antagonist salinomycin. PEITC exposure-associated up-regulation of cPARP (apoptosis-associated cleaved poly [ADP-ribose] polymerase) levels and induction of DR4 and DR5 (death receptor 4 and 5) of TRAIL signaling were observed. Xenotransplantation of hCSCs into mice resulted in greater tumorigenicity than HeLa cells, which was diminished along with serum hVEGF-A (human vascular endothelial growth factor A) levels in the PEITC-pretreated hCSC group. Lung metastasis was observed only in the hCSC-injected group that did not receive PEITC-pretreatment.

The anti-proliferative effects of PEITC in hCSCs may at least partially result from up regulation of DR4 and possibly DR5 of TRAIL-mediated apoptotic pathways. PEITC may offer a novel approach for improving therapeutic outcomes in cancer patients.

Despite considerable improvement in cancer diagnosis and therapy, relapse and metastasis are still common [1]. However, the rise of the cancer stem cell (CSC) hypothesis provides a new approach to eradicating malignancies. Recent studies have shown that CSCs are a small subpopulation of tumor cells that possess self-renewal and tumor-initiation capacity and the ability to give rise to the heterogeneous lineages of malignant cells that comprise a tumor [2]. CSCs have been identified in hematologic and solid cancers and implicated in tumor initiation, development, metastasis, and recurrence. Although the origin(s) and dynamic heterogeneity of CSCs remain unexplained, designing novel approaches to target CSCs has received much attention over the past several years [35].

Phenethyl isothiocyanate (PEITC) is a dietary compound derived from common vegetables such as watercress, broccoli, cabbage, and cauliflower [6]. We and others have shown under experimental conditions that PEITC possesses anti-inflammatory [7, 8] and chemopreventive activity against various cancers, including colon [9], prostate [10], breast [11], cervical [12, 13], ovarian [14], and pancreatic cancer [15]. Safety studies in rats and dogs have shown that PEITC has no apparent toxicity, even when administered in high doses, as determined by NOEL (no-observed-adverse-effect-level) [16], and PEITC is currently in clinical trials in the US for lung cancer (NCT00691132). Cervical cancer is the second-most-fatal cancer in women worldwide, and the incidence rate is significantly higher in developing nations due to the absence of rigorous screening programs [17]. A recent study showed that PEITC can induce the extrinsic apoptosis pathway in a human cervical cancer cell line [12]. However, the chemotherapeutic effects of PEITC in the context of CSCs and more specifically cervical CSCs remain unknown.

Apoptosis, or programmed cell-death, is essential to maintaining tissue homeostasis, and its impairment is implicated in many human diseases, including cancers [18]. The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor super-family, has attracted great interest for clinical applications due to its specific anti-tumor potential without toxic side effects to normal healthy cells [19, 20]. There are two well-characterized apoptosis pathways in mammalian cells. The extrinsic pathway is mediated by death receptors, a subgroup of the TNF receptor superfamily. TRAIL binds to TRAIL-R1 and TRAIL-R2, two death domain-containing receptors, also called DR4 and DR5, to trigger apoptosis. The intrinsic pathway involves mitochondria, and is triggered and controlled by members of the Bcl-2 protein family. Both pathways cause the activation of initiator caspases, which then activate effector caspases [21]. Caspases cause cleavage and inactivation of poly (ADP-ribose) polymerase 1 (PARP)-1, which helps repair single-stranded DNA breaks, and hence PARP-1 cleavage serves as a hallmark of apoptosis [22]. Unfortunately, a variety of human tumors develop resistance to TRAIL-induced apoptosis [23]. But further studies have suggested that TRAIL activity can be sensitized with other chemotherapeutic drugs, such as paclitaxel [24], 5-fluorouracil (5-FU) [25], and cisplatin [26] or dietary bioactive compounds like benzyl isothiocyanate (BITC) [27] or sulforaphane [28, 29]. However, the effects of PEITC on TRAIL pathway in CSCs have not been reported.

In the present study, we investigated the efficacy of PEITC in attenuating the growth of sphere-forming cervical CSCs isolated from HeLa cells (hCSCs) as well as its ability to up regulate death receptors for TRAIL-mediated induction of apoptosis. Furthermore, the in vivo anti-tumorigenicity effects of PEITC were evaluated in a xenograft mouse model.

Phenethyl isothiocyanate (Sigma-Aldrich, St. Louis, MO), 99%, was diluted in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO) to make 0.520-mM stock concentrations and was further diluted in media to obtain 2.520-M final concentrations, which are achievable following oral administration in human [30] and have been used in prior studies by us and others to induce apoptosis in the SW480 colon cancer cell line [9] and cervical cancer cell lines. We used comparable concentrations of salinomycin (2.520M) and lower concentrations (2.520 nM) of paclitaxel (both from Sigma-Aldrich, St. Louis, MO) as positive controls, which are CSC-targeted and CSC-non-specific anti-cancer chemotherapeutics, respectively, following Gupta et al. [31]. For the negative/vehicle control samples, we used DMSO in an amount equivalent to that used with test compounds in test samples.

The human HeLa cell line (ATCC CCL-2, American Type Culture Collection, Manassas, VA) was cultured and maintained in a T-25 flask with Dulbeccos modified eagles medium (DMEM) containing 4mML-glutamine and 4.5g/L glucose (HyClone, Logan, UT), supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Grand Island, NY) and 1% penicillin (25 U/ml)/streptomycin (25g/ml) (Sigma-Aldrich, St. Louis, MO) in a 5% CO2-humidified atmosphere at 37C. HeLa cells were trypsinized with TrypLE (Invitrogen, Grand Island, NY) and then sub-cultured with a 1:5 splitting ratio when the cells reached about 90% confluency. From the parental HeLa cells (termed simply as HeLa in the rest of the document), hCSCs were cultured following a modified protocol described by Gu et al. [5]. Briefly, single-cell suspensions of HeLa cells (4104) were seeded into a 100-mm ultra-low attachment (ULA) petri dish (Corning Inc., Corning, NY) containing 8ml of serum-free mammary epithelial basal medium (MEBM, Lonza, Allendale, NJ), supplemented with 1 B27 (Invitrogen, Grand Island, NY), 4g/ml heparin (Sigma-Aldrich, St. Louis, MO), 20ng/ml hEGF, and 20ng/ml hFGF (Invitrogen, Grand Island, NY). After an initial 4-day culture in suspension at 37C, an additional 9ml of sphere culture medium was added for another 5days of culture. On day 9, spheres were harvested by centrifugation at 500 g for 3minutes, followed by washing with phosphate-buffered saline (PBS), trypsinization with TrypLE for 10minutes at 37C, centrifugation at 500 g for 3minutes, resuspension in 5ml of hCSC culture medium, and counting with a hemocytometer. Both HeLa cells and hCSCs were used for successive experiments.

Around 2106 HeLa cells were seeded into a 60-mm petri dish and incubated overnight at 37C. Cells were washed with 2ml of PBS, trypsinized with 1ml of TrypLE, and resuspended in 1ml of PBS, followed by immunostaining. Similarly, hCSCs were collected after 9days of culture, trypsinized, and resuspended in 2ml of PBS with a density of 1106 cells/ml, followed by immunostaining. Cells were immunostained with anti-CD24FITC (1:500v/v, Millipore, Billerica, MA) or anti-CD44FITC (1:500v/v, Millipore, Billerica, MA) antibodies for 1hour at room temperature. Immunofluorescence was measured using a FACSCalibur cell analyzer (Becton Dickinson, San Jose, CA) with approximately 10,000 events in each sample. Propidium iodide/annexin V staining was performed according to the manufacturers instructions. Briefly, 5105 cells were centrifuged and resuspended in 100l of 1x binding buffer (Invitrogen, Grand Island, NY). The cells were treated with 10M PEITC or vector control (DMSO) for a total of 24h, in the last hour of which 10ng/ml of human recombinant TRAIL (eBioscience, Inc., San Diego, CA) or vector control (DMEM) were added to the cells before harvesting. The cells were then incubated with 5l of annexin VFITC (eBioscience, Inc., San Diego, CA) and 5l of propidium iodide (eBioscience, Inc., San Diego, CA) at room temperature for 5minutes in the dark before analyzing the cells on a FACSCalibur cell analyzer. For DR4 and DR5 expression analysis, 5105 cells were filtered through a filter cap (35m) into a collecting tube (BD Falcon, Franklin Lakes, NJ) and then washed, fixed with 2% paraformaldehyde, and stained with DR4 or DR5 surface markers (1:200v/v) overnight at 4C in a rotating vessel. The immunostained cells were incubated with goat anti-mouse Dylight 488 (1:500v/v) secondary antibody for 2hours at room temperature before acquiring at least 10,000 cells in a flow cytometer.

The fluorescence resulting from interaction of cell DNA with Hoechst 33342 dye was measured to assess the cells ability to efflux the fluorescent dye Hoechst 33342, as most hematopoietic stem cells are able to exclude the dye [32]. HeLa or hCSCs were trypsinized with TrypLE, washed with PBS, and adjusted to 1106 cells/ml in Hanks balanced salt solution (HBSS), before incubating with 5g/ml Hoechst 33342 dye (Life Technologies, Grand Island, NY) for 60minutes at 37C in a 5% CO2 incubator. The cells were then washed three times with HBSS by centrifugation at 300 g for 5minutes. The pellets were resuspended at 1106 cells/ml in HBSS and kept on ice until used for imaging. The Hoechst staining was visualized with an EVOS FL Epifluorescent Microscope (AMG, Bothell, WA) using the DAPI channel. Images were indicated as transmitted (phase contrast images of whole cells), Hoechst-stained (nuclei with Hoechst staining), and merge (an overlay of transmitted and Hoechst staining in the same field). The cells with Hoechst-stained nuclei were counted among 100 cells, and the number of Hoechst-excluded cells was then quantified.

The hCSCs were enriched in spheres in serum-free medium. Sphere culture was carried out as previously described in the sphere culture section. Cells were treated with predetermined doses of 0.5, 1.0, or 2.5M of PEITC or DMSO as control. After 7days incubation, photomicrographs of spheres were acquired under an inverted phase-contrast microscope (Olympus America Inc., Center Valley, PA), and the number of hCSCs was counted using a hemocytometer.

A standard colorimetric method (MTS assay) was used to determine the number of viable cells in samples. For cell-proliferation assays, HeLa and hCSCs were cultured for 4days, and an additional 9ml of sphere culture medium was added for another 5days, as described in the sphere culture section. Viable cells were harvested and counted with a hemocytometer before seeding into 96-well microplates at a density of 2104 cells per well. Cells were cultured in DMEM supplemented with 100 U/ml penicillin, 100g/ml streptomycin, 5% heat-inactivated FBS, and 50M 2-mercaptoethanol. Both hCSCs and HeLa cells were treated with four concentrations of PEITC and salinomycin (2.520M) and paclitaxel (2.520 nM). After 24 and 48hours of incubation, 20l of CellTiter reagent was added directly to the cell-culture wells and incubated for 1hour at 37C, followed by cell viability assessment using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI), containing [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS]. The manufacturers instructions were followed, and treatments were compared with vehicle control (DMSO-treated cells) at 490nm in a BioTek Synergy H4 multimode plate reader (BioTek, Winooski, VT).

hCSCs (1106) were seeded in each well of a 6-well plate and incubated overnight at 37C in a 5% CO2 incubator. Old culture medium was replenished by culture medium containing either 10-M or 15-M concentrations of PEITC for 5hours. The cells were then treated with 10ng/ml human recombinant TRAIL or with 10ng/ml TNF (eBioscience, Inc., San Diego, CA) for additional 1-hour incubation. Cell harvesting and immunoblotting were carried out as we previously reported [9]. Briefly, cells were lysed in ice-cold RIPA buffer containing 150mM NaCl, 50mM Tris (pH8.0), 10% glycerol, 1% Nonidet P-40 (NP-40), and 0.4mM EDTA, followed by a brief vortexing and rotation for 30minutes at 4C. Equal amounts (v/v) of cell lysates were separated by SDS-PAGE through a 12% separating gel, transferred to nitrocellulose membranes, blocked with 5% non-fat dry milk, and double-probed overnight at 4C with mouse anti-human cPARP (1:1000v/v, Millipore, Billerica, MA) and rabbit anti-human -actin (1:5000v/v, Millipore, Billerica, MA) antibodies. Blots were then washed in PBS and further incubated with secondary antibodies, Dylight 680 anti-mouse (1:5000v/v) and Dylight 800 anti-rabbit (1:5000v/v), for 1hour at room temperature. Finally, after rinsing in Tween20 (0.1% in PBS), blots were imaged with a LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE), followed by a densitometric analysis of cPARP levels after normalizing with the -actin housekeeping gene.

Animal studies were carried out in accordance with the guidelines of, and, using an approved protocol by, the Institutional Animal Care and Use Committee (IACUC), South Dakota State University (IACUC protocol approval #12-087A). Twenty female non-obese diabetic, severe combined immunodeficient (NOD/SCID, NOD.CB17-Prkdc scid/J) mice (Jackson Laboratories, Bar Harbor, ME), 17weeks old, were randomly grouped into five groups (four mice per group) in specific pathogen-free (SPF) housing at a constant temperature of 2426C with a 12-h:12-h light/dark cycle. All mice were allowed to acclimatize for 1week and were provided with sterile food and water ad libitum. HeLa and hCSCs were cultured, trypsinized, washed, pre-treated with 10M PEITC where indicated, and resuspended in PBS at the concentration of 1107 cells/ml before injecting into the mice. Each mouse was subcutaneously injected at the neck scruff with one injection of PBS (100l, control group), HeLa (1106), HeLa pretreated with 10M PEITC (1106), hCSCs (1106), or hCSCs pretreated with 10M PEITC (1106). The cell number in each injection was consistent with the study previously carried out by Gu et al. [5]. All mice were routinely monitored for tumor formation, weight loss, pain, and distress. The mice were euthanatized with CO2 asphyxiation 21days post-treatment, and blood was collected through cardiac puncture immediately after sacrifice. Excised tumor and lung samples were kept in 10% formalin for subsequent histopathological examination. The average tumor number or mass per injection was calculated by dividing each groups total number of tumors or total mass by the number of mice in that group.

Excised tumor, lung, and liver were fixed by immersion in 10% buffered formalin for 35 days and then transferred to 70% ethanol for long-term fixation. Representative sections of fixed tissue were trimmed and embedded in paraffin, then sectioned at 3m and stained by hematoxylin and eosin (H&E) [33] for examination performed in a blind manner by a veterinary pathologist, and photomicrographs were captured under a microscope (Leica, Micro Service, St. Michael, MN) at 200 and 400 magnification for illustrative purposes.

Since hCSCs are of human origin, ELISA was carried out to assess the presence of human vascular endothelial growth factor A (hVEGF-A), which promotes tumor angiogenesis in a host. The collected mouse blood samples were kept in a slanted position at room temperature for 1hour, followed by 4C for 24hours, and then centrifuged at 5000rpm for 5minutes. The Platinum ELISA kit (eBioscience, San Diego, CA) was used to quantify the hVEGF-A present in each serum sample (pg/ml) from a single mouse, according to the manufacturers instructions.

Statistical analyses were carried out using Sigma Plot software (Systat Software, Inc., San Jose, CA). Statistical significance between the groups was assessed by multiple mean comparisons using one-way analysis of variance (ANOVA) followed by a post-hoc Dunnetts test. Students t test was applied to compare two groups receiving similar treatments. Data were expressed as meansSEM. Experiments were repeated at least three times. The significance of differences between means is represented by asterisks: *p0.05, **p0.01, ***p0.001.

In this report we used the HeLa cervical cancer cell line to isolate and characterize hCSCs following a previously described sphere culture method [

], which favors self-renewal of CSCs in culture but also causes minimal damage to the cells. In comparison with HeLa cells, the isolated/enriched hCSC population exhibited higher CD44 (90.93% vs. 51.52%) and lower CD24 (0.4% vs. 7.5%) cell-surface marker expression in flow cytometry analyses (Figure

A, B), consistent with results previously reported [

]. Multi-drug resistance characteristic of stem cells was indicated by transporter-mediated efflux of the fluorescent dye Hoechst 33342 [

], and significantly higher numbers of Hoechst-dye-excluded cells in hCSCs (73%) than in HeLa cells (15%) further confirmed their stem-like characteristics (Figure

C, D). Finally, in xenotransplanted mice, greater tumorigenicity was observed in the hCSC group (7 tumors/4 mice) than in the HeLa group (2 tumors/4 mice) (Figure

E). Following validation of hCSC characteristics, we investigated the effects of PEITC and other compounds on hCSCs. The significance of any treatment was compared with untreated/vehicle (DMSO) controls or otherwise specified.

Identification and confirmation of isolated HeLa cancer stem cells (hCSCs). A) Representative FACS histograms showing increased CD44 and decreased CD24 expression in hCSCs compared with HeLa cells B) Summary of FACS analyses showing the percentage of hCSCs expressing CD44 and CD24 (n=3) C) The Hoechst exclusion assay showing transmitted, Hoechst-stained, and overlaid images of HeLa cells and hCSCs. Hoechst 33342 dye emits blue fluorescence when bound to dsDNA. Yellow arrows show Hoechst-excluded cells lacking dark-blue nuclei (200-m scale), which were typically higher in hCSCs than in HeLa cells. D) Quantification of Hoechst-dye-excluded cells showing a higher exclusion rate in hCSCs (n=3). E) In vivo tumorigenicity was compared in NOD/SCID mice (four animals per group) 3weeks after xenotransplantation of HeLa cells, hCSCs, or vehicle (nave control), showing higher tumor counts in the hCSC group. All data are expressed as meansSEM except for in vivo tumor counts. Asterisks indicate statistically significant differences between the groups indicated, ***p0.001.

PEITC attenuated the formation of primary hCSC spheres in a concentration-dependent manner (Figure

A). Addition of PEITC (1.0 and 2.5M) resulted in a 48% and 60% decline in cell numbers, respectively (Figure

B), which is consistent with the corresponding reduction in sphere size (Figure

A). Lower concentrations of PEITC (2.5M) were used in sphere-forming enrichment culture media than in specific assays (2.5M), as shown in the remaining figures. PEITC also significantly reduced proliferation of both HeLa cells and hCSCs in a concentration-dependent manner after 24- and 48-hour exposures, which was a pattern comparable to the effects of salinomycin. The observed effects of 10 nM paclitaxel was limited (Figure

C) in our experiments, which may be due to the slow induction of cell death after low concentrations (10 nM) of paclitaxel, which occurs up to 72hours post treatment. It was previously shown that low concentrations of paclitaxel strongly block mitosis at the metaphase/anaphase transition but could be insufficient to cause immediate cell death in HeLa cells [

].

Effects of PEITC on HeLa cell and hCSC viability. A) Representative micrographs showing PEITC-attenuated sphere formation in hCSCs isolated from HeLa cells in a concentration-dependent manner as observed after 7days of culture in enrichment medium (400-m scale). B) Histogram showing quantification of viable cells on the 7th day of sphere culture from groups shown in A (n=5). C) Concentration-dependent effects of PEITC on the viability of HeLa cells and hCSCs after 24 (i) and 48 (ii) hours. Salinomycin and paclitaxel were used as known reference chemotherapeutic compounds. Absorbance was read at 490nm, and data were expressed as percentage cell viability (n=6). The dotted lines represent the baseline cell viability for DMSO/nave controls, to which all the readings were compared to obtain statistical significance. All data represent meansSEM, and significance was determined by comparing with nave control or as indicated, *p0.05, **p0.01, ***p0.001.

To investigate a potential pro-apoptotic effect of PEITC in triggering hCSC growth inhibition, we carried out western blot experiments on hCSCs treated with different doses of PEITC in the presence or absence of TRAIL and TNF. We observed an increased expression of cPARP with higher doses of PEITC (15M) following exposure for 5hours, which was further augmented by the presence of 10ng/ml TRAIL, which indicated elevated levels of endogenous caspase-mediated apoptosis in hCSCs (Figure

A). After normalizing to the housekeeping gene -actin, densitometric analysis of cPARP levels showed that PEITC induced cPARP and sensitized the TRAIL pathway but not the TNF pathway in hCSCs (Figure

A). It was previously shown that PEITC induces cPARP in HeLa cells [

], which we also observed (data not shown). Next, we carried out an annexin V/propidium iodide (PI) staining with or without TRAIL induction. Dot plot analyses showed that the fraction of annexin-positive cells in hCSCs treated with PEITC was higher than in untreated hCSCs (5.76% vs. 4.12%, Figure

B, C). Similarly, TRAIL-induced hCSCs treated with PEITC showed increased apoptosis relative to TRAIL-induced hCSCs (6.42% vs. 5.81%, Figure

B, C), although the difference was not statistically significant. When compared with the DMSO control, both PEITC- and TRAIL-treated hCSCs showed a trend toward higher apoptotic levels, indicating a potential sensitization of TRAIL-mediated apoptotic pathways by PEITC.

PEITC sensitizes the TRAIL pathway in hCSC apoptosis. A) Representative immunoblot and densitometric analysis (n=3) of cPARP levels in hCSCs after concentration- dependent PEITC exposure in the presence/absence of TRAIL (10ng/ml) and TNF (10ng/ml), normalized to housekeeping -actin expression levels. PEITC independently induced as well as synergized TRAIL induction of cPARP in hCSCs. B) A quantitative bar graph illustrating individual effects as well as synergism between 10M PEITC and TRAIL (10ng/ml) in sensitizing TRAIL-mediated apoptosis (n=3). C) Representative FACS scatter plots of data shown in B with annexin VFITC/propidium iodide staining, confirming individual effects as well as synergism between PEITC (10M) and TRAIL (10ng/ml) in sensitizing TRAIL- mediated apoptosis (iiv). All data represent meansSEM, and significance was determined by comparing with nave control or as indicated, *p0.05, **p0.01, ***p0.001.

To further understand the characteristics of PEITC in the extrinsic apoptosis pathway in hCSCs, we carried out flow cytometry analyses of DR4 and DR5 death receptors. Since both PEITC- and DMSO-treated hCSCs were treated with TRAIL (all treatments included TRAIL), we expected to see greater induction of DR4 and DR5 in PEITC+TRAIL-treated cells compared to TRAIL treatment alone. We observed that PEITC induced overexpression of DR4 in comparison with the DMSO control (69.01% and 52.52%, Figure

A iii, B). Similarly but to a lesser extent, the expression of DR5 in PEITC-treated hCSCs was higher (72.63% and 60.57%) than in the corresponding DMSO control (Figure

A iiiiv, B), showing that the slightly increased overexpression of DR5 was due to PEITC treatment. PEITC was previously shown to upregulate DR4 and DR5 in a different cervical cancer cell line (HEp-2) [

]; hence, we investigated its effect only on hCSCs.

PEITC up-regulated DR4 and DR5 receptors in TRAIL signaling. A) Representative FACS histograms of DR4 and DR5 expression in hCSCs treated with or without 10M PEITC in the presence of TRAIL. PEITC induced overexpression of DR4 (ii) and DR5 (iv) in comparison with DMSO controls (i) and (iii), respectively. The histograms do not show isotype controls. B) Quantitative bar diagrams presenting the groups from A (n=3). All data represent meansSEM, and significance was determined by comparing with nave control as indicated: **p0.01, ***p0.001.

To confirm the higher tumorigenic potential of hCSCs in vivo, we carried out a xenotransplant experiment in NOD-SCID immunodeficient mice that included four treatment groups and a negative/naive control group. Tumor development did not alter food intake and overall well-being of the mice, as evidenced by their normal body weight and activity (data not shown). An equal number of cells (1106) containing either HeLa cells or hCSCs (each with or without 10M PEITC pre-treatment) developed different tumor loads in each group of NOD/SCID mice. The average tumor number per injection was observed to be much higher in the hCSC group (1.75) than in the HeLa group (0.5), while PEITC pre-treatment helped lower tumor formation in both hCSC (1.75 vs. 0.5) and HeLa (0.5 vs. 0.33) groups of mice than in controls (Figure5B). A similar trend was observed when we calculated tumor mass per injection in each group. The hCSC group had a higher average tumor mass than the HeLa group (95mg vs. 60mg, respectively, data not shown). As expected, PEITC-treated hCSCs and HeLa cells produced a lower mass (85mg and 40mg, respectively) than their controls (95mg and 60mg, respectively, data not shown). To further visualize histological differences between tumors driven by CSCs and HeLa cells, the excised tumors were sectioned and stained with H&E. We observed a higher number of differentiated tumor cells with a low mitotic index in the HeLa group (Figure 5Ai). By contrast, the presence of pleomorphic and highly proliferative cells and early signs of neovascularization in the CSC group suggested that the tumors driven by CSCs are highly aggressive (Figure5Aiii). On the other hand, there were more apoptotic cells in the case of HeLa cells treated with PEITC (Figure5Aii) and hCSCs treated with PEITC (Figure5Aiv), suggesting that PEITC induces apoptosis in both HeLa cells and hCSCs.

To validate the human origin of these tumors, we performed ELISA on isolated serum samples. The hCSC group had the highest concentration of human hVEGF-A (12.31pg/ml), followed by hCSCs treated with PEITC (i.e., 4.62pg/ml) and untreated HeLa cells (1.08pg/ml), while we did not detect any hVEGF-A in HeLa cells treated with PEITC (Figure

C). To see whether hCSCs have metastatic potential, we carried out H&E staining of lung sections, which revealed invading tumor cells in the lungs of the hCSC group (Figure

D and Eiii) but not in the other groups. Overall, hCSCs were more tumorigenic than HeLa cells in this model, and their tumorigenicity was attenuated by PEITC pre-treatment prior to xenotransplant.

Effects of 10M PEITC-treated compared with untreated HeLa cells and hCSCs in a xenotransplant NOD/SCID mouse model. A) Representative photomicrographs of H&E-stained and sectioned tumors (3m, 400x) showing greater and more aggressive tumorigenic effects of hCSCs (iii) than HeLa cells (i). Details of native HeLa cells within a small tumor nodule with fairly uniform cell size and shape are shown (ii), and details of a small tumor nodule showing widespread apoptosis are also shown (iv). Empty arrows indicate apoptotic cells (yellow), high mitotic activity (blue), and early signs of neovascularization (white). B) Average tumor number per injection, where the untreated hCSC group showed the highest number of tumors per injection. C) The highest concentration of human serum VEGF-A was in the hCSC group, indicating the human origin of the tumors that were translocated into the blood circulation. D) The metastatic potential among the groups is shown. Metastasis was observed only in the untreated hCSC group. E) Representative photomicrographs of H&E-stained and sectioned lungs (3m, 200x). Filled arrows indicate lung bronchiole (yellow) as a landmark of distant tumor location and invading tumor cells (white) (iii). Overall, hCSCs showed increased tumorigenic activity compared with HeLa cells in this model, which was, however, attenuated upon pre-treatment with PEITC.

Cervical cancer is the second-most-frequent female malignancy worldwide [17]. Concurrent chemoradiotherapy represents the standard of care for patients with advanced-stage cervical cancer, while radical surgery and radiotherapy are widely used for treating early-stage disease. However, the poor control of micrometastases, declining operability, and the high incidence of long-term complications due to radiotherapy underscore the necessity for developing different therapeutic approaches, such as using an adjuvant CAM (complementary and alternative medicine) regimen for improved treatment outcomes [35]. Among cancer patients, the use of alternative treatments ranges between 30 and 75% worldwide and frequently includes dietary approaches, herbals, and other natural products [36]. It is becoming increasingly evident that cancer treatment that fails to eliminate CSCs allows relapse of the tumor [37]. Here we report novel effects of PEITC, a phytochemical that can be derived from a plant-based diet or may be developed as a natural product, in attenuating in vitro hCSC proliferation and in vivo tumorigenicity as well as stimulating intracellular receptors that mediate TRAIL-induced apoptosis.

According to the CSC concept of carcinogenesis, CSCs represent novel and translationally relevant targets for cancer therapy, and the identification, development, and therapeutic use of compounds that selectively target CSCs are major challenges for future cancer treatment [37]. It is proposed that direct targeting of CSCs through their defining surface antigens, such as CD44, is not a rational option, because these antigens are frequently expressed on normal stem cells [38]. On the other hand, triggering tumor cell apoptosis, in general, is the foundation of many cancer therapies. In the case of CSCs, it was suggested that the induction of apoptosis in the CSC fraction of tumor cells by specifically upregulating death receptors or death receptor ligands such as TRAIL is a potential strategy to bypass the refractory response of CSCs to conventional therapies [38]. Preclinical studies have demonstrated the potential of TRAIL to selectively induce apoptosis of tumor cells, because normal cells possess highly expressed decoy receptors that protect them from cell death [20, 39], which has driven the development of TRAIL-based cancer therapies [38, 40]. Unfortunately, a considerable range of cancer cells, especially in some highly malignant tumors, are resistant to TRAIL-induced apoptosis [41]. Therefore, TRAIL synergism using PEITC, a compound with an established low-toxicity profile in healthy animals [16] could offer an important approach to overcoming the current challenges in using TRAIL-targeted therapies, particularly in otherwise-resistant CSCs.

PEITC treatment in hCSCs reduced proliferation and sphere formation and expressed higher levels of cPARP, indicating elevated levels of apoptosis, which is possibly through caspase activation by isothiocyanate in treated cancer cells as reported previously [42]. At similar micromolar concentrations, the effects of PEITC on hCSC proliferation were comparable to salinomycin, which was shown to effectively eliminate CSCs and to induce partial clinical regression of heavily pretreated and therapy-resistant cancers [37]. It is worth mentioning here that salinomycin had considerable cytotoxicity in healthy mammals [37]. PEITC has been well documented for safety to normal mammals. It is interesting to investigate if PEITC is cytotoxic to normal stem cells, which has not been reported. Moreover, the effects of PEITC were significantly better in abrogating hCSC proliferation than paclitaxel, a current cancer chemotherapeutics. This better anti-proliferative effect may be due to the high level of chemoresistance of CSCs to paclitaxel, the overcoming of which by specific targeting of CSCs is hailed as critical. The concentration range of PEITC used (2.520M) was validated in our previous studies [8, 9, 43] and was also shown to be achievable following oral administration in human [30].

We observed that PEITC likely sensitized TRAIL but not the TNF pathway while inducing apoptosis. Although TNF- can trigger apoptosis in some solid tumors, its clinical usage has been limited by the risk of lethal systemic inflammation [44]. By comparing hCSCs treated with PEITC to those without PEITC, we observed PEITC also induced the expression of death receptors DR4 and DR5 in hCSCs, which has not been reported earlier. PEITC was, however, previously shown to upregulate DR4 and DR5 in a different human cervical cancer cell line [12]. The expression levels of either DR4 alone or both death receptors are correlated with TRAIL sensitivity of a cell line [45]. Our result revealed expression of both death receptors were elicited following PEITC treatment, but DR5 expression increase was to a lesser extent compared with DR4s increase. TRAIL is known to trigger apoptosis through binding to DR4 or DR5, which contain cytoplasmic death domains responsible for recruiting adaptor molecules involved in caspase activation [21]. Since all treatments shown in Figure4 included TRAIL treatments, the observations indicate that hCSCs are more prone to TRAIL treatment after incubation with PEITC. While the biological activity of PEITC in inducing apoptosis of cancer cells may involve death receptor signaling, other mechanisms have also been suggested [12, 13]. Finally, to investigate the antagonistic effects of PEITC on hCSC tumorigenicity in vivo, we carried out xenotransplantation in immune-compromised mice. Mice receiving untreated hCSCs produced the highest numbers of tumors and also showed greater invasiveness, as confirmed by the presence of lung metastases. However, given the short 3-week duration of the experiment, metastasis was found in only one of the four animals in the hCSC group but in no other animal in the remaining groups. We observed a marked reduction in tumorigenicity in mice that had received a PEITC-treated hCSC inoculum, and the outcome was comparable to the HeLa-injection group. It should be noted here that the sphere culture approach to isolation of hCSCs that we used in the study followed by cell-surface marker-based characterization helps to identify CSC-enriched subpopulations but did not enable unambiguous isolation of all of the CSCs.

Excerpt from:
Phenethyl isothiocyanate upregulates death receptors 4 and ...

BMC Cell Biology201011:29

DOI: 10.1186/1471-2121-11-29

Cselenyk et al; licensee BioMed Central Ltd.2010

Received: 2September2009

Accepted: 20April2010

Published: 20April2010

Bone marrow derived mesenchymal stem cells (MSCs) are promising candidates for cell based therapies in myocardial infarction. However, the exact underlying cellular mechanisms are still not fully understood. Our aim was to explore the possible role of direct cell-to-cell interaction between ischemic H9c2 cardiomyoblasts and normal MSCs. Using an in vitro ischemia model of 150 minutes of oxygen glucose deprivation we investigated cell viability and cell interactions with confocal microscopy and flow cytometry.

Our model revealed that adding normal MSCs to the ischemic cell population significantly decreased the ratio of dead H9c2 cells (H9c2 only: 0.85 0.086 vs. H9c2+MSCs: 0.16 0.035). This effect was dependent on direct cell-to-cell contact since co-cultivation with MSCs cultured in cell inserts did not exert the same beneficial effect (ratio of dead H9c2 cells: 0.90 0.055). Confocal microscopy revealed that cardiomyoblasts and MSCs frequently formed 200-500 nm wide intercellular connections and cell fusion rarely occurred between these cells.

Based on these results we hypothesize that mesenchymal stem cells may reduce the number of dead cardiomyoblasts after ischemic damage via direct cell-to-cell interactions and intercellular tubular connections may play an important role in these processes.

Cardiovascular diseases represent an enormous medical and social burden [1, 2] and the pathophysiology of most of these diseases, such as myocardial infarction or heart failure, involves death of cardiac myocytes leading to a loss of functional tissue. Cell based therapies are commonly believed to be the next generation of therapies for replacing such lost tissue [35]. Several in vivo animal and human studies have found that implantation of various cell types, typically bone marrow derived stem cells, into damaged myocardium improved cardiac performance. Also where the experimental protocol allowed, surviving grafted cells were detected in the myocardium [6, 7], suggesting that grafting is an effective treatment of acute myocardial infarction [8]. However, the extent of the beneficial effect, the optimal cell type and number, the best method of administration, and the mechanism of action need to be further evaluated [9].

One important issue is the exact mechanism of action, in other words, the interaction between graft and host. Paracrine factors, transdifferentiation and cell fusion are the three generally accepted hypotheses explaining the beneficial effects of stem cell grafting. Paracrine factors through various effects, such as increased angiogenesis or modulation of postinfarct remodeling, may represent an important aspect of the benefits [10, 11]. On the other hand, several studies have found that co-culture of cardiomyocytes with pluripotent stem cells resulted in transdifferentiation of these cells into cardiomyocytes, which raised the hope that in vitro cultured tissue blocks can later be used for cardiac repair [12, 13]. However, although it is possible to construct a tissue in vitro this does not mean that its building blocks will perform similarly when implanted in vivo [14]. Indeed, recent investigations found difficult to reproduce transdifferentiation and that bone marrow derived cells generate cardiomyocytes not by transdifferentiation but rather through cell fusion [15, 16]. Alvarez-Dolado et al demonstrated that bone marrow derived cells fused with cardiomyocytes [17], but the importance of cell fusion events was questioned by an other investigation [18]. Even in studies which found morphologically adequate new cardiomyocytes, the volume of this newly formed tissue seemed to be inadequate to account for the functional benefits. Other hypotheses have also emerged to resolve the apparent controversy among the clinical findings and the cell culture studies, such as the most recently proposed partial cell fusion through direct cell-to-cell interactions. This novel intercellular communication route depends on short cell-to-cell interactions, during which the two connected cells exchange membrane and organelle parts such as mitochondria or other cytoplasmatic components [19]. Recently, it was reported that cardiomyocytes and human mesenchymal stem cells appear to communicate through small diameter nanotubes, and mitochondria can migrate from MSCs to cardiomyocytes [20]. However, the physiological purpose of this constantly changing nanotubular network and its possible role during ischemic conditions is unclear. We hypothesized that stem cells and post-ischemic cardiomyoblasts interact with each other via this novel mechanism and that this mechanism may play a role in the beneficial effect of stem cell transplantation.

The aim of our study was to examine the possibility of rescuing ischemically damaged H9c2 cardiomyoblasts from cell death by adding mesenchymal stem cells to the cultures after ischemia. Furthermore we investigated the importance of direct cell-to-cell interactions during co-cultivation of these cells.

H9c2 rat cardiomyoblasts were obtained from ATCC (Wesel, Germany) and expanded in high glucose (4.5 g/L) DMEM containing 10% fetal bovine serum, 4 mM L-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin. Mouse mesenchymal stem cells (MSCs) were harvested from the femur of C57Bl/6 mice. Isolation and primary culture was performed according to Tropel's method with small alterations [21]. Briefly, animals were anaesthetized with pentobarbital (ip, 50 mg/kg, Nembutal, Ovation, Deerfield, IL, USA), lower limbs were removed and femurs were cleaned of tissue. Bone marrow was collected by flushing femurs with low glucose DMEM containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin. Cells were centrifuged at 1200 rpm and plated in a T75 flask. After 4-5 days, non-adherent cells were removed by washing twice with PBS and adherent cells were then cultured in low glucose DMEM complete medium. Characterization of the cultured MSCs showed that these cells were strongly positive for the specific surface antigen Sca-1 and negative for differentiation markers of other cell lineages (CD34, CD3, CD45R/B220, CD11b, 6G, and TER-119) and were able to differentiate into the osteoblast and adipocyte lineages in vitro, verifying the MSC phenotype [22]. Cell culture media was replaced every 2-3 days thereafter. All investigations conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1985), and were approved by the local ethics committee.

Ischemia-reperfusion was simulated in vitro by performing oxygen glucose deprivation (OGD) on H9c2 cell cultures. Cells were incubated in glucose-free DMEM in an atmosphere of 0.5% O2 and 99.5% N2 for 150 minutes. This procedure was performed on the stage of the confocal microscope (PECON incubation system, Erbach-Bach, Germany) allowing the observation of the cells during OGD. To evaluate cell viability we used calcein-AM (excitation/emission 494/517 nm) to identify live cells, and ethidium-homodimer (excitation/emission 528/617 nm) to stain dead or damaged cells [23].

H9c2 cells and MSCs were labeled before co-cultivation with Vybrant DiO (excitation/emission: 488/501 nm) and DiD (excitation/emission: 633/665 nm) (Molecular Probes, USA) membrane dyes in a dilution of 1:200 according to the manufacturer's description for 30 minutes at 37C. The DiO-labeled H9c2 cells were plated in 12 well-plates at a density of 30,000 cells/well in 2 ml culture medium. Cells were subjected to 150 min OGD, then the medium was changed and 20,000 DiD-labeled MSCs/well were added to the damaged H9c2 cells 30 minutes after the end of OGD either directly or in cell culture inserts (0.4 m pore size, Becton Dickinson, NJ, USA). H9c2 cells not receiving MSCs after OGD were used as controls. Cells were cultivated for a further 24 hours, then labeled with the dead cell stain ethidium homodimer (4 M, 30 minutes, and 37C), then investigated either with confocal microscopy (Zeiss LSM 510 META, Carl Zeiss, Jena, Germany) or with flow cytometry (BD FACSCalibur, Becton Dickinson, NJ, USA).

Time lapse video microscopy was performed during and after in vitro ischemia to investigate morphological changes and possible interactions among the cells over time (1 picture/3 minutes). The H9c2 cardiomyoblasts and MSCs were co-cultured on 42 mm coverslips and stained with Vybrant DiO and DiD, respectively. In experiments to observe mitochondria, all cells were stained after OGD with MitoTracker Red (Molecular Probes, USA) in a dilution of 1:2000 for 10 minutes at 37C according to the manufacturer's description. Flow cytometric measurements were performed on single cell suspensions of trypsinized (0.05% trypsin-EDTA) cell cultures 24 hours after OGD and on normal cell cultures (control) using BD FACSCalibur. DiO-labeled H9c2 cells were identified and gated. Fluorescence data were collected using logarithmic amplification until 10,000 counts were reached.

The evaluation of confocal images for live and dead cells selected by morphology and fluorescence was performed with ImageJ software (National Institutes of Health, USA). In case of co-cultures, MSCs were distinguished from H9c2 cells due to their Vybrant DiD cell labeling. The ratio of dead cells was evaluated in 4 independent fields of view (objective 10) for each culture in a blind fashion. The evaluation of flow cytometry files was carried out using BD CellQuest Pro (Becton Dickinson, NJ, USA). Statistical analysis of data was carried out using one-way analysis of variance with Tukey's multiple comparison post hoc test. Data are expressed as mean SEM.

The optimal duration of OGD to induce cell damage was 150 minutes (Figure

and additional file

: video1.mov). This result is based on microscopic observations of morphological changes in the cell shape and on ethidium homodimer staining which determined whether a particular cell was dying. Flow cytometric analysis was also used to determine that the selected time interval was sufficient to injure the majority of the cardiomyoblasts (Figure

).

Ischemia model on cardiomyoblasts. (A) Follow up of OGD on cardiomyoblasts. Cells were stained with calcein-AM (ex/em 494/517 nm) for live cells (green) and ethidium homodimer (ex/em 528/617 nm) for dead cells (red). (B) Flow cytometry analysis of control and ischemic cardiomyoblasts labeled with ethidium homodimer after OGD. The green curve represents the control cardiomyoblasts and the red shows the ischemic cardiomyoblasts. The complete rightward shift of the red curve based on these representative data indicates that OGD increased the number of dead cells nearly maximally.

Experiments showed that 4-6 hours was not adequate for the added MSCs to attach to the surface of the 12-well plates and exert their effect, 48 hours produced a culture overgrown by cardiomyoblasts and MSCs (data not shown). Therefore, the time point for microscopic evaluation and flow cytometry analysis subsequent to addition to MSCs was determined to be 24 hours.

Confocal microscopy showed that cardiomyoblasts cultured alone displayed the same rounded and blebbed morphology immediately following as well as 24 hours after OGD (Figure

). Flow cytometry analysis showed that OGD significantly increased the cell death rate in this group as shown by the enhanced ethidium homodimer fluorescence intensity (median fluorescence from 19 to 65 units, Figure

). Figure

also shows that a portion of cells remained unstained with ethidium homodimer even after 150 minutes due to the variability of the model. When MSCs were added to post-ischemic cardiomyoblasts, the morphology of the damaged cells was similar to cells cultured in normal conditions without OGD (Figure

). In this group, flow cytometry analysis revealed that the deleterious results of ischemia were decreased (median fluorescence 24 versus 23 units, Figure

). To quantify the effect of added MSCs, confocal images were used. This approach revealed that the ratio of dead H9c2 cells to all H9c2 cells in the wells 24 hours after OGD was significantly higher when the cardiomyoblasts were cultured alone compared to when healthy MSCs were added to the cultures 30 minutes after OGD (0.85 0.086 vs. 0.16 0.035, respectively, p

and

). The absolute number of live H9c2 cells before and after OGD and the number of added MSCs after OGD was also investigated. Before OGD H9c2 cells were close to confluence (63,120 7,694) and there was little increase in cell numbers during the next 24 hours if the cells were left to grow without OGD (76,116 3,396). The number of viable cells 24 hours after OGD was very low when cultured alone or with MSCs in cell insert (1,757 1,081 and 990 608 respectively), but significantly increased (15,174 3,975) if MSCs were added directly. It can be assumed that the injured H9c2 cells were washed out during medium change so only a part of H9c2 cells remained in the wells (Figure

). We also examined the inserts with confocal microscopy to eliminate the possibility of decreased MSC viability on the cell culture inserts. MSCs were labeled with Vybrant DiD before seeding on the cell inserts. We found that Vybrant DiD labeled MSCs were attached to the surface of the inserts and showed normal cell morphology (Figure

). We also investigated whether any of the MSCs had contaminated the underlying H9c2 cardiomyoblast culture, but found no trace of MSCs among the H9c2 cells; therefore no direct cell-to-cell contact could be formed (data not shown).

Morphology and viability of H9c2 cells 24 hours after OGD. (A) DiO-labeled H9c2 cells without MSCs observed 24 hours after OGD were predominantly rounded up and stained with ethidium homodimer indicating cell death in progress. (B) Flow cytometry analysis of control and ischemic H9c2 cells cultured for 24 hours after OGD labeled with ethidium homodimer (ex/em 528/617 nm) showed that the number of dead H9c2 cells was elevated compared to the control group (median fluorescence from 19 to 65 units). (C) Co-cultivation of DiO-labeled H9c2 (ex/em 488/501 nm) cells and DiD-labeled MSCs (ex/em 633/665 nm) for 24 hours after OGD showed that the morphology of ischemically damaged cells were normal after 24 hours. (D) Flow cytometry analysis revealed that after co-cultivation of cells the number of dead H9c2 cells remained on the control level (median fluorescence 24 versus 23 units).

Co-cultivation of H9c2 cells with MSCs decreased cell death. (A) Experimental layouts after in vitro ischemia. (B) The ratio of dead H9c2 cells was significantly smaller when MSCs were added after OGD (0.85 0.086 vs. 0.16 0.035, n = 5), but MSCs added in cell culture inserts did not decrease significantly the ratio of dead H9c2 cells (0.90 0.055, n = 5). Data represent mean SEM. *p < 0.05 C+MSC vs. C and C+MSC vs. C+MSC ins. (C: H9c2 cells only; C+MSC: H9c2 cells and MSCs; C+MSC ins: H9c2 cells and MSCs in cell culture inserts) (C) Absolute number of live H9c2 cells before and after OGD shows that before OGD the H9c2 cells were close to confluence (63,120 7,694) and there was little increase in cell numbers during the next 24 hours if the cells were left to grow without OGD (76,116 3,396). 24 hours after OGD the number of viable cells was very low when cultured alone or with MSCs in cell insert (1,757 1,081 and 990 608 respectively), which was significantly increased (15,174 3,975) if MSCs added directly. (D) MSCs labeled with Vybrant DiD were growing on cell culture inserts in the same manner as under normal culture conditions after 24 hours of cultivation. Scale bar represents 100 m.

Development of intercellular connections, so-called nanotubes, between cardiomyoblasts and MSCs during the 24 hr period after OGD was frequently observed (Figure

). These nanotubes were long enough to span distances of several cell diameters, and their diameters were between 200 and 500 nm. MitoTracker Red staining revealed that these nanotubes connecting stem cells to cardiomyoblasts contained functionally active mitochondria (Figure

). Time lapse video microscopy did not reveal a specific direction for the movement of these mitochondria in the intercellular connections and the typical time frame for the formation of a nanotube was approximately 2 hours (Figure

and additional file

: video2.mov).

Formation of intercellular connections after OGD. (A) Nanotubular network formation was observed among DiO-labeled cardiomyoblasts (green) and DiD-labeled MSCs (red) after 24 hours of co-culture. (B) MitoTracker staining (red) revealed active mitochondria in the nanotubular network (yellow arrows). (C) Time lapse pictures of the formation of a nanotube between a DiO-labeled cardiomyoblast (green) and DiD-labeled stem cell (red).

Addition to the formation of intercellular communications, double labeled and double nuclei cells indicated that cell fusion events were present in the co-cultures. The typical time frame for a cell fusion was approximately 4 hours (Figure

and additional file

: video3.mov). To examine whether cell fusion occurs in normal, non-ischemic conditions among H9c2 cells and MSCs we co-cultured these cells without OGD and found that such phenomenon also occurs among healthy cells. Flow cytometry analysis showed beside 59.42% of DiO-labeled H9c2 cells and 30.1% of DiD-labeled MSCs also 8.14% of double labeled cells. However, according to the forward and side scatter plot the distribution demonstrates that most of the double labeled cells are the same size as H9c2 cells or MSCs, suggesting that these cells are picking up the other marker through direct cell-to-cell contact (Figure

and

).

Cell fusion of a H9c2 cell and a mesenchymal stem cell. The time lapse pictures demonstrate steps of the fusion of a H9c2 cell (green) and a mesenchymal stem cell (red). Fused cells with double nuclei exhibit a combined yellow staining.

Co-culture of H9c2 and stem cells in normal conditions. (A) Cardiomyoblasts (green) and MSCs (red) after one day co-cultivation. (B) A representative double labeled cell with double nuclei (nuclei were stained with Hoechst). (C) DiO-labeled cardiomyoblasts (green) and DiD-labeled MSCs (red) analyzed with flow cytometry after one day co-cultivation. We found three different cell populations: 59.42% of DiO-labeled H9c2 cells, 30.1% of DiD-labeled MSCs and 8.14% of double labeled cells. (D) The distribution according to the forward scatter plot demonstrates that several double labeled yellow cells are mostly the same size as the green H9c2 cells or the red MSCs disapproving complete cell fusion.

We also considered the possibility that MSCs may incorporate cell debris and thus acquire double labeling of fluorescent dyes. During the observations performed with time-lapse video microscopy we frequently saw that healthy cells contacted and moved around cell debris and apoptotic bodies in the culture dish, however, phagocytosis was not observed. The cells which contacted differentially stained cell debris did not pick up any fluorescent signal from the other, indicating that the double labeling of cells arose from a specific and controlled mechanism rather than cross-contamination. This is further strengthened by the observation that not all cell-to-cell connection resulted in dye transfer. (additional file 4: video4.mov).

We report that healthy mesenchymal stem cells are capable of rescuing post-ischemic cardiomyoblasts from cell death through a mechanism not yet implicated in the effects of stem cells after ischemic conditions. Thus, the beneficial effect of stem cell grafting may be based not only on improved neovascularisation and replacement of lost cells but on rescuing the damaged cells of the host as well.

The most likely explanation of the beneficial effects of MSC co-culture is that these cells improve the chances of the damaged H9c2 cells to restore their function and prevent later cell death. Ethidium homodimer has been shown to stain not only dead but damaged cells as well [23]. Thus, although most H9c2 cells were stained by ethidium homodimer after OGD (Figure 1B), many cells were probably only reversibly damaged. An alternative explanation of our results could be an increased replication of surviving H9c2 cells. However, the nearly ten-fold difference in the number of viable H9c2 cells between our experimental groups 24 hr after OGD (Figure 3C) and the normal doubling time of these cells make this possibility unlikely to explain the difference.

The used in vitro ischemia model demonstrates that the beneficial effects of MSC co-culture seem to be dependent on direct cell-to-cell connections and intercellular nanotubes.

Nanotube formation has already been shown to occur among endothelial progenitor cells, cardiomyocytes [20, 24], immune cells and other lineages [25, 26]. The characterization of nanotubes revealed that these filaments contain actin and in some cases, microsomes or mitochondria [27]. We found that this phenomenon occurs frequently between cardiomyoblasts and mesenchymal stem cells. Stem cells failed to rescue post-ischemic cardiomyoblasts when intercellular connections were blocked by a physical barrier. These observations indicate that intercellular connections work toward the survival of cells both during and after ischemia, however, the underlying mechanisms may be slightly different.

One plausible mechanism for the rescuing effect is that transplanted cells improve regeneration through secreting paracrine factors [14, 2831]. However, results from our experiments with the plate insert show that paracrine factors secreted by the cells are probably too low in our system to have any beneficial effect on these severely damaged cells. This in vitro experimental setup allows the investigation of cell-to-cell contacts, however, it cannot rule out that paracrine effects play a significant role in a more physiological in vivo setting. The time frame of the experimental protocol is also important. In our experiments we added the cells at an early time point and terminated the experiment before significant differentiation can occur. During a later time point the effect of paracrine factors is probably much more important especially in the differentiation process as shown by several other authors [6, 11].

Cell fusion is another phenomenon which is frequently observed in co-culture studies and in some cases, in in vivo experiments as well [15, 32, 33]. Several studies have shown that cell fusion can result in transdifferentiation, thus offering an alternative mechanism by which grafted cells improve the infarcted myocardium. Using videomicroscopy we also found several double labeled, double nuclei cells indicative of cell fusion. However, cell fusion showed high variations among different culture and detection techniques, and therefore extensive cell fusion as an in vitro artefact cannot be ruled out [14, 34, 35]. During our investigations we only observed a few unquestionable cell fusions which cannot account for the rescue of the high number of damaged cardiomyoblasts [36].

We also found double labeled cells without double nuclei in the co-culture of cardiomyoblasts and stem cells after 24 hours. The double labeling of these cells may be the result of direct cell-to cell connections. During these periods of connection, cells are able to exchange membrane parts and Vybrant dye molecules can drift from one cell to another. Movement of dye molecules from one cell to another through gap junction connections is precluded because the lipophilic Vybrant dyes are high molecular weight stains and cannot permeate through gap junctions [34]. Driesen et al. [19] showed that low molecular weight tracers such as calcein-AM get from one cell to another through gap junctions, and high molecular weight tracers by partial cell fusion, thus the conclusion may be drawn that dye transfers after 24 hours in our experiment are most probably the results of direct cell-to-cell connections. Still, gap junctions may create an opportunity for grafted cells to interact with the host tissues [37, 38]. In the present experimental model most of cell-to-cell interactions were short-lived tubular connections, which formed a constantly changing web between the two investigated cell types.

Our experimental model was devised to investigate acute effects with high temporal and spatial resolution, therefore ruling out differentiation, which occurs over time. Moreover, an in vitro transplantation model in a cell culture system cannot mimic the 3-dimensional tissue where cell-to-cell connections are different. These circumstances obviously limit the conclusions drawn from our results. On the other hand, this experimental setting was necessary and favorable to investigate short-term cellular interactions.

The present study highlights that stem cell grafting may be beneficial through an acute, direct mechanism which saves damaged cardiomyoblasts. Novel grafting protocols can harness this effect, which raises the possibility that stem cells given early and locally can preserve heart tissue rather than simply help to replace what is already lost.

This work was supported by OTKA (Hungarian Scientific Research Fund) D45933, T049621, TT (Hungarian Science and Technology Foundation) A4/04 and Arg-17/2006, Bolyai, veges Fellowships and TMOP 4.2.2-08/1/KMR-2008-0004. We would like to thank Dr. Ferenc Uher for providing the mouse mesenchymal stem cells. We are grateful to Nancy Busija for copyediting the manuscript and we thank David Busija and Mrk Kollai for critically revising the manuscript.

Below are the links to the authors original submitted files for images.

ACS and ZSL conceived the study. ACS completed the majority of the confocal microscopy experiments, made the flow cytometry measurements, performed statistical analysis, and helped draft the manuscript. EP helped draft the manuscript and performed statistical analysis. EMH participated in the flow cytometer measurements. KL participated in the time lapse video microscopy experiments and helped draft the manuscript. In addition to collaborating on the conception of the study, ZSL participated in the study design, provided coordination among the researchers and experiments, and helped draft the manuscript. All authors read and approved the final manuscript.

Institute of Human Physiology and Clinical Experimental Research, Semmelweis University

Cselenyk et al; licensee BioMed Central Ltd.2010

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Research article

Open Access

BMC Cancer201414:591

DOI: 10.1186/1471-2407-14-591

Wang et al.; licensee BioMed Central Ltd.2014

Received: 21April2014

Accepted: 11August2014

Published: 15August2014

The cytokine TRAIL (tumor necrotic factor-related apoptosis-inducing ligand) selectively induces apoptosis in cancer cells, but cancer stem cells (CSCs) that contribute to cancer-recurrence are frequently TRAIL-resistant. Here we examined hitherto unknown effects of the dietary anti-carcinogenic compound phenethyl isothiocyanate (PEITC) on attenuation of proliferation and tumorigenicity and on up regulation of death receptors and apoptosis in human cervical CSC.

Cancer stem-like cells were enriched from human cervical HeLa cell line by sphere-culture method and were characterized by CSC-specific markers analyses (flow cytometry) and Hoechst staining. Cell proliferation assays, immunoblotting, and flow cytometry were used to assess anti-proliferative as well as pro-apoptotic effects of PEITC exposure in HeLa CSCs (hCSCs). Xenotransplantation study in a non-obese diabetic, severe combined immunodeficient (NOD/SCID) mouse model, histopathology, and ELISA techniques were further utilized to validate our results in vivo.

PEITC attenuated proliferation of CD44high/+/CD24low/, stem-like, sphere-forming subpopulations of hCSCs in a concentration- and time-dependent manner that was comparable to the CSC antagonist salinomycin. PEITC exposure-associated up-regulation of cPARP (apoptosis-associated cleaved poly [ADP-ribose] polymerase) levels and induction of DR4 and DR5 (death receptor 4 and 5) of TRAIL signaling were observed. Xenotransplantation of hCSCs into mice resulted in greater tumorigenicity than HeLa cells, which was diminished along with serum hVEGF-A (human vascular endothelial growth factor A) levels in the PEITC-pretreated hCSC group. Lung metastasis was observed only in the hCSC-injected group that did not receive PEITC-pretreatment.

The anti-proliferative effects of PEITC in hCSCs may at least partially result from up regulation of DR4 and possibly DR5 of TRAIL-mediated apoptotic pathways. PEITC may offer a novel approach for improving therapeutic outcomes in cancer patients.

Despite considerable improvement in cancer diagnosis and therapy, relapse and metastasis are still common [1]. However, the rise of the cancer stem cell (CSC) hypothesis provides a new approach to eradicating malignancies. Recent studies have shown that CSCs are a small subpopulation of tumor cells that possess self-renewal and tumor-initiation capacity and the ability to give rise to the heterogeneous lineages of malignant cells that comprise a tumor [2]. CSCs have been identified in hematologic and solid cancers and implicated in tumor initiation, development, metastasis, and recurrence. Although the origin(s) and dynamic heterogeneity of CSCs remain unexplained, designing novel approaches to target CSCs has received much attention over the past several years [35].

Phenethyl isothiocyanate (PEITC) is a dietary compound derived from common vegetables such as watercress, broccoli, cabbage, and cauliflower [6]. We and others have shown under experimental conditions that PEITC possesses anti-inflammatory [7, 8] and chemopreventive activity against various cancers, including colon [9], prostate [10], breast [11], cervical [12, 13], ovarian [14], and pancreatic cancer [15]. Safety studies in rats and dogs have shown that PEITC has no apparent toxicity, even when administered in high doses, as determined by NOEL (no-observed-adverse-effect-level) [16], and PEITC is currently in clinical trials in the US for lung cancer (NCT00691132). Cervical cancer is the second-most-fatal cancer in women worldwide, and the incidence rate is significantly higher in developing nations due to the absence of rigorous screening programs [17]. A recent study showed that PEITC can induce the extrinsic apoptosis pathway in a human cervical cancer cell line [12]. However, the chemotherapeutic effects of PEITC in the context of CSCs and more specifically cervical CSCs remain unknown.

Apoptosis, or programmed cell-death, is essential to maintaining tissue homeostasis, and its impairment is implicated in many human diseases, including cancers [18]. The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor super-family, has attracted great interest for clinical applications due to its specific anti-tumor potential without toxic side effects to normal healthy cells [19, 20]. There are two well-characterized apoptosis pathways in mammalian cells. The extrinsic pathway is mediated by death receptors, a subgroup of the TNF receptor superfamily. TRAIL binds to TRAIL-R1 and TRAIL-R2, two death domain-containing receptors, also called DR4 and DR5, to trigger apoptosis. The intrinsic pathway involves mitochondria, and is triggered and controlled by members of the Bcl-2 protein family. Both pathways cause the activation of initiator caspases, which then activate effector caspases [21]. Caspases cause cleavage and inactivation of poly (ADP-ribose) polymerase 1 (PARP)-1, which helps repair single-stranded DNA breaks, and hence PARP-1 cleavage serves as a hallmark of apoptosis [22]. Unfortunately, a variety of human tumors develop resistance to TRAIL-induced apoptosis [23]. But further studies have suggested that TRAIL activity can be sensitized with other chemotherapeutic drugs, such as paclitaxel [24], 5-fluorouracil (5-FU) [25], and cisplatin [26] or dietary bioactive compounds like benzyl isothiocyanate (BITC) [27] or sulforaphane [28, 29]. However, the effects of PEITC on TRAIL pathway in CSCs have not been reported.

In the present study, we investigated the efficacy of PEITC in attenuating the growth of sphere-forming cervical CSCs isolated from HeLa cells (hCSCs) as well as its ability to up regulate death receptors for TRAIL-mediated induction of apoptosis. Furthermore, the in vivo anti-tumorigenicity effects of PEITC were evaluated in a xenograft mouse model.

Phenethyl isothiocyanate (Sigma-Aldrich, St. Louis, MO), 99%, was diluted in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO) to make 0.520-mM stock concentrations and was further diluted in media to obtain 2.520-M final concentrations, which are achievable following oral administration in human [30] and have been used in prior studies by us and others to induce apoptosis in the SW480 colon cancer cell line [9] and cervical cancer cell lines. We used comparable concentrations of salinomycin (2.520M) and lower concentrations (2.520 nM) of paclitaxel (both from Sigma-Aldrich, St. Louis, MO) as positive controls, which are CSC-targeted and CSC-non-specific anti-cancer chemotherapeutics, respectively, following Gupta et al. [31]. For the negative/vehicle control samples, we used DMSO in an amount equivalent to that used with test compounds in test samples.

The human HeLa cell line (ATCC CCL-2, American Type Culture Collection, Manassas, VA) was cultured and maintained in a T-25 flask with Dulbeccos modified eagles medium (DMEM) containing 4mML-glutamine and 4.5g/L glucose (HyClone, Logan, UT), supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Grand Island, NY) and 1% penicillin (25 U/ml)/streptomycin (25g/ml) (Sigma-Aldrich, St. Louis, MO) in a 5% CO2-humidified atmosphere at 37C. HeLa cells were trypsinized with TrypLE (Invitrogen, Grand Island, NY) and then sub-cultured with a 1:5 splitting ratio when the cells reached about 90% confluency. From the parental HeLa cells (termed simply as HeLa in the rest of the document), hCSCs were cultured following a modified protocol described by Gu et al. [5]. Briefly, single-cell suspensions of HeLa cells (4104) were seeded into a 100-mm ultra-low attachment (ULA) petri dish (Corning Inc., Corning, NY) containing 8ml of serum-free mammary epithelial basal medium (MEBM, Lonza, Allendale, NJ), supplemented with 1 B27 (Invitrogen, Grand Island, NY), 4g/ml heparin (Sigma-Aldrich, St. Louis, MO), 20ng/ml hEGF, and 20ng/ml hFGF (Invitrogen, Grand Island, NY). After an initial 4-day culture in suspension at 37C, an additional 9ml of sphere culture medium was added for another 5days of culture. On day 9, spheres were harvested by centrifugation at 500 g for 3minutes, followed by washing with phosphate-buffered saline (PBS), trypsinization with TrypLE for 10minutes at 37C, centrifugation at 500 g for 3minutes, resuspension in 5ml of hCSC culture medium, and counting with a hemocytometer. Both HeLa cells and hCSCs were used for successive experiments.

Around 2106 HeLa cells were seeded into a 60-mm petri dish and incubated overnight at 37C. Cells were washed with 2ml of PBS, trypsinized with 1ml of TrypLE, and resuspended in 1ml of PBS, followed by immunostaining. Similarly, hCSCs were collected after 9days of culture, trypsinized, and resuspended in 2ml of PBS with a density of 1106 cells/ml, followed by immunostaining. Cells were immunostained with anti-CD24FITC (1:500v/v, Millipore, Billerica, MA) or anti-CD44FITC (1:500v/v, Millipore, Billerica, MA) antibodies for 1hour at room temperature. Immunofluorescence was measured using a FACSCalibur cell analyzer (Becton Dickinson, San Jose, CA) with approximately 10,000 events in each sample. Propidium iodide/annexin V staining was performed according to the manufacturers instructions. Briefly, 5105 cells were centrifuged and resuspended in 100l of 1x binding buffer (Invitrogen, Grand Island, NY). The cells were treated with 10M PEITC or vector control (DMSO) for a total of 24h, in the last hour of which 10ng/ml of human recombinant TRAIL (eBioscience, Inc., San Diego, CA) or vector control (DMEM) were added to the cells before harvesting. The cells were then incubated with 5l of annexin VFITC (eBioscience, Inc., San Diego, CA) and 5l of propidium iodide (eBioscience, Inc., San Diego, CA) at room temperature for 5minutes in the dark before analyzing the cells on a FACSCalibur cell analyzer. For DR4 and DR5 expression analysis, 5105 cells were filtered through a filter cap (35m) into a collecting tube (BD Falcon, Franklin Lakes, NJ) and then washed, fixed with 2% paraformaldehyde, and stained with DR4 or DR5 surface markers (1:200v/v) overnight at 4C in a rotating vessel. The immunostained cells were incubated with goat anti-mouse Dylight 488 (1:500v/v) secondary antibody for 2hours at room temperature before acquiring at least 10,000 cells in a flow cytometer.

The fluorescence resulting from interaction of cell DNA with Hoechst 33342 dye was measured to assess the cells ability to efflux the fluorescent dye Hoechst 33342, as most hematopoietic stem cells are able to exclude the dye [32]. HeLa or hCSCs were trypsinized with TrypLE, washed with PBS, and adjusted to 1106 cells/ml in Hanks balanced salt solution (HBSS), before incubating with 5g/ml Hoechst 33342 dye (Life Technologies, Grand Island, NY) for 60minutes at 37C in a 5% CO2 incubator. The cells were then washed three times with HBSS by centrifugation at 300 g for 5minutes. The pellets were resuspended at 1106 cells/ml in HBSS and kept on ice until used for imaging. The Hoechst staining was visualized with an EVOS FL Epifluorescent Microscope (AMG, Bothell, WA) using the DAPI channel. Images were indicated as transmitted (phase contrast images of whole cells), Hoechst-stained (nuclei with Hoechst staining), and merge (an overlay of transmitted and Hoechst staining in the same field). The cells with Hoechst-stained nuclei were counted among 100 cells, and the number of Hoechst-excluded cells was then quantified.

The hCSCs were enriched in spheres in serum-free medium. Sphere culture was carried out as previously described in the sphere culture section. Cells were treated with predetermined doses of 0.5, 1.0, or 2.5M of PEITC or DMSO as control. After 7days incubation, photomicrographs of spheres were acquired under an inverted phase-contrast microscope (Olympus America Inc., Center Valley, PA), and the number of hCSCs was counted using a hemocytometer.

A standard colorimetric method (MTS assay) was used to determine the number of viable cells in samples. For cell-proliferation assays, HeLa and hCSCs were cultured for 4days, and an additional 9ml of sphere culture medium was added for another 5days, as described in the sphere culture section. Viable cells were harvested and counted with a hemocytometer before seeding into 96-well microplates at a density of 2104 cells per well. Cells were cultured in DMEM supplemented with 100 U/ml penicillin, 100g/ml streptomycin, 5% heat-inactivated FBS, and 50M 2-mercaptoethanol. Both hCSCs and HeLa cells were treated with four concentrations of PEITC and salinomycin (2.520M) and paclitaxel (2.520 nM). After 24 and 48hours of incubation, 20l of CellTiter reagent was added directly to the cell-culture wells and incubated for 1hour at 37C, followed by cell viability assessment using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI), containing [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS]. The manufacturers instructions were followed, and treatments were compared with vehicle control (DMSO-treated cells) at 490nm in a BioTek Synergy H4 multimode plate reader (BioTek, Winooski, VT).

hCSCs (1106) were seeded in each well of a 6-well plate and incubated overnight at 37C in a 5% CO2 incubator. Old culture medium was replenished by culture medium containing either 10-M or 15-M concentrations of PEITC for 5hours. The cells were then treated with 10ng/ml human recombinant TRAIL or with 10ng/ml TNF (eBioscience, Inc., San Diego, CA) for additional 1-hour incubation. Cell harvesting and immunoblotting were carried out as we previously reported [9]. Briefly, cells were lysed in ice-cold RIPA buffer containing 150mM NaCl, 50mM Tris (pH8.0), 10% glycerol, 1% Nonidet P-40 (NP-40), and 0.4mM EDTA, followed by a brief vortexing and rotation for 30minutes at 4C. Equal amounts (v/v) of cell lysates were separated by SDS-PAGE through a 12% separating gel, transferred to nitrocellulose membranes, blocked with 5% non-fat dry milk, and double-probed overnight at 4C with mouse anti-human cPARP (1:1000v/v, Millipore, Billerica, MA) and rabbit anti-human -actin (1:5000v/v, Millipore, Billerica, MA) antibodies. Blots were then washed in PBS and further incubated with secondary antibodies, Dylight 680 anti-mouse (1:5000v/v) and Dylight 800 anti-rabbit (1:5000v/v), for 1hour at room temperature. Finally, after rinsing in Tween20 (0.1% in PBS), blots were imaged with a LI-COR Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE), followed by a densitometric analysis of cPARP levels after normalizing with the -actin housekeeping gene.

Animal studies were carried out in accordance with the guidelines of, and, using an approved protocol by, the Institutional Animal Care and Use Committee (IACUC), South Dakota State University (IACUC protocol approval #12-087A). Twenty female non-obese diabetic, severe combined immunodeficient (NOD/SCID, NOD.CB17-Prkdc scid/J) mice (Jackson Laboratories, Bar Harbor, ME), 17weeks old, were randomly grouped into five groups (four mice per group) in specific pathogen-free (SPF) housing at a constant temperature of 2426C with a 12-h:12-h light/dark cycle. All mice were allowed to acclimatize for 1week and were provided with sterile food and water ad libitum. HeLa and hCSCs were cultured, trypsinized, washed, pre-treated with 10M PEITC where indicated, and resuspended in PBS at the concentration of 1107 cells/ml before injecting into the mice. Each mouse was subcutaneously injected at the neck scruff with one injection of PBS (100l, control group), HeLa (1106), HeLa pretreated with 10M PEITC (1106), hCSCs (1106), or hCSCs pretreated with 10M PEITC (1106). The cell number in each injection was consistent with the study previously carried out by Gu et al. [5]. All mice were routinely monitored for tumor formation, weight loss, pain, and distress. The mice were euthanatized with CO2 asphyxiation 21days post-treatment, and blood was collected through cardiac puncture immediately after sacrifice. Excised tumor and lung samples were kept in 10% formalin for subsequent histopathological examination. The average tumor number or mass per injection was calculated by dividing each groups total number of tumors or total mass by the number of mice in that group.

Excised tumor, lung, and liver were fixed by immersion in 10% buffered formalin for 35 days and then transferred to 70% ethanol for long-term fixation. Representative sections of fixed tissue were trimmed and embedded in paraffin, then sectioned at 3m and stained by hematoxylin and eosin (H&E) [33] for examination performed in a blind manner by a veterinary pathologist, and photomicrographs were captured under a microscope (Leica, Micro Service, St. Michael, MN) at 200 and 400 magnification for illustrative purposes.

Since hCSCs are of human origin, ELISA was carried out to assess the presence of human vascular endothelial growth factor A (hVEGF-A), which promotes tumor angiogenesis in a host. The collected mouse blood samples were kept in a slanted position at room temperature for 1hour, followed by 4C for 24hours, and then centrifuged at 5000rpm for 5minutes. The Platinum ELISA kit (eBioscience, San Diego, CA) was used to quantify the hVEGF-A present in each serum sample (pg/ml) from a single mouse, according to the manufacturers instructions.

Statistical analyses were carried out using Sigma Plot software (Systat Software, Inc., San Jose, CA). Statistical significance between the groups was assessed by multiple mean comparisons using one-way analysis of variance (ANOVA) followed by a post-hoc Dunnetts test. Students t test was applied to compare two groups receiving similar treatments. Data were expressed as meansSEM. Experiments were repeated at least three times. The significance of differences between means is represented by asterisks: *p0.05, **p0.01, ***p0.001.

In this report we used the HeLa cervical cancer cell line to isolate and characterize hCSCs following a previously described sphere culture method [

], which favors self-renewal of CSCs in culture but also causes minimal damage to the cells. In comparison with HeLa cells, the isolated/enriched hCSC population exhibited higher CD44 (90.93% vs. 51.52%) and lower CD24 (0.4% vs. 7.5%) cell-surface marker expression in flow cytometry analyses (Figure

A, B), consistent with results previously reported [

]. Multi-drug resistance characteristic of stem cells was indicated by transporter-mediated efflux of the fluorescent dye Hoechst 33342 [

], and significantly higher numbers of Hoechst-dye-excluded cells in hCSCs (73%) than in HeLa cells (15%) further confirmed their stem-like characteristics (Figure

C, D). Finally, in xenotransplanted mice, greater tumorigenicity was observed in the hCSC group (7 tumors/4 mice) than in the HeLa group (2 tumors/4 mice) (Figure

E). Following validation of hCSC characteristics, we investigated the effects of PEITC and other compounds on hCSCs. The significance of any treatment was compared with untreated/vehicle (DMSO) controls or otherwise specified.

Identification and confirmation of isolated HeLa cancer stem cells (hCSCs). A) Representative FACS histograms showing increased CD44 and decreased CD24 expression in hCSCs compared with HeLa cells B) Summary of FACS analyses showing the percentage of hCSCs expressing CD44 and CD24 (n=3) C) The Hoechst exclusion assay showing transmitted, Hoechst-stained, and overlaid images of HeLa cells and hCSCs. Hoechst 33342 dye emits blue fluorescence when bound to dsDNA. Yellow arrows show Hoechst-excluded cells lacking dark-blue nuclei (200-m scale), which were typically higher in hCSCs than in HeLa cells. D) Quantification of Hoechst-dye-excluded cells showing a higher exclusion rate in hCSCs (n=3). E) In vivo tumorigenicity was compared in NOD/SCID mice (four animals per group) 3weeks after xenotransplantation of HeLa cells, hCSCs, or vehicle (nave control), showing higher tumor counts in the hCSC group. All data are expressed as meansSEM except for in vivo tumor counts. Asterisks indicate statistically significant differences between the groups indicated, ***p0.001.

PEITC attenuated the formation of primary hCSC spheres in a concentration-dependent manner (Figure

A). Addition of PEITC (1.0 and 2.5M) resulted in a 48% and 60% decline in cell numbers, respectively (Figure

B), which is consistent with the corresponding reduction in sphere size (Figure

A). Lower concentrations of PEITC (2.5M) were used in sphere-forming enrichment culture media than in specific assays (2.5M), as shown in the remaining figures. PEITC also significantly reduced proliferation of both HeLa cells and hCSCs in a concentration-dependent manner after 24- and 48-hour exposures, which was a pattern comparable to the effects of salinomycin. The observed effects of 10 nM paclitaxel was limited (Figure

C) in our experiments, which may be due to the slow induction of cell death after low concentrations (10 nM) of paclitaxel, which occurs up to 72hours post treatment. It was previously shown that low concentrations of paclitaxel strongly block mitosis at the metaphase/anaphase transition but could be insufficient to cause immediate cell death in HeLa cells [

].

Effects of PEITC on HeLa cell and hCSC viability. A) Representative micrographs showing PEITC-attenuated sphere formation in hCSCs isolated from HeLa cells in a concentration-dependent manner as observed after 7days of culture in enrichment medium (400-m scale). B) Histogram showing quantification of viable cells on the 7th day of sphere culture from groups shown in A (n=5). C) Concentration-dependent effects of PEITC on the viability of HeLa cells and hCSCs after 24 (i) and 48 (ii) hours. Salinomycin and paclitaxel were used as known reference chemotherapeutic compounds. Absorbance was read at 490nm, and data were expressed as percentage cell viability (n=6). The dotted lines represent the baseline cell viability for DMSO/nave controls, to which all the readings were compared to obtain statistical significance. All data represent meansSEM, and significance was determined by comparing with nave control or as indicated, *p0.05, **p0.01, ***p0.001.

To investigate a potential pro-apoptotic effect of PEITC in triggering hCSC growth inhibition, we carried out western blot experiments on hCSCs treated with different doses of PEITC in the presence or absence of TRAIL and TNF. We observed an increased expression of cPARP with higher doses of PEITC (15M) following exposure for 5hours, which was further augmented by the presence of 10ng/ml TRAIL, which indicated elevated levels of endogenous caspase-mediated apoptosis in hCSCs (Figure

A). After normalizing to the housekeeping gene -actin, densitometric analysis of cPARP levels showed that PEITC induced cPARP and sensitized the TRAIL pathway but not the TNF pathway in hCSCs (Figure

A). It was previously shown that PEITC induces cPARP in HeLa cells [

], which we also observed (data not shown). Next, we carried out an annexin V/propidium iodide (PI) staining with or without TRAIL induction. Dot plot analyses showed that the fraction of annexin-positive cells in hCSCs treated with PEITC was higher than in untreated hCSCs (5.76% vs. 4.12%, Figure

B, C). Similarly, TRAIL-induced hCSCs treated with PEITC showed increased apoptosis relative to TRAIL-induced hCSCs (6.42% vs. 5.81%, Figure

B, C), although the difference was not statistically significant. When compared with the DMSO control, both PEITC- and TRAIL-treated hCSCs showed a trend toward higher apoptotic levels, indicating a potential sensitization of TRAIL-mediated apoptotic pathways by PEITC.

PEITC sensitizes the TRAIL pathway in hCSC apoptosis. A) Representative immunoblot and densitometric analysis (n=3) of cPARP levels in hCSCs after concentration- dependent PEITC exposure in the presence/absence of TRAIL (10ng/ml) and TNF (10ng/ml), normalized to housekeeping -actin expression levels. PEITC independently induced as well as synergized TRAIL induction of cPARP in hCSCs. B) A quantitative bar graph illustrating individual effects as well as synergism between 10M PEITC and TRAIL (10ng/ml) in sensitizing TRAIL-mediated apoptosis (n=3). C) Representative FACS scatter plots of data shown in B with annexin VFITC/propidium iodide staining, confirming individual effects as well as synergism between PEITC (10M) and TRAIL (10ng/ml) in sensitizing TRAIL- mediated apoptosis (iiv). All data represent meansSEM, and significance was determined by comparing with nave control or as indicated, *p0.05, **p0.01, ***p0.001.

To further understand the characteristics of PEITC in the extrinsic apoptosis pathway in hCSCs, we carried out flow cytometry analyses of DR4 and DR5 death receptors. Since both PEITC- and DMSO-treated hCSCs were treated with TRAIL (all treatments included TRAIL), we expected to see greater induction of DR4 and DR5 in PEITC+TRAIL-treated cells compared to TRAIL treatment alone. We observed that PEITC induced overexpression of DR4 in comparison with the DMSO control (69.01% and 52.52%, Figure

A iii, B). Similarly but to a lesser extent, the expression of DR5 in PEITC-treated hCSCs was higher (72.63% and 60.57%) than in the corresponding DMSO control (Figure

A iiiiv, B), showing that the slightly increased overexpression of DR5 was due to PEITC treatment. PEITC was previously shown to upregulate DR4 and DR5 in a different cervical cancer cell line (HEp-2) [

]; hence, we investigated its effect only on hCSCs.

PEITC up-regulated DR4 and DR5 receptors in TRAIL signaling. A) Representative FACS histograms of DR4 and DR5 expression in hCSCs treated with or without 10M PEITC in the presence of TRAIL. PEITC induced overexpression of DR4 (ii) and DR5 (iv) in comparison with DMSO controls (i) and (iii), respectively. The histograms do not show isotype controls. B) Quantitative bar diagrams presenting the groups from A (n=3). All data represent meansSEM, and significance was determined by comparing with nave control as indicated: **p0.01, ***p0.001.

To confirm the higher tumorigenic potential of hCSCs in vivo, we carried out a xenotransplant experiment in NOD-SCID immunodeficient mice that included four treatment groups and a negative/naive control group. Tumor development did not alter food intake and overall well-being of the mice, as evidenced by their normal body weight and activity (data not shown). An equal number of cells (1106) containing either HeLa cells or hCSCs (each with or without 10M PEITC pre-treatment) developed different tumor loads in each group of NOD/SCID mice. The average tumor number per injection was observed to be much higher in the hCSC group (1.75) than in the HeLa group (0.5), while PEITC pre-treatment helped lower tumor formation in both hCSC (1.75 vs. 0.5) and HeLa (0.5 vs. 0.33) groups of mice than in controls (Figure5B). A similar trend was observed when we calculated tumor mass per injection in each group. The hCSC group had a higher average tumor mass than the HeLa group (95mg vs. 60mg, respectively, data not shown). As expected, PEITC-treated hCSCs and HeLa cells produced a lower mass (85mg and 40mg, respectively) than their controls (95mg and 60mg, respectively, data not shown). To further visualize histological differences between tumors driven by CSCs and HeLa cells, the excised tumors were sectioned and stained with H&E. We observed a higher number of differentiated tumor cells with a low mitotic index in the HeLa group (Figure 5Ai). By contrast, the presence of pleomorphic and highly proliferative cells and early signs of neovascularization in the CSC group suggested that the tumors driven by CSCs are highly aggressive (Figure5Aiii). On the other hand, there were more apoptotic cells in the case of HeLa cells treated with PEITC (Figure5Aii) and hCSCs treated with PEITC (Figure5Aiv), suggesting that PEITC induces apoptosis in both HeLa cells and hCSCs.

To validate the human origin of these tumors, we performed ELISA on isolated serum samples. The hCSC group had the highest concentration of human hVEGF-A (12.31pg/ml), followed by hCSCs treated with PEITC (i.e., 4.62pg/ml) and untreated HeLa cells (1.08pg/ml), while we did not detect any hVEGF-A in HeLa cells treated with PEITC (Figure

C). To see whether hCSCs have metastatic potential, we carried out H&E staining of lung sections, which revealed invading tumor cells in the lungs of the hCSC group (Figure

D and Eiii) but not in the other groups. Overall, hCSCs were more tumorigenic than HeLa cells in this model, and their tumorigenicity was attenuated by PEITC pre-treatment prior to xenotransplant.

Effects of 10M PEITC-treated compared with untreated HeLa cells and hCSCs in a xenotransplant NOD/SCID mouse model. A) Representative photomicrographs of H&E-stained and sectioned tumors (3m, 400x) showing greater and more aggressive tumorigenic effects of hCSCs (iii) than HeLa cells (i). Details of native HeLa cells within a small tumor nodule with fairly uniform cell size and shape are shown (ii), and details of a small tumor nodule showing widespread apoptosis are also shown (iv). Empty arrows indicate apoptotic cells (yellow), high mitotic activity (blue), and early signs of neovascularization (white). B) Average tumor number per injection, where the untreated hCSC group showed the highest number of tumors per injection. C) The highest concentration of human serum VEGF-A was in the hCSC group, indicating the human origin of the tumors that were translocated into the blood circulation. D) The metastatic potential among the groups is shown. Metastasis was observed only in the untreated hCSC group. E) Representative photomicrographs of H&E-stained and sectioned lungs (3m, 200x). Filled arrows indicate lung bronchiole (yellow) as a landmark of distant tumor location and invading tumor cells (white) (iii). Overall, hCSCs showed increased tumorigenic activity compared with HeLa cells in this model, which was, however, attenuated upon pre-treatment with PEITC.

Cervical cancer is the second-most-frequent female malignancy worldwide [17]. Concurrent chemoradiotherapy represents the standard of care for patients with advanced-stage cervical cancer, while radical surgery and radiotherapy are widely used for treating early-stage disease. However, the poor control of micrometastases, declining operability, and the high incidence of long-term complications due to radiotherapy underscore the necessity for developing different therapeutic approaches, such as using an adjuvant CAM (complementary and alternative medicine) regimen for improved treatment outcomes [35]. Among cancer patients, the use of alternative treatments ranges between 30 and 75% worldwide and frequently includes dietary approaches, herbals, and other natural products [36]. It is becoming increasingly evident that cancer treatment that fails to eliminate CSCs allows relapse of the tumor [37]. Here we report novel effects of PEITC, a phytochemical that can be derived from a plant-based diet or may be developed as a natural product, in attenuating in vitro hCSC proliferation and in vivo tumorigenicity as well as stimulating intracellular receptors that mediate TRAIL-induced apoptosis.

According to the CSC concept of carcinogenesis, CSCs represent novel and translationally relevant targets for cancer therapy, and the identification, development, and therapeutic use of compounds that selectively target CSCs are major challenges for future cancer treatment [37]. It is proposed that direct targeting of CSCs through their defining surface antigens, such as CD44, is not a rational option, because these antigens are frequently expressed on normal stem cells [38]. On the other hand, triggering tumor cell apoptosis, in general, is the foundation of many cancer therapies. In the case of CSCs, it was suggested that the induction of apoptosis in the CSC fraction of tumor cells by specifically upregulating death receptors or death receptor ligands such as TRAIL is a potential strategy to bypass the refractory response of CSCs to conventional therapies [38]. Preclinical studies have demonstrated the potential of TRAIL to selectively induce apoptosis of tumor cells, because normal cells possess highly expressed decoy receptors that protect them from cell death [20, 39], which has driven the development of TRAIL-based cancer therapies [38, 40]. Unfortunately, a considerable range of cancer cells, especially in some highly malignant tumors, are resistant to TRAIL-induced apoptosis [41]. Therefore, TRAIL synergism using PEITC, a compound with an established low-toxicity profile in healthy animals [16] could offer an important approach to overcoming the current challenges in using TRAIL-targeted therapies, particularly in otherwise-resistant CSCs.

PEITC treatment in hCSCs reduced proliferation and sphere formation and expressed higher levels of cPARP, indicating elevated levels of apoptosis, which is possibly through caspase activation by isothiocyanate in treated cancer cells as reported previously [42]. At similar micromolar concentrations, the effects of PEITC on hCSC proliferation were comparable to salinomycin, which was shown to effectively eliminate CSCs and to induce partial clinical regression of heavily pretreated and therapy-resistant cancers [37]. It is worth mentioning here that salinomycin had considerable cytotoxicity in healthy mammals [37]. PEITC has been well documented for safety to normal mammals. It is interesting to investigate if PEITC is cytotoxic to normal stem cells, which has not been reported. Moreover, the effects of PEITC were significantly better in abrogating hCSC proliferation than paclitaxel, a current cancer chemotherapeutics. This better anti-proliferative effect may be due to the high level of chemoresistance of CSCs to paclitaxel, the overcoming of which by specific targeting of CSCs is hailed as critical. The concentration range of PEITC used (2.520M) was validated in our previous studies [8, 9, 43] and was also shown to be achievable following oral administration in human [30].

We observed that PEITC likely sensitized TRAIL but not the TNF pathway while inducing apoptosis. Although TNF- can trigger apoptosis in some solid tumors, its clinical usage has been limited by the risk of lethal systemic inflammation [44]. By comparing hCSCs treated with PEITC to those without PEITC, we observed PEITC also induced the expression of death receptors DR4 and DR5 in hCSCs, which has not been reported earlier. PEITC was, however, previously shown to upregulate DR4 and DR5 in a different human cervical cancer cell line [12]. The expression levels of either DR4 alone or both death receptors are correlated with TRAIL sensitivity of a cell line [45]. Our result revealed expression of both death receptors were elicited following PEITC treatment, but DR5 expression increase was to a lesser extent compared with DR4s increase. TRAIL is known to trigger apoptosis through binding to DR4 or DR5, which contain cytoplasmic death domains responsible for recruiting adaptor molecules involved in caspase activation [21]. Since all treatments shown in Figure4 included TRAIL treatments, the observations indicate that hCSCs are more prone to TRAIL treatment after incubation with PEITC. While the biological activity of PEITC in inducing apoptosis of cancer cells may involve death receptor signaling, other mechanisms have also been suggested [12, 13]. Finally, to investigate the antagonistic effects of PEITC on hCSC tumorigenicity in vivo, we carried out xenotransplantation in immune-compromised mice. Mice receiving untreated hCSCs produced the highest numbers of tumors and also showed greater invasiveness, as confirmed by the presence of lung metastases. However, given the short 3-week duration of the experiment, metastasis was found in only one of the four animals in the hCSC group but in no other animal in the remaining groups. We observed a marked reduction in tumorigenicity in mice that had received a PEITC-treated hCSC inoculum, and the outcome was comparable to the HeLa-injection group. It should be noted here that the sphere culture approach to isolation of hCSCs that we used in the study followed by cell-surface marker-based characterization helps to identify CSC-enriched subpopulations but did not enable unambiguous isolation of all of the CSCs.

We have provided the first evidence that PEITC is effective in abolishing human cervical CSCs in vitro, and PEITC-treated hCSC xenotransplants were less tumorigenic in a relevant mouse model. PEITC, in combination with TRAIL, upregulated the death receptor-induced extrinsic pathway of apoptosis and resulted in the increase in cPARP proteins. It should be noted that in the current study we did not evaluate the individual effectiveness of TRAIL against hCSCs, but TRAIL is currently in clinical trials in the US (NCT00508625). Importantly, PEITC is anti-proliferative in both HeLa cancer cells and hCSCs, suggesting that it may contribute to eradication of cancer more efficiently than compounds targeting either CSCs or regular cancer cells alone. Collectively, our data strongly justify future clinical trials of PEITC, individually or in combination with recombinant TRAIL therapy, for improved treatment outcomes in cancer patients.

Cleaved poly adenosine diphosphate-ribose polymerase

Cancer stem cells

Death receptors

HeLa cervical cancer stem cells

Human vascular endothelial growth factor A

Non-obese diabetic, severe combined immunodeficient

No-observed-adverse-effect-level

Phenethyl isothiocyanate

Tumor necrotic factor-alpha

Tumor necrotic factor-related apoptosis-inducing ligand.

We acknowledge Qingming Song for his help with mice work. Support for this work came from National Institutes of Health grant R00AT4245 and SD-Agriculture Experiment Station grant 3AH360 to MD. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors declare that they have no competing interests.

Study conception: MD; Designed research: DW, YL, BU, MD; Conducted Research: DW, BU, YL, DK; Project direction/supervision and provision of reagents/materials/equipment: MD; Data analyses: YL, BU; Manuscript writing: MD, BU, YL; All authors read, provided comments and approved the manuscript.

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Phenethyl isothiocyanate upregulates death ... - BMC Cancer

November 10, 2004

Tags: Jaenisch LabStem Cells + Therapeutic Cloning

Your doctor has some bad news. Turns out your heart isnt working right. In fact, due to deterioration in the muscle tissue, its only operating at 10 percent capacity. That explains your chest pains, difficulty breathing, and inability to exert yourself without getting winded. Unfortunately, you know what the diagnosis means: getting on a donor list, staying at home, and waiting for the hospital beeper to go off if a donor organ becomes available. And even if that does happenand the chances are slimyoull always be wondering how long the transplant will last, worrying that your immune system will wise up to this foreign mass of muscle and attack it with everything its got.

But your doctor has another idea. He will collect cells from the surface of your skin and put them in a dish. Youll go home, with orders to stay in bed and rest. About six weeks later, you will arrive at the hospital and be wheeled into the operating room. The last thing youll remember is the anesthesiologist placing a mask on your face and asking you to count backward from 10. When you wake up in recovery, groggy and achy, your doctor will say that youre going to be fine. Even as the two of you speak, your heart muscle will be renewing itself. Tissue will have been engrafted into your hearttissue created from your very own DNA. No red flags to alert your immune system. In a few weeks, youll be completely restored.

For now, the above scenario is speculative fictionhighly controversial speculative fiction. Politicians, lawyers, ethicists, religious leaders, United Nations delegates, and scientists are embroiled in a debate over whether the process used to heal your heart is morally flawed.

For that new heart tissue to be created, researchers would need to remove the nucleus from one of your skin cells and implant it into a donor egg cell from which the nucleus had been removed. They would coax the egg cell to divide into a blastocyst, a mass of about 100 cells. In the center of that mass theyd find the payloadembryonic stem cells, microscopic dots with nothing but pure potential. The cells are able to form any type of cell in the human body, including those from which scientists could conceivably grow your heart tissue. Or liver tissue. Or pancreatic tissue. Or brain tissue. Or spinal cord tissue. And so on. To do that, they would need to destroy the cloned blastocyst, and thats where it gets messy.

If, rather than harvesting it for stem cells, scientists instead placed that blastocyst, grown from your skin cells, inside a human uterus, it would have the potential to develop into a fetus. Nine months later, if all went well, a baby would be delivered. But not just any baby. It would be a carbon copy of you, cell for cell. It would be your clone, the twin you never had.

Just the prospect of creating a human being in this way is an ethical minefield in and of itself. But so is destroying the blastocyst. And so is creating it in the first place. To make matters worse, for researchers today to learn how to create your heart muscle tomorrow, they need to experiment on human embryonic stem cells. Until now, scientists in the field have used leftover blastocysts that stock the freezers of fertility clinics for their studies. These blastocysts are fertilized embryos that have the potential to develop into healthy babies.

Welcome to the ethical bouillabaisse known as embryonic stem cell research, where issues related to religion, abortion, cloning, and human disease are dumped together into a single scientific stew. Rarely has an issue of basic science been so hotly debated on every imaginable front, from family dinner tables to political platforms.

The Bush administration remains firmly behind the stem cell research policy announced in 2001, which restricted federally funded embryonic stem cell research to existing stem cell lines. But last May, Nancy Reagan, Republican icon and wife of the late President Ronald Reagan, asked the sitting president to change his policy on embryonic stem cell research, calling it the best hope for people with Alzheimers disease, the illness that plagued her husband in his final years. And in July, the Reagans son, Ron, carried the same message to the Democratic National Convention.

But behind all the political sparring, where is the science? Critics claim that embryonic stem cell advocates are inflating their case; advocates say it is the most exciting development in biology in decades. Still, fundamental questions remain: How advanced is the research? Can therapeutic cloning actually work, delivering on its promise to cure the incurable? And what of the arguments both camps cite to prove their points? Do the current findings somehow manage to achieve a weird combination of ambiguity and promise in such a way that both sides can claim science is on their side?

In 1953, cancer researcher Leroy Stevens discovered teeth and hair in mouse testicles, and the field of stem cell biology was born. A major tobacco company had awarded a grant to Jackson Laboratory in Bar Harbor, Maine, where Stevens was a scientist, for a study the company hoped would prove that the paper in cigarettesnot tobaccocaused cancer. After exposing mice to large amounts of cigarette ingredients, Stevens noticed that a few were developing gigantic scrotums. When he dissected the scrotums, he was taken aback by what he found inside: a hodgepodge of random tissue, including cartilage, teeth, and hair.

This particular type of tumor is called a teratoma, taken from the Greek word teraton, which means monster. Its a tumor that originates from a germ cell (precursors for both egg and sperm cells), hence its ability to form such a bizarre array of tissue. Stevens quickly abandoned his tobacco research and spent the next few decades studying these teratomas, trying to get at their cellular roots. Eventually he came across what he called a pluripotent embryonic stem cell, that is, a cell that can give rise to a variety of tissues. Stevens work was limited in that the cell lines he discovered always maintained the potential to form these monster-like cancers.

Nearly 30 years after Stevens initial discovery, scientists in the United States and the United Kingdom iso-lated embryonic stem cells from a mouse blastocyst, a find that energized the field. Still, research in the area remained safely cloistered in the walls of academic study. Then, in 1998, two groups independently announced that they had isolated human embryonic stem cells. One group from the Wisconsin Regional Primate Research Center had used leftover blastocysts from a fertility clinic. The second team, from Johns Hopkins University School of Medicine, harvested their stem cells from aborted fetuses.

For researchers, this was a watershed discovery. For opponents of embryonic stem cell research, it was a call to arms. The ethical and political question of should we find therapies this way? came head to head with the scientific question can we find therapies this way? The stew began to bubble.

Whitehead Institutes Rudolf Jaenisch knows a thing or two about mice. Years ago he was among the first scientists to incorporate foreign DNA into a mouses genome in such a way that the new genetic information could be passed down to subsequent generations. Called transgenics, this procedure is now commonplace in labs around the world. For well over a decade, Jaenisch, who also is a professor of biology at MIT, has cloned thousands of mice, trying to decipher all the factors involved in what he calls reprogrammingthe process by which the host egg cell reactivates the entire genome of the donor nucleus. While much of the basic biology of how cloning works remains a mystery, one thing is clear to Jaenisch: There is no such thing as a normal clone.

The vast majority of cloned embryos die in utero, he says. Others are stillbirths. The slim percentage that grow to adulthood are ridden with all sorts of genetic-related health conditions. Theyre obese; they die young. I suspect many have neurological damage which is hard for us to detect. Out of all the animals ever cloned, Im not sure whether any normal clone has yet been produced.

The problem, Jaenisch says, is that its impossible for an egg cell to reactivate every single gene in the donor nucleus. Something inevitably goes wrong. This isnt a technical issue, he maintains. Its not like the early days of in vitro fertilization, where we simply needed to improve the techniques. This is a principal biological issue. For this reason, he and most other scientists in the field believe that human reproductive cloning should be universallyand permanentlybanned. Human reproductive cloning would be the conscious and willful creation of a grossly malformed person. The very thought of doing it is reprehensible.

While the fetus created from a cloned blastocyst is not normal, the embryonic stem cells derived from it are. In 2002, Jaenisch collaborated with George Daley, then a Whitehead Fellow, on a study of a mouse that had no functional immune system due to a genetic defectfor all intents and purposes, a bubble boy. The team removed a cell from the tip of the mouses tail, extracted the nucleus, and placed it into a de-nucleated egg cell. It became a blastocyst from which they culled embryonic stem cells. The stem cells, because they were taken from the diseased mouse, contained that same genetic flaw. The scientists corrected the defect in the stem cells and grew them into mature blood stem cells, which they then injected into the mouse. It was, essentially, the same kind of procedure used in the hypothetical repair of your damaged heart. And it had the same outcome: The mouse was cured.

This study, published in the journal Cell, was the first proof-of-principle experiment proving that therapeutic cloning can work, says Jaenisch.

Last summer, Mayo Clinic scientists reported in the American Journal of Physiology that they used embryonic stem cells to repair damaged heart tissue in rats.

Obviously, neither mice nor rats are men. Still, Human cells are no more complex than mouse cells, says Lawrence Goldstein, a professor of cellular and molecular medicine at the University of California, San Diego. Its like a Cadillac versus a Volkswagen. The parts dont necessarily go in the same places, but the principles are the same.

But figuring out which parts go where requires a steep learning curve.

We know a tremendous amount about mouse embryonic stem cells and how to culture and differentiate them, says Daley, now a professor at Harvard Medical School. But for now, our understanding of how to do the same in human embryonic stem cells is much more primitive. There are issues of cell viability and engraftability that have yet to be explored in greater detail. Im sure there are challenges that we dont even know yet.

Still, researchers have begun to see some success in creating mature tissue from human embryonic stem cells. So far, theyve derived heart cells called cardiomyocytes, blood precursors (which can become either red or white blood cells), and certain classes of neurons. Goldstein is using human embryonic stem cells to create Alzheimers cells. Our goal is to make human embryonic stem cells that carry the mutations that cause hereditary Alzheimers disease and use those cells to test hypotheses that weve gotten from animal models of the disease, says Goldstein. Using funding from Howard Hughes Medical Institute allows him to take advantage of human embryonic stem cells outside the limited number approved for federal funding in 2001 by President Bush.

But what about human therapeutic cloning, performing in a person the same kind of procedure Jaenisch and Daley performed in a mouse?

The firstand so far onlybreakthrough here occurred earlier this year when Woo Suk Hwang and Shin Yong Moon of Seoul National University reported in the journal Science that they had successfully cloned a human blastocyst and removed viable embryonic stem cells from it. Notes Jaenisch, This paper proves that human therapeutic cloning is possible.

The American Medical Association, the National Academy of Sciences, and such publications as the New England Journal of Medicine have issued statements supporting this work, creating the impression that all scientists stand united against those trying to prevent embryonic stem cell research on moral and religious grounds.

But first impressions can be deceiving.

James Sherley is blunt. I do not subscribe to the majority view at all, the MIT associate professor says. Im just one of many scientists who feels this way. Ask yourself, What are we destroying? It really is nonsensical to debate the whole question of when life begins. We know that embryos are alive. With therapeutic cloning, were talking about destroying one human being for another human beings gain. Thats something that we as a society must not do.

This argument essentially is the same as the one posed by the anti-therapeutic-cloning, anti-embryonic-stem-cell research faction: Whether the blastocyst is cloned or taken from a fertility clinic, they claim, acquiring embryonic stem cells destroys a human life. (Jaenisch counters by pointing out that a cloned blastocyst has little, if any, chance of ever developing into a normal baby.)

But Sherley has another problem with this area of research, one that his fellow critics seldom, if ever, mention.

A researcher at MITs Biological Engineering Division, Sherley works with adult stem cells. Unlike embry-onic stem cells, adult stem cells are generally thought to become only the type of tissue from which theyve been taken. A familiar example: bone marrow transplants in which the adult stem cells from the donor marrow help the cancer patient. Ideally, a persons own adult stem cells could be used in treatment. A cancer patient could have adult stem cells taken from his blood samples, multiplied in a dish, and administered without any danger of rejection.

Adult stem cell researchers have hit two significant roadblocks: These cells are hard to identify and difficult to grow. But according to Sherley, embryonic stem cell researchers soon will face the same obstacles.

You have to ask, What do you need in order to produce tissue for long-term replacement therapy? The answer is, You need adult stem cells, Sherley says. If these embryonic stem cell therapies will be successful, they must produce adult stem cells. So these researchers will soon have the same problems that we have. Theyll have to figure out ways to locate and then multiply the adult stem cells from the tissue cultures that they created using embryonic stem cells.

Sherley says that mature tissue alone wont suffice for long-term replacement therapy. Even with bone marrow transplants, if the marrow doesnt contain adult stem cells, the procedure fails.

The solution, as he sees it, is to bypass altogether the moral quagmire of experimenting with human blastocysts and focus exclusively on adult stem cells. Besides, I just cant accept that reproductive clones are unhealthy but stem cells from reproductive clones are fine, he says. The data arent convincing.

But many of his fellow scientists arent persuaded. The real issue, says Jaenisch, is that so far, its impossible to propagate and grow adult stem cells. And adult stem cells havent been shown to have therapeutic value, except for blood cells.

Whats more, Daley notes, not every tissue has adult stem cells. For the pancreas, the heart, and much of the brain, there does not appear to be active regeneration from adult stem cells. For these tissues, embryonic stem cells are likely to be the best source of replacement cells.

As for the moral question regarding when life begins, I just spent the other day working with a number of ethicists and philosophers discussing this very issue, says Goldstein, and very smart, experienced people with different viewpoints confront the issue differently and arrive at different answers. This sort of debate is a standard thing to happen when we have new technologies that test our conceptions of who we are and what were about.

In 2002, Bernard Siegel was channel surfing when he stumbled on a press conference in which spokespersons for the UFO cult the Raelians announced that they had cloned the first human baby. Siegel, an attorney, decided that the manner in which the cult members were manipulating this alleged baby was evidence for a child abuse investigation. So, he filed for guardianship.

Then came the media firestorm, he says. (Because of this case, the Raelians refused to do a DNA test on the childwho Siegel is certain does not exist.)

Even after the case was dropped, Siegel noticed how the Raelians had affected the world of stem cell research. Rael, their leader, had testified in a congressional hearing and appeared before the National Academy of Sciences to make his case in favor of human reproductive cloning. Conservatives seized on his testimony and used it as evidence that all forms of cloningincluding therapeutic cloningshould be banned.

There was no single, unified group of scientists that could answer to this, says Siegel. And so he founded the Genetics Policy Institute (GPI), a Coral Gables, Florida-based science advocacy group whose membership includes many top stem cell researchers.

This fall will mark the first real test of the groups effectiveness.

Toward the end of this year, delegates with the United Nations will renew a debate on two competing treaties that were tabled last year. The first, the Costa Rican treatywhich is supported by the U.S.bans all forms of cloning, including therapeutic. The second, the Belgium treaty, would allow therapeutic cloning while banning the procedure for reproduction.

It is too early to tell how the vote will go. If delegates adopted the Costa Rican treaty, it would cast a pall on the research, declaring it an affront to human dignity and morally reproachable, Siegel says. But what he fears most is that it would breathe life in the Brownback Bill, a bill authored by United States senator Sam Brownback (R-Kan.), that proposes to make the very process of nuclear transfer with human cells a criminal offense, punishable with mandatory jail time for any scientist who attempts it.

This fall, were heading straight toward a public-policy train wreck, says Siegel. Coming to a head are the U.N. vote, a U.S. presidential election in which embryonic stem cell research has been a key issue, and a California initiative that would provide up to $295 million annually for embryonic stem cell research. These will all, in one fell swoop, influence the landscape of stem cell research, he says.

Meanwhile, both scientists and the public must be patient. It will be many years before we see whether therapeutic cloning will ever treat, for example, your heart muscle. And there still is the possibility that researchers will find ways to cure myriad diseases in mice and rats, yet never apply those techniques successfully in people. Until someone does, in fact, make the transition to humans, the debate will rage on, forcing scientists to work under a cloud of public controversy.

But researchers push forward, confident that this field eventually will deliver on some of its promises.

Goldstein, for one, is optimistic that his efforts one day will yield treatments to rid the body of cancer, diabetes, and other ailments. Sure, its possible for this to be a huge failure, but I dont see that, he predicts. The science and the data are sound enough so that a guy like me, whos done this for 25 years and has a reasonably good scientific track record, is willing to put substantial resources and energy into this. Im willing to take risks, but I wouldnt do this if I thought there was a high likelihood it would fail.

Written by David Cameron.

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Whitehead Institute - News - 2004 - Life, death and stem cells

Diabetes exacts its toll on many Americans, young and old. For years, researchers have painstakingly dissected this complicated disease caused by the destruction of insulin producing islet cells of the pancreas. Despite progress in understanding the underlying disease mechanisms for diabetes, there is still a paucity of effective therapies. For years investigators have been making slow, but steady, progress on experimental strategies for pancreatic transplantation and islet cell replacement. Now, researchers have turned their attention to adult stem cells that appear to be precursors to islet cells and embryonic stem cells that produce insulin.

For decades, diabetes researchers have been searching for ways to replace the insulin-producing cells of the pancreas that are destroyed by a patient's own immune system. Now it appears that this may be possible. Each year, diabetes affects more people and causes more deaths than breast cancer and AIDS combined. Diabetes is the seventh leading cause of death in the United States today, with nearly 200,000 deaths reported each year. The American Diabetes Association estimates that nearly 16 million people, or 5.9 percent of the United States population, currently have diabetes.

Diabetes is actually a group of diseases characterized by abnormally high levels of the sugar glucose in the bloodstream. This excess glucose is responsible for most of the complications of diabetes, which include blindness, kidney failure, heart disease, stroke, neuropathy, and amputations. Type 1 diabetes, also known as juvenile-onset diabetes, typically affects children and young adults. Diabetes develops when the body's immune system sees its own cells as foreign and attacks and destroys them. As a result, the islet cells of the pancreas, which normally produce insulin, are destroyed. In the absence of insulin, glucose cannot enter the cell and glucose accumulates in the blood. Type 2 diabetes, also called adult-onset diabetes, tends to affect older, sedentary, and overweight individuals with a family history of diabetes. Type 2 diabetes occurs when the body cannot use insulin effectively. This is called insulin resistance and the result is the same as with type 1 diabetesa build up of glucose in the blood.

There is currently no cure for diabetes. People with type 1 diabetes must take insulin several times a day and test their blood glucose concentration three to four times a day throughout their entire lives. Frequent monitoring is important because patients who keep their blood glucose concentrations as close to normal as possible can significantly reduce many of the complications of diabetes, such as retinopathy (a disease of the small blood vessels of the eye which can lead to blindness) and heart disease, that tend to develop over time. People with type 2 diabetes can often control their blood glucose concentrations through a combination of diet, exercise, and oral medication. Type 2 diabetes often progresses to the point where only insulin therapy will control blood glucose concentrations.

Each year, approximately 1,300 people with type 1 diabetes receive whole-organ pancreas transplants. After a year, 83 percent of these patients, on average, have no symptoms of diabetes and do not have to take insulin to maintain normal glucose concentrations in the blood. However, the demand for transplantable pancreases outweighs their availability. To prevent the body from rejecting the transplanted pancreas, patients must take powerful drugs that suppress the immune system for their entire lives, a regimen that makes them susceptible to a host of other diseases. Many hospitals will not perform a pancreas transplant unless the patient also needs a kidney transplant. That is because the risk of infection due to immunosuppressant therapy can be a greater health threat than the diabetes itself. But if a patient is also receiving a new kidney and will require immunosuppressant drugs anyway, many hospitals will perform the pancreas transplant.

Over the past several years, doctors have attempted to cure diabetes by injecting patients with pancreatic islet cellsthe cells of the pancreas that secrete insulin and other hormones. However, the requirement for steroid immunosuppressant therapy to prevent rejection of the cells increases the metabolic demand on insulin-producing cells and eventually they may exhaust their capacity to produce insulin. The deleterious effect of steroids is greater for islet cell transplants than for whole-organ transplants. As a result, less than 8 percent of islet cell transplants performed before last year had been successful.

More recently, James Shapiro and his colleagues in Edmonton, Alberta, Canada, have developed an experimental protocol for transplanting islet cells that involves using a much larger amount of islet cells and a different type of immunosuppressant therapy. In a recent study, they report that [17], seven of seven patients who received islet cell transplants no longer needed to take insulin, and their blood glucose concentrations were normal a year after surgery. The success of the Edmonton protocol is now being tested at 10 centers around the world.

If the success of the Edmonton protocol can be duplicated, many hurdles still remain in using this approach on a wide scale to treat diabetes. First, donor tissue is not readily available. Islet cells used in transplants are obtained from cadavers, and the procedure requires at least two cadavers per transplant. The islet cells must be immunologically compatible, and the tissue must be freshly obtainedwithin eight hours of death. Because of the shortage of organ donors, these requirements are difficult to meet and the waiting list is expected to far exceed available tissue, especially if the procedure becomes widely accepted and available. Further, islet cell transplant recipients face a lifetime of immunosuppressant therapy, which makes them susceptible to other serious infections and diseases.

Before discussing cell-based therapies for diabetes, it is important to understand how the pancreas develops. In mammals, the pancreas contains three classes of cell types: the ductal cells, the acinar cells, and the endocrine cells. The endocrine cells produce the hormones glucagon, somatostatin, pancreatic polypeptide (PP), and insulin, which are secreted into the blood stream and help the body regulate sugar metabolism. The acinar cells are part of the exocrine system, which manufactures digestive enzymes, and ductal cells from the pancreatic ducts, which connect the acinar cells to digestive organs.

In humans, the pancreas develops as an outgrowth of the duodenum, a part of the small intestine. The cells of both the exocrine systemthe acinar cellsand of the endocrine systemthe islet cellsseem to originate from the ductal cells during development. During development these endocrine cells emerge from the pancreatic ducts and form aggregates that eventually form what is known as Islets of Langerhans. In humans, there are four types of islet cells: the insulin-producing beta cells; the alpha cells, which produce glucagon; the delta cells, which secrete somatostatin; and the PP-cells, which produce pancreatic polypeptide. The hormones released from each type of islet cell have a role in regulating hormones released from other islet cells. In the human pancreas, 65 to 90 percent of islet cells are beta cells, 15 to 20 percent are alpha-cells, 3 to 10 percent are delta cells, and one percent is PP cells. Acinar cells form small lobules contiguous with the ducts (see Figure 7.1. Insulin Production in the Human Pancreas). The resulting pancreas is a combination of a lobulated, branched acinar gland that forms the exocrine pancreas, and, embedded in the acinar gland, the Islets of Langerhans, which constitute the endocrine pancreas.

Figure 7.1. Insulin Production in the Human Pancreas. The pancreas is located in the abdomen, adjacent to the duodenum (the first portion of the small intestine). A cross-section of the pancreas shows the islet of Langerhans which is the functional unit of the endocrine pancreas. Encircled is the beta cell that synthesizes and secretes insulin. Beta cells are located adjacent to blood vessels and can easily respond to changes in blood glucose concentration by adjusting insulin production. Insulin facilitates uptake of glucose, the main fuel source, into cells of tissues such as muscle.

( 2001 Terese Winslow, Lydia Kibiuk)

During fetal development, new endocrine cells appear to arise from progenitor cells in the pancreatic ducts. Many researchers maintain that some sort of islet stem cell can be found intermingled with ductal cells during fetal development and that these stem cells give rise to new endocrine cells as the fetus develops. Ductal cells can be distinguished from endocrine cells by their structure and by the genes they express. For example, ductal cells typically express a gene known as cytokeratin-9 (CK-9), which encodes a structural protein. Beta islet cells, on the other hand, express a gene called PDX-1, which encodes a protein that initiates transcription from the insulin gene. These genes, called cell markers, are useful in identifying particular cell types.

Following birth and into adulthood, the source of new islet cells is not clear, and some controversy exists over whether adult stem cells exist in the pancreas. Some researchers believe that islet stem cell-like cells can be found in the pancreatic ducts and even in the islets themselves. Others maintain that the ductal cells can differentiate into islet precursor cells, while others hold that new islet cells arise from stem cells in the blood. Researchers are using several approaches for isolating and cultivating stem cells or islet precursor cells from fetal and adult pancreatic tissue. In addition, several new promising studies indicate that insulin-producing cells can be cultivated from embryonic stem cell lines.

In developing a potential therapy for patients with diabetes, researchers hope to develop a system that meets several criteria. Ideally, stem cells should be able to multiply in culture and reproduce themselves exactly. That is, the cells should be self-renewing. Stem cells should also be able to differentiate in vivo to produce the desired kind of cell. For diabetes therapy, it is not clear whether it will be desirable to produce only beta cellsthe islet cells that manufacture insulinor whether other types of pancreatic islet cells are also necessary. Studies by Bernat Soria and colleagues, for example, indicate that isolated beta cellsthose cultured in the absence of the other types of islet cellsare less responsive to changes in glucose concentration than intact islet clusters made up of all islet cell types. Islet cell clusters typically respond to higher-than-normal concentrations of glucose by releasing insulin in two phases: a quick release of high concentrations of insulin and a slower release of lower concentrations of insulin. In this manner the beta cells can fine-tune their response to glucose. Extremely high concentrations of glucose may require that more insulin be released quickly, while intermediate concentrations of glucose can be handled by a balance of quickly and slowly released insulin.

Isolated beta cells, as well as islet clusters with lower-than-normal amounts of non-beta cells, do not release insulin in this biphasic manner. Instead insulin is released in an all-or-nothing manner, with no fine-tuning for intermediate concentrations of glucose in the blood [5, 18]. Therefore, many researchers believe that it will be preferable to develop a system in which stem or precursor cell types can be cultured to produce all the cells of the islet cluster in order to generate a population of cells that will be able to coordinate the release of the appropriate amount of insulin to the physiologically relevant concentrations of glucose in the blood.

Several groups of researchers are investigating the use of fetal tissue as a potential source of islet progenitor cells. For example, using mice, researchers have compared the insulin content of implants from several sources of stem cellsfresh human fetal pancreatic tissue, purified human islets, and cultured islet tissue [2]. They found that insulin content was initially higher in the fresh tissue and purified islets. However, with time, insulin concentration decreased in the whole tissue grafts, while it remained the same in the purified islet grafts. When cultured islets were implanted, however, their insulin content increased over the course of three months. The researchers concluded that precursor cells within the cultured islets were able to proliferate (continue to replicate) and differentiate (specialize) into functioning islet tissue, but that the purified islet cells (already differentiated) could not further proliferate when grafted. Importantly, the researchers found, however, that it was also difficult to expand cultures of fetal islet progenitor cells in culture [7].

Many researchers have focused on culturing islet cells from human adult cadavers for use in developing transplantable material. Although differentiated beta cells are difficult to proliferate and culture, some researchers have had success in engineering such cells to do this. For example, Fred Levine and his colleagues at the University of California, San Diego, have engineered islet cells isolated from human cadavers by adding to the cells' DNA special genes that stimulate cell proliferation. However, because once such cell lines that can proliferate in culture are established, they no longer produce insulin. The cell lines are further engineered to express the beta islet cell gene, PDX-1, which stimulates the expression of the insulin gene. Such cell lines have been shown to propagate in culture and can be induced to differentiate to cells, which produce insulin. When transplanted into immune-deficient mice, the cells secrete insulin in response to glucose. The researchers are currently investigating whether these cells will reverse diabetes in an experimental diabetes model in mice [6, 8].

These investigators report that these cells do not produce as much insulin as normal islets, but it is within an order of magnitude. The major problem in dealing with these cells is maintaining the delicate balance between growth and differentiation. Cells that proliferate well do not produce insulin efficiently, and those that do produce insulin do not proliferate well. According to the researchers, the major issue is developing the technology to be able to grow large numbers of these cells that will reproducibly produce normal amounts of insulin [9].

Another promising source of islet progenitor cells lies in the cells that line the pancreatic ducts. Some researchers believe that multipotent (capable of forming cells from more than one germ layer) stem cells are intermingled with mature, differentiated duct cells, while others believe that the duct cells themselves can undergo a differentiation, or a reversal to a less mature type of cell, which can then differentiate into an insulin-producing islet cell.

Susan Bonner-Weir and her colleagues reported last year that when ductal cells isolated from adult human pancreatic tissue were cultured, they could be induced to differentiate into clusters that contained both ductal and endocrine cells. Over the course of three to four weeks in culture, the cells secreted low amounts of insulin when exposed to low concentrations of glucose, and higher amounts of insulin when exposed to higher glucose concentrations. The researchers have determined by immunochemistry and ultrastructural analysis that these clusters contain all of the endocrine cells of the islet [4].

Bonner-Weir and her colleagues are working with primary cell cultures from duct cells and have not established cells lines that can grow indefinitely. However the cells can be expanded. According to the researchers, it might be possible in principle to do a biopsy and remove duct cells from a patient and then proliferate the cells in culture and give the patient back his or her own islets. This would work with patients who have type 1 diabetes and who lack functioning beta cells, but their duct cells remain intact. However, the autoimmune destruction would still be a problem and potentially lead to destruction of these transplanted cells [3]. Type 2 diabetes patients might benefit from the transplantation of cells expanded from their own duct cells since they would not need any immunosuppression. However, many researchers believe that if there is a genetic component to the death of beta cells, then beta cells derived from ductal cells of the same individual would also be susceptible to autoimmune attack.

Some researchers question whether the ductal cells are indeed undergoing a dedifferentiation or whether a subset of stem-like or islet progenitors populate the pancreatic ducts and may be co-cultured along with the ductal cells. If ductal cells die off but islet precursors proliferate, it is possible that the islet precursor cells may overtake the ductal cells in culture and make it appear that the ductal cells are dedifferentiating into stem cells. According to Bonner-Weir, both dedifferentiated ductal cells and islet progenitor cells may occur in pancreatic ducts.

Ammon Peck of the University of Florida, Vijayakumar Ramiya of Ixion Biotechnology in Alachua, FL, and their colleagues [13, 14] have also cultured cells from the pancreatic ducts from both humans and mice. Last year, they reported that pancreatic ductal epithelial cells from adult mice could be cultured to yield islet-like structures similar to the cluster of cells found by Bonner-Weir. Using a host of islet-cell markers they identified cells that produced insulin, glucagon, somatostatin, and pancreatic polypeptide. When the cells were implanted into diabetic mice, the diabetes was reversed.

Joel Habener has also looked for islet-like stem cells from adult pancreatic tissue. He and his colleagues have discovered a population of stem-like cells within both the adult pancreas islets and pancreatic ducts. These cells do not express the marker typical of ductal cells, so they are unlikely to be ductal cells, according to Habener. Instead, they express a marker called nestin, which is typically found in developing neural cells. The nestin-positive cells do not express markers typically found in mature islet cells. However, depending upon the growth factors added, the cells can differentiate into different types of cells, including liver, neural, exocrine pancreas, and endocrine pancreas, judged by the markers they express, and can be maintained in culture for up to eight months [20].

The discovery of methods to isolate and grow human embryonic stem cells in 1998 renewed the hopes of doctors, researchers, and diabetes patients and their families that a cure for type 1 diabetes, and perhaps type 2 diabetes as well, may be within striking distance. In theory, embryonic stem cells could be cultivated and coaxed into developing into the insulin-producing islet cells of the pancreas. With a ready supply of cultured stem cells at hand, the theory is that a line of embryonic stem cells could be grown up as needed for anyone requiring a transplant. The cells could be engineered to avoid immune rejection. Before transplantation, they could be placed into nonimmunogenic material so that they would not be rejected and the patient would avoid the devastating effects of immunosuppressant drugs. There is also some evidence that differentiated cells derived from embryonic stem cells might be less likely to cause immune rejection (see Chapter 10. Assessing Human Stem Cell Safety). Although having a replenishable supply of insulin-producing cells for transplant into humans may be a long way off, researchers have been making remarkable progress in their quest for it. While some researchers have pursued the research on embryonic stem cells, other researchers have focused on insulin-producing precursor cells that occur naturally in adult and fetal tissues.

Since their discovery three years ago, several teams of researchers have been investigating the possibility that human embryonic stem cells could be developed as a therapy for treating diabetes. Recent studies in mice show that embryonic stem cells can be coaxed into differentiating into insulin-producing beta cells, and new reports indicate that this strategy may be possible using human embryonic cells as well.

Last year, researchers in Spain reported using mouse embryonic stem cells that were engineered to allow researchers to select for cells that were differentiating into insulin-producing cells [19]. Bernat Soria and his colleagues at the Universidad Miguel Hernandez in San Juan, Alicante, Spain, added DNA containing part of the insulin gene to embryonic cells from mice. The insulin gene was linked to another gene that rendered the mice resistant to an antibiotic drug. By growing the cells in the presence of an antibiotic, only those cells that were activating the insulin promoter were able to survive. The cells were cloned and then cultured under varying conditions. Cells cultured in the presence of low concentrations of glucose differentiated and were able to respond to changes in glucose concentration by increasing insulin secretion nearly sevenfold. The researchers then implanted the cells into the spleens of diabetic mice and found that symptoms of diabetes were reversed.

Manfred Ruediger of Cardion, Inc., in Erkrath, Germany, is using the approach developed by Soria and his colleagues to develop insulin-producing human cells derived from embryonic stem cells. By using this method, the non-insulin-producing cells will be killed off and only insulin-producing cells should survive. This is important in ensuring that undifferentiated cells are not implanted that could give rise to tumors [15]. However, some researchers believe that it will be important to engineer systems in which all the components of a functioning pancreatic islet are allowed to develop.

Recently Ron McKay and his colleagues described a series of experiments in which they induced mouse embryonic cells to differentiate into insulin-secreting structures that resembled pancreatic islets [10]. McKay and his colleagues started with embryonic stem cells and let them form embryoid bodiesan aggregate of cells containing all three embryonic germ layers. They then selected a population of cells from the embryoid bodies that expressed the neural marker nestin (see Appendix B. Mouse Embryonic Stem Cells). Using a sophisticated five-stage culturing technique, the researchers were able to induce the cells to form islet-like clusters that resembled those found in native pancreatic islets. The cells responded to normal glucose concentrations by secreting insulin, although insulin amounts were lower than those secreted by normal islet cells (see Figure 7.2. Development of Insulin-Secreting Pancreatic-Like Cells From Mouse Embryonic Stem Cells). When the cells were injected into diabetic mice, they survived, although they did not reverse the symptoms of diabetes.

Figure 7.2. Development of Insulin-Secreting Pancreatic-Like Cells From Mouse Embryonic Stem Cells. Mouse embryonic stem cells were derived from the inner cell mass of the early embryo (blastocyst) and cultured under specific conditions. The embryonic stem cells (in blue) were then expanded and differentiated. Cells with markers consistent with islet cells were selected for further differentiation and characterization. When these cells (in purple) were grown in culture, they spontaneously formed three-dimentional clusters similar in structure to normal pancreatic islets. The cells produced and secreted insulin. As depicted in the chart, the pancreatic islet-like cells showed an increase in release of insulin as the glucose concentration of the culture media was increased. When the pancreatic islet-like cells were implanted in the shoulder of diabetic mice, the cells became vascularized, synthesized insulin, and maintained physical characteristics similar to pancreatic islets.

( 2001 Terese Winslow, Caitlin Duckwall)

According to McKay, this system is unique in that the embryonic cells form a functioning pancreatic islet, complete with all the major cell types. The cells assemble into islet-like structures that contain another layer, which contains neurons and is similar to intact islets from the pancreas [11]. Several research groups are trying to apply McKay's results with mice to induce human embryonic stem cells to differentiate into insulin-producing islets.

Recent research has also provided more evidence that human embryonic cells can develop into cells that can and do produce insulin. Last year, Melton, Nissim Benvinisty of the Hebrew University in Jerusalem, and Josef Itskovitz-Eldor of the Technion in Haifa, Israel, reported that human embryonic stem cells could be manipulated in culture to express the PDX-1 gene, a gene that controls insulin transcription [16]. In these experiments, researchers cultured human embryonic stem cells and allowed them to spontaneously form embryoid bodies (clumps of embryonic stem cells composed of many types of cells from all three germ layers). The embryoid bodies were then treated with various growth factors, including nerve growth factor. The researchers found that both untreated embryoid bodies and those treated with nerve growth factor expressed PDX-1. Embryonic stem cells prior to formation of the aggregated embryoid bodies did not express PDX-1. Because expression of the PDX-1 gene is associated with the formation of beta islet cells, these results suggest that beta islet cells may be one of the cell types that spontaneously differentiate in the embryoid bodies. The researchers now think that nerve growth factor may be one of the key signals for inducing the differentiation of beta islet cells and can be exploited to direct differentiation in the laboratory. Complementing these findings is work done by Jon Odorico of the University of Wisconsin in Madison using human embryonic cells of the same source. In preliminary findings, he has shown that human embryonic stem cells can differentiate and express the insulin gene [12].

More recently, Itskovitz-Eldor and his Technion colleagues further characterized insulin-producing cells in embryoid bodies [1]. The researchers found that embryonic stem cells that were allowed to spontaneously form embryoid bodies contained a significant percentage of cells that express insulin. Based on the binding of antibodies to the insulin protein, Itskovitz-Eldor estimates that 1 to 3 percent of the cells in embryoid bodies are insulin-producing beta-islet cells. The researchers also found that cells in the embryoid bodies express glut-2 and islet-specific glucokinase, genes important for beta cell function and insulin secretion. Although the researchers did not measure a time-dependent response to glucose, they did find that cells cultured in the presence of glucose secrete insulin into the culture medium. The researchers concluded that embryoid bodies contain a subset of cells that appear to function as beta cells and that the refining of culture conditions may soon yield a viable method for inducing the differentiation of beta cells and, possibly, pancreatic islets.

Taken together, these results indicate that the development of a human embryonic stem cell system that can be coaxed into differentiating into functioning insulin-producing islets may soon be possible.

Ultimately, type 1 diabetes may prove to be especially difficult to cure, because the cells are destroyed when the body's own immune system attacks and destroys them. This autoimmunity must be overcome if researchers hope to use transplanted cells to replace the damaged ones. Many researchers believe that at least initially, immunosuppressive therapy similar to that used in the Edmonton protocol will be beneficial. A potential advantage of embryonic cells is that, in theory, they could be engineered to express the appropriate genes that would allow them to escape or reduce detection by the immune system. Others have suggested that a technology should be developed to encapsulate or embed islet cells derived from islet stem or progenitor cells in a material that would allow small molecules such as insulin to pass through freely, but would not allow interactions between the islet cells and cells of the immune system. Such encapsulated cells could secrete insulin into the blood stream, but remain inaccessible to the immune system.

Before any cell-based therapy to treat diabetes makes it to the clinic, many safety issues must be addressed (see Chapter 10. Assessing Human Stem Cell Safety). A major consideration is whether any precursor or stem-like cells transplanted into the body might revert to a more pluripotent state and induce the formation of tumors. These risks would seemingly be lessened if fully differentiated cells are used in transplantation.

But before any kind of human islet-precursor cells can be used therapeutically, a renewable source of human stem cells must be developed. Although many progenitor cells have been identified in adult tissue, few of these cells can be cultured for multiple generations. Embryonic stem cells show the greatest promise for generating cell lines that will be free of contaminants and that can self renew. However, most researchers agree that until a therapeutically useful source of human islet cells is developed, all avenues of research should be exhaustively investigated, including both adult and embryonic sources of tissue.

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7. Stem Cells and Diabetes [Stem Cell Information]

About blood stem cells

Blood stem cells are also known as haematopoietic stem cells. Like other stem cells, they can self-renew, or copy themselves. They also produce the different types of specialized cells found in the blood: both red blood cells and the many kinds of white blood cells needed by the bodys immune system.

The tree of blood: Blood stem cells are at the origin of all blood cell types. Once a blood stem cell divides, its daughter cells take various differentiation routes to produce different types of specialized blood cells.

Specialized blood cells do not live very long, so the body needs to replace them continuously. Blood stem cells do this job. They are found in the bone marrow of long bones such as the femurs (thigh bones), and in the hips or pelvis, the vertebrae (backbones) and the rib cage. They can also be obtained from the umbilical cord blood and the placenta at birth.

Blood stem cells need to make just the right number of each type of blood cell to keep the body healthy. This is a carefully controlled process. When it goes wrong, the result may be a blood disease such as leukaemia or anaemia.

Blood stem cells are already widely used to treat such diseases. A survey in 2008 showed that more than 26,000 patients are treated with blood stem cells in Europe each year. These blood stem cells come from three different sources bone marrow, the bloodstream of an adult or umbilical cord blood.

Scientists are still learning about how blood stem cells develop in the embryo, how they are controlled in the adult body and what goes wrong in certain blood diseases. But they are also using todays understanding of blood stem cells to investigate new ways to treat patients. A bone marrow transplant is only possible if a compatible donor is available. The patient and donor must be very carefully matched to avoid immune rejection of the transplant. Even when a suitable donor can be found, there is still a small risk of rejection. Umbilical cord blood does not need to be matched quite so precisely to the patient, but there are not enough stem cells in an umbilical cord to treat an adult. So we need to find alternatives.

Researchers are investigating ways to produce large numbers of blood stem cells in the laboratory. They are also developing methods for growing specialized blood cells from blood stem cells, for example to produce red blood cells for blood transfusions.

Red blood cells frompluripotent stem cells Red blood cells carry oxygen around the body. Patients who lose a lot of blood need to have it replaced straight away by a blood transfusion. There are not enough blood donors to meet patient needs, so researchers are looking for an alternative solution. Sincepluripotent stem cells have the potential to make any cell type of the body, they could potentially provide an unlimited supply of red blood cells. It is already possible to make small numbers of red blood cells frompluripotent stem cells in the lab. Now the real challenge is to develop techniques for producing the large numbers of red blood cells that are needed for transfusion.

Growing blood stem cells in the lab Red blood cells, like other mature blood cells, are short-lived and specialized for a particular job. To cure disease in the long-term, doctors need to transplant something that can keep producing new blood cells throughout the patients life: blood stem cells. Scientists are searching for ways to grow a limitless supply of blood stem cells. One possibility might be to collect stem cells from the bone marrow then grow and multiply them in the lab. Researchers are also trying to make blood stem cells from embryonic stem cells or induced pluripotent stem (iPS) cells. iPS cells could be made from a patients own skin and then used to produce blood stem cells. This would overcome the problem of immune rejection.

Stem cells for blood - making red blood cells from embryonic stem cells EuroStemCell FAQ page on umbilical cord blood banking The European Group for Blood and Marrow Transplantation UK National Health Service information on bone marrow transplantations Original scientific paper by Till and Mcculloch identifying blood stem cells for the first time

This factsheet was created by Christle Gonneau and reviewed by Lesley Forrester and Cristina Pina.

Lead image of blood cells by Anne Weston/Wellcome Images. Blood stem cell photograph reproduced with permission from Taoudi et al. (2005) "Progressive divergence of definitive haematopoietic stem cells from the endothelial compartment does not depend on contact with the foetal liver", Development 132: 4179- 4191. 'Tree of blood' diagram by Christele Gonneau, with blood cell drawings courtesy of Jonas Larsson, Lund Univeristy, Sweden. All other images courtesy of Joanne Mountford at the University of Glasgow.

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Blood stem cells: the pioneers of stem cell research ...

Botanical Stem Cell Nutrition

Stem cell nutrition from aqua-botanical source, has shown to support the release of millions of adult stem cells from the bone marrow very quickly. These stem cells can then migrate and attach to any cells, tissue, bone, muscle, cartilage, organ anywhere in the body needing repair. Once they attach, they become that tissue and multiply 3 to 5000 times.

When there is an injury or a stress to an organ, compounds are released that reach the bone marrow and trigger the release of stem cells. Stem Cells can be thought of as master cells. Stem cells circulate and function to replace dysfunctional cells, thus fulfilling the natural process of maintaining optimal health

When Christian Drapeau first posited that Adult Stem Cells were the very foundation of the body's natural healing system, scientific study in the field was in its infancy. His hypothesis that Adult Stem Cells, created by bone marrow, flowed to any tissue or organ needing regeneration and morphed into healthy cells of that location, was initially ridiculed by medical science. Since 2006 however, and at a geometrically increasing pace, Christian Drapeau's position gained not just momentum but widespread interest in scientific circles as study after study reveals that Adult Stem Cell science holds phenomenal promise in all areas of human healing.

Stem cell nutrition are typically aquatic botanicals and support wellness by assisting the body in its ability to maintain healthy stem cell physiology, production, and placement. Just as antioxidants are important to protect your cells from free radical damage, stem cell nutrition is equally important to support your stem cells in maintaining proper organ and tissue functioning in your body.

The health benefits of having more stem cells in the blood circulation have been demonstrated by numerous scientific studies. It would be too long here to summarize this vast body of scientific data. I simply suggest you research the work of Dr. Donald Orlic at the NIH.

Christian's theory that Adult Stem Cells are nothing less than the human body's natural self-renewal system has profound implications for every area of modern medicine. The idea that heart disease, diabetes, liver degeneration, and other conditions could be things of the past is no longer science fiction; because of recent Adult Stem Cell research breakthroughs, these are real possibilities in the short term.

Stem cells are defined as cells with the unique capacity to self-replicate throughout the entire life of an organism and to differentiate into cells of various tissues. Most cells of the body are specialized and play a well-defined role in the body. For example, brain cells respond to electrical signals from other brain cells and release neurotransmitters; cells of the retina are activated by light, and pancreatic -cells produce insulin. These cells, called somatic cells, will never differentiate into other types of cells or even proliferate. By contrast, stem cells are primitive cells that remain undifferentiated until they receive a signal prompting them to become various types of specialized cells.

Botanical stem cell nutrition are available in countries around the world.

The Stem Cell Theory of Renewal proposes that stem cells are naturally released by the bone marrow and travel via the bloodstream toward tissues to promote the body's natural process of renewal. When an organ is subjected to a process that requires renewal, such as the natural aging process, this organ releases compounds that trigger the release of stem cells from the bone marrow. The organ also releases compounds that attracts stem cells to this organ. The released stem cells then follow the concentration gradient of these compounds and leave the blood circulation to migrate to the organ where they proliferate and differentiate into cells of this organ, supporting the natural process of renewal.

Most of the cells in the human body are specialists assigned to a specific organ or type of tissue, such as the neuronal cells that wire the brain and central nervous system. Stem cells are different. When they divide, they can produce either more stem cells, or they can serve as progenitors that differentiate into specialized cells as they mature. Hence the name, because specialist cells can "stem" from them. The potential to differentiate into specialist cells whose populations in the body have become critically depleted as the result of illness or injury is what makes stem cells so potentially valuable to medical research.

The idea is that if the fate of a batch of stem cells could be directed down specific pathways, they could be grown, harvested, and then transplanted into a problem area. If all went according to plan, these new cells would overcome damaged or diseased cells, leading to healing and recovery. "The life of a stem cell can be viewed as a hierarchical branching process, where the cell is faced with a series of fate switches," Schaffer says. "Our goal is to identify the cell fate switches, and then provide stem cells with the proper signals to guide them down a particular developmental trajectory."

Stem cells have the remarkable potential to develop into many different cell types in the body. Serving as a sort of repair system for the body, they can theoretically divide without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell.

When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function. Scientists believe it should be possible to harness this ability to turn stem cells into a super "repair kit" for the body.

Scientist and author Christian Drapeau explains how the Stem Cell enhancers function to maximize human performance - Supporting the release of stem cells from the bone marrow and increasing the number of circulating stem cells improves various aspects of human health. For very active and sports focused people, Stem Cells are the raw materials to repair micro-tears and micro-injuries created during training. The results, according to Drapeau, are that active people, whether former NBA stars or amateur weekenders, can exercise more intensely at each training session with the ultimate consequence of greater performance.

Theoretically, it should be possible to use stem cells to generate healthy tissue to replace that either damaged by trauma, or compromised by disease. Among the conditions which scientists believe may eventually be treated by stem cell therapy are Parkinson's disease, Alzheimer's disease, heart disease, stroke, arthritis, diabetes, burns and spinal cord damage.

Do you have a question about holistic health or need assistance? Email Shirley Call 323-522-4521

Aquatic plant-based stem cell nutrition have been shown to support the release of millions of stem cells from the bone marrow very quickly. These stem cells can then migrate and attach to any cells, tissue, bone, muscle, cartilage, organ anywhere in the body needing repair. Once they attach, they become that tissue and multiply 3 to 5000 times. When there is an injury or a stress to an organ of your beloved pet or horse, compounds are released that reach the bone marrow and trigger the release of stem cells. Stem Cells can be thought of as master cells. Stem cells circulate and function to replace dysfunctional cells, thus fulfilling the natural process of maintaining optimal health.

As they do in humans, adult stem cells reside in animals bone marrow, where they are released whenever there is a problem somewhere in the body. Looking back on stem cell research, we realize that most studies have been done with animals, mostly mice, but also with dogs, horses, pigs, sheep and cattle. These studies have revealed that animal stem cells conduct themselves the same way human stem cells do. When there is an injury or a stress to an organ of your beloved pet or horse, compounds are released that reach the bone marrow and trigger the release of stem cells. The stem cells then travel to tissues and organs in need of help to regain optimal health.

Eve-Marie Lucerne - Eve-Marie keeps nine horses, all older thoroughbreds, and was eager to participate in the trials of a new stem cell enhancer for horses. She shared her allotment of test products with a few large commercial thoroughbred farms, veterinarians and other horse people she knows, and has been pleased with the consistently excellent results she has seen and others have reported to her. This product will help so many animals, she says, adding, People and animals are more alike than we are different. So it makes sense that a stem cell enhancer for animals with promote their health, too.

Eve-Marie's Equine Stem Cells Nutrition show dramatic results. For several horses facing serious physical challenges, cases where the animals might have to be put down, we saw a return to quality of life. This did not happen before Equine Stem Cell Nutrition. Eve-Marie says that this turnaround was quick, less than two weeks in many cases, and that the subject horses were back to health and enjoying pasture life within a month. One of the unofficial trial subjects for the equine stem cell nutrition was a 30-year old donkey who was in bad shape, Eve-Marie reports. He hadchronic respiratory difficulty and could move about only haltingly. His owner had stem cell enhancer supplements to help with her own serious health challenges and shared it with the donkey. The donkey's owner says this is the first time she wasn't sick, and her donkey is walking all around, feeling great an enjoying life again!

Farrier and National Hoof practitioner Stephen Dick received some of the trial product from Eve-Marie, and had good results with the two horses he selected for trial. For a 12-year-old quarterhorse stallion, the equine product brought dramatic results. This horse used to lie down twenty-two hours of the day, because he suffered discomfort whenever he stood, Steve reports, continuing, after a couple of weeks with Equine Stem Cell Nutrition, he was getting up and moving around, showing no discomfort. For a high-spirited mare with a leg problem, the equine product brought about a whole new lease on life, Steve says. This horse had been in a stall for 8 months. After about 6 weeks taking the equine product with her grain, her condition had improved and she was out of the stall, walking around in the pasture again.

Little Joe, a small 18-year-old quarterhorse that Judy Fisher bought when he was nearly 400 pounds underweight. You could count his ribs, Judy says, remembering, and his backbone stuck up like a ridge all along his back. He was very, very thin! Little Joe also suffered from breathing problems that kept him lethargic and inactive. Vet-recommended remedies were unsuccessful in changing Little Joe's physical problems, and the vet told Judy he didn't expect Little Joe to live through the winter. I figured Little Joe was in such bad shape that anything was worth a try, she says. She began giving the horse stem cell nutrition with his feed and grain twice a day. Within a couple of weeks, Judy was surprised to see Little Joe beginning to gain weight and run, buck, snort and kick. His breathing was no longer labored and his skin and coat were improving. Within six weeks Little Joe's overall appearance had changed dramatically. He had put on almost 300 pounds. When his former owner came to visit, Judy says, he didn't recognize Little Joe. That's how different he looked!

Sara participated in the stem cell nutrition product trials with her two horses and her 80-pound mixed-breed dog. She noted significant improvement in the health and quality of life for all three animals during the time of the trials. For JJ, Sara's 18-year old quarterhorse, the equine product brought about improvements in his overall mood, appearance and alertness quickly. He really liked the product from the beginning, Sara reports, pointing out that Hank, her 16-year-old thoroughbred/quarterhorse, had not taken to the taste of it too readily. I was able to slowly wean him on it though, she says. For Hank, the equine product was a balm for the skin problems resulting from his allergy to fly bites. His skin condition improved dramatically. Sara reports, noting that before the equine product the horse had scratched and bitten himself into ope wounds; after the equine product, the scratching and biting dropped off to almost nothing. Sara also noticed an increase in Hank's energy and liveliness in the first week on the equine product. The horse's foot and hip discomforts also responded well, leading to a noticeable increase in his mobility and an overall improvement in his quality of life throughout the two-month study.

Sara gave the pet product to her dog, Roxy, who had suffered for two years with ear problems that led to scratching, often until her skin was raw. Vet-recommended remedies had been temporary, quick-fixes, Sara says, but the discomfort always returned with a vengeance. For the pet trials, Sara gave Roxy two tabs of the product a day for two months, noting this is the only supplement she was getting. Sara says Roxy's problem with her ears definitely improved, the hair as grown back on her head and ears, and the ear problem has not recurred, adding that Roxy is happier and engaging, more playful.

Sonya had originally planned to use only her six-year-old Doberman Pinscher, Ginger, as a test subject for the pet product to see if the product could help with discomfort in her hips that had limited her mobility. That was until the day when Sonya's 14-year-old Irish Setter/Lab, Rowdy, took it upon himself to scarf up some large crumbs from Ginger's test dose of the pet product. Before dining on Ginger's leftovers, Rowdy was best characterized as the lazy dog type, very lethargic and he never left my side, Sonya reports.

Using the nutritional stem cell pet product , the first day Rowdy found the puppy back to his old self again, says Sonya and wanted to stay out all night, tracking who-knows-what, but having a great time. Sonya added Rowdy to her trial regimen and says that only two-three trial chewables made the difference in Rowdy's energy level, returning him to youthful activity. Meanwhile, the original trial subject, Ginger, has a new life and is able to keep up with 2-year-old Norwich Terrier mix Scrappy for the first time ever. Ginger has regained her ability to run at top speeds, and is able to dig for hours with Scrappy. Previously unable to get up from a prone position without some difficulty, Ginger now leaps to standing or chasing positions without a moment's hesitation. Stem Cell Nutrition for dogs has helped both of my big dogs to have their youth back, Sonya reports, adding I am a real believer in the canine product, as it has provided a spectacular change in both Ginger and Rowdy.

For dogs, depending on the size of your companion, you simply give half or one full tablet, which has a tasty dried beef and liver meal flavor. For horses, to the animal's daily grain you add one scoop of scrumptious molasses based Stem Cell Enhancer.

My German Shepherd was not able to get around, but within 2 weeks of using Stem Cell Nutrition for pets, her hips were not as stiff and she started running like a puppy. Jan, IN

Both of my big dogs have gained their youth back. I am a true believer in Stem Cell Nutrition for pets as it has provided a spectacular change in both Ginger and Rowdy. Sonya, IN

Stem cell nutrition for dogs, horses and other animals are specially formulated to be a delectable treat for your animal. The pet chewables and equine blends make it easy to provide your animals with this valuable nutritional supplement. The most common story is that of old, tired and sluggish dogs turned within a week or so into active, alert dogs running around like puppies. The same was observed in horses. Old horses who used to remain standing in the barn or under a tree, sluggish or stricken by too much discomfort to walk around, suddenly began moving about, and at times running and bucking like young colts. One of the most common reports was obvious improvements in hoof health and coat appearance.

Botanical stem cell nutrition are available in countries around the world.

Frequently Asked Questions about Stem Cell Enhancer Bibliography

Do you have a question about holistic health or need assistance? Email Shirley Call 323-522-4521

The National Health Institute lists seventy-four treatable diseases using ASCs in therapy - an invasive and costly procedure of removing the stem cells from one's bone marrow (or a donor's bone marrow) and re-injecting these same cells into an area undergoing treatment. For example, this procedure is sometimes done before a cancer patient undergoes radiation. Healthy stem cells from the bone marrow are removed and stored, only to be re-inserted after radiation into the area of the body in need of repair. This is a complex and expensive procedure, not accessible to the average person. However, there is now a way that every single person, no matter what their health condition, can have access to the benefits of naturally supporting their body's innate ability to repair every organ and tissue using stem cell nutrition.

David A. Prentice, Ph.D. - "Within just a few years, the possibility that the human body contains cells that can repair and regenerate damaged and diseased tissue has gone from an unlikely proposition to a virtual certainty. Adult stem cells have been isolated from numerous adult tissues, umbilical cord, and other non-embryonic sources, and have demonstrated a surprising ability for transformation into other tissue and cell types and for repair of damaged tissues.

A new U.S. study involving mice suggests the brain's own stem cells may have the ability to restore memory after an injury. These neural stem cells work by protecting existing cells and promoting neuronal connections. In their experiments, a team at the University of California, Irvine,were able to bring the rodents' memory back to healthy levels up to three months after treatment. The finding could open new doors for treatment of brain injury, stroke and dementia, experts say.

"This is one of the first reports that you can take a stem cell transplantation approach and restore memory," said lead researcher Mathew Blurton-Jones, a postdoctorate fellow at the university. "There is a lot of awareness that stem cells might be useful in treating diseases that cause loss of motor function, but this study shows that they might benefit memory in stroke or traumatic brain injury, and potentially Alzheimer's disease."

In the study, published in the Oct. 31 issue of the Journal of Neuroscience, Blurton-Jones and his colleagues used genetically engineered mice that naturally develop brain lesions. The researchers destroyed cells in a brain area called the hippocampus. These cells are known to be vital to memory formation and it is in this region that neurons often die after injury, the researchers explained. To test the mice's memory, Blurton-Jones's group conducted place and object recognition tests with both healthy mice and brain-injured mice.

Healthy mice remembered their surroundings about 70 percent of the time, while brain-injured mice remembered it only 40 percent of the time. For objects, healthy mice recalled objects about 80 percent of the time, but injured mice remembered them only 65 percent of the time. The researchers then injected each mouse with about 200,000 neural stem cells. They found that mice with brain injuries that received the stem cells now remembered their surroundings about 70 percent of the time -- the same as healthy mice. However, mice that didn't receive stem cells still had memory deficits.

The researchers also found that in healthy mice injected with stem cells, the stem cells traveled throughout the brain. In contrast, stem cells given to injured mice lingered in the hippocampus. Only about 4 percent of those stem cells became neurons, indicating that the stem cells were repairing existing cells to improve memory, rather than replacing the dead brain cells, Blurton-Jones's team noted. The researchers are presently doing another study with mice stricken with Alzheimer's. "The initial results are promising," Blurton-Jones said. "This has a huge potential, but we have to be cautious about not rushing into the clinic too early."

One expert is optimistic about the findings. "Putting in these stem cells could eventually help in age-related memory decline," said Dr. Paul R. Sanberg, director of the Center of Excellence for Aging and Brain Repair at the University of South Florida College of Medicine. "There is clearly a therapeutic potential to this." Sanberg noted that for the process to work with Alzheimer's it has to work with older brains. "There is clearly therapeutic potential in humans, but there are a lot of hurdles to overcome," he said. "This is another demonstration of the potential for neural stem cells in brain disorders.".

Botanical stem cell nutrition are available in countries around the world. (Select market/country at the top)

Dr. Nancy White Ph.D.- " I've always been interested in health generally and in particular the brain, focusing on the balance of neurotransmitters. I often do quantitative EEG's for assessment of my patients. I'm impressed with the concept of a natural product like stem cell nutrition that could help release adult stem cells from the bone mass where the body would have no objection and no rejection. I've tried stem cell nutrition for general health anti-aging. After taking it for a time, I fell more agile and my joints are far more flexible. I was astounded while doing yoga that I was suddenly able to bend over and touch my forehead to my knees. I haven't been able to do that comfortably in probably twenty years. I noticed how much better my balance has become. I believe stem cell nutrition is responsible for these effects, because I certainly haven't been trained extensively in yoga. Also since taking stem cell nutrition, I feel better and my skin is more moist and has a finer texture.

A bald friend of mine, who is also taking the stem cell nutrition, had several small cancers on top of his head. His doctor had removed one from his arm already, and his dermatologist set a date to remove those from his scalp. Before the appointment, my friend was shaving one morning and, looking in the mirror, saw that the cancers were all gone. They had disappeared within a few weeks of starting the stem cell nutrition and his skin is better overall. Also, his knee, which he'd strained playing tennis, was like new. Stem cell nutrition seems to go where the body's priority is. You never know what the affect is going to be, but you notice something is changing. Another friend of mine seems to be dropping years. Her skin looks smoother and her face younger. After about six weeks on the stem cell nutrition, she looks like she's ten years younger. A woman who gives her regular facials asked what she was doing, because her skin looked so much different. Stem cell nutrition is remarkable and could help anybody. Everybody should try it, because it's natural and there are no risks. As we grow older in years, we still can have good health. That's the ideal. Even if you don't currently have a problem, stem cell nutrition is a preventative." Dr. White holds a Ph. D. in Clinical Psychology, an MA in Behavioral Science, and a B.F.A. in Fine Arts, Magna Cum Laude. In addition, she is licensed in the State of Texas as a Psychologist , a Marriage and Family Therapist and as a Chemical Dependency Counselor.

Dr. Cliff Minter - "Stem cells are the most powerful cells in the body. We know that stem cells, once they're circulating in the bloodstream, will travel to any area of the body that has been compromised or damaged and turn into healthy cells. There have been controversial discussions about the new stem cells found in embryos, but the truth is that everyone has adult stem cells in their own bodies. We are all created from stem cells. As a child or a young adult, your body automatically releases stem cells whenever you injure yourself. That's why you heal so fast when you are younger. After about age 35, we don't heal as fast anymore, because the stem cells aren't released the same way as when we are younger. Stem cell nutrition helps all of us heal our bodies. If you look at the New England Journal of Medicine, you'll find that the number one indicator of a healthy heart is the number of stem cells circulating in the body. Stem cell nutrition is the organic and all-natural way to stimulate the bone marrow to release adult stem cells into the bloodstream.

By taking stem cell nutrition, you can maintain optimum health and aid your body in healing itself. It's certainly a better way to recuperate from an illness than using prescription drugs, because even when a medication works, it can often be hard on your liver and the rest of your body. Stem cell nutrition has no negative side effects. This makes it a powerful approach to healing and good health in general. I found out about stem cell nutrition after someone asked for my opinion on it. I did some research and found it to be one of the greatest ways to slow down aging that we have. Aging is nothing more than the breakdown of cells. Stem cell nutrition combats that action. As cells break down, stem cell nutrition replaces them with healthy cells. This is the greatest, most natural anti-aging method I know. I was skeptical at first, but the results I've personally seen in people I've talked with have been wide-ranged. Lots of people have reported an increase in energy and better sleeping patterns.

I've seen people with arthritis in various parts of their bodies reverse the disease, and people with asthma end up with their lungs totally clear. One person that was on oxygen almost 24/7 is now totally off of oxygen. Two ladies who suffered badly from PMS told me they were 100 percent symptom-free within weeks of starting the stem cell nutrition. Two people I know had tennis elbow which usually takes about six to nine months to heal. Within weeks of taking stem cell nutrition, both report their "tennis elbow" is gone. It makes sense, because stem cells go to whatever area is compromised and turn into healthy cells.

I use stem cell nutrition as a preventative. I've noticed an increase in my energy level and an improved sleeping pattern. Stem cell nutrition has zero negative side effects, is very powerful, and we know how it works. It's good for children as well as adults. This is the best, most natural way I know to optimum health. If you just want to use it for prevention, this is the best thing I know for staying healthy. And if you do those and regaining optimum health. I recommend it to everybody." Dr. Cliff Minter (retired) graduated from Illinois College of Podiatric Medicine. He completed his residency at the Hugar Surgery Center in the Hines Veteran Administration Hospital in Illinois before going into private practice in Ventura, CA. Dr. Minter is a national and international speaker on the subjects of business and nutritional products.

Fernando Aguila, M.D. - "Due to a heavy patient load, I have recently found that I tire more easily, my legs are cramping, and by the time I get home, even my shoulders and rib cage hurt. I knew I had to find a way to increase my stamina, energy and vitality. A friend gave me information about stem cell nutrition and how it promotes the release of stem cells in the body. One of the components apparently promotes the migration of the stem cells to tissues or organs where regeneration and repair is needed most. My attention was drawn to the fact that it can increase energy, vitality, wellness, concentration, and much more. It sounded just like what I needed. Since then, I've heard reports of people experiencing excellent results in a number of different areas in their health. The improvements sounded dramatic. Because of all of their testimonies, I was willing to believe it could promote wellness in the human body.

I tried stem cell nutrition myself. After a day, of hard work, I realized I wasn't tired at all, my legs were not aching, and I didn't have any shoulder pain. I decided the stem cell nutrition must be working. I continued to take it, and was able to work so efficiently and steadily that one surgeon commented that I was moving like a ball of fire. Stem cell nutrition gives me support physically and mentally. I look forward to seeing what the major medical journals have to say about the studies being done with this new approach to wellness." Fernando Aguila, M.D., graduated from the University of Santa Thomas in Manila , Philippines. He finished his internship at Cambridge City Hospital, Cambridge, MA and completed his residency at the New England Medical Center in Boston, MA. He obtained a fellowship in OB-GYN anesthesia at the Brigham and Women's Hospital in Boston and a fellowship in cardio-thoracic anesthesia at the Cleveland Clinic Foundation in Cleveland, OH.

Botanical stem cell nutrition are available in countries around the world

View Dr. Christian Drapeau's introduction to Adult Stem Cell Bone Marrow Release

Christian Drapeau is America's best known advocate for Adult Stem Cell science health applications and the founder of the field of Stem Cell Nutrition. He holds a BS in Neurophysiology from McGill University and a Master of Science in Neurology and Neurosurgery from the Montreal Neurological Institute.

One particular stem cell enhancers that was studied was found to contain a polysaccharide fraction that was shown to stimulate the migration of Natural Killer (NK) cells out of the blood into tissues. The same polysaccharide fraction was also shown to strongly stimulate the activation of NK cells. NK cells play the very important role in the body of identifying aberrant or defective cells and eliminating them. NK cells are especially known for their ability to detect and destroy virally infected cells and cells undergoing uncontrolled cellular division. The same polysaccharide fraction was also shown to stimulate macrophage activity. Macrophages constitute the front line of the immune system. They first detect an infection or the presence of bacteria or virally infected cells, and they then call for a full immune response. Adult Stem Cell Nutritional Enhancer also contains a significant concentration of chlorophyll and phycocyanin, the blue pigment in AFA. Phycocyanin has strong anti-inflammatory properties and therefore can assist the immune system.

The release of stem cells from the bone marrow and their migration to tissues is a natural process that happens everyday. Stem cell enhancers simply support that natural process and tips the balance toward health everyday. It does not do anything that the body does not already do everyday. So far, no instances of cancer or any similar problem have ever been observed when using in vivo natural release of stem cells from the bone marrow.

Each day, stem cells in the bone marrow evolve to produce red blood cells, white blood cells, and platelets. These mature cells are then released into the bloodstream where they perform their vital life-supporting functions. When bone marrow stem cell activity is interfered with, diseases such as anemia (red blood cell deficit), neutropenia (specialized white blood cell deficit), or thrombocytopenia (platelet deficit) are often diagnosed. Any one of these conditions can cause death if not corrected.

Scientists have long known that folic acid, vitamin B12, and iron are required for bone marrow stem cells to differentiate into mature red blood cells.3-7 Vitamin D has been shown to be crucial in the formation of immune cells,8-11 whereas carnosine has demonstrated a remarkable ability to rejuvenate cells approaching senescence and extend cellular life span.12-28

Other studies of foods such as blueberries show this fruit can prevent and even reverse cell functions that decline as a result of normal aging.29-36 Blueberry extract has been shown to increase neurogenesis in the aged rat brain.37,38 Green tea compounds have been shown to inhibit the growth of tumor cells, while possibly providing protection against normal cellular aging.39,40

Based on these findings, scientists are now speculating that certain nutrients could play important roles in maintaining the healthy renewal of replacement stem cells in the brain, blood, and other tissues. It may be possible, according to these scientists, to use certain nutrient combinations in the treatment of conditions that warrant stem cell replacement

These studies demonstrate for the first time that various natural compounds can promote the proliferation of human bone marrow cells and human stem cells. While these studies were done in vitro, they provide evidence that readily available nutrients may confer a protective effect against today's epidemic of age-related bone marrow degeneration.

Dr. Robert Sampson, MD on stem cell nutrition - "... we have a product that has been shown and demonstrated in the patent to increase the level of adult circulating stem cells by up to 30%. It seems to me we're having a great opportunity here to optimize the body's natural ability to create health." Recent scientific developments have revealed that stem cells derived from the bone marrow, travel throughout the body, and act to support optimal organ and tissue function. Stem cell enhancers supports the natural role of adult stem cells. Stem cell enhancer are typically derived from certain edible algae that grows in fresh water.

Botanical stem cell nutrition are available in countries around the world. (Select market/country at the top)

Do you have a question about holistic health or need assistance? Email Shirley Call 323-522-4521

The possibility that a decline in the numbers or plasticity of stem cell populations contributes to aging and age-related disease is suggested by recent findings. The remarkable plasticity of stem cells suggests that endogenous or transplanted stem cells can be tweaked' in ways that will allow them to replace lost or dysfunctional cell populations in diseases ranging from neurodegenerative and hematopoietic disorders to diabetes and cardiovascular disease.

As you age, the number and quality of stem cells that circulate in your body gradually decrease, leaving your body more susceptible to injury and other age-related health challenges. Just as antioxidants are important to protect your cells from free radical damage, stem cell nutrition is equally important to support your stem cells in maintaining proper organ and tissue functioning in your body.

A fundamental breakthrough in our understanding of nervous system development was the identification of multipotent neural stem cells (neurospheres) about ten years ago. Dr. Weiss and colleagues showed that EGF (epidermal growth factor) dependent stem cells could be harvested from different brain regions at different developmental stages and that these could be maintained over multiple passages in vitro. This initial finding has lead to an explosion of research on stem cells, their role in normal development and their potential therapeutic uses. Many investigators have entered this field and the progress made has been astounding.

How does an increase in the number of circulating stem cells lead to optimal health? Circulating stem cells can reach various organs and become cells of that organ, helping such organ regain and maintain optimal health. Recent studies have suggested that the number of circulating stem cells is a key factor; the higher the number of circulating stem cells the greater is the ability of the body at healing itself. Scientific interest in adult stem cells has centered on their ability to divide or self-renew indefinitely, and generate all the cell types of the organ from which they originate, potentially regenerating the entire organ from a few cells. Adult stem cells are already being used clinically to treat many diseases. These include as reparative treatments with various cancers, autoimmune disease such as multiple sclerosis, lupus and arthritis, anemias including sickle cells anemia and immunodeficiencies. Adult stem cells are also being used to treat patients by formation of cartilage, growing new corneas to restore sight to blind patients, treatments for stroke, and several groups are using adult stem cells to repair damage after heart attacks. Early clinical trials have shown initial success in patient treatments for Parkinsons disease and spinal cord injury. The first FDA approved trial to treat juvenile diabetes in human patients is ready to begin at Harvard Medical School, using adult stem cells. An advantage of using adult stem cells is that in most cases, the patients own stem cells can be used for the treatment, circumventing the problems of immune rejection, and without tumor formation.

Why do we hear much in the news about embryonic stem cells and very little about adult stem cells? The first human embryonic stem cells were grown in vitro, in a petri dish, in the mid 1990s. Rapidly, scientists were successful at growing them for many generations and to trigger their differentiation into virtually any kind of cells, i.e. brain cells, heart cells, liver cells, bone cells, pancreatic cells, etc. When scientists tried growing adult stem cells, the endeavor was met with less success, as adult stem cells were difficult to grow in vitro for more than a few generations. This led to the idea that embryonic stem cells have more potential than adult stem cells. In addition, the ethical concerns linked to the use of embryonic stem cells have led to a disproportionate representation of embryonic stem cells in the media. But recent developments over the past 2-3 years have established that adult stem cells have capabilities comparable to embryonic stem cells in the human body, not in the test tube. Many studies have indicated that simply releasing stem cells from the bone marrow can help support the body's natural process for renewal of tissues and organs.

The bone marrow constantly produces stem cells for the entire life of an individual. Stem cells released by the bone marrow are responsible for the constant renewal of red blood cells and lymphocytes (immune cells). A 25-30% increase in the number of circulating stem cells is well within physiological range and does not constitute stress on the bone marrow environment. The amount of active bone marrow amounts to about 2,600 g (5.7 lbs), with about 1.5 trillion marrow cells. Stem cells that do not reach any tissue or become blood cells return to the bone marrow.

Effectiveness of stem cell "enhancers" was demonstrated in a triple-blind study. Volunteers rested for one hour before establishing baseline levels. After the first blood samples, volunteers were given stem cell "enhancers"or placebo. Thereafter, blood samples were taken at 30, 60 and 120 minutes after taking the consumables. The number of circulating stem cells was quantified by analyzing the blood samples using Fluorescence-Activated Cell Sorting (FACS). Consumption of stem cell "enhancers" triggered a significant 25-30% increase in the number of circulating stem cells.

Botanical stem cell nutrition are available in countries around the world. (Select market/country at the top)

Do you have a question about holistic health or need assistance? Email Shirley Call 323-522-4521

See original here:
Adult Stem Cells Nutrition Boost Healing in Humans, Animals

1. Introduction

Bone marrow is a complex tissue containing hematopoietic cell progenitors and their progeny integrated within a connective-tissue network of mesenchymal-derived cells known as the stroma (1). The stroma cells, or Mesenchymal stem cells (MSCs), are multi-potent progenitor cells that constitute a minute proportion of the bone marrow, represented as a rare population of cells that makes up 0.001 to 0.01% of the total nucleated cells. They represent 10-fold less abundance than the haematopoietic stem cells (2), which contributes to the organization of the microenvironment supporting the differentiation of hematopoietic cells (3). MSC are present in tissues of young, as well as, adult individuals (4, 5), including the adipose tissue, umbilical cord blood, amniotic fluid and even peripheral blood (1, 6-8). MSCs were characterized over thirty years ago as fibroblast-like cells with adhesive properties in culture (9, 10). The term MSC has become the predominant term in the literature since the early 90s (11), after which their research field has grown rapidly due to the promising therapeutic potential, resulting in an increased frequency of clinical trials in the new millennium at an explosive rate.

As data accumulated, there was a need to establish a consensus on the proper definition of the MSCs. The International Society for Cellular Therapy has recommended the minimum criteria for defining multi-potent human MSCs (12, 13). The criteria included: (i) cells being adherent to plastic under standard culture conditions; (ii) MSC being positive for the expression of CD105, CD73 and CD90 and negative for expression of the haematopoietic cell surface markers CD34, CD45, CD11a, CD19 or CD79a, CD14 or CD11b and histocompatibility locus antigen (HLA)-DR; (iii) under a specific stimulus, MSC differentiate into osteocytes, adipocytes and chondrocytes in vitro. These criteria presented properties to purify MSC and to enable their expansion by several-fold in-vitro, without losing their differentiation capacity. When plated at low density, MSCs form small colonies, called colony-forming units of fibroblasts (CFU-f), and which correspond to the progenitors that can differentiate into one of the mesenchymal cell lineages (14, 15). It has been reported lately that MSCs are able to differentiate into both mesenchymal, as well as, non-mesenchymal cell lineages, such as adipocytes, osteoblasts, chondrocytes, tenocytes, skeletal myocytes, neurones and cells of the visceral mesoderm, both in vitro and in vivo (16, 17).

All cells have half-lives and their natural expiration must be matched by their replacement; MSCs, by proliferating and differentiating, can be the proposed source of these new replacement cells as characterized in their differentiation capacity. This replacement hypothesis mimics the known sequence of events involved in the turnover and maintenance of blood cells that are formed from haematopoietic stem cells (HSCs) (18). Unlike HSCs, MSCs can be culture-expanded ex vivo in up to 40 or 50 cell doublings without differentiation (19). A dramatic decrease in MSC per nucleated marrow cell can be observed when the results are grouped by decade, thus showing a 100-fold decrease from birth to old age. Being pericytes present in all vascularized tissues, the local availability of MSC decreases substantially as the vascular density decreases by one or two orders of magnitude with age (20). In recent years, the discovery of MSCs with properties similar, but not identical, to BM-MSCs has been demonstrated in the stromal fraction of the connective tissue from several organs, including adipose tissue, trabecular bone, derma, liver and muscle (21-24). It is important to note that the origin of MSCs might determine their fate and functional characteristics (25). Studies of human bone marrow have revealed that about one-third of the MSC clones are able to acquire phenotypes of pre-adipocytes, osteocytes and chondrocytes (16). This is in concordance with data showing that 30% of the clones from bone marrow have been found to exhibit a trilineage differentiation potential, whereas the remainder display a bi-lineage (osteo-chondro) or uni-lineage (osteo) potential (26). Moreover, MSC populations derived from adipose tissue and derma present a heterogeneous differentiation potential; indeed, only 1.4% of single cells obtained from adipose-derived adult stem cell (ADAS) populations were tri-potent, the others being bi-potent or unipotent (27).

Mesenchymal Stem Cells have been shown to possess immunomodulatory characteristics, as described through the inhibition of T-cell proliferation in vitro (28-30). These observations have triggered a huge interest in the immunomodulatory effects of MSCs. The in vitro studies have been complemented in vivo, where both confirmed the immunosuppressive effect of MSC. MSC activating stimuli in vitro, appears to include the secretion of cytokines and the interaction with other immune cells in the presence of proinflammatory cytokines (Fig 1) (31). Primarily, the in vivo effect has been originally shown in a baboon model, in which infusion of ex vivoexpanded matched donor or third-party MSCs delayed the time to rejection of histo-incompatible skin grafts (29). The delay indicated a potential role for MSC in the prevention and treatment of graft-versus-host disease (GVHD) in ASCT, in organ transplantation to prevent rejection, and in autoimmune disorders. Recently, MSCs were used to successfully treat a 9-year-old boy with severe treatment-resistant acute GVHD, further confirming the potent immunosuppressive effect in humans (32). The immunosuppressive potential has no immunologic restriction, whether the MSCs are autologous with the stimulatory or the responder lymphocytes or the MSCs are derived from a third party. The degree of MSC suppression is dose dependent, where high doses of MSC are inhibitory, whereas low doses enhance lymphocyte proliferation in MLCs (33). Broadly, MSC modulate cytokine production by the dendritic and T cell subsets DC/Th1 and DC/Th2 (34), block the antigen presenting cell (APC) maturation and activation (35), and increase the proportion of CD4+CD25+ regulatory cells in a mixed lymphocyte reaction (36).

Potential mechanisms of the MSC interactions with immune cells. Mesenchymal stem cells (MSCs) can inhibit both the proliferation and cytotoxicity of resting natural killer (NK) cells, as well as, their cytokine production by releasing prostaglandin E2 (PGE2), indoleamine 2,3-dioxygenase (IDO) and soluble HLA-G5 (sHLA-G5). Killing of MSCs by cytokine-activated NK cells involves the engagement of cell-surface receptors (Thick blue line) expressed by NK cells with its ligands expressed on MSCs. MSCs inhibit the differentiation of monocytes to immature myeloid dendritic cells (DCs), bias mature DCs to an immature DC state, inhibit tumour-necrosis factor (TNF) production by DCs and increase interleukin-10 (IL-10) production by plasmacytoid DCs (pDCs). MSC-derived PGE2 is involved in all of these effects. Immature DCs are susceptible to activated NK cell-mediated lysis. MSC Direct inhibition of CD4+ T-cell function depends on their release of several soluble molecules, including PGE2, IDO, transforming growth factor-1 (TGF1), hepatocyte growth factor (HGF), inducible nitric-oxide synthase (iNOS) and haem-oxygenase-1 (HO1). MSC inhibition of CD8+ T-cell cytotoxicity and the differentiation of regulatory T cells mediated directly by MSCs are related to the release of sHLA-G5 by MSCs. In addition, the upregulation of IL-10 production by pDCs results in the increased generation of regulatory T cells through an indirect mechanism. MSC-driven inhibition of B-cell function seems to depend on soluble factors and cellcell contact. Finally, MSCs dampen the respiratory burst and delay the spontaneous apoptosis of neutrophils by constitutively releasing IL-6.

Dendritic cells have the elementary role of antigen presentation to nave T cells upon maturation, which in turn induce the proinflammatory cytokines. Immature DCs acquire the expression of co-stimulatory molecules and upregulate expression of MHC-I and II, as well as, other cell-surface markers (CD11c and CD83). Mesenchymal stem cells have profound effect on the development of DC, where in the presence of MSC, the percentage of DC with conventional phenotype is reduced, while that of plasmacytoid DC is increased, therefore biasing the immune system toward Th2 and away from Th1 responses in a PGE-2 dependent mechanism (37). Furthermore, MSCs inhibit the up-regulation of CD1a, CD40, CD80, CD86, and HLA-DR during DC differentiation and prevent an increase of CD40, CD86, and CD83 expression during DC maturation (38). When mature DCs are incubated with MSCs they have a decreased cell-surface expression of MHC class II molecules, CD11c, CD83 and co-stimulatory molecules, as well as, decreased interleukin-12 (Il-12) production, thereby impairing the antigen-presenting function of the DCs (Fig 1) (39, 40). MSCs can also decrease the pro-inflammatory potential of DCs by inhibiting their production of tumour-necrosis factor (TNF-) (40). Furthermore, plasmacytoid DCs (pDCs), which are specialized cells for the production of high levels of type-I IFN in response to microbial stimuli, upregulate production of the anti-inflammatory cytokine IL-10 after incubation with MSCs (34). These observations indicate a potent anti-inflammatory and immunoregulatory effect for MSC in vitro and potentially in vivo.

Natural killer (NK) cells are key effector cells of the innate immunity in anti-viral and anti-tumor immune responses through their Granzyme B mediated cytotoxicity and the production of pro-inflammatory cytokines (41). NK-mediated target cell lysis results from an antigen-ligand interaction realized by activating NK-cell receptors, and associated with reduced or absent MHC-I expression by the target cell (42). MSCs can inhibit the cytotoxic activity of resting NK cells by down-regulating expression of NKp30 and natural-killer group 2, member D (NKG2D), which are activating receptors involved in NK-cell activation and target-cell killing (Fig 1) (43). Resting NK cells proliferate and acquire strong cytotoxic activity when cultured with IL-2 or IL-15, but when incubated with MSC in the presence of these cytokines, resting NK-cell, as well as, pre-activated NK cell proliferation and IFN- production are almost completely abrogated (44, 45). It is worth noting that although the susceptibility of NK cells to MSC mediated inhibition is potent, the pre-activated NK cells showed more resistance to the immunosuppressive effect of MSC compared to resting NK cells (43). The susceptibility of human MSCs to NK-cell-mediated cytotoxicity depends on the low level of cell-surface expression of MHC class I molecules by MSCs and the expression of several ligands, that are recognized by activating NK-cell receptors. Autologous and allogeneic MSC were susceptible to lysis by NK cells (43), where NK cell-mediated lysis was inversely correlated with the expression of HLA class I on MSC (46). Incubation of MSCs with IFN- partially protected them from NK-cell-mediated cytotoxicity, through the up-regulation of expression of MHC-I molecules on MSCs (43). Taken together, a possible dynamic interaction between NK cells and MSC in vivo exists, where the latter partially inhibit activated MSC, without compromising their ability to kill MSC, reflecting on an interaction tightly regulated by IFN- concentration.

Neutrophils play a major role in innate immunity during the course of bacterial infections, where they are activated to kill foreign infectious agents and accordingly undergo a respiratory burst. MSCs have been shown to dampen the respiratory burst and to delay the spontaneous apoptosis of resting and activated neutrophils through an IL-6-dependent mechanism (47). MSC had no effect on neutrophil phagocytosis, expression of adhesion molecules, and chemotaxis in response to IL-8, f-MLP, or C5a (47). Stimulation with bacterial endotoxin induces chemokine receptor expression and mobility of MSCs, which secrete large amounts of inflammatory cytokines and recruit neutrophils in an IL-8 and MIF-dependent manner. Recruited and activated neutrophils showed a prolonged lifespan, an increased expression of inflammatory chemokines, and an enhanced responsiveness toward subsequent challenge with LPS, which suggest a role for MSCs in the early phases of pathogen challenge, when classical immune cells have not been recruited yet (48). Furthermore, MSC have shown the capability to mediate the preservation of resting neutrophils, a phenomenon that might be important in those anatomical sites, where large numbers of mature and functional neutrophils are stored, such as the bone marrow and lungs (49).

T-cells are major players of the adaptive immune response. After T-cell receptor (TCR) engagement, T cells proliferate and undergo numerous effector functions, including cytokine release and, in the case of CD8+ T cells (CTL), cytotoxicity. Abundant reports have shown that T-cell proliferation stimulated with polyclonal mitogens, allogeneic cells or specific antigen is inhibited by MSCs (28, 29, 50-56). The observation that MSCs can reduce T cell proliferation in vitro is mirrored by the in vivo finding through infusions of hMSCs that control GVHD following bone marrow transplantation. Nevertheless, there is no demonstrable correlation between the measured effects of MSCs in vitro and their counter effect in vivo due to the lack of universality of methodology correlating the in vitro findings with the in vivo therapeutic potential.

MSC inhibition of T-cell proliferation is not MHC restricted, since it can be mediated by both autologous and allogeneic MSCs and depends on the arrest of T-cells in the G0/G1 phase of the cell cycle (55, 57). Thus, MSCs do not promote T-cell apoptosis, but instead maintain T cell survival upon subjection to overstimulation through the TCR and upon commitment to undergo CD95CD95-ligand-dependent activation-induced cell death (57). MSC effects on T cell proliferation in vitro appear to have both contact-dependent and contact-independent components (58). Inhibition of T-cell proliferation by MSCs leads to decreased IFN- secretion in vitro and in vivo associated with increased IL-4 production by T helper 2 (TH2) cells (34, 59). Taken together, there is an implication for a shift from a pro-inflammatory state characterized by IFN- secretion to an anti-inflammatory state characterized by IL-4 secretion (Fig 1). An imperative role for effector T-cell is the MHC restricted killing of virally-infected or of allogeneic cells mediated via CD8+ CTLs, and which is down-regulated by MSC (60).

Regulatory T cells (Tregs), a subpopulation of T cells, are vital to keep the immune system in check, help avoid immune-mediated pathology and contain unrestricted expansion of effector T-cell populations, which results in maintaining homeostasis and tolerance to self antigens. Tregs are currently identified by co-expression of CD4 and CD25, expression of the transcription factor FoxP3, production of regulatory cytokines IL-10 and TGF-, and ability to suppress proliferation of activated CD4+CD25+ T cells in co-culture experiments. Differential expression of CD127 (-chain of the IL-7 receptor) enable flow cytometry-based separation of human Tregs from CD127+ non-regulatory T-cells (61). MSCs have been reported to induce the production of IL-10 by pDCs, which, in turn, trigger the generation of regulatory T cells (Fig 1) (34, 40). Furthermore, Tregs secrete TGF- and when used in vitro, TGF- in combination with IL-2 directs the differentiation of T-cells into Tregs, while Tregs suppress the proliferation of TCR-dependent proliferation of effector CD25null or CD25low T-cells in a non-autologous fashion. Also TGF- alters angiogenesis following injury in experiments using MSC (62). In addition, after co-culture with antigen-specific T-cells, MSCs can directly induce the proliferation of regulatory T-cells through release of the immunomodulatory HLA-G isoform HLA-G5 (Fig 1) (63). Taken together, MSCs can modulate the intensity of an immune response by inhibiting antigen-specific T-cell proliferation and cytotoxicity and promoting the generation of regulatory T-cells.

Antibody producing B-cells constitute the second main cell type involved in adaptive immunity. Interactions between MSCs and B-cells have produced controversial results attributable to the inconsistent experimental conditions used (31, 55, 64). Initial reports on mice suggested that MSC exercise a dampening effect on the proliferation of B-cells (64), which is in concordance with most published works to date (31, 55, 64). Furthermore, MSCs can also inhibit B-cell differentiation and constitutive expression of chemokine receptors via the release of soluble factors and cell-cell contact mediated possibly by the Programmed Cell Death 1 (PD-1) and its ligand (31, 64). The addition of MSCs, at the beginning of a mixed lymphocyte reaction (MLC), considerably inhibited immunoglobulin production in standard MLC, irrespective of the MSC dose employed, which suggests that third-party MSC are able to suppress allo-specific antibody production, consequently, overcoming a positive cross-match in sensitized transplant recipients (65). However, other in vitro studies have shown that MSCs could support the survival, proliferation and differentiation to antibody-secreting cells of B-cells from normal individuals and from pediatric patients with systemic lupus erythematosus (66, 67). A major mechanism of B-cell suppression was hMSC production of soluble factors, as indicated by transwell experiments, where hMSCs inhibited B-cell differentiation shown as significant impairment of IgM, IgG, and IgA production. CXCR4, CXCR5, and CCR7 B-cell expression, as well as chemotaxis to CXCL12, the CXCR4 ligand, and CXCL13, the CXCR5 ligand, were significantly down-regulated by hMSCs, suggesting that these cells affect chemotactic properties of B-cells (Fig 1). B-cell costimulatory molecule expression and cytokine production were unaffected by hMSCs (64). Regardless of the controversial in vitro effects, B-cell response is mainly a T-cell dependent mechanism, and thus its outcome is significantly influenced by the MSC-mediated inhibition of T-cell functions. More recently, Corcione et al have shown that systemic administration of MSCs to mice affected by experimental autoimmune encephalomyelitis (EAE), a prototypical disease mediated by self-reactive T cells, results in striking disease amelioration mediated by the induction of peripheral tolerance (52). In addition, it has been shown that tolerance induction by MSCs may occur also through the inhibition of dendritic-cell maturation and function (34, 35), thus suggesting that activated T cells are not the only targets of MSCs.

Low concentrations of IFN- upregulate the expression of MHC-II molecules by MSCs, which indicates that they could act as antigen presenting cells (APCs) early in an immune response, when the level of IFN- are low (68, 69). However, this process of MHC-II expression by MSCs, along with the potential APC characteristics, was reversed as IFN- concentrations increased. These observations could suggest that MSCs function as conditional APC in the early phase of an immune response, while later switch into an immunosuppressive function (68). Since bone marrow might be a site for the induction of T-cell responses to blood-borne antigens (70), and since MSC are derived from the stromal progenitor cells that reside in the bone marrow, therefore, MSC express a yet unidentified role in the control of the immune response physiology of the bone marrow. Dendritic cells are the main APC for T-cell responses, and MSC-mediated suppression of DC maturation would prohibit efficient antigen presentation and thus, the clonal expansion of T-cells. Direct interactions of MSCs with T-cells in vivo could lead to the arrest of T-cell proliferation, inhibition of CTL-mediated cytotoxicity and generation of CD4+ regulatory T-cells. As a consequence, impaired CD4+ T-cell activation would translate into defective T-cell help for B-cell proliferation and differentiation to antibody-secreting cells.

The hMSCs express few to none of the B7-1/B7-2 (CD80/CD86) costimulatorytype molecules; this appears to contribute, at least in part, to their immune privilege characteristic. Mechanisms that lead to immune tolerance rely on interrelated pathways that involve complex cross talk and cross regulation of T-cells and APCs by one another. Both soluble mediators and modulation exerted via complex networks of cytokines and costimulatory molecules appear to play a role in MSC regulation of T cells, and these mechanisms invoke both contact-dependent and -independent pathways.

Although many of the studies use MSC-conditioned medium, both contact-dependent and -independent mechanisms are probably invoked in the therapeutic use of MSCs (20, 71). In addition to cytokines, the network of costimulatory molecules is hypothesized to play a prominent role in modulating tolerance and inflammation. MSCs down-regulate the expression of costimulatory molecules (30, 72, 73). The discovery of new functions for B7 family members, together with the identification of additional B7 and CD28 family members, is revealing new ways in which the B7:CD28 family may regulate T-cell activation and tolerance. Not only do CD80/86:CD28 interactions promote initial T-cell activation, they also regulate self-tolerance by supporting CD4+CD25+ Treg homeostasis (74-76). Cytotoxic T-lymphocyte antigen 4 (CTLA-4) can exert inhibitory effects in both B7-1/B7-2dependent and independent fashions. B7-1 and B7-2 can signal bi-directionally through engaging CD28 and CTLA-4 on T cells and by delivering signals into B7-expressing cells (77). The B7 family membersinducible co-stimulator (ICOS) ligand, PD-L1 (B7-H1), PD-L2 (B7-DC), B7-H3, and B7-H4 (B7x/B7-S1)are expressed on professional APC cells, while B7-H4 and B7-H1 are expressed on hMSCs and on cells within non-lymphoid organs. These observations may provide a new means for regulating T-cell activation and tolerance in peripheral tissues (31, 71, 78). ICOS and PD-1 are expressed upon T-cell-induction, and they regulate previously activated T-cells (79). Both the ICOS:ICOSL pathway and the PD-1:PD-L1/PD-L2 pathway play a critical role in regulating T-cell activation and tolerance (79). There is consensus that both CTLA-4 and PD-1 inhibit T-cell and B-cell activation and may play a crucial role in peripheral tolerance (79, 80). Both CTLA-4 and PD-1 functions are associated with Rheumatoid Arthritis (RA) and other autoimmune diseases. PD-1 is over expressed on CD4+ T cells in the synovial fluid of RA patients (81). Whether or not these costimulatory molecules are critical mediators of MSC-mediated immune suppression and/or tolerance in vivo is still under current investigation.

Studies have shown that MSCs escape the immune system, and this makes them a potential therapeutic tool for various transplantation procedures. MSCs express intermediate levels of HLA major histocompatibility complex (MHC) class I molecules (16, 50, 82, 83), while they do not express HLA class II antigens of the cell surface. However, HLA class II is readily detectable by Western blot on whole-cell lysates of unstimulated adult MSCs, thus suggesting that MSCs contain intracellular deposits of HLA class II allo-antigens (83). Cell-surface expression can be induced by treatment of the cells with IFN- for 1 or 2 days. Unlike adult MSCs, the fetal liver derived cells have no intracellular nor cell surface HLA class II expression (84), but incubation with IFN- initiated their intracellular expression followed by surface expression. Differentiation of MSCs into their mesodermal lineages of bone, cartilage, or adipose tissue, both in adult and fetal MSCs continued to express HLA-I, but not class II (84). Undifferentiated MSCs in vitro fail to elicit a proliferative response from allogeneic lymphocytes, thus suggesting that the cells are not inherently immunogenic (28, 30, 50). When pre-cultured with IFN- for full HLA class II expression, MSCs still escape recognition by allo-reactive T-cells, (83, 84) as is the case with MSCs differentiated adipocytes, osteoblasts, and chondrocytes. Limited in vivo data demonstrate the persistence of allogeneic MSCs into immunocompetent hosts after transplantation. In one patient treated with MSCs, DNA of donor MSC could not be detected in any organ at autopsy few weeks after the infusion, while in another patient receiving MSCs from two donors, the donor DNA from both donors was detected in lymph node and colon, the target organs of GVHD, within weeks after infusion (85). Data from our lab indicated that MSC were undetectable after two weeks in an allogeneic system (86). Therefore, the question of whether MSCs are recognized by an intact allogeneic immune system in vivo remains open, although the in vitro data support the theory that MSCs escape the immune system. MSCs do not express FAS ligand or costimulatory molecules, such as B7-1, B7-2, CD40, or CD40L (50). When costimulation is inadequate, T-cell proliferation can be induced by the addition of exogenous costimulation. However, MSCs differ from other cell types, and no T-cell proliferation can be observed when they are cultured with HLA-mismatched lymphocytes in the presence of a CD28-stimulating antibody (50). However, in agreement with the in vitro data, infusion or implantation of allogeneic and MHC-mismatched MSCs into baboons has been well tolerated (87-89). Unique immunologic properties of MSCs were also suggested by the fact that engraftment of human MSCs occurred after intra-uterine transplantation into sheep, even when the transplantation was performed after the fetuses became immunocompetent (90). MSC mainly fail to activate T-cells and show to be targets for CD8+ T cell-cytotoxicity, althought controversial (60). Phyto-hemagglutinin (PHA) blasts, generated to react against a specific donor, will lyse chromium-labeled mononuclear cells from that individual but it will not lyse MSCs derived from the same donor. Furthermore, killer cell inhibitory receptor (KIR ligand)mismatched natural killer cells do not lyse MSCs (60). Thus, MSCs, although incompatible at the MHC, tend to escape the immune system.

Although MSCs are transplantable across allogeneic barriers, a delayed type hypersensitivity reaction can lead to rejection in xenogenic models of human MSCs injected into immunocompetent rats (91). In this study, MSCs were identified in the heart muscle of severe compromised immune deficiency rats, in contrast to that of immunocompetent rats. In the latter group, peripheral blood lymphocytes proliferated after re-stimulation with human MSCs in vitro, thus suggesting cellular immunization. Such a proliferative response in vitro has not been detected in humans treated with intravenous (IV) infusion of allogeneic MSCs (Le Blanc and Ringdn, unpublished data, 2004).

Several studies have acknowledged the immunosuppressive activities of MSCs, but the underlying mechanisms are far from being fully characterized. The initial step in the interaction between MSCs and their target cells involves cellcell contact mediated by adhesion molecules, in concordance with studies showing the dependence of T-cell proliferation on the engagement of PD-1 by its ligand (31). Several soluble immunosuppressive factors, either produced constitutively by MSCs or released following cross-talk with target cells have been reported, including nitric oxide and indoleamine 2,3-dioxygenase (IDO), which are only released by MSC after IFN- stimulation with target cells (92, 93), and thus not in a constitutive manner. IDO induces the depletion of tryptophan from the local environment, which is an essential amino acid for lymphocyte proliferation. MSC-derived IDO was reported as a requirement to inhibit the proliferation of IFN--producing TH1 cells (92) and together with prostaglandin E2 (PGE-2) to block NK-cell activity (Fig 1) (44). In addition, IFN-, alone or in combination with TNF-, IL-1 or IL-1, stimulates the production of chemokines by mouse MSCs that attract T-cells and stimulate the production of inducible nitric-oxide synthase (iNOS), which in turn inhibits T-cell activation through the production of nitric oxide (56). It is worth noting that MSCs from IFN- receptor (IFN--R1) deficient mice do not have immunosuppressive activity, which highlights the vital role of IFN- in the immunosuppressive function of MSC (56).

Additional soluble factors, such as transforming growth factor-1 (TGF-1), hepatocyte growth factor (HGF), IL-10, PGE-2, haem-oxygenase-1 (HO1), IL-6 and soluble HLA-G5, are constitutively produced by MSCs (28, 34, 51, 63, 94) or secreted in response to cytokines released by target cells upon interacting with MSC. TNF- and IFN- have been shown to stimulate the production of PGE-2 by MSCs above constitutive level (34). Furthermore, IL-6 was shown to dampen the respiratory burst and to delay the apoptosis of human neutrophils by inducing phosphorylation of the transcription factor signal transducer and activator of transcription 3 (47), and to inhibit the differentiation of bone-marrow progenitor cells into DCs (95).

The failure to reverse suppression, when neutralizing antibodies against IL-10, TGF- and IGF were added to MLR reactions does point to the possibility that MSC may secrete as yet uncharacterized immunosuppressive factors (93). Galectin-1 and Galectin-3, newly characterized lectins, are constitutively expressed and secreted by human bone marrow MSC. Inhibition of galectin-1 and galectin-3 gene expression with small interfering RNAs abrogated the suppressive effect of MSC on allogeneic T-cells (Fig 1) (96). The restoration of T-cell proliferation in the presence of - lactose indicates that the carbohydrate-recognition domain of galectins is responsible for the immunosuppression of T-cells and highlights an extracellular mechanism of action for the MSC-secreted galectins. In this respect, the inhibition of T-cell proliferation could result from either direct effects of galectin-1 and galectin-3 on T cells and/or through a direct or an indirect on effect on dendritic cells (97).

HLA-G5 represents another important molecule involved in MSC mediated regulation of the immune response, where its production has been shown to suppress T-cell proliferation, as well as NK-cell and T-cell cytotoxicity, while promoting the generation of Tregs (63, 98). HLA-G protein expression is constitutive and the level is not modified upon stimulation by allogeneic lymphocytes in MSC/MLR. HLA-G5 is detected on MSCs by real-time reverse-phase polymerase chain reaction, immune-fluorescence, flow cytometry and enzyme-linked immunosorbent assay in the supernatant (99). Cell contact between MSCs and activated T-cells induces IL-10 production, which, in turn, stimulates the release of soluble HLA-G5 by MSCs (63). It is worth nothing that none of these molecules have an exclusive role and that MSC-mediated immune-regulation is a redundant system that is mediated by several molecules.

One important characteristic of hMSCs is their ability to suppress inflammation resulting from injury, as well as, resulting from allogeneic solid organ transplants, and autoimmune disease. Consistent with in vitro studies, murine allogeneic MSCs are effective cellular therapy models in the treatment of murine models of human disease (52, 100-102). Several studies have documented the substantial clinical improvements observed in animal models, when MSC were systemically introduced as a therapy in mouse models of multiple sclerosis (102, 103), inflammatory bowel disease (104-106), infarct, stroke, and other neurologic diseases (107, 108), as well as diabetes (109). These findings strongly suggest that xenogeneic hMSCs are not immunologically recognized by various immunocompetent mouse models of disease and are able to home to sites of inflammation. However, the mechanisms behind the immunosuppressive actions at the site of inflammation and its association with the homing activity have not yet been completely elucidated.

Nitric Oxide (NO) mediate its effect partly through phosphorylation of Stat-5, which results in suppression of T- cell proliferation, partly through the inhibition of NO synthase or the inhibition of prostaglandin synthesis. This reveals the MSC-dependent effects on proliferation. Although indoleamine-2, 3-dioxygenase (IDO) has been hypothesized to be critical in mediating the effect of NO, neutralizing IDO by using a blocking antibody did not interfere with NOs suppressive effects (93, 110).

Within an in vivo setting, injury, inflammation, and/or foreign cells can lead to T-cell activation, which results in those T-cells producing proinflammatory cytokines including, but not limited to, TNF-, IFN-, IL-1, and IL-1. Combinations of cytokines may also induce cell production of chemokines, some of which bind to CXCR3-R expressing cells (including T cells) that co-localize with MSCs. MSCs then produce NO, which inhibits Stat-5 phosphorylation, thereby leading to cell-cycle arrest (and thus halting T cell proliferation) (Fig 1) (110). In addition, iNOS appears to be important in mouse MSC in vivo effects. MSCs from mice that lack iNOS (or IFN-R1) are unable to suppress GVHD. In contrast to mouse MSCs that use NO in mediating their immune-suppressive effect, hMSCs and MSCs from non-human primates appear to mediate their immune-suppressive effects via IDO (56). There is some controversy about whether the effect of IDO results from local depletion of tryptophan, or from the accumulation of tryptophan metabolites (which is suggested by data showing that use of a tryptophan antagonist, 1-methyl-L tryptophan, restored allo-reactivity that would otherwise have been suppressed by MSCs). In addition to its effect on the JAK-STAT pathway, NO may also influence mitogen activated protein kinase and nuclear factor B, which would cause a reduction in the gene expression of proinflammatory cytokines.

The clinical experience with and the safety of MSCs is of utmost interest for their wide therapeutic applications. The pioneering in vivo studies with MSC focused on the engraftment facilitation for the haematopoietic stem cells (111). Further work also focused on the regenerative functions of MSC in terms of functional repair of damaged tissues (112). Hypoimmunogenicity of MSC provided a critical advantage in their use for clinical and therapeutic purposes in vitro (50), followed by pre-clinical studies (29) and reaching the human clinical studies (32) with the use of allogeneic donors. Allogeneic MSC have proved to be an option with major advantages in clinical use, since the use of autologous MSC is hindered by the limited time frame for clonal expansion and the costly in vitro proliferation. However, some sub-acute conditions, such as autoimmune diseases, might allow the use of autologous MSCs and their culture in vitro. It is worth noting that some reports have recently challenged the belief that allogeneic MSCs are poorly immunogenic (113, 114), indicating that in some cases an autologous MSC source could be advantageous. Recent reports have shown that MSCs from patients with autoimmune disease have a normal capability to support hematopoiesis, (115) and to exercise immunomodulatory functions (116), and to show a normal phenotypic characteristics (117).

The perspective role of adult stem cells in degenerative disease conditions, where there is progressive tissue damage and an inability to repair, possibly due to the depletion of stem cell populations or functional alteration, has been considered. In cases of osteoarthritis, a disease of the joints where there is progressive and irreversible loss of cartilage characterized by changes in the underlying bone, Murphy et al showed that the proliferative capacity of the MSC was substantially reduced, and this was independent of the harvest site from patients with end-stage OA undergoing joint replacement surgery (118). In this study the marrow samples were harvested both from the site of surgery (either the hip or the knee) and also from the iliac crest. These effects were apparently disease-related, and not age-related. However, the data suggest that susceptibility to OA and perhaps other degenerative diseases may be due to the reduced mobilization or proliferation of stem cells. In addition, successfully recruited cells may have a limited capacity to differentiate, leading to defective tissue repair. Alternatively, the altered stem cell activity may be in response to the elevated levels of inflammatory cytokines seen in OA, which was confirmed by several other investigators (119, 120).

Similarly, the functional impairment of the anti-proliferative effect of MSCs derived from patients with aplastic anaemia (121) or multiple myeloma (122) might be resulting from an intrinsic abnormality in the microenvironment of the bone marrow, which is consistent with the possible use of autologous MSC for therapeutic purposes.

With the knowledge of the homing capacity of MSC and their capacity to engraft into the recipients bone after systemic administration, MSCs have been utilized to treat children with severe osteogenesis imperfecta, leading to improved parameters of increased growth velocity and total body mineral content associated with fewer fractures (123). Systemic infusion of allogeneic MSCs also led to encouraging bone marrow recovery in patients with tumors following chemotherapy (123). The immunosuppressive effect of infused MSCs has been successfully shown in acute, severe graft-versus-host disease (GvHD) (32). The probable effect of MSC was the inhibition of donor T-cell reactivity to histocompatibility antigens of the recipient tissue. Currently, there is no successful therapy for steroid-refractory acute GVHD. The possible role of MSCs in this context is therefore of potential interest. Le Blanc et al reported a case of grade IV acute GVHD of the gut and liver in a patient who had undergone ASCT with cells from an unrelated female donor (32). The patient was unresponsive to all types of immunosuppression drugs. When the patient was infused with 2x 106 MSCs per kilogram from his HLA-haploidentical mother, his GVHD responded with a decline in bilirubin and normalization of stools. After the MSC infusion, DNA analysis of his bone marrow showed the presence of minimal residual disease (124). When immunosuppression was discontinued, the patient again developed severe acute GVHD, with its associated symptoms within a few weeks.

Modulation of host allo-reactivity led to accelerated bone-marrow recovery in patients co-transplanted with MSCs and haplo-identical HSCs (125). Clinical trials are being conducted on the immunomodulatory potential of MSCs in the treatment of Crohns disease, with the potential for those cells to contribute to the regeneration of gastrointestinal epithelial cells (126).

As described previously, MSCs are characterized by their hypoimmunogenicity. In 2000, data from several research groups demonstrated long-term allo-MSC engraftment in a variety of non-cardiac tissues in the absence of immunosuppression (88, 90). On the basis of these observations, investigators began to look into the possibility of allo-MSCs engraftment into affected myocardium in rats, and later in swine, where allo-MSCs were found to readily engraft in necrotic myocardium and favorably alter ventricular function (2). The allo-MSC engraftment occurred without evidence of immunologic rejection or lymphocytic infiltration in the absence of assisted immunosuppressive therapy emphasizing some of the apparent advantages of these cells over other cell populations for cellular cardiomyoplasty. The immunologically privileged status of MSCs was also observed in xenogeneic setting, where Saito et al injected MSC intravenously from C57BL/6 mice into immunocompetent adult Lewis rats (127). When these animals were later subjected to MIs, murine MSCs could be identified in the region of necrosis, and these cells expressed muscle specific proteins not present before coronary ligation.

Consistent with results from in vitro studies, murine allogeneic MSCs are effective in the treatment of murine models of human disease (52, 103, 128). Several studies have reported clinical improvements in mouse models of multiple sclerosis and amyotrophic lateral sclerosis, inflammatory bowel disease, stroke, diabetes, infarct and GVHD using I.V. injected xenogeneic hMSCs rather than allogeneic MSCs (108, 109). A major advantage in using hMSCs in mouse models of human disease is that the possibility of gathering mechanistic data through measuring biomarkers from body fluids or using noninvasive imaging technology, which may prove to be an advantage in clinical studies when applied on humans.

In experiments designed to study the trafficking of hMSCs, investigators used mouse models of severe erosive polyarthritis characterized by an altered transgene allele that results in chronic over-expression of TNF- and which resemble human RA patients (60, 72). The motive behind utilizing these mice models was to investigate similarities in MSC homing with mouse models of chronic asthma and acute lung injury. Injected hMSC revealed a reduction in ankle arthritis parameters associated with decrease appendage related erythema, possibly due to the MSC localization to ankle joints as revealed by bioluminescence (129). Similar observations for inducing tolerance were made using adipose-derived MSC, where Treg were induced in RA PBMC and in mouse models of arthritis (36, 130). Furthermore, studies of rheumatoid arthritis T-cells showed a down-regulation of effector response using adipose-derived MSCs (131). Variations in this potential described by the capability of MSCs to down-regulate collagen-induced arthritis, and in the ability to induce Tregs, depend on the source of MSC (mouse vs. human) and its characteristics (primary isolate of MSC line), which reflect on difference in function compared to primary expanded MSC (132). Other studies reported that in the collagen-induced model of arthritis, mice infused with MSCs have increased numbers of CD4+CD25+ cells that express FoxP3 and thus reveal a Treg phenotype (20). Recent data on collagen-induced arthritis model, where murine MSCs did not reveal therapeutic benefits against arthritis in vivo, but did show anti-proliferative effect in vitro suggest that there is no appropriate in vitro measures that can be accurately extrapolated into a potential therapeutic utility of MSCs in vivo, and that mouse MSCs show difference in functional characteristics to hMSC in terms of immunoregulatory capacity (133).

MSCs immunological properties appeared to have potential therapeutic advantages in other forms of autoimmune diseases, especially in type 1 diabetes. In NOD mouse model, several physiological defects that aim to maintain peripheral and central tolerance contribute to the development of autoimmune diabetes. These defects are summed up as a combination of immune cell dysfunction (including T-cell, NK cells, B-cells, and dendritic cells), associated with the presence of inflammatory cytokine milieu (134). MSCs possess specific immunomodulatory properties capable of halting autoimmunity through immunomodulation processes described in this chapter. The processes might be through a direct effect via the presentation of differential levels of negative costimulatory molecules and the secretion of regulatory cytokines that affect regulatory T-cells/autoreactive T-cells. Furthermore, MSCs could modulate the immunological dysregulation observed in antibody producing B-cells and cytotoxic NK cells. Dendritic cells have been shown to be defective in NOD mice characterized by higher levels of costimulation with a potential capability to shift to a TH-1 type of immune response.

In an experimental mouse model of diabetes induced by streptozotocin, it was observed that MSCs promoted the endogenous repair of pancreatic islets and renal glomeruli (109). Similarly, co-infusion of MSCs and bone-marrow cells inhibited the proliferation of -cell-specific T-cells isolated from the pancreas of diabetic mice and restored insulin and glucose levels through the induction of recipient-derived pancreatic -cell regeneration in the absence of trans-differentiation of MSCs (135). These studies show that the in vivo administration of MSCs is clinically efficacious through the modulation of pathogenic - and T-cell responses and through potent bystander effects on the target tissue.

The timing of MSC infusion seems to be a critical parameter in their therapeutic efficacy. In the EAE mouse model of multiple sclerosis, MSC systematically injected at disease onset ameliorated myelin oligodendrocyte glycoprotein (MOG)-induced EAE and further decreased the infiltration T-cells, B-cells and macrophages into the central nervous system (CNS). Furthermore, T cells isolated from the lymph nodes of MSC-treated mice did not proliferate after in vitro re-challenge with MOG peptide, which is an indication of the induction of T-cell anergy (52). Systematic injection of MSCs was found to inhibit the in vivo production of pathogenic plp-specific antibodies and to suppress the encephalitogenic potential of plp-specific T cells in passive-transfer experiments. In this model, the MSCs migrated to the lymphoid organs, as well as, to the inflamed CNS, where they exercised a protective effect on the neuronal axons in situ (135, 136). In these studies, the therapeutic effect of MSCs depended on the release of anti-apoptotic, anti-inflammatory and trophic molecules, as occurred in the case of stroke in rats (137), and, possibly, on the recruitment of local progenitors and their subsequent induction to differentiate into neural cells (138). As trophic effect, the MSCs appeared to favor oligo-dendrogenisis by neural precursor cells (139).

Several other studies have provided insights into the effects of MSCs mediated by cytokines. In a model of acute renal failure, the administration of MSCs increased the recovery of renal function through the inhibition of production of proinflammatory cytokines, such as Il-1, TNF and IFN, and through an anti-apoptotic effect on target cells (140). Along the same line, the anti-inflammatory activity of MSCs was revealed in a mouse model of lung fibrosis, where they inhibited the effects of IL-1-producing T cells and TNF-producing macrophages through the release of IL-1 receptor antagonist (IL-1RA) (141). The release of trophic factors such as the WNT-associated molecule secreted frizzled-related protein 2 (SFRp2), which leads to the rescue of ischemic cardiomyocytes and the restoration of ventricular functions represent another important function for MSC (142).

With all the promising therapeutic potential of MSC, there seems to be a growing concern about their association with tumors. The immunoregulatory and anti-proliferative effects of MSCs led to several studies investigating the inhibitory effect of MSCs on tumor growth. Inhibition or, more frequently, stimulation of tumor-cell proliferation in vitro and/or tumor growth in vivo by MSCs has been reported (143-145). The heterogeneous nature of the MSC populations and the different experimental tumor models used, contribute to the effect of tumors on MSC in which the microenvironment generated by tumors influence the behavior of MSCs (146). Two main mechanisms are probably involved in the enhancement of tumor growth by MSCs. First, the cell-to-cell cross-talk between MSCs and tumor cells contribute to tumor progression, thus integrating within the tumor stroma (147), and second, the suppressive effects of MSCs on the immune system of tumor-bearing hosts might facilitate tumorigenesis, as shown for the inhibition of melanoma rejection, possibly mediated by regulatory CD8+ T cells (144). Irrespective of the possible interactions between cancer cells, immune cells and MSCs, the potential risk of stimulating the growth cancer by MSCs must be considered.

As a whole, the data accumulated from preclinical and clinical data indicate that bone marrow-derived MSCs have, in addition to their therapeutic purposes in regenerative medicine, effects that can result from other characteristics, such as their anti-proliferative and anti-inflammatory properties. The immuno suppressive activity of MSCs provides means for inducing peripheral tolerance following systemic injection mediated through the inhibition of cell division, thereby preventing their responsiveness to antigenic triggers while maintaining them in a quiescent state. In addition, the clinical efficacy of MSCs in different experimental model seems to occur almost only during the acute phase of disease associated with limited trans-differentiation, which indicates that the therapeutic effectiveness of MSCs relies heavily on their ability to modify microenvironments. These modifications occur through the release of anti-inflammatory cytokines, and anti-apoptotic and trophic molecules that promote the repair and protection of damaged tissues, as well as, maintain the integrity of the immune cells.

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Mesenchymal Stem Cells: Immunology and Therapeutic ...

Death is the termination of all biological functions that sustain an organism. Phenomena which commonly bring about death include biological aging (senescence), predation, malnutrition, disease, suicide, homicide, starvation, dehydration, and accidents or trauma resulting in terminal injury.[1] Bodies of living organisms begin to decompose shortly after death. Death has commonly been considered a sad or unpleasant occasion, particularly for humans, due to the affection for the being that has died and/or the termination of social and familial bonds with the deceased. Other concerns include fear of death, necrophobia, anxiety, sorrow, grief, emotional pain, depression, sympathy, compassion, solitude, or saudade. The potential for an afterlife is of concern for humans and the possibility of reward or judgement and punishment for past sin with people of certain religions.

The word death comes from Old English dea, which in turn comes from Proto-Germanic dauthuz (reconstructed by etymological analysis). This comes from the Proto-Indo-European stem dheu- meaning the "Process, act, condition of dying".[2]

The concept and symptoms of death, and varying degrees of delicacy used in discussion in public forums, have generated numerous scientific, legal, and socially acceptable terms or euphemisms for death. When a person has died, it is also said they have passed away, passed on, expired, or are gone, among numerous other socially accepted, religiously specific, slang, and irreverent terms. Bereft of life, the dead person is then a corpse, cadaver, a body, a set of remains, and when all flesh has rotted away, a skeleton. The terms carrion and carcass can also be used, though these more often connote the remains of non-human animals. As a polite reference to a dead person, it has become common practice to use the participle form of "decease", as in the deceased; another noun form is decedent. The ashes left after a cremation are sometimes referred to by the neologism cremains, a portmanteau of "cremation" and "remains".

Senescence refers to a scenario when a living being is able to survive all calamities, but eventually dies due to old age. Human, animal, and plant cells normally reproduce and function during the whole period of natural existence, but the aging process derives from deterioration of cellular activity and ruination of regular functioning. Aptitude of cells for gradual deterioration and mortality means that cells are naturally sentenced to stable and long-term loss of living capacities, even despite continuing metabolic reactions and viability. In the United Kingdom, for example, nine out of ten of all the deaths that occur on a daily basis relates to senescence, while around the world it accounts for two-thirds of 150,000 deaths that take place daily (Hayflick & Moody, 2003).

Almost all animals who survive external hazards to their biological functioning eventually die from biological aging, known in life sciences as "senescence". Some organisms experience negligible senescence, even exhibiting biological immortality. These include the jellyfish Turritopsis dohrnii,[3] the hydra, and the planarian. Unnatural causes of death include suicide and homicide. From all causes, roughly 150,000 people die around the world each day.[4] Of these, two thirds die directly or indirectly due to senescence, but in industrialized countriessuch as the United States, the United Kingdom, and Germanythe rate approaches 90%, i.e., nearly nine out of ten of all deaths are related to senescence.[4]

Physiological death is now seen as a process, more than an event: conditions once considered indicative of death are now reversible.[5] Where in the process a dividing line is drawn between life and death depends on factors beyond the presence or absence of vital signs. In general, clinical death is neither necessary nor sufficient for a determination of legal death. A patient with working heart and lungs determined to be brain dead can be pronounced legally dead without clinical death occurring. As scientific knowledge and medicine advance, a precise medical definition of death becomes more problematic.[6]

Signs of death or strong indications that a warm-blooded animal is no longer alive are:

The concept of death is a key to human understanding of the phenomenon.[7] There are many scientific approaches to the concept. For example, brain death, as practiced in medical science, defines death as a point in time at which brain activity ceases.[7][8][9][10]

One of the challenges in defining death is in distinguishing it from life. As a point in time, death would seem to refer to the moment at which life ends. Determining when death has occurred requires drawing precise conceptual boundaries between life and death. This is problematic because there is little consensus over how to define life. This general problem applies to the particular challenge of defining death in the context of medicine.

It is possible to define life in terms of consciousness. When consciousness ceases, a living organism can be said to have died. One of the flaws in this approach is that there are many organisms which are alive but probably not conscious (for example, single-celled organisms). Another problem is in defining consciousness, which has many different definitions given by modern scientists, psychologists and philosophers. Additionally, many religious traditions, including Abrahamic and Dharmic traditions, hold that death does not (or may not) entail the end of consciousness. In certain cultures, death is more of a process than a single event. It implies a slow shift from one spiritual state to another.[11]

Other definitions for death focus on the character of cessation of something.[12][clarification needed] In this context "death" describes merely the state where something has ceased, for example, life. Thus, the definition of "life" simultaneously defines death.

Historically, attempts to define the exact moment of a human's death have been problematic. Death was once defined as the cessation of heartbeat (cardiac arrest) and of breathing, but the development of CPR and prompt defibrillation have rendered that definition inadequate because breathing and heartbeat can sometimes be restarted. Events which were causally linked to death in the past no longer kill in all circumstances; without a functioning heart or lungs, life can sometimes be sustained with a combination of life support devices, organ transplants and artificial pacemakers.

Today, where a definition of the moment of death is required, doctors and coroners usually turn to "brain death" or "biological death" to define a person as being dead; people are considered dead when the electrical activity in their brain ceases. It is presumed that an end of electrical activity indicates the end of consciousness. Suspension of consciousness must be permanent, and not transient, as occurs during certain sleep stages, and especially a coma. In the case of sleep, EEGs can easily tell the difference.

The category of "brain death" is seen by some scholars to be problematic. For instance, Dr. Franklin Miller, senior faculty member at the Department of Bioethics, National Institutes of Health, notes: "By the late 1990s... the equation of brain death with death of the human being was increasingly challenged by scholars, based on evidence regarding the array of biological functioning displayed by patients correctly diagnosed as having this condition who were maintained on mechanical ventilation for substantial periods of time. These patients maintained the ability to sustain circulation and respiration, control temperature, excrete wastes, heal wounds, fight infections and, most dramatically, to gestate fetuses (in the case of pregnant "brain-dead" women)."[13]

Those people maintaining that only the neo-cortex of the brain is necessary for consciousness sometimes argue that only electrical activity should be considered when defining death. Eventually it is possible that the criterion for death will be the permanent and irreversible loss of cognitive function, as evidenced by the death of the cerebral cortex. All hope of recovering human thought and personality is then gone given current and foreseeable medical technology. At present, in most places the more conservative definition of death irreversible cessation of electrical activity in the whole brain, as opposed to just in the neo-cortex has been adopted (for example the Uniform Determination Of Death Act in the United States). In 2005, the Terri Schiavo case brought the question of brain death and artificial sustenance to the front of American politics.

Even by whole-brain criteria, the determination of brain death can be complicated. EEGs can detect spurious electrical impulses, while certain drugs, hypoglycemia, hypoxia, or hypothermia can suppress or even stop brain activity on a temporary basis. Because of this, hospitals have protocols for determining brain death involving EEGs at widely separated intervals under defined conditions.

The death of a person has legal consequences that may vary between different jurisdictions. A death certificate is issued in most jurisdictions, either by a doctor himself or by an administrative office upon presentation of a doctor's declaration of death.

There are many anecdotal references to people being declared dead by physicians and then "coming back to life", sometimes days later in their own coffin, or when embalming procedures are about to begin. From the mid-18th century onwards, there was an upsurge in the public's fear of being mistakenly buried alive,[14] and much debate about the uncertainty of the signs of death. Various suggestions were made to test for signs of life before burial, ranging from pouring vinegar and pepper into the corpse's mouth to applying red hot pokers to the feet or into the rectum.[15] Writing in 1895, the physician J.C. Ouseley claimed that as many as 2,700people were buried prematurely each year in England and Wales, although others estimated the figure to be closer to 800.[16]

In cases of electric shock, cardiopulmonary resuscitation (CPR) for an hour or longer can allow stunned nerves to recover, allowing an apparently dead person to survive. People found unconscious under icy water may survive if their faces are kept continuously cold until they arrive at an emergency room.[17] This "diving response", in which metabolic activity and oxygen requirements are minimal, is something humans share with cetaceans called the mammalian diving reflex.[17]

As medical technologies advance, ideas about when death occurs may have to be re-evaluated in light of the ability to restore a person to vitality after longer periods of apparent death (as happened when CPR and defibrillation showed that cessation of heartbeat is inadequate as a decisive indicator of death). The lack of electrical brain activity may not be enough to consider someone scientifically dead. Therefore, the concept of information-theoretic death[18] has been suggested as a better means of defining when true death occurs, though the concept has few practical applications outside of the field of cryonics.

There have been some scientific attempts to bring dead organisms back to life, but with limited success.[19] In science fiction scenarios where such technology is readily available, real death is distinguished from reversible death.

The leading cause of human death in developing countries is infectious disease. The leading causes in developed countries are atherosclerosis (heart disease and stroke), cancer, and other diseases related to obesity and aging. By an extremely wide margin, the largest unifying cause of death in the developed world is biological aging,[4] leading to various complications known as aging-associated diseases. These conditions cause loss of homeostasis, leading to cardiac arrest, causing loss of oxygen and nutrient supply, causing irreversible deterioration of the brain and other tissues. Of the roughly 150,000 people who die each day across the globe, about two thirds die of age-related causes.[4] In industrialized nations, the proportion is much higher, approaching 90%.[4] With improved medical capability, dying has become a condition to be managed. Home deaths, once commonplace, are now rare in the developed world.

In developing nations, inferior sanitary conditions and lack of access to modern medical technology makes death from infectious diseases more common than in developed countries. One such disease is tuberculosis, a bacterial disease which killed 1.7M people in 2004.[21]Malaria causes about 400900M cases of fever and 13M deaths annually.[22]AIDS death toll in Africa may reach 90100M by 2025.[23][24]

According to Jean Ziegler (United Nations Special Reporter on the Right to Food, 2000Mar 2008), mortality due to malnutrition accounted for 58% of the total mortality rate in 2006. Ziegler says worldwide approximately 62M people died from all causes and of those deaths more than 36M died of hunger or diseases due to deficiencies in micronutrients.[25]

Tobacco smoking killed 100million people worldwide in the 20th century and could kill 1billion people around the world in the 21st century, a World Health Organization report warned.[20]

Many leading developed world causes of death can be postponed by diet and physical activity, but the accelerating incidence of disease with age still imposes limits on human longevity. The evolutionary cause of aging is, at best, only just beginning to be understood. It has been suggested that direct intervention in the aging process may now be the most effective intervention against major causes of death.[26]

Selye proposed a unified non-specific approach to many causes of death. He demonstrated that stress decreases adaptability of an organism and proposed to describe the adaptability as a special resource, adaptation energy. The animal dies when this resource is exhausted.[27] Selye assumed that the adaptability is a finite supply, presented at birth. Later on, Goldstone proposed the concept of a production or income of adaptation energy which may be stored (up to a limit), as a capital reserve of adaptation.[28] In recent works, adaptation energy is considered as an internal coordinate on the "dominant path" in the model of adaptation. It is demonstrated that oscillations of well-being appear when the reserve of adaptability is almost exhausted.[29]

In 2012, suicide overtook car crashes for leading causes of human injury deaths in America, followed by poisoning, falls and murder.[30] Causes of death are different in different parts of the world. In high-income and middle income countries nearly half up to more than two thirds of all people live beyond the age of 70 and predominantly die of chronic diseases. In low-income countries, where less than one in five of all people reach the age of 70, and more than a third of all deaths are among children under 15, people predominantly die of infectious diseases.[31]

An autopsy, also known as a postmortem examination or an obduction, is a medical procedure that consists of a thorough examination of a human corpse to determine the cause and manner of a person's death and to evaluate any disease or injury that may be present. It is usually performed by a specialized medical doctor called a pathologist.

Autopsies are either performed for legal or medical purposes. A forensic autopsy is carried out when the cause of death may be a criminal matter, while a clinical or academic autopsy is performed to find the medical cause of death and is used in cases of unknown or uncertain death, or for research purposes. Autopsies can be further classified into cases where external examination suffices, and those where the body is dissected and an internal examination is conducted. Permission from next of kin may be required for internal autopsy in some cases. Once an internal autopsy is complete the body is generally reconstituted by sewing it back together. Autopsy is important in a medical environment and may shed light on mistakes and help improve practices.

A "necropsy" is an older term for a postmortem examination, unregulated, and not always a medical procedure. In modern times the term is more often used in the postmortem examination of the corpses of animals.

Cryonics (from Greek 'kryos-' meaning 'icy cold') is the low-temperature preservation of animals and humans who cannot be sustained by contemporary medicine, with the hope that healing and resuscitation may be possible in the future.[32][33]

Cryopreservation of people or large animals is not reversible with current technology. The stated rationale for cryonics is that people who are considered dead by current legal or medical definitions may not necessarily be dead according to the more stringent information-theoretic definition of death.[18][34] It is proposed that cryopreserved people might someday be recovered by using highly advanced technology.[35][36]

Some scientific literature supports the feasibility of cryonics.[35][36][37] Many other scientists regard cryonics with skepticism.[38] By 2015, more than 300 people have undergone cryopreservation procedures since cryonics was first proposed in 1962.[39]

Life extension refers to an increase in maximum or average lifespan, especially in humans, by slowing down or reversing the processes of aging. Average lifespan is determined by vulnerability to accidents and age or lifestyle-related afflictions such as cancer, or cardiovascular disease. Extension of average lifespan can be achieved by good diet, exercise and avoidance of hazards such as smoking. Maximum lifespan is also determined by the rate of aging for a species inherent in its genes. Currently, the only widely recognized method of extending maximum lifespan is calorie restriction. Theoretically, extension of maximum lifespan can be achieved by reducing the rate of aging damage, by periodic replacement of damaged tissues, or by molecular repair or rejuvenation of deteriorated cells and tissues.

A United States poll found that religious people and irreligious people, as well as men and women and people of different economic classes have similar rates of support for life extension, while Africans and Hispanics have higher rates of support than white people.[40] 38 percent of the polled said they would desire to have their aging process cured.

Researchers of life extension are a subclass of biogerontologists known as "biomedical gerontologists". They try to understand the nature of aging and they develop treatments to reverse aging processes or to at least slow them down, for the improvement of health and the maintenance of youthful vigor at every stage of life. Those who take advantage of life extension findings and seek to apply them upon themselves are called "life extensionists" or "longevists". The primary life extension strategy currently is to apply available anti-aging methods in the hope of living long enough to benefit from a complete cure to aging once it is developed.

"One of medicine's new frontiers: treating the dead", recognizes that cells that have been without oxygen for more than five minutes die,[41] not from lack of oxygen, but rather when their oxygen supply is resumed. Therefore, practitioners of this approach, e.g., at the Resuscitation Science institute at the University of Pennsylvania, "aim to reduce oxygen uptake, slow metabolism and adjust the blood chemistry for gradual and safe reperfusion."[42]

Before about 1930, most people in Western countries died in their own homes, surrounded by family, and comforted by clergy, neighbors, and doctors making house calls.[43] By the mid-20th century, half of all Americans died in a hospital.[44] By the start of the 21st century, only about 20 to 25% of people in developed countries died outside a medical institution.[44][45][46] The shift away from dying at home, towards dying in a professionalized medical environment, has been termed the "Invisible Death."[44]

In society, the nature of death and humanity's awareness of its own mortality has for millennia been a concern of the world's religious traditions and of philosophical inquiry. This includes belief in resurrection or an afterlife (associated with Abrahamic religions), reincarnation or rebirth (associated with Dharmic religions), or that consciousness permanently ceases to exist, known as eternal oblivion (associated with atheism).[47]

Commemoration ceremonies after death may include various mourning, funeral practices and ceremonies of honouring the deceased. The physical remains of a person, commonly known as a corpse or body, are usually interred whole or cremated, though among the world's cultures there are a variety of other methods of mortuary disposal. In the English language, blessings directed towards a dead person include rest in peace, or its initialism RIP.

Death is the center of many traditions and organizations; customs relating to death are a feature of every culture around the world. Much of this revolves around the care of the dead, as well as the afterlife and the disposal of bodies upon the onset of death. The disposal of human corpses does, in general, begin with the last offices before significant time has passed, and ritualistic ceremonies often occur, most commonly interment or cremation. This is not a unified practice; in Tibet, for instance, the body is given a sky burial and left on a mountain top. Proper preparation for death and techniques and ceremonies for producing the ability to transfer one's spiritual attainments into another body (reincarnation) are subjects of detailed study in Tibet.[48]Mummification or embalming is also prevalent in some cultures, to retard the rate of decay.

Legal aspects of death are also part of many cultures, particularly the settlement of the deceased estate and the issues of inheritance and in some countries, inheritance taxation.

Capital punishment is also a culturally divisive aspect of death. In most jurisdictions where capital punishment is carried out today, the death penalty is reserved for premeditated murder, espionage, treason, or as part of military justice. In some countries, sexual crimes, such as adultery and sodomy, carry the death penalty, as do religious crimes such as apostasy, the formal renunciation of one's religion. In many retentionist countries, drug trafficking is also a capital offense. In China, human trafficking and serious cases of corruption are also punished by the death penalty. In militaries around the world courts-martial have imposed death sentences for offenses such as cowardice, desertion, insubordination, and mutiny.[49]

Death in warfare and in suicide attack also have cultural links, and the ideas of dulce et decorum est pro patria mori, mutiny punishable by death, grieving relatives of dead soldiers and death notification are embedded in many cultures. Recently in the western world, with the increase in terrorism following the September 11 attacks, but also further back in time with suicide bombings, kamikaze missions in World War II and suicide missions in a host of other conflicts in history, death for a cause by way of suicide attack, and martyrdom have had significant cultural impacts.

Suicide in general, and particularly euthanasia, are also points of cultural debate. Both acts are understood very differently in different cultures. In Japan, for example, ending a life with honor by seppuku was considered a desirable death, whereas according to traditional Christian and Islamic cultures, suicide is viewed as a sin. Death is personified in many cultures, with such symbolic representations as the Grim Reaper, Azrael, the Hindu God Yama and Father Time.

In Brazil, a human death is counted officially when it is registered by existing family members at a cartrio, a government-authorized registry. Before being able to file for an official death, the deceased must have been registered for an official birth at the cartrio. Though a Public Registry Law guarantees all Brazilian citizens the right to register deaths, regardless of their financial means, of their family members (often children), the Brazilian government has not taken away the burden, the hidden costs and fees, of filing for a death. For many impoverished families, the indirect costs and burden of filing for a death lead to a more appealing, unofficial, local, cultural burial, which in turn raises the debate about inaccurate mortality rates.[50]

Talking about death and witnessing it is a difficult issue with most cultures. Western societies may like to treat the dead with the utmost material respect, with an official embalmer and associated rites. Eastern societies (like India) may be more open to accepting it as a fait accompli, with a funeral procession of the dead body ending in an open air burning-to-ashes of the same.

Much interest and debate surround the question of what happens to one's consciousness as one's body dies. The belief in the permanent loss of consciousness after death is often called eternal oblivion. Belief that consciousness is preserved after physical death is described by the term afterlife.

After death the remains of an organism become part of the biogeochemical cycle. Animals may be consumed by a predator or a scavenger. Organic material may then be further decomposed by detritivores, organisms which recycle detritus, returning it to the environment for reuse in the food chain. Examples of detritivores include earthworms, woodlice and dung beetles.

Microorganisms also play a vital role, raising the temperature of the decomposing matter as they break it down into yet simpler molecules. Not all materials need to be decomposed fully. Coal, a fossil fuel formed over vast tracts of time in swamp ecosystems, is one example.

Contemporary evolutionary theory sees death as an important part of the process of natural selection. It is considered that organisms less adapted to their environment are more likely to die having produced fewer offspring, thereby reducing their contribution to the gene pool. Their genes are thus eventually bred out of a population, leading at worst to extinction and, more positively, making the process possible, referred to as speciation. Frequency of reproduction plays an equally important role in determining species survival: an organism that dies young but leaves numerous offspring displays, according to Darwinian criteria, much greater fitness than a long-lived organism leaving only one.

Extinction is the cessation of existence of a species or group of taxa, reducing biodiversity. The moment of extinction is generally considered to be the death of the last individual of that species (although the capacity to breed and recover may have been lost before this point). Because a species' potential range may be very large, determining this moment is difficult, and is usually done retrospectively. This difficulty leads to phenomena such as Lazarus taxa, where species presumed extinct abruptly "reappear" (typically in the fossil record) after a period of apparent absence. New species arise through the process of speciation, an aspect of evolution. New varieties of organisms arise and thrive when they are able to find and exploit an ecological niche and species become extinct when they are no longer able to survive in changing conditions or against superior competition.

Inquiry into the evolution of aging aims to explain why so many living things and the vast majority of animals weaken and die with age (exceptions include Hydra and the already cited jellyfish Turritopsis dohrnii, which research shows to be biologically immortal). The evolutionary origin of senescence remains one of the fundamental puzzles of biology. Gerontology specializes in the science of human aging processes.

Organisms showing only asexual reproduction (e.g. bacteria, some protists, like the euglenoids and many amoebozoans) and unicellular organisms with sexual reproduction (colonial or not, like the volvocine algae Pandorina and Chlamydomonas) are "immortal" at some extent, dying only due to external hazards, like being eaten or meeting with a fatal accident. In multicellular organisms (and also in multinucleate ciliates),[52] with a Weismannist development, that is, with a division of labor between mortal somatic (body) cells and "immortal" germ (reproductive) cells, death becomes an essential part of life, at least for the somatic line.[53]

The Volvox algae are among the simplest organisms to exhibit that division of labor between two completely different cell types, and as a consequence include death of somatic line as a regular, genetically regulated part of its life history.[53][54]

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Death - Wikipedia, the free encyclopedia

Last year, Patricia Beals was told she'd need a double knee replacement to repair her severely arthritic knees or she'd probably spend the rest of her life in a wheelchair.

Hoping to avoid surgery, Beals, 72, opted instead for an experimental treatment that involved harvesting bone marrow stem cells from her hip, concentrating the cells in a centrifuge and injecting them back into her damaged joints.

"Almost from the moment I got up from the table, I was able to throw away my cane," Beals says. "Now I'm biking and hiking like a 30-year-old."

A handful of doctors around the country are administering treatments like the one Beals received to stop or even reverse the ravages of osteoarthritis. Stem cells are the only cells in the body able to morph into other types of specialized cells. When the patient's own stem cells are injected into a damaged joint, they appear to transform into chondrocytes, the cells that go on to produce fresh cartilage. They also seem to amplify the body's own natural repair efforts by accelerating healing, reducing inflammation, and preventing scarring and loss of function.

Christopher J. Centeno, M.D., the rehab medicine specialist who performed Beals' procedure, says the results he sees from stem cell therapy are remarkable. Of the more-than-200 patients his Bloomfield, Colo., clinic treated over a two-year period, he says, "two thirds of them reported greater than 50 percent relief and about 40 percent reported more than 75 percent relief one to two years afterward."

According to Centeno, knees respond better to the treatment than hips. Only eight percent of his knee patients opted for a total knee replacement two years after receiving a stem cell injection. The complete results from his clinical observations will be published in a major orthopedic journal later this year.

The Pros and Cons

The biggest advantage stem cell injections seem to offer over more invasive arthritis remedies is a quicker, easier recovery. The procedure is done on an outpatient basis and the majority of patients are up and moving within 24 hours. Most wear a brace for several weeks but still can get around. Many are even able to do some gentle stationary cycling by the end of the first week.

There are also fewer complications. A friend who had knee replacement surgery the same day Beals had her treatment developed life-threatening blood clots and couldn't walk for weeks afterwards. Six months out, she still hasn't made a full recovery.

Most surgeries don't go so awry, but still: Beals just returned from a week-long cycling trip where she covered 20 to 40 miles per day without so much as a tweak of pain.

As for risks, Centeno maintains they are virtually nonexistent.

"Because the stem cells come from your own body, there's little chance of infection or rejection," he says.

Not all medical experts are quite so enthusiastic, however. Dr. Tom Einhorn, chairman of the department of orthopedic surgery at Boston University, conducts research with stem cells but does not use them to treat arthritic patients. He thinks the idea is interesting but the science is not there yet.

"We need to have animal studies and analyze what's really happening under the microscope. Then, and only then, can you start doing this with patients," he says.

The few studies completed to date have examined how stem cells heal traumatic injuries rather than degenerative conditions such as arthritis. Results have been promising but, as Einhorn points out, the required repair mechanisms in each circumstance are very different.

Another downside is cost: The injections aren't approved by the FDA, which means they aren't covered by insurance. At $4,000 a pop -- all out of pocket -- they certainly aren't cheap, and many patients require more than one shot.

Ironically, one thing driving up the price is FDA involvement. Two years ago, the agency stepped in and stopped physicians from intensifying stem cells in the lab for several days before putting them back into the patient. This means all procedures must be done on the same day, no stem cells may be preserved and many of the more expensive aspects of the treatment must be repeated each time.

Centeno says same day treatments often aren't as effective, either.

But despite the sky-high price tag and lack of evidence, patients like Beals believe the treatment is nothing short of a miracle. She advises anyone who is a candidate for joint replacement to consider stem cells first.

"Open your mind up and step into it," she says. "Do it. It's so effective. It's the future and it works."

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Stem Cells: Alternative to Knee Replacement?

Immortality is the ability to live forever or eternal life.[2]Natural selection has developed potential biological immortality in at least one species, Turritopsis dohrnii.[3]

Certain scientists, futurists, and philosophers have theorized about the immortality of the human body (either through an immortal cell line researched or else deeper contextual understanding in advanced fields that have certain scope in the proposed long term reality that can be attained such as per mentioned in the reading of an article or scientific documentation of such a proposed idea would lead to), and advocate that human immortality is achievable in the first few decades of the 21st century, whereas other advocates believe that life extension is a more achievable goal in the short term, with immortality awaiting further research breakthroughs into an indefinite future. The absence of aging would provide humans with biological immortality, but not invulnerability to death by physical trauma; although mind uploading could solve that issue. Whether the process of internal endoimmortality would be delivered within the upcoming years depends chiefly on research (and in neuron research in the case of endoimmortality through an immortalized cell line) in the former view and perhaps is an awaited goal in the latter case.[4]

In religious contexts, immortality is often stated to be one of the promises of God (or other deities) to human beings who show goodness or else follow divine law. What form an unending human life would take, or whether an immaterial soul exists and possesses immortality, has been a major point of focus of religion, as well as the subject of speculation, fantasy, and debate.

Life extension technologies promise a path to complete rejuvenation. Cryonics holds out the hope that the dead can be revived in the future, following sufficient medical advancements. While, as shown with creatures such as hydra and planarian worms, it is indeed possible for a creature to be biologically immortal, it is not known if it is possible for humans.

Mind uploading is the transference of brain states from a human brain to an alternative medium providing similar functionality. Assuming the process to be possible and repeatable, this would provide immortality to the computation of the original brain, as predicted by futurists such as Ray Kurzweil.[5]

The belief in an afterlife is a fundamental tenet of most religions, including Hinduism, Buddhism, Jainism, Sikhism, Christianity, Zoroastrianism, Islam, Judaism, and the Bah' Faith; however, the concept of an immortal soul is not. The "soul" itself has different meanings and is not used in the same way in different religions and different denominations of a religion. For example, various branches of Christianity have disagreeing views on the soul's immortality and its relation to the body.

Physical immortality is a state of life that allows a person to avoid death and maintain conscious thought. It can mean the unending existence of a person from a physical source other than organic life, such as a computer. Active pursuit of physical immortality can either be based on scientific trends, such as cryonics, digital immortality, breakthroughs in rejuvenation or predictions of an impending technological singularity, or because of a spiritual belief, such as those held by Rastafarians or Rebirthers.

There are three main causes of death: aging, disease and physical trauma.[6] Such issues can be resolved with the solutions provided in research to any end providing such alternate theories at present that require unification.

Aubrey de Grey, a leading researcher in the field,[7] defines aging as "a collection of cumulative changes to the molecular and cellular structure of an adult organism, which result in essential metabolic processes, but which also, once they progress far enough, increasingly disrupt metabolism, resulting in pathology and death." The current causes of aging in humans are cell loss (without replacement), DNA damage, oncogenic nuclear mutations and epimutations, cell senescence, mitochondrial mutations, lysosomal aggregates, extracellular aggregates, random extracellular cross-linking, immune system decline, and endocrine changes. Eliminating aging would require finding a solution to each of these causes, a program de Grey calls engineered negligible senescence. There is also a huge body of knowledge indicating that change is characterized by the loss of molecular fidelity.[8]

Disease is theoretically surmountable via technology. In short, it is an abnormal condition affecting the body of an organism, something the body shouldn't typically have to deal with its natural make up.[9] Human understanding of genetics is leading to cures and treatments for myriad previously incurable diseases. The mechanisms by which other diseases do their damage are becoming better understood. Sophisticated methods of detecting diseases early are being developed. Preventative medicine is becoming better understood. Neurodegenerative diseases like Parkinson's and Alzheimer's may soon be curable with the use of stem cells. Breakthroughs in cell biology and telomere research are leading to treatments for cancer. Vaccines are being researched for AIDS and tuberculosis. Genes associated with type 1 diabetes and certain types of cancer have been discovered, allowing for new therapies to be developed. Artificial devices attached directly to the nervous system may restore sight to the blind. Drugs are being developed to treat a myriad of other diseases and ailments.

Physical trauma would remain as a threat to perpetual physical life, as an otherwise immortal person would still be subject to unforeseen accidents or catastrophes. The speed and quality of paramedic response remains a determining factor in surviving severe trauma.[10] A body that could automatically repair itself from severe trauma, such as speculated uses for nanotechnology, would mitigate this factor. Being the seat of consciousness, the brain cannot be risked to trauma if a continuous physical life is to be maintained. This aversion to trauma risk to the brain would naturally result in significant behavioral changes that would render physical immortality undesirable.

Organisms otherwise unaffected by these causes of death would still face the problem of obtaining sustenance (whether from currently available agricultural processes or from hypothetical future technological processes) in the face of changing availability of suitable resources as environmental conditions change. After avoiding aging, disease, and trauma, you could still starve to death.

If there is no limitation on the degree of gradual mitigation of risk then it is possible that the cumulative probability of death over an infinite horizon is less than certainty, even when the risk of fatal trauma in any finite period is greater than zero. Mathematically, this is an aspect of achieving "actuarial escape velocity"

Biological immortality is an absence of aging, specifically the absence of a sustained increase in rate of mortality as a function of chronological age. A cell or organism that does not experience aging, or ceases to age at some point, is biologically immortal.

Biologists have chosen the word immortal to designate cells that are not limited by the Hayflick limit, where cells no longer divide because of DNA damage or shortened telomeres. The first and still most widely used immortal cell line is HeLa, developed from cells taken from the malignant cervical tumor of Henrietta Lacks without her consent in 1951. Prior to the 1961 work of Leonard Hayflick and Paul Moorhead, there was the erroneous belief fostered by Alexis Carrel that all normal somatic cells are immortal. By preventing cells from reaching senescence one can achieve biological immortality; telomeres, a "cap" at the end of DNA, are thought to be the cause of cell aging. Every time a cell divides the telomere becomes a bit shorter; when it is finally worn down, the cell is unable to split and dies. Telomerase is an enzyme which rebuilds the telomeres in stem cells and cancer cells, allowing them to replicate an infinite number of times.[11] No definitive work has yet demonstrated that telomerase can be used in human somatic cells to prevent healthy tissues from aging. On the other hand, scientists hope to be able to grow organs with the help of stem cells, allowing organ transplants without the risk of rejection, another step in extending human life expectancy. These technologies are the subject of ongoing research, and are not yet realized.[citation needed]

Life defined as biologically immortal is still susceptible to causes of death besides aging, including disease and trauma, as defined above. Notable immortal species include:

As the existence of biologically immortal species demonstrates, there is no thermodynamic necessity for senescence: a defining feature of life is that it takes in free energy from the environment and unloads its entropy as waste. Living systems can even build themselves up from seed, and routinely repair themselves. Aging is therefore presumed to be a byproduct of evolution, but why mortality should be selected for remains a subject of research and debate. Programmed cell death and the telomere "end replication problem" are found even in the earliest and simplest of organisms.[16] This may be a tradeoff between selecting for cancer and selecting for aging.[17]

Modern theories on the evolution of aging include the following:

There are some known naturally occurring and artificially produced chemicals that may increase the lifetime or life-expectancy of a person or organism, such as resveratrol.[20][21]

Some scientists believe that boosting the amount or proportion of telomerase in the body, a naturally forming enzyme that helps maintain the protective caps at the ends of chromosomes,[22] could prevent cells from dying and so may ultimately lead to extended, healthier lifespans. A team of researchers at the Spanish National Cancer Centre (Madrid) tested the hypothesis on mice. It was found that those mice which were genetically engineered to produce 10 times the normal levels of telomerase lived 50% longer than normal mice.[23]

In normal circumstances, without the presence of telomerase, if a cell divides repeatedly, at some point all the progeny will reach their Hayflick limit. With the presence of telomerase, each dividing cell can replace the lost bit of DNA, and any single cell can then divide unbounded. While this unbounded growth property has excited many researchers, caution is warranted in exploiting this property, as exactly this same unbounded growth is a crucial step in enabling cancerous growth. If an organism can replicate its body cells faster, then it would theoretically stop aging.

Embryonic stem cells express telomerase, which allows them to divide repeatedly and form the individual. In adults, telomerase is highly expressed in cells that need to divide regularly (e.g., in the immune system), whereas most somatic cells express it only at very low levels in a cell-cycle dependent manner.

Technological immortality is the prospect for much longer life spans made possible by scientific advances in a variety of fields: nanotechnology, emergency room procedures, genetics, biological engineering, regenerative medicine, microbiology, and others. Contemporary life spans in the advanced industrial societies are already markedly longer than those of the past because of better nutrition, availability of health care, standard of living and bio-medical scientific advances. Technological immortality predicts further progress for the same reasons over the near term. An important aspect of current scientific thinking about immortality is that some combination of human cloning, cryonics or nanotechnology will play an essential role in extreme life extension. Robert Freitas, a nanorobotics theorist, suggests tiny medical nanorobots could be created to go through human bloodstreams, find dangerous things like cancer cells and bacteria, and destroy them.[24] Freitas anticipates that gene-therapies and nanotechnology will eventually make the human body effectively self-sustainable and capable of living indefinitely in empty space, short of severe brain trauma. This supports the theory that we will be able to continually create biological or synthetic replacement parts to replace damaged or dying ones. Future advances in nanomedicine could give rise to life extension through the repair of many processes thought to be responsible for aging. K. Eric Drexler, one of the founders of nanotechnology, postulated cell repair devices, including ones operating within cells and utilizing as yet hypothetical biological machines, in his 1986 book Engines of Creation. Raymond Kurzweil, a futurist and transhumanist, stated in his book The Singularity Is Near that he believes that advanced medical nanorobotics could completely remedy the effects of aging by 2030.[25] According to Richard Feynman, it was his former graduate student and collaborator Albert Hibbs who originally suggested to him (circa 1959) the idea of a medical use for Feynman's theoretical micromachines (see nanobiotechnology). Hibbs suggested that certain repair machines might one day be reduced in size to the point that it would, in theory, be possible to (as Feynman put it) "swallow the doctor". The idea was incorporated into Feynman's 1959 essay There's Plenty of Room at the Bottom.[26]

Cryonics, the practice of preserving organisms (either intact specimens or only their brains) for possible future revival by storing them at cryogenic temperatures where metabolism and decay are almost completely stopped, can be used to 'pause' for those who believe that life extension technologies will not develop sufficiently within their lifetime. Ideally, cryonics would allow clinically dead people to be brought back in the future after cures to the patients' diseases have been discovered and aging is reversible. Modern cryonics procedures use a process called vitrification which creates a glass-like state rather than freezing as the body is brought to low temperatures. This process reduces the risk of ice crystals damaging the cell-structure, which would be especially detrimental to cell structures in the brain, as their minute adjustment evokes the individual's mind.

One idea that has been advanced involves uploading an individual's habits and memories via direct mind-computer interface. The individual's memory may be loaded to a computer or to a new organic body. Extropian futurists like Moravec and Kurzweil have proposed that, thanks to exponentially growing computing power, it will someday be possible to upload human consciousness onto a computer system, and exist indefinitely in a virtual environment. This could be accomplished via advanced cybernetics, where computer hardware would initially be installed in the brain to help sort memory or accelerate thought processes. Components would be added gradually until the person's entire brain functions were handled by artificial devices, avoiding sharp transitions that would lead to issues of identity, thus running the risk of the person to be declared dead and thus not be a legitimate owner of his or her property. After this point, the human body could be treated as an optional accessory and the program implementing the person could be transferred to any sufficiently powerful computer. Another possible mechanism for mind upload is to perform a detailed scan of an individual's original, organic brain and simulate the entire structure in a computer. What level of detail such scans and simulations would need to achieve to emulate awareness, and whether the scanning process would destroy the brain, is still to be determined.[27] Whatever the route to mind upload, persons in this state could then be considered essentially immortal, short of loss or traumatic destruction of the machines that maintained them.[clarification needed]

Transforming a human into a cyborg can include brain implants or extracting a human processing unit and placing it in a robotic life-support system. Even replacing biological organs with robotic ones could increase life span (i.e., pace makers) and depending on the definition, many technological upgrades to the body, like genetic modifications or the addition of nanobots would qualify an individual as a cyborg. Some people believe that such modifications would make one impervious to aging and disease and theoretically immortal unless killed or destroyed.

Another approach, developed by biogerontologist Marios Kyriazis, holds that human biological immortality is an inevitable consequence of evolution. As the natural tendency is to create progressively more complex structures,[28] there will be a time (Kyriazis claims this time is now[29]), when evolution of a more complex human brain will be faster via a process of developmental singularity[30] rather than through Darwinian evolution. In other words, the evolution of the human brain as we know it will cease and there will be no need for individuals to procreate and then die. Instead, a new type of development will take over, in the same individual who will have to live for many centuries in order for the development to take place. This intellectual development will be facilitated by technology such as synthetic biology, artificial intelligence and a technological singularity process.

As late as 1952, the editorial staff of the Syntopicon found in their compilation of the Great Books of the Western World, that "The philosophical issue concerning immortality cannot be separated from issues concerning the existence and nature of man's soul."[31] Thus, the vast majority of speculation regarding immortality before the 21st century was regarding the nature of the afterlife.

Immortality in ancient Greek religion originally always included an eternal union of body and soul as can be seen in Homer, Hesiod, and various other ancient texts. The soul was considered to have an eternal existence in Hades, but without the body the soul was considered dead. Although almost everybody had nothing to look forward to but an eternal existence as a disembodied dead soul, a number of men and women were considered to have gained physical immortality and been brought to live forever in either Elysium, the Islands of the Blessed, heaven, the ocean or literally right under the ground. Among these were Amphiaraus, Ganymede, Ino, Iphigenia, Menelaus, Peleus, and a great part of those who fought in the Trojan and Theban wars. Some were considered to have died and been resurrected before they achieved physical immortality. Asclepius was killed by Zeus only to be resurrected and transformed into a major deity. In some versions of the Trojan War myth, Achilles, after being killed, was snatched from his funeral pyre by his divine mother Thetis, resurrected, and brought to an immortal existence in either Leuce, the Elysian plains, or the Islands of the Blessed. Memnon, who was killed by Achilles, seems to have received a similar fate. Alcmene, Castor, Heracles, and Melicertes were also among the figures sometimes considered to have been resurrected to physical immortality. According to Herodotus' Histories, the 7th century BC sage Aristeas of Proconnesus was first found dead, after which his body disappeared from a locked room. Later he was found not only to have been resurrected but to have gained immortality.

The philosophical idea of an immortal soul was a belief first appearing with either Pherecydes or the Orphics, and most importantly advocated by Plato and his followers. This, however, never became the general norm in Hellenistic thought. As may be witnessed even into the Christian era, not least by the complaints of various philosophers over popular beliefs, many or perhaps most traditional Greeks maintained the conviction that certain individuals were resurrected from the dead and made physically immortal and that others could only look forward to an existence as disembodied and dead, though everlasting, souls. The parallel between these traditional beliefs and the later resurrection of Jesus was not lost on the early Christians, as Justin Martyr argued: "when we say... Jesus Christ, our teacher, was crucified and died, and rose again, and ascended into heaven, we propose nothing different from what you believe regarding those whom you consider sons of Zeus." (1 Apol. 21).

The goal of Hinayana is Arhatship and Nirvana. By contrast, the goal of Mahayana is Buddhahood.

According to one Tibetan Buddhist teaching, Dzogchen, individuals can transform the physical body into an immortal body of light called the rainbow body.

Christian theology holds that Adam and Eve lost physical immortality for themselves and all their descendants in the Fall of Man, although this initial "imperishability of the bodily frame of man" was "a preternatural condition".[32] Christians who profess the Nicene Creed believe that every dead person (whether they believed in Christ or not) will be resurrected from the dead at the Second Coming, and this belief is known as Universal resurrection.[citation needed]

N.T. Wright, a theologian and former Bishop of Durham, has said many people forget the physical aspect of what Jesus promised. He told Time: "Jesus' resurrection marks the beginning of a restoration that he will complete upon his return. Part of this will be the resurrection of all the dead, who will 'awake', be embodied and participate in the renewal. Wright says John Polkinghorne, a physicist and a priest, has put it this way: 'God will download our software onto his hardware until the time he gives us new hardware to run the software again for ourselves.' That gets to two things nicely: that the period after death (the Intermediate state) is a period when we are in God's presence but not active in our own bodies, and also that the more important transformation will be when we are again embodied and administering Christ's kingdom."[33] This kingdom will consist of Heaven and Earth "joined together in a new creation", he said.

Hindus believe in an immortal soul which is reincarnated after death. According to Hinduism, people repeat a process of life, death, and rebirth in a cycle called samsara. If they live their life well, their karma improves and their station in the next life will be higher, and conversely lower if they live their life poorly. After many life times of perfecting its karma, the soul is freed from the cycle and lives in perpetual bliss. There is no place of eternal torment in Hinduism, although if a soul consistently lives very evil lives, it could work its way down to the very bottom of the cycle.[citation needed]

There are explicit renderings in the Upanishads alluding to a physically immortal state brought about by purification, and sublimation of the 5 elements that make up the body. For example, in the Shvetashvatara Upanishad (Chapter 2, Verse 12), it is stated "When earth, water fire, air and akasa arise, that is to say, when the five attributes of the elements, mentioned in the books on yoga, become manifest then the yogi's body becomes purified by the fire of yoga and he is free from illness, old age and death." This phenomenon is possible when the soul reaches enlightenment while the body and mind are still intact, an extreme rarity, and can only be achieved upon the highest most dedication, meditation and consciousness.[citation needed]

Another view of immortality is traced to the Vedic tradition by the interpretation of Maharishi Mahesh Yogi:

That man indeed whom these (contacts) do not disturb, who is even-minded in pleasure and pain, steadfast, he is fit for immortality, O best of men.[34]

To Maharishi Mahesh Yogi, the verse means, "Once a man has become established in the understanding of the permanent reality of life, his mind rises above the influence of pleasure and pain. Such an unshakable man passes beyond the influence of death and in the permanent phase of life: he attains eternal life... A man established in the understanding of the unlimited abundance of absolute existence is naturally free from existence of the relative order. This is what gives him the status of immortal life."[34]

An Indian Tamil saint known as Vallalar claimed to have achieved immortality before disappearing forever from a locked room in 1874.[35][36]

Many Indian fables and tales include instances of metempsychosisthe ability to jump into another bodyperformed by advanced Yogis in order to live a longer life.[citation needed]

The traditional concept of an immaterial and immortal soul distinct from the body was not found in Judaism before the Babylonian Exile, but developed as a result of interaction with Persian and Hellenistic philosophies. Accordingly, the Hebrew word nephesh, although translated as "soul" in some older English Bibles, actually has a meaning closer to "living being".[citation needed]Nephesh was rendered in the Septuagint as (psch), the Greek word for soul.[citation needed]

The only Hebrew word traditionally translated "soul" (nephesh) in English language Bibles refers to a living, breathing conscious body, rather than to an immortal soul.[37] In the New Testament, the Greek word traditionally translated "soul" () has substantially the same meaning as the Hebrew, without reference to an immortal soul.[38] Soul may refer to the whole person, the self: three thousand souls were converted in Acts 2:41 (see Acts 3:23).

The Hebrew Bible speaks about Sheol (), originally a synonym of the grave-the repository of the dead or the cessation of existence until the Resurrection. This doctrine of resurrection is mentioned explicitly only in Daniel 12:14 although it may be implied in several other texts. New theories arose concerning Sheol during the intertestamental literature.

The views about immortality in Judaism is perhaps best exemplified by the various references to this in Second Temple Period. The concept of resurrection of the physical body is found in 2 Maccabees, according to which it will happen through recreation of the flesh.[39] Resurrection of the dead also appears in detail in the extra-canonical books of Enoch,[40] and in Apocalypse of Baruch.[41] According to the British scholar in ancient Judaism Philip R. Davies, there is little or no clear reference either to immortality or to resurrection from the dead in the Dead Sea scrolls texts.[42] Both Josephus and the New Testament record that the Sadducees did not believe in an afterlife,[43] but the sources vary on the beliefs of the Pharisees. The New Testament claims that the Pharisees believed in the resurrection, but does not specify whether this included the flesh or not.[44] According to Josephus, who himself was a Pharisee, the Pharisees held that only the soul was immortal and the souls of good people will be reincarnated and pass into other bodies, while the souls of the wicked will suffer eternal punishment. [45]Jubilees seems to refer to the resurrection of the soul only, or to a more general idea of an immortal soul.[46]

Rabbinic Judaism claims that the righteous dead will be resurrected in the Messianic age with the coming of the messiah. They will then be granted immortality in a perfect world. The wicked dead, on the other hand, will not be resurrected at all. This is not the only Jewish belief about the afterlife. The Tanakh is not specific about the afterlife, so there are wide differences in views and explanations among believers.[citation needed]

It is repeatedly stated in Lshi Chunqiu that death is unavoidable.[47]Henri Maspero noted that many scholarly works frame Taoism as a school of thought focused on the quest for immortality.[48] Isabelle Robinet asserts that Taoism is better understood as a way of life than as a religion, and that its adherents do not approach or view Taoism the way non-Taoist historians have done.[49] In the Tractate of Actions and their Retributions, a traditional teaching, spiritual immortality can be rewarded to people who do a certain amount of good deeds and live a simple, pure life. A list of good deeds and sins are tallied to determine whether or not a mortal is worthy. Spiritual immortality in this definition allows the soul to leave the earthly realms of afterlife and go to pure realms in the Taoist cosmology.[50]

Zoroastrians believe that on the fourth day after death, the human soul leaves the body and the body remains as an empty shell. Souls would go to either heaven or hell; these concepts of the afterlife in Zoroastrianism may have influenced Abrahamic religions. The Persian word for "immortal" is associated with the month "Amurdad", meaning "deathless" in Persian, in the Iranian calendar (near the end of July). The month of Amurdad or Ameretat is celebrated in Persian culture as ancient Persians believed the "Angel of Immortality" won over the "Angel of Death" in this month.[51]

The possibility of clinical immortality raises a host of medical, philosophical, and religious issues and ethical questions. These include persistent vegetative states, the nature of personality over time, technology to mimic or copy the mind or its processes, social and economic disparities created by longevity, and survival of the heat death of the universe.

The Epic of Gilgamesh, one of the first literary works, is primarily a quest of a hero seeking to become immortal.[7]

Physical immortality has also been imagined as a form of eternal torment, as in Mary Shelley's short story "The Mortal Immortal", the protagonist of which witnesses everyone he cares about dying around him. Jorge Luis Borges explored the idea that life gets its meaning from death in the short story "The Immortal"; an entire society having achieved immortality, they found time becoming infinite, and so found no motivation for any action. In his book "Thursday's Fictions", and the stage and film adaptations of it, Richard James Allen tells the story of a woman named Thursday who tries to cheat the cycle of reincarnation to get a form of eternal life. At the end of this fantastical tale, her son, Wednesday, who has witnessed the havoc his mother's quest has caused, forgoes the opportunity for immortality when it is offered to him.[52] Likewise, the novel Tuck Everlasting depicts immortality as "falling off the wheel of life" and is viewed as a curse as opposed to a blessing. In the anime Casshern Sins humanity achieves immortality due to advances in medical technology, however the inability of the human race to die causes Luna, a Messianic figure, to come forth and offer normal lifespans because she had believed that without death, humans could not live. Ultimately, Casshern takes up the cause of death for humanity when Luna begins to restore humanity's immortality. In Anne Rice's book series "The Vampire Chronicles", vampires are portrayed as immortal and ageless, but their inability to cope with the changes in the world around them means that few vampires live for much more than a century, and those who do often view their changeless form as a curse.

Although some scientists state that radical life extension, delaying and stopping aging are achievable,[53] there are no international or national programs focused on stopping aging or on radical life extension. In 2012 in Russia, and then in the United States, Israel and the Netherlands, pro-immortality political parties were launched. They aimed to provide political support to anti-aging and radical life extension research and technologies and at the same time transition to the next step, radical life extension, life without aging, and finally, immortality and aim to make possible access to such technologies to most currently living people.[54]

There are numerous symbols representing immortality. The ankh is an Egyptian symbol of life that holds connotations of immortality when depicted in the hands of the gods and pharaohs, who were seen as having control over the journey of life. The Mbius strip in the shape of a trefoil knot is another symbol of immortality. Most symbolic representations of infinity or the life cycle are often used to represent immortality depending on the context they are placed in. Other examples include the Ouroboros, the Chinese fungus of longevity, the ten kanji, the phoenix, the peacock in Christianity,[55] and the colors amaranth (in Western culture) and peach (in Chinese culture).

Immortal species abound in fiction, especially in fantasy literature.

Link:
Immortality - Wikipedia, the free encyclopedia

Multipotent Cell-Secreted Extracellular Matrix Supports Cartilage Formation Histogen to present at International Cartilage Repair Society 2015

CHICAGO, May 8, 2015 - Histogen, Inc., a regenerative medicine company developing solutions based on the products of cells grown under simulated embryonic conditions, will present new research on its human extracellular matrix (hECM) material in the promotion of cartilage regeneration during the International Cartilage Repair Society (ICRS) 2015 Meeting, taking place May 8-11, 2015 in Chicago, IL. The orthobiologic applications of all of Histogen's products are being developed by its worldwide joint venture, PUR Biologics LLC.

Histogen has previously shown that hypoxia-induced multipotent cells produce soluble and insoluble materials that contain components associated with stem cell niches in the body and with scarless healing. These proteins include a variety of laminins, osteonectin, decorin, hyaluronic acid, collagen type IV, SPARC, CXCL12, NID1, NID2, NOTCH2, tenascin, thrombospondin, fibronectin, versican, and fibrillin-2. In vitro studies further demonstrated that the CCM and ECM promote the adhesion, proliferation and migration of bone marrow-derived human mesenchymal stem cells (MSCs).

In this latest research, in vivo studies with the hECM were undertaken to determine their potential as orthobiologics. Rabbit studies demonstrated the potential of the hECM to promote regeneration and repair of full-thickness articular cartilage defects. Eight weeks following hECM treatment of femoral osteochondral defects, mature bone and hyaline cartilage formation was seen, exemplified by the presence of a tide mark and integration into the adjacent cartilage. This work is currently being repeated in a goat cartilage defect model, with similar results to date.

"The efficacy we have seen with the multipotent cell-secreted ECM in bone and cartilage regeneration is unprecedented," said Ryan Fernan, CEO of PUR Biologics. "The preclinical work overwhelmingly supports use of the material as an orthobiologic to reduce inflammation and promote cartilage regeneration in the articulating joint and intervertebral spinal disc. We look forward to entering human trials for these indications, as well as to continuing our research on utilizing the product for soft tissue repair in a variety of sports injuries."

Histogen's cell conditioned media (CCM) and hECM were also evaluated in an ex vivo rabbit intervertebral spinal disc model to study the effects of these materials in an environment where an extensive inflammatory response was induced by thrombin injection. Compared to untreated controls, both the CCM and ECM treatment significantly down regulated the expression of the inflammatory cytokine genes IL-1, IL-6, TNF-alpha, as well as the genes encoding the extracellular matrix degrading enzymes MMP3, and ADAMTS4, while upregulating aggrecan expression in the annulus fibrosus and nucleus pulposus tissue.

Dr. Gail Naughton, CEO of Histogen, will present "Human Cell Conditioned Media and Extracellular Matrix Reduce Inflammation and Support Hyaline Cartilage Formation" at the ICRS 2015 meeting in Chicago on May 9, 2015. Following the event, the presentation will be available upon request.

About PUR Biologics PUR Biologics is dedicated to providing regenerative biologic solutions to address musculoskeletal surgical needs, including spine, dental, ligament and medical device coating applications. In addition to distribution of approved allograft and biologic products, PUR is focused on development of next-generation orthopedic products based upon human protein and growth factor materials for bone and tissue regeneration. For more information visit http://www.purbiologics.com.

About Histogen Histogen is a regenerative medicine company developing solutions based upon the products of cells grown under proprietary conditions that mimic the embryonic environment, including low oxygen and suspension. Through this unique technology process, newborn cells are encouraged to naturally produce the vital proteins and growth factors from which the Company has developed its rich product portfolio. Histogen's technology focuses on stimulating a patient's own stem cells by delivering a proprietary complex of multipotent human proteins that have been shown to support stem cell growth and differentiation. For more information, please visit http://www.histogen.com.

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Novel Immunomodulatory Treatment Induces Apoptosis in Melanoma Histogen to present data at 2015 Society of Investigative Dermatology Annual Meeting

ATLANTA, May 6, 2015 - Histogen, Inc., a regenerative medicine company developing solutions based on the products of cells grown under simulated embryonic conditions, will present new research on its 105F immunomodulatory treatment candidate for melanoma during the 2015 Society of Investigative Dermatology (SID) Annual Meeting, taking place May 6-9, 2015 in Atlanta, GA.

Histogen has previously shown that hypoxia-induced multipotent cells produce a soluble material with anti-oncologic properties, with potential benefit in the treatment of a wide range of cancers. Studies to characterize the active components of the material have identified a low molecular weight fraction (105F) which directly induces apoptosis, or controlled cell death, in 21 human cancer cell lines. In its latest research, Histogen sought to further examine the mechanism of action of 105F in melanoma through in vitro and in vivo studies.

After treatment with 105F, melanoma cells were shown to release Interleukin 6 (IL-6) and TNF a, pro-inflammatory cytokines acting as signals to the immune system. This induction of an immune "flare" in combination with tumor cell apoptosis could be critically important in recruiting immune cells to the tumor for cytotoxic attack.

"We were excited to see the dual activity of 105F, both directly inducing cancer cell death and activating an anti-tumorigenic immune response to reduce metastatic disease," said Dr. Gail Naughton, CEO and Chairman of the Board of Histogen. "These results represent a potential treatment for melanoma and other solid tumors that works through multiple channels to eliminate cancer cells, but is not toxic to the body's healthy cells."

An in vivo model of lung metastasis in C57Bl/6 mice further showed the efficacy of 105F in the treatment of melanoma. Daily intravenous injections of 105F over 14 days resulted in a significant (p=0.0049) reduction in lesions and marked immune cell infiltration as compared to controls.

Dr. Naughton will present "105F is a novel immunoadaptive treatment candidate for melanoma that induces apoptosis and the secretion of pro-inflammatory IL-6" at the 2015 SID Annual Meeting in Atlanta beginning May 6, 2015. Following the event, the presentation will be available upon request.

About Histogen Histogen is a regenerative medicine company developing solutions based upon the products of cells grown under proprietary conditions that mimic the embryonic environment, including low oxygen and suspension. Through this unique technology process, newborn cells are encouraged to naturally produce the vital proteins and growth factors from which the Company has developed its rich product portfolio. Histogen's technology focuses on stimulating a patient's own stem cells by delivering a proprietary complex of multipotent human proteins that have been shown to support stem cell growth and differentiation. For more information, please visit http://www.histogen.com.

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Histogen's Composition for Oncology Treatments Receives US Patent

SAN DIEGO, October 8, 2014 - Histogen Oncology, a company developing innovative cancer therapies based on Histogen's regenerative medicine technology, today announced that the United States Patent & Trademark Office has issued patent 12/363,479 entitled "Extracellular matrix compositions for the treatment of cancer" to Histogen.

The patent, which is the fifth U.S. patent issued to Histogen, covers the soluble and insoluble compositions of proteins and cofactors that are secreted by multipotent stem cells through Histogen's technology process for use in the treatment of cancer. The patent claims support of the use of the compositions alone or as a delivery system for traditional chemotherapeutic agents.

Through the recent formation and funding of the Histogen Oncology joint venture, research and development of the unique, naturally secreted compositions is progressing toward a Phase I clinical trial for end stage pancreatic cancer.

"We are pleased about the timely issuance of our U.S. patent for the treatment of cancer," said Dr. Gail K. Naughton, Histogen CEO and Chairman of the Board. "Our collaborations with top institutions continue to produce mounting evidence supporting the unique mechanism of action of our secreted material in preventing metastasis and reducing tumor load while having no toxic affect on normal cells."

Histogen's composition has shown effectiveness in inhibiting over 21 human cancer cell lines both in vitro as well as in animal models. The mechanism of action of the secreted material is through the induction of apoptosis (controlled cell death) primarily in malignant cells, so there is little to no toxicity to normal cells. Histogen Oncology is studying the efficacy of a small molecular weight fraction of the cell secreted composition as a stand alone treatment as well as in combination therapy to evaluate whether effectiveness can be demonstrated with less toxic drug doses.

About Histogen Histogen is a regenerative medicine company developing solutions based upon the products of cells grown under proprietary conditions that mimic the embryonic environment, including low oxygen and suspension. Through this unique technology process, newborn cells are encouraged to naturally produce the vital proteins and growth factors from which the Company has developed its rich product portfolio. Histogen's technology focuses on stimulating a patient's own stem cells by delivering a proprietary complex of multipotent human proteins that have been shown to support stem cell growth and differentiation. For more information, please visit http://www.histogen.com.

Contacts Eileen Brandt, (858) 200-9520 ebrandt@histogeninc.com

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Histogen Oncology Created to Develop Novel Biologic Cancer Treatments Histogen, Inc. and Wylde, LLC Form Joint Venture

SAN DIEGO, July 8, 2014 - Histogen Inc., a regenerative medicine company developing solutions based on the products of cells grown under simulated embryonic conditions, has partnered with Southern California medical device group Wylde, LLC to create Histogen Oncology. This joint venture will focus on the development of unique cell-derived materials for cancer applications.

Under this joint venture, Histogen Oncology has acquired exclusive rights to Histogen's human multipotent cell conditioned media (CCM) and extracellular matrix (ECM) materials, as well as their derivatives, for oncology applications throughout North America. Histogen Oncology's initial clinical focus is pancreatic cancer, a highly treatment-resistant cancer in which a sub-fraction of the CCM has shown substantial preclinical promise.

"We have been very impressed with the results of Histogen's preliminary oncology work, not only because of the significant survival benefit but also because it is a naturally-derived material that is showing no toxicity," said Christopher Wiggins of Wylde, LLC. "There are so many patients out there who are not candidates for existing therapies due to the toxic nature of available drugs. This is particularly true in pancreatic cancer, where 80% of people diagnosed already have stage four disease."

In post-resection nude mouse models, intravenous treatment with the CCM sub-fraction resulted in prolonged survival by more than three fold in a majority of treated animals. In non-resection models, more than 50% of treated mice lived twice as long as the control. These results point to a potentially significant outcome for pancreatic cancer patients, and Histogen Oncology intends to progress the material toward a Phase I clinical trial for no-option pancreatic cancer patients in the coming 18 months.

Research on the mechanism responsible for cancer cell inhibition by the CCM shows the upregulation of Caspase 9 and cleaved Caspase 3, which causes cancer cells to enter apoptosis, or programmed cell death.

"The activity of the CCM sub-fraction is unique in a number of ways. Whereas most cancer therapies target rapidly dividing cells but not cancer stem cells, the inhibitory effect of this material is seen in malignant cells and circulating tumor cells as well," said Dr. Gail Naughton, CEO and Chairman of the Board of Histogen, Inc. "In addition, the activity is selective for malignant cells, supporting the proliferation of human dermal fibroblasts, embryonic stem cells and mesenchymal stem cells, while inhibiting tumor growth."

Histogen Oncology will be supported by Histogen's research group and funded by Wylde, LLC., made up of experts from the surgery and medical device industries. The creation of this joint venture allows for dedicated development of the CCM sub-fraction as a cancer treatment, as Histogen continues to allocate resources to the Company's revenue-generating aesthetic and promising therapeutic programs.

"We are extremely excited to fuel and push the next stage of development for this innovative and potentially life-saving therapy," said Wiggins. "The next generation of cancer treatment will have cell-signaling at its core, be beneficial in combination with existing therapies as well as stand alone, and provide an option to patients who currently have none. We believe Histogen's material has all of those characteristics and more."

About Histogen Aesthetics Histogen is a regenerative medicine company developing solutions based upon the products of cells grown under proprietary conditions that mimic the embryonic environment, including low oxygen and suspension. Through this unique technology process, newborn cells are encouraged to naturally produce the vital proteins and growth factors from which the Company has developed its rich product portfolio. Histogen's technology focuses on stimulating a patient's own stem cells by delivering a proprietary complex of multipotent human proteins that have been shown to support stem cell growth and differentiation. For more information, please visit http://www.histogen.com.

Contacts Eileen Brandt, (858) 200-9520 ebrandt@histogeninc.com

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Histogen Aesthetics Acquires CellCeuticals Biomedical Skin Treatments

SAN DIEGO, March 10, 2014 - Histogen Aesthetics, a subsidiary of regenerative medicine company Histogen, Inc. focused on skin care and cosmeceuticals, announced today that the Company has acquired the CellCeuticals Biomedical Skin Treatments line of skincare products.

Histogen Aesthetics will continue sales of the eleven existing CellCeuticals Biomedical Skin Treatments skincare products, while bringing new innovation to the line through the addition of a unique regenerative medicine technology, working to improve skin aging at a cellular level.

"We have long admired the science, clinical data and elegant formulas behind the CellCeuticals line, and see it as an ideal fit for our recently revitalized aesthetics subsidiary," said Dr. Gail K. Naughton, CEO and Chairman of Histogen, Inc. "We are very excited to begin infusing unique cell-signaling factors into the CellCeuticals regimen, to truly transform skin one cell at a time."

Dr. Naughton has spent more than 30 years in tissue engineering and regenerative medicine, and holds over 100 patents in the field. She founded Histogen in 2007, focused on developing therapies that work to stimulate the stem cells in the body to regenerate tissues and organs. Through this work, she has also seen how different compositions of human proteins can have cosmetic benefits, particularly in anti-aging and rejuvenation.

"I am pleased that the CellCeuticals Biomedical Skin Treatments will evolve, and see Histogen Aesthetics as an excellent home for this innovative product line," said Paul Scott Premo, co-founder of CellCeuticals Skin Care, Inc. "I believe the addition of this regenerative medicine technology will be the opportunity to introduce a new generation of products that are the vanguard of regenerative skin care."

The CellCeuticals system is made up of eleven distinctive products including the Extremely Gentle Skin Cleanser, CellGenesis Regenerative Skin Treatment, and PhotoDefense Color Radiance SPF55+ with proprietary and patented PhotoPlex technology. The line is currently available at retailers including QVC.com, Dermstore.com, and Nordstrom.com, as well as http://www.cellceuticalskincare.com.

About Histogen Aesthetics Histogen Aesthetics LLC, formed in 2008 as a subsidiary of Histogen, Inc., focuses on the development of innovative skin care products utilizing regenerative medicine technology. Histogen Aesthetics' technology is based on the expertise of founder Dr. Gail K. Naughton, in which fibroblasts are grown under unique conditions, producing a complex of naturally-secreted proteins and synergistic bio-products known to stimulate skin cells to regenerate and rejuvenate tissues. In 2014, Histogen Aesthetics acquired CellCeuticals Biomedical Skin Treatments, a line of scientifically-proven products that reactivate cells to help aging skin perform and look healthier and younger. For more information, visit http://www.cellceuticalskincare.com.

About Suneva Medical, Inc. Histogen is a regenerative medicine company developing solutions based upon the products of cells grown under proprietary conditions that mimic the embryonic environment, including low oxygen and suspension. Through this unique technology process, newborn cells are encouraged to naturally produce the vital proteins and growth factors from which the Company has developed its rich product portfolio. Histogen's lead product, Hair Stimulating Complex (HSC) has shown success in two Company-sponsored clinical trials as an injectable treatment for alopecia. In addition, the human multipotent cell conditioned media produced through Histogen's process is also being researched for oncology applications, and in orthopedics through joint venture PUR Biologics, LLC. For more information, please visit http://www.histogen.com.

Contacts Eileen Brandt, (858) 200-9520 ebrandt@histogeninc.com

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Histogen and Suneva Medical Expand License for Cell Conditioned Media-based Aesthetic Products Internationally

SAN DIEGO, CA, January 14, 2014 - Histogen, Inc., a regenerative medicine company developing solutions based on the products of cells grown under simulated embryonic conditions, today announced that they have entered into an international license agreement with Suneva Medical, Inc. for physician-dispensed aesthetic products containing Histogen's proprietary multipotent cell conditioned media (CCM).

This agreement is an amendment to the existing license between Histogen and Suneva Medical, through which Suneva has exclusively licensed the Regenica skincare line within the United States since February 2012. Under the terms of the international agreement, Suneva Medical is now the exclusive licensee for the distribution of Regenica through the physician-dispensed channel in Europe, most of Asia, South America, Canada, Australia, and the Middle East.

"Not only has Suneva had sales success, but they have generated enthusiasm around the Regenica product line and our technology here in the US," said Gail K. Naughton, Ph.D., CEO and Chairman of the Board of Histogen. "We are excited about expanding our skincare partnership internationally, and look forward to an exciting year for Regenica."

Regenica contains Histogen's proprietary Multipotent Cell Conditioned Media, made up of soluble cell-signaing proteins and growth factors which support the body's epidermal stem cells and renew skin throughout life. Through Histogen's technology process, which mimics the embryonic environment including conditions of low oxygen and suspension, cells are triggered to become multipotent, and naturally produce these proteins associated with skin renewal and scarless healing.

"We believe that Regenica truly is the next generation in growth factor technology, and we are extremely pleased that the products will now have a presence around the world," said Nicholas L. Teti, Jr., Chairman and Chief Executive Officer of Suneva Medical. "Our relationship with Histogen in the US physician market has been a valuable asset to Suneva, and has laid the groundwork for international success."

About Histogen Histogen is a regenerative medicine company developing solutions based upon the products of cells grown under proprietary conditions that mimic the embryonic environment, including low oxygen and suspension. Through this unique technology process, newborn cells are encouraged to naturally produce the vital proteins and growth factors from which the Company has developed its rich product portfolio. Histogen's lead product, Hair Stimulating Complex (HSC) has shown success in two Company-sponsored clinical trials as an injectable treatment for alopecia. In addition, the human multipotent cell conditioned media produced through Histogen's process can be found in skincare products including ReGenica, which is distributed by Suneva Medical in partnership with Obagi Medical Products. For more information, please visit http://www.histogen.com.

About Suneva Medical, Inc. Suneva Medical, Inc. is a privately-held aesthetics company focused on developing, manufacturing and commercializing novel, differentiated products for the general dermatology and aesthetic markets. The company currently markets Artefill in the US, Korea, Singapore and Vietnam; Refissa and Regenica Skincare in the U.S.; and Bellafill in Canada. For more information, visit http://www.sunevamedical.com.

Regenica is a trademark of Suneva Medical, Inc. The Multipotent Cell Conditioned Media Complex is covered by U.S. patents #8,257,947 and #8,524,494.

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Multipotent Stem Cell Proteins Support Soft Tissue Regeneration Histogen to present data at TERMIS AM Annual Conference in Atlanta

ATLANTA, November 13, 2013 - Histogen, Inc., a regenerative medicine company developing solutions based on the products of cells grown under simulated embryonic conditions, announced that Dr. Michael Zimber will give a podium presentation entitled "Human Multipotent Stem Cell Proteins Support Soft Tissue Regeneration" today at the Tissue Engineering and Regenerative Medicine International Society (TERMIS) Americas Annual Meeting in Atlanta, GA.

Through its proprietary technology process that simulates the conditions of the embryonic environment, Histogen has developed a human extracellular matrix (hECM) material composed of stem cell-associated proteins including SPARC, decorin, collagens I,III,IV, V, fibronectin, fibrillin, laminins, and hyaluronic acid. The hECM's distinctive composition of growth factors and other proteins are known to stimulate stem cells in the body, regenerate tissues, and promote scarless healing.

Histogen sought to examine whether the hECM may promote scarless healing in full thickness wounds, similar to that seen in fetal healing, using a variety of forms of the material, including hollow spheres to maximize void fill volume. In preclinical studies, all hECM-treated wounds healed rapidly with minimum contractions, and the hECM microspheres had a statistically significant improvement in healing as compared to the controls (p<0.05) and produced a 25% thicker dermis. In addition, hECM applied topically after microneedling resulted in up to a 3X dermal thickening.

"We are very pleased that our propriety materials produced by hypoxia-induced human multipotent stem cells have shown significant healing results in both soft and hard tissues," said Dr. Gail Naughton, CEO and Chairman of the Board of Histogen. "These results open new therapeutic markets, show tremendous potential for our material in cutaneous wound care and orthopedics, as well as support the expansion of our aesthetic pipeline to include soft tissue fillers."

In addition to "Human Multipotent Stem Cell Proteins Support Soft Tissue Regeneration", Dr. Zimber will also be presenting "Human Multipotent Stem Cell Proteins Support Osteogenesis In Vitro" during the TERMIS AM Annual Meeting taking place November 10-13, 2013 in Atlanta. Following the event, these presentations will be available upon request.

About Histogen Histogen is a regenerative medicine company developing solutions based upon the products of cells grown under proprietary conditions that mimic the embryonic environment, including low oxygen and suspension. Through this unique technology process, newborn cells are encouraged to naturally produce the vital proteins and growth factors from which the Company has developed its rich product portfolio. Histogen's lead product, Hair Stimulating Complex (HSC) has shown success in two Company-sponsored clinical trials as an injectable treatment for alopecia. In addition, the human multipotent cell conditioned media produced through Histogen's process can be found in skincare products including ReGenica, which is distributed by Suneva Medical in partnership with Obagi Medical Products. For more information, please visit http://www.histogen.com.

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Multipotent Stem Cell Proteins Support Rejuvenation while Inhibiting Skin Cancer Histogen to present data at TERMIS AP Annual Conference in Shanghai

San Diego, October 24, 2013 - Histogen, Inc., a regenerative medicine company developing solutions based on the products of cells grown under simulated embryonic conditions, announced that the Company's Chairman and CEO, Dr. Gail Naughton, will present today at the Tissue Engineering and Regenerative Medicine International Society (TERMIS) Asia Pacific Annual Meeting in Shanghai, China.

Through its proprietary technology process that simulates the conditions of the embryonic environment, Histogen is uniquely able to trigger the de-differentiation of skin cells into multipotent stem cells without genetic manipulation. The cells express key stem cell markers including Oct4, Sox2 and Nanog, and secrete a distinctive composition of growth factors and other proteins known to stimulate stem cells in the body, regenerate tissues, and promote scarless healing.

It is the soluble and insoluble compositions of multipotent proteins and growth factors resulting from this process that have been shown to both promote skin regeneration and induce controlled cell death in multiple skin cancers.

"The anti-aging and rejuvenation benefits of human multipotent stem cell proteins have been shown in several clinical studies, and have resulted in the material's use as a thriving next-generation ingredient for skin care," said Dr. Naughton. "In parallel, we have also been studying the anti-cancer activity of these proteins, and have shown that, just as in the embryonic environment, they support normal tissue growth while resulting in the controlled death of cancer cells".

In vitro studies performed with Histogen's material have shown reduction in Squamous Cell Carcinoma (SCC), Basal Cell Carcinoma, and Melanoma cell number through the mechanism of apoptosis, or controlled cell death, induced by the upregulation of Caspase in these cancer cells. In one in vivo model, melanoma load was reduced by up to 80% versus the control (p<0.05) by the addition of the insoluble multipotent stem cell proteins, and a dose response curve was seen. Similar inhibition was seen with SCC. In subcutaneous mouse experiments, tumor growth was inhibited by 70-90%.

"Human Multipotent Stem Cell Proteins Stimulate Skin Regeneration While Inducing Skin Cancer Cell Apotosis" will be presented by Dr. Naughton during the TERMIS AP Annual Meeting taking place October 23-26, 2013 in Shanghai. Further information and data on the ability of multipotent stem cell proteins to induce apoptosis in skin cancers can be found in the publication Journal of Cancer Therapy at file.scirp.org/Html/1-8901700_33923.htm.

About Histogen Histogen is a regenerative medicine company developing solutions based upon the products of cells grown under proprietary conditions that mimic the embryonic environment, including low oxygen and suspension. Through this unique technology process, newborn cells are encouraged to naturally produce the vital proteins and growth factors from which the Company has developed its rich product portfolio. Histogen's lead product, Hair Stimulating Complex (HSC) has shown success in two Company-sponsored clinical trials as an injectable treatment for alopecia. In addition, the human multipotent cell conditioned media produced through Histogen's process can be found in skincare products including ReGenica, which is distributed by Suneva Medical in partnership with Obagi Medical Products. For more information, please visit http://www.histogen.com.

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Histogen to present at 2013 STEM CELL MEETING ON THE MESA

San Diego, October 11, 2013 - Histogen, Inc., a regenerative medicine company developing therapies for conditions including hair loss and cancer, announced today that Histogen CEO Gail K. Naughton, Ph.D. will give a company presentation at the 3rd Annual Regen Med Partnering Forum, part of the Stem Cell Meeting on the Mesa to be held October 14-16 in La Jolla, CA.

Histogen's solutions are based upon the products of cells grown under proprietary conditions that mimic the embryonic environment, including low oxygen and suspension. The technology focuses on stimulating a patient's own stem cells by delivering a proprietary complex of proteins that have been shown to support stem cell growth and differentiation.

"It is an exciting time for Histogen, as we continue to move the technology forward with expanded partnerships in skincare, compelling clinical data in both male and female hair loss, and early but exciting results in orthopedics," said Dr. Naughton. "We look forward to sharing our story during the Stem Cell Meeting on the Mesa, and to progressing our products even further through growing relationships with industry leaders and through our potential merger with Stratus Media to form publicly-traded Restorgenex."

Organized by the Alliance for Regenerative Medicine (ARM), the California Institute for Regenerative Medicine (CIRM) and the Sanford Consortium for Regenerative Medicine, the 2013 Stem Cell Meeting on the Mesa is a three-day conference aimed at bringing together senior members of the regenerative medicine industry with the scientific research community to advance stem cell science into cures. The Regen Med Partnering Forum, held October 14 &15 at the Estancia La Jolla Hotel, is the only partnering meeting organized specifically for the regenerative medicine and advanced therapies industry.

The following are specific details regarding Histogen's presentation at the conference:

Event: Regen Med Partnering Forum - 2013 Stem Cell Meeting on the Mesa Date: October 14, 2013 Time: 3:15pm Location: Estancia La Jolla Hotel & Spa, 9700 North Torrey Pines Road, La Jolla

A live video webcast of all company presentations will be available at: stemcellmeetingonthemesa.com/webcast and will also be published on ARM's website shortly after the event. Histogen will also make a copy of Dr. Naughton's presentation available at http://www.histogen.com.

About Histogen Histogen is a regenerative medicine company developing solutions based upon the products of cells grown under proprietary conditions that mimic the embryonic environment, including low oxygen and suspension. Through this unique technology process, newborn cells are encouraged to naturally produce the vital proteins and growth factors from which the Company has developed its rich product portfolio. Histogen's lead product, Hair Stimulating Complex (HSC) has shown success in two Company-sponsored clinical trials as an injectable treatment for alopecia. In addition, the human multipotent cell conditioned media produced through Histogen's process can be found in skincare products including ReGenica, which is distributed by Suneva Medical in partnership with Obagi Medical Products. For more information, please visit http://www.histogen.com.

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Stratus Media Group and Histogen Execute Letter of Intent for Biotechnology Merger

LOS ANGELES, October 07, 2013 - Stratus Media Group, Inc. (OTCQB:SMDI) announced today that it was planning to expand its entrance into the biotechnology industry with the execution of a letter of intent between the Company and Histogen, Inc., a regenerative medicine company developing innovative therapies for conditions including hair loss and cancer.

The non-binding letter of intent outlines the primary terms of a merger of San Diego-based Histogen into Stratus, to be renamed Restorgenex Corporation. The letter of intent has been approved by the board of directors of both companies, and the parties are engaged in completing a formal merger agreement.

Histogen's solutions are based upon the products of cells grown under proprietary conditions that mimic the embryonic environment, including low oxygen and suspension. The technology focuses on stimulating a patient's own stem cells by delivering a proprietary complex of proteins that have been shown to support stem cell growth and differentiation. Histogen's lead product, Hair Stimulating Complex (HSC) has shown success in two Company-sponsored clinical trials as an injectable treatment for alopecia. In addition, the human multipotent cell conditioned media produced through Histogen's process can be found in skincare products including ReGenica, which is distributed by Suneva Medical in partnership with Obagi Medical Products.

"Histogen's technology platform opens a spectrum of potential product opportunities in both aesthetics and therapeutics, an ideal fit with our vision for Restorgenex," said Sol J. Barer, Ph.D., who will assume the position of Chairman of the Board of Restorgenex effective November 1, 2013. "The expertise of the Histogen team in developing regenerative products from concept to market, along with the success Histogen has already found in skincare partnering, will add significant value to our Company."

Following successful completion of this proposed merger, the company's goal is to build Restorgenex into a world-class cosmeceutical and pharmaceutical company in the large and expanding fields of dermatology and hair restoration. The parties intend to move toward a formal merger agreement in which Histogen would become a wholly-owned subsidiary, Histogen founder Gail K. Naughton, Ph.D. would assume the position of Chief Executive Officer of Restorgenex, and the corporate headquarters of Restorgenex would be located in San Diego. The merger will require, among other things, the satisfaction of customary closing conditions including the approval of Histogen's shareholders.

"I am very excited about the potential of a merger between Histogen and Restorgenex, and look forward to moving into the next stage," said Dr. Naughton. "It is an honor to be working with biotechnology visionaries Dr. Sol Barer and Isaac Blech, and to have them recognize the promise of Histogen's products is a true testament to the unique and exciting nature of our technology."

Dr. Naughton has spent more than 25 years extensively researching the tissue engineering process, holds more than 95 U.S. and foreign patents, and has been honored for her pioneering work in the field by prestigious organizations including receiving the Intellectual Property Owners Association Inventor of the Year Award.

Prior to founding Histogen in 2007, Dr. Naughton oversaw the design and development of the world's first up-scaled manufacturing facility for tissue engineered products, was pivotal in raising over $350M from the public market and corporate partnerships, and brought four human cell-based products from concept through FDA approval and market launch as President of Advanced Tissue Sciences.

"I believe the potential acquisition of Histogen, and the expertise and vision Dr. Naughton will bring as Chief Executive Officer will be a tremendous asset in ushering the Company into the biotechnology industry," said Jerold Rubinstein, current Chairman and Chief Executive Officer of Stratus.

http://www.histogen.com http://www.stratusmediagroup.com

Forward-Looking Statements Statements in this press release relating to plans, strategies, projections of results, and other statements that are not descriptions of historical facts may be forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995 and the Securities Acts of 1933 and 1934. Forward-looking information is inherently subject to risks and uncertainties, and actual results could differ materially from those currently anticipated due to a number of factors. Although the company's management believes that the expectations reflected in the forward-looking statements are reasonable, it cannot guarantee future results, performance or achievements. The company has no obligation to update these forward-looking statements.

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Histogen - About Histogen - Latest news, upcoming events ...

Since the beginning of time, mankind has pondered the question of what it means to be alive. When, precisely, can one be considered a human being? With the advent of stem cell research, we are forced to confront this question head-on. Human embryonic stem cells have enormous medical potential; by harnessing the power of their undifferentiated state, we may be able to cure diseases and disabilities that have plagued mankind for millennia. Rather than simply treating the symptoms of debilitating conditions, we may be able to attack the diseases at their source, working from within the body itself. However, obtaining embryonic stem cells, despite the many benefits that may result, poses many new ethical questions. Embryonic stem cells are generated during the early stages of the formation of a human embryo. These cells adhere to the wall of the blastocyst and, in the process of obtaining them, the embryo is destroyed. The ethical dilemma is this; does this tiny but powerful group of cells constitute a human life, and, if so, it is justifiable to take it in order to save others?

What are Embryonic Stem Cells?

A fertilized embryo must undergo a series of divisions in order to grow. There are many different types of cells in an adult human, but, with the exception of red blood cells and lymph cells, every cell in the human body contains an identical genome. [5] If all cells contain an identical DNA sequence and originate from the same source, how does each cell know what to become? The answer lies in a population of cells known as stem cells. To be classified as a stem cell requires the possession of two key abilities

Unlike somatic cells, stem cells begin, not with a single fate, but in an undifferentiated state. This initial lack of specificity is crucial, for their division has the ability to field more unspecified cells or ones that will eventually become any cell in the organism. [3] The earliest stem cells are known as totipotent, meaning that they have the ability to differentiate into any cell in the embryo or the resulting adult. As the cells divide, they progressively lose their totipotent abilities, becoming more and more specified. The egg-sperm unit divides every 12-18 hours; first from two cells into four, then eight, then sixteen. [5] After this third cell division, totipotent cells give rise to pluripotent stem cells, which can become nearly any cell in the body. The unit is now a hollow ball known as a blastula, and with pluripotent cells adhered to the wall in a clump known as the Inner Cell Mass. The wall will become the supportive placenta as the embryo grows, with the ICM becoming the embryo itself. [7] As the pluripotent cells divide, they develop into cells called Lineage Restricted Stem Cells, then Progenitor Cells, and finally Differentiated Cells with pre-determined function. [See Figure 1] Each time the cell divides, it becomes more highly specified, and less plastic in terms of medical potential.

Why use Embryonic Stem Cells?

Because of their unique ability to generate so many different kinds of cells, and potential to reside in several areas of the human body, stem cells may eventually establish themselves as a cornerstone of 21st century medicine. Stem cell research has created an entirely new branch of medicine, called Regenerative Medicine. The specialty of this new discipline would be to repair organs or tissues affected or destroyed by age, disease or injury. [5] [6] [10] In at least one instance, experimental techniques have been highly successful. Scientists have developed large sheets of epidermal cells, which can be used to repair burns that have destroyed the full thickness of the skin. [10] Researchers are hoping to branch out and use the self-renewal and differentiating abilities of embryonic stem cells to treat diseases such as Parkinsons Disease or Type 1 Diabetes, or even paralysis resulting from damage to the spinal cord. [5] The hope is to learn to culture the cells and to manipulate their differentiation prior to inserting them into a patient. The cells would, in theory, be used to repair or re-grow the damaged tissues without being rejected by the patients immune system. In diabetic patients, the cells may be used to replace non-functioning pancreatic cells, while in paraplegic individuals the cells may replace the damaged components of the spine, allowing them to walk again.

An Ethical Mess: Are we taking a life in order to give life?

Obtaining embryonic stem cells for research purposes invariably results in the destruction of the embryo. Many individuals pose the question of whether this constitutes taking one human beings life in order to preserve the life of another. Currently, there are five major views concerning whether this ball of cells is in fact alive. Each viewpoint suggests that ones life begins at a different point in development.

Genetic View: Fertilization

Embryological View: Gastrulation A second position posits that one becomes human at gastrulation. [5] [12] Between 12 and 14 days after fertilization, the embryo begins to form germ layers, which will eventually develop into the three major tissue types found in adults. [17] Scientists view this as a turning point in development for, at the onset of gastrulation, the embryo can no longer divide to form twins. [12] If it survives, it is committed to forming a single individual. The blastula, now called a gastrula, develops three distinct layers of cells; the ectoderm, the mesoderm and the endoderm. The outermost layer, the ectoderm, will develop into the central nervous system, hair, fingernails and the epidermis of the skin. The endoderm, the innermost layer, gives rise to the lining of the digestive and respiratory tracts, and the glands such as the pancreas and liver. The mesoderm, the middle layer, is perhaps the most diverse, for it will eventually yield the muscles, the gonads, cartilage and the circulatory system, to name only a few. [17] Cells that are beginning to form the germ layers are too far along the differentiation pathway to be as useful as their predecessors. Considering the embryo to be alive only once gastrulation occurs is consistent with views in favor of Embryonic Stem Cell research. If one chooses this viewpoint, experimenting with embryos prior to this would not constitute taking a human life, for researchers would be obtaining the cells much earlier than the time of gastrulation.

Neurological View: EEG Activity The third major viewpoint is that human life begins with the acquisition of recognizable brain activity. At approximately, 24 weeks of age, there is a sufficient amount of coherence in the fetus developing brain that its activity can be seen via an electroencephalogram (EEG). [5] [18] In the United States, death is often determined by brain function. As stated in the Uniform Determination of Death Act, so-called brain death is defined as when the entire brain ceas[es] to function, irreversibly. The entire brain includes the brain stem, as well as the neocortex. The concept of entire brain distinguishes determination of death under this Act from neocortical death or persistent vegetative state. " [16] An individual whose cardiovascular and respiratory systems still function, but who produces no brain activity is considered to be dead. The fetal heart beat is present from approximately 7 weeks of gestational age, [4] but brain activity is not present until 24 weeks. This follows the logic present in US law; if we choose to define death in terms of the cessation of brain activity, we may choose to define life by its onset. This position also supports the use of stem-cell research, as the cells would be obtained months before the commencement of any recognizable brain activity.

Ecological View: Survival A fourth standpoint in terms of human life is viability. Some individuals choose to define human life as the point where the fetus is viable outside the mothers womb. [18] This has often been determined by lung function, as the respiratory system is both crucial for survival and one of the last systems to finish developing in the human fetus. Development of the lungs begins as early as week 4 of gestation, and continues until birth with the proliferation of the alveolar sacs. [8] [14] Surfactant, a compound produced in the alveoli beginning at about 34 weeks of age, reduces surface tension in the lungs and allows them to expand. Without this compound, infants have severely decreased lung function, which may prove fatal. Premature infants also have underdeveloped brain and immune function, which makes them highly susceptible to both apnea and infection as well as a host of other health problems. [14] Historically, many infants born before 28 weeks of age were unable to survive. [5] However, with the continued development of neonatal intensive care and cardio-pulmonary life support, the cutoff line for viability has become increasingly blurred. One is now forced to question whether a neonate born at 25 weeks of age with severely impaired brain and body function and kept alive only with assistance of machines is truly living.

The Birth View

Conclusion

Stem cells hold a power never before seen in medicine. If properly controlled, they may allow us to fight diseases that are now considered incurable. Their use, however, remains highly controversial, owing to the destruction of embryos in the process. Advocates against embryonic stem-cell research and use argue that the tiny ball of cells inside the blastula is alive. These individuals take the Fertilization viewpoint, maintaining that a human, no matter how small, is still a human. To them, the use of embryonic stem cells, even to save many others, can never justify the destruction of a human life. Supporters of embryonic stem cells maintain that zygotes are not truly human prior to gastrulation, brain function or even birth itself. Use of the inner cell mass in its earliest stages does not constitute ethical wrongdoing. The cells are obtained so early that the mass does not yet possess any human qualities, such as differentiated tissues or brain function. These individuals also point out that the majority of the blastocysts being used would not survive to begin with, and that anti-stem cell groups should see that the ends justify the means. Clearly stem cells have many potential benefits for mankind, but at the moment they are surrounded by a controversy that is unlikely to resolve itself any time soon. In the future, perhaps we will find a way to manipulate differentiated cells to have undifferentiated properties, thereby avoiding the ethics of embryonic stem cell use. However, until that day arrives, we must continue to ask ourselves the question of what it means to be human.

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Life and Death in Terms of Embryonic Stem Cells

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but so far there is a huge rejection factor and the death ra...

" rea-from what i have read the donor stem cell transplant is the only "cure" but so far there is a huge rejection factor and the death rate was too high for them to continue this as... "

ssage. I have found out that the death rate for stem cell tr...

" ...for your message. I have found out that the death rate for stem cell transplants is less than 1%. It's ...the process. I have been taking vitamin E and Beta Carotene. I'll... "

time of her death (unfortunately suicide). I also talked...

" ...type of BC than I do and had a stem cell transplant at UCLA over 10 years ago and was NED at the time of her death (unfortunately suicide). I also talked to someone who had... "

and had great success with it. The rate of death i...

" ...heard a woman here in BC speak about her stem cell transplant. She is 3 years post treatment and had great success with it. The rate of death is mostly attributed to liver problems brought on by... "

on the death of your mother. My mother died just...

" ...all, my condolences on the death of your mother. My mother died just over 9 years ago of GBM, a primary brain cancer. ...for any other Lymphoma included Stem Cell Transplant for very aggressive forms of... "

the time of his death he was being treated in Boston for...

" ...4/20/13 from flu after a stem cell transplant; M, 57 yrs. Source: http://obits.mlive.com/obituaries/an...37#fbLoggedOut ...2013. At the time of his death he was being treated in ...He had recently received a... "

more complications and a higher death rate. The mi...

" ...Hutchinson Cancer Research Center said: "Allogeneic stem cell transplants have the advantage of a ...immune reconstitution and "graft-versus-host disease", have more complications and a higher... "

did not cause her death.Kellie van Meurs suffered from a ra...

" ...say it did not cause her death. Kellie van Meurs suffered from ...to undergo an autologous hematopoietic stem cell transplant (HSCT) under the care of ...cells after high-dose chemotherapy. Ms van Meurs was... "

the UK has been suspended following the death of a patient,...

" ...in the bowel. Also, with Stem Cell Transplant, you absolutely cannot have any ...trial in the UK has been suspended following the death of a patient, so it looks... "

rd year. Getting over the death of a parent is difficult en...

" ...that you have had such a hard year. Getting over the death of a parent is difficult enough without having to cope ...joining. My husband had his stem cell transplant January 2011 and although he... "

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Death and Stem Cell Transplant - Posts about Drugs, Side ...

The Philippine Medical Association (PMA) is reportedly(article fromABS-CBN News) investigating the recent deaths of 3 prominent politicians due to stem cell therapies via stem cell tourism in Germany at an as yet unidentified clinic.

The names of the politicians are unknown, but strangely enough today theres another separate article on stem cells from the Philippine Daily Inquirerthat mentions 3 politicians who have received stem cell treatments:

A number of politicians have been reported to have used stem cell therapy, including former President Joseph Estrada, Sen. Juan Ponce Enrile and former Sen. Ernesto Maceda.

Of course these men may not be the politicians referred to in the ABS-CBN piece. Maceda was quoted:

I am now convinced that my stem cell therapy is effective and thats the reason why Ive been able to keep up with the rigorous campaign schedule, he said. I feel 20 years younger.

In the past the German stem cell clinic X-cell was notorious for being linked to a babys death.

PMA president Dr. Leo Olarte commented on the more recent case:

They were given stem cells from sheep, rabbits and animals. They died after one year, they had late hypersensitivity reaction, he said.

It sounds like a very horrible situation. More information is needed to get the bottom of this.

The ABS-CBN Foundation, presumably the outfit responsible for ABS-CBN News that reported the 3 politician deaths, is an advocacy group of some kind in the Philippines that works with Olarte ( see him with Gina Lopez, Managing Director of ABS-CBN in the pic below).

Stem cells are generating a lot of buzz in the Philippines and apparently stem cell interventions of various kinds are becoming more common including a supposed aphrodisiac stem cell potion called Soup. No. 7. Thus, efforts to reign in dangerous stem cell interventions there are very important. At the same time some in the Philippines such as Olarte want to promote stem cell tourism as well it seems. Its a fine line to walk

For example, in commenting toABS-CBN News in the same article as about the German deaths, Olarte seems to be playing up the state of stem cell interventions in that country more generally:

Olarte said the country already has experts, who are members of the Philippine Society for Stem Cell Medicine (PSSCM), competent to perform the treatment in the Philippines.

We have more or less 400 specialists, he said, even noting that the stem cell treatment in the country is much cheaper by 50% than what is being sold abroad.

Im not so convinced that even these supposedly okay stem cell treatments promoted by Olarte are proven safe or effective either even if they are cheaper.

The deaths of the three politicians in Germany are disturbing news and highlight the care that must be taken in regulating stem cell interventions to keep patients safe.

. Bookmark the

.

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deaths of 3 politicians - Knoepfler Lab Stem Cell Blog

Brain stem death is a clinical syndrome defined by the absence of reflexes with pathways through the brain stem - the stalk of the brain, which connects the spinal cord to the mid-brain, cerebellum and cerebral hemispheres - in a deeply comatose, ventilator-dependent patient. Identification of this state carries a very grave prognosis for survival; cessation of heartbeat often occurs within a few days although it may continue for weeks or even months if intensive support is maintained.[1]

In the United Kingdom, the formal diagnosis of brain stem death by the procedure laid down in the official Code of Practice[1] permits the diagnosis and certification of death on the premise that a person is dead when consciousness and the ability to breathe are permanently lost, regardless of continuing life in the body and parts of the brain, and that death of the brain stem alone is sufficient to produce this state.[2]

This concept of brain stem death is also accepted as grounds for pronoucing death for legal purposes in India[3] and Trinidad & Tobago.[4] Elsewhere in the world the concept upon which the certification of death on neurological grounds is based is that of permanent cessation of all function in all parts of the brain - whole brain death - with which the reductionist United Kingdom concept should not be confused. The United States' President's Council on Bioethics made it clear, in its White Paper of December 2008, that the United Kingdom concept and clinical criteria are not considered sufficient for the diagnosis of death in the United States of America.[5]

The United Kingdom (UK) criteria were first published by the Conference of Medical Royal Colleges (with advice from the Transplant Advisory Panel) in 1976, as prognostic guidelines.[6] They were drafted in response to a perceived need for guidance in the management of deeply comatose patients with severe brain damage who were being kept alive by mechanical ventilators but showing no signs of recovery. The Conference sought to establish diagnostic criteria of such rigour that on their fulfilment the mechanical ventilator can be switched off, in the secure knowledge that there is no possible chance of recovery. The published criteria negative responses to bedside tests of some reflexes with pathways through the brain stem and a specified challenge to the brain stem respiratory centre, with caveats about exclusion of endocrine influences, metabolic factors and drug effects were held to be sufficient to distinguish between those patients who retain the functional capacity to have a chance of even partial recovery and those where no such possibility exists. Recognition of that state required the withdrawal of fruitless further artificial support so that death might be allowed to occur, thus sparing relatives from the further emotional trauma of sterile hope.[6]

In 1979, the Conference of Medical Royal Colleges promulgated its conclusion that identification of the state defined by those same criteria then thought sufficient for a diagnosis of brain death means that the patient is dead [7]Death certification on those criteria has continued in the United Kingdom (where there is no statutory legal definition of death) since that time, particularly for organ transplantation purposes, although the conceptual basis for that use has changed.

In 1995, after a review by a Working Group of the Royal College of Physicians of London, the Conference of Medical Royal Colleges [2] formally adopted the more correct term for the syndrome, "brain stem death" - championed by Pallis in a set of 1982 articles in the British Medical Journal [8] and advanced a new definition of human death as the basis for equating this syndrome with the death of the person. The suggested new definition of death was the irreversible loss of the capacity for consciousness, combined with irreversible loss of the capacity to breathe. It was stated that the irreversible cessation of brain stem function will produce this state and therefore brain stem death is equivalent to the death of the individual.[2]

In the UK, the formal rules for the diagnosis of brain stem death have undergone only minor modifications since they were first published [6] in 1976. The most recent revision of the UK's Department of Health Code of Practice governing use of that procedure for the diagnosis of death [1] reaffirms the preconditions for its consideration. These are:

With these pre-conditions satisfied, the definitive criteria are:

Two doctors, of specified status and experience, are required to act together to diagnose death on these criteria and the tests must be repeated after a short period of time ... to allow return of the patients arterial blood gases and baseline parameters to the pre-test state. These criteria for the diagnosis of death are not applicable to infants below the age of two months

With due regard for the cause of the coma, and the rapidity of its onset, testing for the purpose of diagnosing death on brain stem death grounds may be delayed beyond the stage where brain stem reflexes may be absent only temporarily because the cerebral blood flow is inadequate to support synaptic function although there is still sufficient blood flow to keep brain cells alive [9] and capable of recovery. There has recently been renewed interest in the possibility of neuronal protection during this phase by use of moderate hypothermia and by correction of the neuroendocrine abnormalities commonly seen in this early stage.[13]

Published studies of patients meeting the criteria for brain stem death or whole brain death the American standard which includes brain stem death diagnosed by similar means record that even if ventilation is continued after diagnosis, the heart stops beating within only a few hours or days.[14] However, there have been some very long-term survivals[15] and it is noteworthy that expert management can maintain the bodily functions of pregnant brain dead women for long enough to bring them to term.[16]

The management of patients pronounced dead on meeting the brain stem death criteria depends upon the reason for diagnosing death on that basis. If the intent is to take organs from the body for transplantation, the ventilator is reconnected and life-support measures are continued, perhaps intensified, with the addition of procedures designed to protect the wanted organs until they can be removed. Otherwise, the ventilator is left disconnected on confirmation of the lack of respiratory centre response.

The diagnostic criteria were originally published for the purpose of identifying a clinical state associated with a fatal prognosis (see above). The change of use, in the UK, to criteria for the diagnosis of death itself was protested from the first.[17][18] The initial basis for the change of use was the claim that satisfaction of the criteria sufficed for the diagnosis of the death of the brain as a whole, despite the persistence of demonstrable activity in parts of the brain.[19] In 1995, that claim was abandoned[7] and the diagnosis of death (acceptable for legal purposes in the UK in the context of organ procurement for transplantation) by the specified testing of brain stem functions was based on a new definition of death, viz. the permanent loss of the capacity for consciousness and spontaneous breathing. There are doubts that this concept is generally understood and accepted and that the specified testing is stringent enough to determine that state. It is, however, associated with substantial risk of exacerbating the brain damage and even causing the death of the apparently dying patient so tested (see "the apnoea test" above). This raises ethical problems which seem not to have been addressed.

It has been argued that sound scientific support is lacking for the claim that the specified purely bedside tests have the power to diagnose true and total death of the brain stem, the necessary condition for the assumption of permanent loss of the intrinsically untestable consciousness-arousal function of those elements of the reticular formation which lie within the brain stem (there are elements also within the higher brain).[19] Knowledge of this arousal system is based upon the findings from animal experiments[20][21][22] as illuminated by pathological studies in humans.[23] The current neurological consensus is that the arousal of consciousness depends upon reticular components which reside in the midbrain, diencephalon and pons.[24][25] It is said that the midbrain reticular formation may be viewed as a driving centre for the higher structures, loss of which produces a state in which the cortex appears, on the basis of electroencephalographic (EEG) studies, to be awaiting the command or ability to function. The role of diencephalic (higher brain) involvement is stated to be uncertain and we are reminded that the arousal system is best regarded as a physiological rather than a precise anatomical entity. There should, perhaps, also be a caveat about possible arousal mechanisms involving the first and second cranial nerves (serving sight and smell) which are not tested when diagnosing brain stem death but which were described in cats in 1935 and 1938.[20] In humans, light flashes have been observed to disturb the sleep-like EEG activity persisting after the loss of all brain stem reflexes and of spontaneous respiration.[26]

There is also concern about the permanence of consciousness loss, based on studies in cats, dogs and monkeys which recovered consciousness days or weeks after being rendered comatose by brain stem ablation and on human studies of brain stem stroke raising thoughts about the plasticity of the nervous system.[23] Other theories of consciousness place more stress on the thalamocortical system.[27] Perhaps the most objective statement to be made is that consciousness is not currently understood. That being so, proper caution must be exercised in accepting a diagnosis of its permanent loss before all cerebral blood flow has permanently ceased.

The ability to breathe spontaneously depends upon functioning elements in the medulla the respiratory centre. In the UK, establishing a neurological diagnosis of death involves challenging this centre with the strong stimulus offered by an unusually high concentration of carbon dioxide in the arterial blood, but it is not challenged by the more powerful drive stimulus provided by anoxia although the effect of that ultimate stimulus is sometimes seen after final disconnection of the ventilator in the form of agonal gasps.

No testing of testable brain stem functions such as oesophageal and cardiovascular regulation is specified in the UK Code of Practice for the diagnosis of death on neurological grounds. There is published evidence[28][29][30] strongly suggestive of the persistence of brain stem blood pressure control in organ donors.

A small minority of medical practitioners working in the UK have argued that neither requirement of the UK Health Department's Code of Practice basis for the equation of brain stem death with death is satisfied by its current diagnostic protocol[1] and that in terms of its ability to diagnose de facto brain stem death it falls far short.

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Brain stem death - Wikipedia, the free encyclopedia

Stem Cell Death caused by Common Medications used by Physicians

In yet another study, researchers have again determined that steroid medication and local anesthetics are really bad for cells.Steroid is a strong anti-inflammatory that iscommonlyused by physicians to treat swelling. The most common steroid medications are Cortisone, Hydrocortisone, Depomedrol, and Betamethasone. Local anesthetics are medications injected by physicians that help numb an area. The most common are Lidocaine, Novacaine, and Marcaine (bupivicane). Several years ago we tested local anesthetics with stem cells in our stem cell culture lab and thisexperiencechanged the way we harvest cells.However, we still see fat based stem cell clinics using common liposuction methods to collect stem cells. The problem with the most common aspiration method for fat liposuction is the use of large amounts of stem cell killing local anesthetic infiltrated into the adipose tissues. While this helps with patient comfort and the technique is easy and straightforward for the doctor, since the doctor has no idea about the viability of the cells being harvested, he has no way of knowing if he is killing the cells with the anesthetic. As discussed, several years ago we investigated the effects of these anesthetics with stem cells after we began to notice that certain harvest techniques used by certain clinic doctors would lead to much lower stem cell yields in culture. In addition, we began to see stem cell culture failures in certain patients on steroid medications. To determine why this was happening, we began to isolate the slight variables in technique used by different physicians and patient medications and ultimately this lead us to test various anesthetics with stem cells. We were blown away by how toxic these anaesthetics like lidocaine and bupivicane (the two most common used by doctors) were to cells. In fact, even at doses about 100 to 1,000 times less that what physicians normally use, some of these anesthetics are still effective at killing stem cells. So if youre planning an adipose or bone marrow stem cell procedure and your doctor wants to use generous amounts of local anesthetics that will come in contact with your cells, the stem cells the doctor is harvesting are likely DOA. While there are ways to keep patients comfortable with local anesthetics and keep cells safe, these procedures have to be carefully developed over time with viability being periodically checked in an advanced culture facility. Since our first 4 years of stem cell experience involved a check for cell viability every time (live cells grow in culture, dead cells dont), we developed our harvest techniques to keep cells alive. In addition, if youre on steroid medications, you may want to speak with your doctor about getting off these medications for your stem cellprocedure, as our experience (and this recent study) show that these medications are also hard on cells.

Disclaimer: Like all medical procedures, Regenexx Procedures have a success & failure rate. Not all patients will experience the same results.

If you liked this post, you may really enjoy this book by the same author - Dr. Chris Centeno

Written by Regenexx Founder, Dr. Chris Centeno, this 150 page book explains the Regenexx approach to patients and orthopedic conditions. Whether youre are an existing patient or simply interested in the human body and how everything in the body ties together, you will enjoy exploring this book in-depth. With hyperlinks to more detailed information, related studies and commentary, this book condenses a huge amount of data and resources into an enjoyable and entertaining read.

Chris Centeno, M.D. is a specialist in regenerative medicine and the new field of Interventional Orthopedics.

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Stem Cell Death caused by Common Medications used by ...

Center for Lung Disease

We recently opened the Center for Lung Disease.Thedepartment specializes inChronic Obstructive Pulmonary Disease (COPD),Chronic Bronchitis,Emphysema, and other lung related illnesses. Our goal is to introduce and educate patients to the world of adult (autologous) stem cell therapies.

More information about COPD Treatment

COPD is an insidious and complex disease that causes serious treatment issues for the patient. It manifests itself in rampant inflammation of lung tissue. This inflammation kills lung cells and destroys the structure of the air sacs (alveoli) where oxygen and carbon dioxide are exchanged. Once alveolar structure is destroyed, it leads to the over inflation and dysfunction of the lung tissue, which is manifested in the patient as emphysema.

Rampant inflammation also causes swelling of the bronchial airways. The airways narrow and cause interference with air movement out of the lungs (bronchiolitis and chronic bronchitis). This leads to difficulty breathing and abnormal spirometric results characteristic of the disease.

The continuous killing of lung cells and the attendant lung tissue structural damage in COPD is both progressive (even if you stop smoking), and irreversible with current treatment protocols (bronchodilators, corticosteroids and supplemental oxygen). Understanding why COPD is both progressive and irreversible is the key to understanding the true nature of COPD and its inherent connection to stem cells.

Science has shown that adult stem cells reside in human bone marrow throughout life and are found throughout the human body. Adult stem cells heal our bodies by replacing dead and dying cells and are attracted to injured tissue by elevated chemical signals. Injured cells also chemically signal adult stem cells to transform (differentiate) into the cells needed to rebuild and repair damaged tissue (engraftment). This bone marrow adult stem cell healing system resides in each and every one of us.

COPD is irreversible because it constantly interferes with the chemical signals necessary for the adult stem cell healing system to do its work. COPD is progressive because in many heavy smokers the inflammation becomes entwined within the mechanisms of the immune system. In other words COPD can be viewed as an autoimmune disease, in which the patients own immune system perpetuates the inflammation and lung damage even after the smoking irritants and toxins are removed. The progression of lung cell damage and cell death continues, and the interference in the adult stem cell healing system continues in the Chronic Obstructive Pulmonary Disease process.

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COPD Stem Cell Treatment | Analytical Stem Cell

A new method for delivering stem cells to damaged heart muscle has shown early promise in treating severe heart failure, researchers report.

In a preliminary study, they found the tactic was safe and feasible for the 48 heart failure patients they treated. And after a year, the patients showed a modest improvement in the heart's pumping ability, on average.

It's not clear yet whether those improvements could be meaningful, said lead researcher Dr. Amit Patel, director of cardiovascular regenerative medicine at the University of Utah.

He said larger clinical trials are underway to see whether the approach could be an option for advanced heart failure.

Other experts stressed the bigger picture: Researchers have long studied stem cells as a potential therapy for heart failure -- with limited success so far.

"There's been a lot of promise, but not much of a clinical benefit yet," said Dr. Lee Goldberg, who specializes in treating heart failure at the University of Pennsylvania.

Researchers are still sorting through complicated questions, including how to best get stem cells to damaged heart muscle, said Goldberg, who was not involved in the new study.

What's "novel" in this research, he said, is the technique Patel's team used to deliver stem cells to the heart. They took stem cells from patients' bone marrow and infused them into the heart through a large vein called the coronary sinus.

Patel agreed that the technique is the advance.

"Most other techniques have infused stem cells through the arteries," Patel explained. One obstacle, he said, is that people with heart failure generally have hardened, narrowed coronary arteries, and the infused stem cells "don't always go to where they should."

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Stem Cells Show Promise in Heart Failure Treatment