StemCell Therapy

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This case study was developed as one of a set of three studies, focusing on somewhat mature but rapidly evolving technologies. These case studies are intended to draw out lessons for the development of a cross-sectoral governance framework for emerging technologies in health and medicine. The focus of the case studies is the governance ecosystem in the United States, though where appropriate, the international landscape is included to provide context. Each of these case studies:

Each case study begins with two short vignettes designed to highlight and make concrete a subset of the ethical issues raised by the case (seeBox 1andBox 2). These vignettes are not intended to be comprehensive but rather to provide a sense of the kinds of ethical issues being raised today by the technology in question.

The cases are structured by a set of guiding questions, outlined subsequently. These questions are followed by the historical context for the case to allow for clearer understanding of the trajectory and impact of the technology over time and the current status (status quo) of the technology. The bulk of the case consists of a cross-sectoral analysis organized according to the following sectors: academia, health care/nonprofit, government, private sector, and volunteer/consumer. Of note, no system of dividing up the world will be perfectthere will inevitably be overlap and imperfect fits. For example, government could be broken into many categories, including international, national, tribal, sovereign, regional, state, city, civilian, or military. The sectoral analysis is further organized into the following domains: science and technology, governance and enforcement, affordability and reimbursement, private companies, and social and ethical considerations. Following the cross-sectoral analysis is a broad, nonsectoral list of additional questions regarding the ethical and societal implications raised by the technology.

The next section of the case is designed to broaden the lens beyond the history and current status of the technology at the center of the case. The Beyond section highlights additional technologies in the broad area the focal technology occupies (e.g., neurotechnology), as well as facilitating technologies that can expand the capacity or reach of the focal technology. The Visioning section is designed to stretch the imagination to envision the future development of the technology (and society), highlighting potential hopes and fears for one possible evolutionary trajectory that a governance framework should take into account.

Finally, lessons learned from the case are identifiedincluding both the core case and the visioning exercise. These lessons will be used, along with the cases themselves, to help inform the development of a cross-sectoral governance framework, intended to be shaped and guided by a set of overarching principles. This governance framework will be created by a committee of the National Academies of Sciences, Engineering, and Medicine (https://www.nationalacademies.org/our-work/creating-a-framework-for-emerging-science-technology-and-innovation-in-health-and-medicine).

Regenerative medicine as a field is quite broad but is generally understood to focus on the regeneration, repair, and replacement of cells, tissues, and organs to restore function (Mason and Dunnill, 2008). The aspect of regenerative medicine on which this case study focuses relates to the ability to treator curegenetic hematologic disease safely and effectively, and the significant trade-offs that come with these novel therapies.

The story of this therapy begins in the history of bone marrow transplants. The medicinal value of bone marrow has long been recognized and was first discussed in the 1890s as a potential treatment (administered orally) of diseases believed to be characterized by defective hemogenesis (Quine, 1896).

While allogeneic bone marrow transplant (in which stem cells from a donor are collected and transplanted into the recipient) may be the most broadly known form of hematopoietic stem and progenitor cell (HSPC) transplant, a range of other cell types are also used. HSPCs used in transplant can be either allogeneic (i.e., from a donor) or autologous (i.e., from the person who will also receive the transplant). The cells used in transplant research and clinical care can come from bone marrow, peripheral blood stem cells (PBSCs), umbilical cord blood, and pluripotent stem cell-derived cells.

A major challenge throughout the history of HSPC transplantation has been the dire risks associated with these transplants, including the morbidity and mortality caused by immunological reactions between the transplanted cells and the tissues of the recipient. In particular, graft-versus-host disease (GVHD) is a serious response in which the transplanted stem cells view the recipients tissues as foreign and mount an immune response, attacking the recipients body. If an autologous transplant is not possible given the nature of the disease to be treated, an immunologically well-matched healthy donor for allogeneic transplant is critical. For genetic hematologic disease, a new approach that would not only treat but cure the condition is now being tested: genetic modification of the patients own HSPCs to correct or compensate for the defect, followed by transplantation of the corrected autologous cells.

This challenge of matching transplantable cells to patients has driven evolution within the field of regenerative medicine, including logistical fixes in the form of HSPC registries and banks to technological approaches including the use of pluripotent stem cell-derived cell sources and genome editing (e.g., clustered regularly interspaced short palindromic repeats [CRISPR]).

This challenge of immunological matching has also driven significant ethical challenges, even beyond the substantial risks of HSPC transplantation itself. In contrast to many novel technologies, where finances are a primary barrier to access, in the case of regenerative medicine, there is the additional barrier of biology. People who are not of European descent have a lower probability of finding well-matched donors than do people of European descent. Furthermore, genetic hematologic diseases like sickle cell disease (SCD) and thalassemia, for which HSPC transplant is the only established cure (and a fraught one, at that), have struggled to garner the financial and grant support needed to move research forward. This challenge persists despite SCD being three times more prevalent in the United States than cystic fibrosis, which has historically benefited from generous public and private funding (Farooq et al., 2020; Wailoo and Pemberton, 2006). All of this stands on a background of long-understood barriers even to standard of care (e.g., adequate pain management) for individuals with SCD in particular (Haywood et al., 2009). Together, these facts raise concerns related to equity and access at multiple stages of research, development, and clinical care.

Finally, advances in this science have also attracted the attention of those who are willing to take advantage of patients under the guise of cutting-edge therapy, creating a robust market of direct-to-consumer (DTC) cell-based services and interventions that at best waste time and money and at worst cause serious harm or death (Bauer et al., 2018).

The following guiding questions were used to frame and develop this case study.

Additional guiding questions to consider include the following:

HSPC transplant was initially only attempted in terminally ill patients (Thomas, 1999). The first recorded bone marrow transfusion was given to a 19-year-old woman with aplastic anemia in 1939 (Osgood et al., 1939). This was long before the Nuremburg Code, the Declaration of Helsinki, or the Belmont Report and anything like current understandings of informed consent (NCPHSBBR, 1979; Rickham, 1964; International Military Tribunal, 1949). There was also little understanding of the factors associated with graft failureno attempts at bone marrow transfusions succeeded, and all patients died. Despite this early experience, the consequences of World War II, particularly the need to improve radiation and burn injury treatment, propelled this work forward (de la Morena and Gatti, 2010).

As human transplant work continued, experiments in mice and dogs in the 1950s and 1960s showed that after lethal radiation, these animals would recover if given autologous bone marrow. However, if given allogeneic marrow, the animal would reject the graft and die or accept the graft but then die from wasting syndrome, which later came to be understood as GVHD (Mannick et al., 1960; Billingham and Brent, 1959; Barnes et al., 1956; Rekers et al., 1950). It became clear that close immunologic matching between donor and recipient and management of GVHD in the recipient would be vital to the success of allogeneic bone marrow transplants (de la Morena and Gatti, 2010).

A 1970 accounting of the reported experience with HSPC transplants to date described approximately 200 allogeneic stem cell graft attempts (six involving fetal tissue) in subjects aged less than 1 to over 80 years, most of which had taken place between 1959 and 1962 (Bortin, 1970). (Of note, there were likely scores of unreported cases; in fact, the author ended the article with a call for reporting of all HSPC transplant attempts to the newly established American College of Surgeons-National Institutes of Health Organ Transplant Registry.) Of the reported cases (which often included the subjects initials), only 11 individuals were unequivocal allogeneic chimeras, and of those, only five were still alive at the time their case was reported. Many of the reported subjects died of opportunistic infections or GVHD, the noting of which often did not capture the true human toll of these deaths. For many years, even success (i.e., engraftment of the transplanted marrow) ended in death due to these other causes (Math et al., 1965; Thomas et al., 1959). As Donnall Thomas, a pioneer and leader in the field who won the Nobel Prize for discoveries concerning organ and cell transplantation in the treatment of human disease in 1990, reflected years later, the experience with allogeneic transplants had been so dismal that questions were raised about whether or not such studies should be continued (Thomas, 2005; Nobel Prize, 1990). In fact, the dismal experience with HSPC transplant eventually led most investigators to discontinue this work in humans, the focus returning for a time to animal studies (Little and Storb, 2002).

However, the discovery of human leukocyte antigen (HLA) in 1958 by Jean Dausset, which helps the immune system differentiate between what is self and what is foreign, and subsequent advances in the understanding of HLA matching and immunosuppression during the 1960s and 1970s led to a resumption of human clinical trials (Nobel Prize, 1980). In 1971, the first successful use of HSPC transplant to treat leukemia was reported (Granot and Storb, 2020). The following decades saw additional developments in HSPC transplant, improving the safety of the intervention, thus enabling its consideration for treatment of a broader array of blood diseases, including the hemoglobinopathies (Granot and Storb, 2020; Apperley, 1993).

The first use of HSPC transplant to cure thalassemia was in 1981, in a 16-month-old child, with an HLA-identical sibling donorthis patient was alive and thalassemia-free more than 20 years later (Bhatia and Walters, 2008; Thomas et al., 1982). Thalassemia major (the most serious form of the disease) requires chronic blood transfusion and chelation for life, a process which leads to gradual iron buildup and related organ damage, including heart failure, which is a common cause of death. Life expectancy for treated patients has increased substantially and varies by thalassemia type and treatment compliance, but patients can now live into their 40s and beyond (Pinto et al., 2019).

The first cure of SCD via HSPC transplant was incidental. An 8-year-old girl with acute myeloid leukemia (AML) was successfully treated for her leukemia with a bone marrow transplant, curing her SCD in the process (Johnson et al., 1984). By this time, life expectancy for an individual with SCD had improved substantially, reaching the mid-20s due to advances in understanding and treatment of the disease (particularly the use of antibiotics to manage the frequent infections that plagued those with the disease) (Wailoo, 2017; Prabhakar et al., 2010). The first five patients, all children, in whom HSPC transplants were used intentionally to treat SCD were reported in 1988 (Vermylen et al., 1988). As Vermylen and colleagues reported, In all cases there was complete cessation of vaso-occlusive episodes and haemolysis (Vermylen et al., 1988).

Around this same time, there were also advancements in the sources of transplantable hematopoietic cells, expanding beyond bone marrow to include peripheral blood stem cells and umbilical cord blood (Gluckman et al., 1989; Kessinger et al., 1988). Cord blood was particularly appealing for a number of reasons, including that it is less immunogenic than the other cell sources, reducing the risk of GVHD.

The development of cord blood transplant has a very different origin story to that of bone marrow, beginning with a hypothesis and the founding of a company (Ballen et al., 2013). The company, Biocyte Corporation (later PharmaStem Therapeutics), funded the early work and held two short-lived patents over the isolation, preservation, and culture of umbilical cord blood (Shyntum and Kalkreuter, 2009). The longevity of the science has thankfully surpassed that of the company that launched it. The first cord bloodbased HSPC transplant was conducted with the approval of the relevant institutional review boards (IRBs) and the French National Ethics Committee, to treat a 5-year-old boy with Fanconi anemia using cells from the birth of an unaffected, HLA-matched sister (Ballen et al., 2013; Gluckman et al., 1989). The success of the early cases (the 5-year-old boy was still alive and well 25 years later) led to the use of unrelated cord blood transplant and expansion of use beyond malignant disease (Ballen et al., 2013; Kurtzberg et al., 1996). Benefits of cord blood include noninvasive collection, ability to cryopreserve characterized tissue for ready use, reduced likelihood of transmitting infections, and lower immunogenicity relative to bone marrow, enabling imperfect HLA matching and expanding access, in particular for people not of European descent (Barker et al., 2010; Gluckman et al., 1997). Cord blood HSPC transplant was first used primarily in children, because it was thought that the relatively low number of cells in a cord blood unit would limit its use in adults, but over time, as techniques and supportive care have improved, so has success of cord blood transplant in adults (Eapen et al., 2010; Ballen et al., 2007). Today, cord blood is widely used for HSPC transplants in both children and adults, with outcomes as good as or better than with bone marrow. Despite these advancements, however, allogeneic HSPC transplant continued to depend on the availability of HLA-matched donors.

Unfortunately, only about 35 percent of patients have HLA-matched siblings, so patients have needed to look beyond their immediate family for matched donors. This need led to the creation of HLA-typed donor registries, starting with the founding of the Europdonor registry in the Netherlands in 1970 and the International Blood and Marrow Transplant Registry at the Medical College of Wisconsin in 1972 (McCann and Gale, 2018). In 1986, the National Marrow Donor Program (NMDP), which operates the Be the Match registry, was founded by the U.S. Navy. Other registries in the United States and Europe followed, and by 1988, there were eight active registries around the world with more than 150,000 donors (van Rood and Oudshoorn, 2008). The Bone Marrow Donors Worldwide network, which connected these registries, was formed in 1988 to facilitate the identification of potential donors, and in 2017 its activities were taken over by the World Marrow Donor Association (WMDA) (Oudshoorn et al., 1994). Today, the combined registry includes more than 37,600,000 donors and more than 800,000 cord blood units from 54 different countries (seeFigure 1) (WMDA, 2021; Petersdorf, 2010).

However, even with tremendous global collaboration to identify and make available donor information, access is not equal. The NMDP estimates suggest that while approximately 90 percent of people of European descent will identify a well-matched unrelated marrow donor, the same will be true for only about 70 percent of people of Asian or Hispanic descent and 60 percent of those of African descent (Pidala et al., 2013). Causes for this disparity include higher HLA diversity among these populations compared to those of European descent and smaller numbers of racial and ethnic minority volunteers in donor registries and ultimately available for transplant (Sacchi et al., 2008; Kollman et al., 2004).

Alongside the public registries, trading on the success of cord blood HSPC transplants and playing on the fears of new parents, a thriving market of private cord blood banks has developed (Murdoch et al., 2020). These for-profit private banks market their servicescollecting and storing cord blood for potential future personal useas insurance policies for the health of ones newborn, without much data to support the claim. While donation of cord blood to a public bank is free to the donor, costs associated with private banking include a collection fee (US$1,350$2,300) and annual storage fees ($100$175/year), which are unlikely to be covered by health insurance (Shearer et al., 2017). At the same time, public banks are held to transparent, rigorous storage and quality standards that do not apply to private banks, leading to lower overall quality of cord blood in private banks (Shearer et al., 2017; Sun et al., 2010; Committee on Obstetric Practice, 2008). Finally, cord blood stored in public banks is 30 times more likely to be accessed for clinical use than samples stored in private banks, and there is broad professional consensus, and associated professional guidance, that public banking is preferable to private banking (Shearer et al., 2017; Ballen et al., 2015). Despite these differences, in 2017, there were about 800,000 cord blood units in public banks, compared with more than 5 million in private banks (Kurtzberg, 2017).

While adult stem cell sources (bone marrow, peripheral blood, and cord blood) have dominated research and clinical care for many decades, in the late 1990s and mid-2000s, new tools were added in the form of several pluripotent stem cell types, including embryonic stem cells, embryonic germ cells, nuclear transfer (NT)-derived stem cells, and most recently, induced pluripotent stem cells (iPSCs) (Tachibana et al., 2013; Yu et al., 2007; Takahashi and Yamanaka, 2006; Shamblott et al., 1998; Thomson et al., 1998). In contrast to the previous cell sources, which are restricted to repopulating blood cell types, these new pluripotent stem cells can turn into any of the approximately 220 cell types in the human body and have a correspondingly diverse array of potential applications. For the purposes of this case, the authors focus on the use of these cells in hematologic disease, but understanding some of the history of the development and use of these cells is helpful for the broader goals of the case. Importantly, these new cell types emerged in a very different regulatory and societal environment than the environment in which bone marrow transplants were first being developed.

The first derivations of human embryonic stem cells (ESCs) and embryonic germ cells (EGCs) were published in 1998 (Shamblott et al., 1998; Thomson et al., 1998). Both of these seminal papers concluded with discussion of the potential for the use of these cells in transplantation-based treatments and cures and emphasized the need to address the challenge of immune rejection, either through the development of cell banks, akin to the registries described previously, or through the genetic modification of the cells to create universal donor cells or to match the particular cellular therapy to the particular patient.

Unlike bone marrow or cord blood, however, the source of these cells was human embryos and fetal tissue, and at the time of these publications, there was already a notable history of governance of these tissues (Matthews and Yang, 2019; Green, 1995; NIH, 1994). In addition, the Dickey-Wicker Amendment had been in place for 3 years, prohibiting the use of federal funds to create human embryos for research or to conduct research in which human embryos are destroyed, discarded, or knowingly subjected to risk of injury or death (104th Congress, 1995). Within weeks of the papers publication, a legal opinion was issued from the Department of Health and Human Services (HHS) interpreting Dickey-Wicker with regard to the new research (Rabb, 1995). Though federal dollars could not be used to create ESCs or EGCs, it was determined that federal dollars could be used to conduct research with pluripotent stem cells thus derived. This interpretation was supported later that year by a report of the National Bioethics Advisory Commission (NBCA, 1999). This did not, however, settle the issue.

A year later, President George W. Bush was elected following a campaign in which he made clear his opposition to this research (Cimons, 2001). In August 2001, in his first address to the nation, President Bush announced that federal funding would be permitted for research using the approximately 60 ESC lines already in existence at the time of his announcement, but not for research with newly derived lines (CNN, 2001). The president seemed to be attempting to walk a fine line between allowing promising research to move forward and not causing the federal government (and taxpayers) to be complicit in the destruction of human embryos. Ultimately, many of these 60 approved Bush lines proved impossible to access or difficult to work with. Furthermore, the accounting required in institutions and laboratories working with both Bush lines and newer lines was daunting (Murugan, 2009).

As ethical and policy debates raged, states began passing their own legislation governing human ESC research, beginning with California, and creating over time a patchwork of state-level policy that ranged from providing government funding for ESC research, as in California, to classifying the work as a felony, such as in Arizona (CIRM.ca.gov, n.d.; Justia US Law, 2020). In 2005, Congress passed its own bill that would permit federal funding of research with an expanded number of human ESC lines, but the bill was subsequently vetoed by President Bush (109th Congress, 2005). The same year, the National Research Council and the Institute of Medicine published its tremendously influential report titled Guidelines for Human Embryonic Stem Cell Research (IOM and NRC, 2005). These guidelines led to highly effective self-regulation in the field, as the Guidelines were adopted across the United States at institutions conducting human ESC research (Robertson, 2010). The Guidelines recommended the creation of a new institutional oversight committee to review ESC research, similar to IRBs, among other recommendations. The Guidelines remained the primary source of governance for ESC research through the end of the Bush administration.

An additional scientific innovation during this time was the announcement of the creation of iPSCs in 2006 (Nobel Prize, 2012; Takahashi and Yamanaka, 2006). iPSCs are derived from somatic tissue, not embryonic or fetal tissue, through the introduction of a small set of transcription factors that effectively reset the mature cell back to a pluripotent state. This concept had actually been introduced as an alternative to ESCs by President Bushs bioethics commission, though it had been met with skepticism, and Shinya Yamanakas announcement at the 2006 International Society for Stem Cell Research (ISSCR) annual meeting stunned the assembled scientists (Scudellari, 2016; The Presidents Council on Bioethics, 2005). This scientific end-run around the destruction of human embryos led to a flood of new researchers, as scientists now needed only somatic cells, rather than highly regulated embryonic or fetal tissue, to participate in this new wave of regenerative medicine research.

By the end of President Bushs second term, in addition to the National Academies Guidelines, guidelines were also issued from the ISSCR and a number of other academic groups (ISSCR, n.d.; The Hinxton Group, 2006). Internationally, as in the United States, a patchwork of policy responses had emerged, ranging from very restrictive to permissive to supportive, leading both domestically and internationally to a degree of brain drain as some scientists relocated to jurisdictions that permitted this research (Verginer and Riccaboni, 2021; Levine, 2012).

When President Barack Obama took office in 2009, he issued an Executive Order reversing former president Bushs prior actions (White House, 2009). Rather than establishing the final rules himself, he permitted funding of ESC research to the extent permitted by law (a nod to the Dickey-Wicker Amendment) and charged the National Institutes of Health (NIH) with developing guidelines for such funding. The NIH guidelines, which largely followed the Guidelines, were finalized in July 2009 and were promptly tied up in a years-long battle in the courts until the Supreme Court declined to hear the final appeal in 2013, leaving the NIH guidelines intact (NIH, 2013, 2009).

The final piece of the regenerative medicine puzzle is the need to overcome immune rejection of transplanted cells. As noted in the initial HPSC papers, potential ways to overcome immune rejection (in the absence of iPSCs) included both banking of a large number of diverse cell lines and genetic modification of the cells intended for transplant, although at the time the technology to do so did not exist (Faden et al., 2003). Gene therapy of this sort had been contemplated for years, and gene transfer trials had begun in the 1990s using the tools scientists had at the time (IOM, 2014). Governance structures grew up around these trials, including the transition of the Recombinant DNA Advisory Committee (RAC) from reviewing NIH-funded research involving recombinant DNA (rDNA) to reviewing gene transfer protocols (IOM, 2014). Of note, though the RAC served as a model internationally for the governance of rDNA research, its mandate was repeatedly questioned and its work critiqued, even as its role evolved (IOM, 2014). As the pace and volume of gene transfer research picked up, the pace of review slowed. Responding not only to the resulting critiques but also the accumulated experience and data, the RAC relaxed restrictions and expedited reviews where possible, ultimately pivoting again to a focus on novel protocols, and leaving more straightforward protocols to the U.S. Food and Drug Administration (FDA) to approve or deny (IOM, 2014). But the original vision of genetically tailored cellular therapy articulated in the 1998 papers did not become possible until almost 15 years later.

In 2012, the publication of the paper that introduced clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) launched a new era of genetic modification (Jinek et al., 2012). This new tool dramatically improved upon prior gene editing tools with respect to technical ease, speed, and cost, putting the kind of editing imagined in the 1998 papers within reach.

Today, median health care costs for HSPC (including the procedure and 3 months of follow-up) in the United States are approximately $140,000$290,000, depending on the type of procedure (Broder et al., 2017). While 200-day nonremission mortality has decreased substantially since 2000, it remains high (11%) (McDonald et al., 2020). The risks of transplant remain a significant barrier to access, in particular for those with nonmalignant disease, such as SCD. Beyond this, and as noted previously, there are significant ethnic and racial disparities in access to HSPC transplant, largely due to the relatively lower probability of identifying a well-matched HSPC donor (Barker et al., 2019). A recent study demonstrated that while White patients of European descent have a 75 percent chance of finding a well-matched (8/8 HLA-matched) donor, for White Americans of Middle Eastern or North African descent, the probability is 46 percent (Gragert et al., 2014). For Hispanic, Asian, Pacific Islander, and Native American individuals, the probability of such a match ranges from 27 to 52 percent, and for Black Americans, the probability is 1619 percent (Gragert et al., 2014). Contributing to these disparities for racial and ethnic minority groups are higher HLA diversity, smaller numbers of racial and ethnic minority volunteers in donor registries, and the higher rates at which matched minority volunteers become unavailable for donation (e.g., due to inability to reach the volunteer or medical deferral due to diabetes, asthma, infectious disease, or other identified condition) (Sacchi et al., 2008; Kollman et al., 2004). Giving preference to 8/8 HLA-matched pairs therefore benefits White patients and disadvantages patients of color, but removing this preference might result in higher rates of graft failure. Attempts to balance these competing considerations raise ethical questions about justice and beneficence.

Another ethical question in HSPC transplantation revolves around compensation or incentives for donation. Increasing the number and availability of HSCP donors would improve the probability of identifying an appropriate unrelated match for patients in need of a transplant, but the 1984 National Organ Transplant Act (NOTA) banned the sale of bone marrow and organs, making the provision of financial incentives to donate illegal (98th Congress, 1983). Nonetheless, debates over the ethics of providing incentives to encourage the donation of bone marrow and HSCs persist among bioethicists and health economists. In an effort to reduce disincentives to donate, the federal government offers up to 1 work week of leave for federal employees who donate bone marrow, and most states have followed suit for state employees (Lacetera et al., 2014). Some states also offer tax deductions for nonmedical donation-related costs, and there is some evidence that these types of legislation do lead to modest increases in donation rates (Lacetera et al., 2014).

Although removing disincentives to donation is generally considered ethically acceptable, there is more debate about whether offering financial incentives for donation equates to a morally problematic commodification of the human body. In 2011, the 9th Circuit held in Flynn v. Holder that compensation for the collection of PBSCs does not violate NOTAs ban on compensation (Cohen, 2012). In response, a coalition of cell therapy organizations published a statement arguing that this decision would mean that donors would no longer be motivated by altruism, and that people seeking to sell PBSCs might withhold important health information (Be the Match, 2012). After a regulatory back-and-forth over the status of PBSCs, HHS withdrew a proposed rule that would have effectively reversed Flynn v. Holder, so the current state of the law allows compensation for PBSCs (Todd, 2017).

Although Linus Pauling declared sickle cell disease (SCD) to be the first molecular disease (i.e., the first disease understood at the molecular level) in 1949, and it has long been considered an ideal target for gene therapy given that it is predominantly caused by a single mutation in the HBB gene and its phenotypic consequences are in a circulating cell type, developing a cure has not been as straightforward as hoped (Pauling et al., 1949). Though the presentation of SCD can vary significantly, clinical effects include anemia, painful vaso-occlusive crises, acute chest syndrome, splenic sequestration, stroke, chronic pulmonary and renal dysfunction, growth retardation, and premature death (OMIM, n.d.a.).

Standard treatment for SCD consists primarily of preventative and supportive care, including prophylactic penicillin, opioids for severe chronic pain, hydroxyurea, and transfusion therapy (Yawn et al., 2014). Such care has dramatically increased the life expectancy of those living with SCD (median survival in the United States is in the mid- to late 40s) (Wailoo, 2017; Ballas et al., 2016; Prabhakar et al., 2010). At the same time, this care costs more than $35,000 annually, and many patients have difficulty accessing such high-quality care, particularly adequate pain management (Bergman and Diamond, 2013; Haywood, 2013; Haywood et al., 2009; Kauf et al., 2009; Smith et al., 2006). Until recently, the only evidence-based cure for SCD and beta-thalassemia major was allogeneic hematopoietic cell transplantation (HCT), which comes with significant costs and risks (Bhatia and Walters, 2008).

Despite the fact that SCD is one of the most common genetic diseases worldwide and it was the first genetic disease to be molecularly defined, it has received relatively little research funding over the years, an observation that has been a frequent subject of critique (Farooq et al., 2020; Demirci et al., 2019; Benjamin, 2011; Smith et al., 2006; Scott, 1970). In contrast to better-funded diseases, such as cystic fibrosis and Duchenne muscular dystrophy, which are more common in White individuals of European descent, in the United States, SCD predominantly affects non-Hispanic Black and Hispanic populations, including 1 in 365 Black individuals and 1 in 16,300 Hispanic individuals (OMIM, n.d.b., n.d.c.; CDC, 2022). This disparity in research funding despite disease prevalence is part of the larger story of the impacts of structural racism in the United States and on its medical system (The New York Times, 2019; IOM, 2003; HHS and AHRQ, 2003).

Furthermore, as noted previously, those of African and Hispanic ancestry are less likely to be able to identify a suitable match in the existing registries. Due to this difficulty, the improvements in treatment not focused on an HSPC transplant, and the risks of such a transplant, relatively few patients with SCD are treated with HSPC transplant (Yawn et al., 2014; Benjamin, 2011). Gene therapy delivered in the context of an autologous HSPC transplant offers the possibility not only of a safer cure but also broader access by eliminating the need to identify a matched donor.

Recently, the promise of regenerative medicine and gene therapy for genetic hematologic disease appears to be coming to fruition (Ledford, 2020; Stein, 2020; Kolata, 2019). While a number of approaches are currently in various stages of preclinical and clinical research, two promising clinical trials involve the induction of fetal hemoglobin (rather than direct correction of the disease-causing mutation in the HBB gene) (Demirci et al., 2019). Fetal hemoglobin is the predominant globin type in the second and third trimester fetus and for the first few months of life, at which point production shifts from fetal to adult hemoglobin. It has long been recognized that SCD does not present until after this shift occurs (Watson et al., 1948). Furthermore, some patients with the causative SCD mutation are nonetheless asymptomatic, due to also having inherited hereditary persistence of fetal hemoglobin mutations (Stamatoyannopoulos et al., 1975). These findings and others suggested that inducing fetal hemoglobin, even in the presence of a faulty HBB gene, could mitigate the disease.

The first trial uses a viral vector to introduce into autologous bone marrow a short hairpin RNA (shRNA) that inhibits the action of the BCL11A gene. BCL11A is an inhibitor of fetal hemoglobin, so when BCL11A is inhibited, fetal hemoglobin can be produced (Esrick et al., 2021). The second trialthe first published study to use CRISPR to treat a genetic diseaseincludes both patients with SCD and with transfusion-dependent -thalassemia (Frangoul et al., 2021). In this trial, CRISPR-Cas9 is used to target the BCL11A gene to affect the same de-repression of fetal hemoglobin as in the first trial. Both trials, which have collectively enrolled more than 15 patients, have reduced or eliminated the clinical manifestation of disease in all patients thus far, though it remains to be seen how long-lasting this effect will be. However, the first trial was recently suspended after participants in the first trial and a related trial developed acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) (Liu, 2021); an investigation is under way regarding the cause of the AML and MDS. Marketing of a treatment for transfusion-dependent -thalassemia currently approved and available in the European Union (EU) was also suspended, as that treatment is manufactured using the same vector (BB305 lentiviral vector) used in the current trials, and it is possible that the vector is the source of the serious adverse events in the research participants.

Further challenges remain, including technical challenges, such as the possibility that gene editing tools, as they are derived from bacterial systems, will provoke an immune response; and concerns about financial access, given the anticipated cost of such curative therapies (ICER, 2021; Kim et al., 2018). In addition, despite the technical ease of the technology and designing new nucleic acid targets, intellectual property protecting CRISPR has, to date, narrowed the number of developers actively pursuing CRISPR-based clinical trials (Sherkow, 2017). At the same time, this new technology might also solve a number of ethical issues around HSPC transplants, including by expanding biological access to HSPC transplant and mitigating the concerns raised by the creation of savior siblings for HLA-matched cord blood transplantation for older siblings (Kahn and Mastroianni, 2004).

A long-standing challenge in the field of regenerative medicine is the DTC marketing of unproven cell-based interventions. Since at least the 2000s, unscrupulous scientists and health professionals in the United States and internationally have been offering stem cell therapy at significant cost, often to vulnerable individuals, and without a legitimate scientific or medical basis (Knoepfler and Turner, 2018; Murdoch et al., 2018; Regenberg et al., 2009; Enserink, 2006). From 2009 to 2016, the number of such clinics in the United States doubled annually (Knoepfler and Turner, 2018). While the clinics look legitimate, their claims are fantastical, promising to treat or cure everything from knee pain to Parkinsons disease. Such clinics are often vague about the cell sources involved in the interventions offered, but sometimes they claim to use bone marrow, cord blood, embryonic stem cells, and iPSCs, as well as other types of autologous adult stem cells (e.g., adipose, olfactory) and a range of other cell types, cell sources, and cell mixtures (Murdoch et al., 2018). While such interventions launch from legitimate science and scientific potential, the claims exceed and diverge from what is proven. The interventions are at best very expensive placebos and at worst could cause serious harm or death (Bauer et al., 2018).

Over time, attempts have been made to rein in these clinics by the FDA, the Federal Trade Commission (FTC), the ISSCR, individual customers and their lawyers, and others, but these attempts have faced a number of challenges (Pearce, 2020). The ISSCR, the primary professional society for those engaged in regenerative medicine, has struggled for years against such clinics. Early on, they attempted to establish a mechanism to publicly vet these clinics, though the effort was abandoned in part due to push back from the clinics lawyers (Taylor et al., 2010; personal communication from ISSCR Leadership, n.d.). In part because the majority of US-based clinics offer autologous interventions (removing and then reintroducing the patients own cells), the FDA struggled to clarify the line between medical practice and their regulatory authority. The FDA began issuing occasional warning letters to these clinics starting in 2011, though the letters were issued infrequently (Knoepfler and Turner, 2018). Under this relatively weak enforcement, the market expanded dramatically, and pressure increased on the FDA to take meaningful action (Knoepfler, 2018; Turner and Knoepfler, 2016).

In late 2017, the FDA took several significant steps to curtail these clinics, including using U.S. marshals to seize product from a California clinic, bringing a lawsuit against a Florida clinic, and publishing largely celebrated finalized guidance outlining a risk-based approach to the regulation of regenerative medicine products (FDA, 2019; Pew Research Center, 2019). The following year, the FTC took independent action against clinics making false claims about their interventions, and Google banned advertising for unproven or experimental medical techniques such as most stem cell therapy, cellular (non-stem) therapy, and gene therapy (Biddings, 2019; Fair, 2018). In 2019, the FDA won their case against US Stem Cells in Florida, significantly strengthening their ability to regulate these clinics (Wan and McGinley, 2019). Following the establishment of clear regulatory authority over at least a subset of clinics, FDA has begun to step up its enforcement (Knoepfler, 2020; Wan and McGinley, 2019; FDA, 2018). Increased action is anticipated following the end of the 3-year grace period established in the 2017 guidance, though there is some concern about the capacity of the agency to make significant headway against the more than 600 clinics now in operationa worry bolstered by a 2019 study suggesting that despite increased enforcement, the unproven stem cell market seems to have shifted rather than contracted (Knoepfler, 2019; Pew Research Center, 2019). What seems clear is that it will take a collective and multipronged approach to ensure that the cell-based interventions to which patients have access are safe and effective (Lomax et al., 2020; Pew Research Center, 2019; Master et al., 2017; Zarzeczny et al., 2014).

The cross-sectoral analysis is structured according to sectors (seeFigure 2) and domains (science and technology, governance and enforcement, end-user affordability and insurance reimbursement [affordability and reimbursement], private companies, and social and ethical considerations). The sectors described subsequently are intended to be sufficiently broad to encompass a number of individuals, groups, and institutions that have an interest or role in regenerative medicine. Health care is the primary nonprofit actor of interest, and so in this structure, health care has replaced nonprofit, though other nonprofit actors may have a role in this and other emerging technologies, and, of course, not all health care institutions are nonprofits.

Today, many regenerative medicine technologies are researched, developed, and promoted by a scientific-industrial complex largely driven by market-oriented goals. The development of various components of regenerative medicine may be altered by differing intellectual property regimes. This larger ecosystem is also embedded in a broad geopolitical context, in which the political and the economic are deeply intertwined, shaping national and regional investment and regulation. The political economy of emerging technologies involves and affects not only global markets and regulatory systems across different levels of government but also nonstate actors and international governance bodies. Individuals and societies subsequently adopt emerging technologies, adjusting their own values, attitudes, and norms as necessary, even as these technologies begin to shape the environments where they are deployed or adopted. Furthermore, individual and collective interests may change as the hype cycle of an emerging technology evolves (Gartner, 2022). Stakeholders in this process may include scientific and technological researchers, business firms and industry associations, government officials, civil society groups, worker safety groups, privacy advocates, and environmental protection groups, as well as economic and social justicefocused stakeholders (Marchant et al., 2014).

This intricate ecosystem of stakeholders and interests may be further complicated by the simultaneous introduction of other technologies and platforms with different constellations of ethical issues, modes of governance, and political economy contexts. In the following sections, this ecosystem is disaggregated and organized for ease of presentation. It is important to keep in mind that there are entanglements and feedback loops between and among the different sectors, such that pulling on a single thread in one sector often affects multiple areas and actors across the broader ecosystem.

For the purposes of this case study, the primary actors within the academic sector are academic and clinical researchers and the professional societies that represent them.

Science and technology:This case involves a tremendous amount of research and development that has taken place in and grown out of academia, including preclinical and clinical HSPC transplant research; human ESC, EGC, and iPSC research; and genome editing.

Governance and enforcement:Current work at research institutions is governed by IRBs and REBs, stem cell research oversight committees, and institutional animal care and use committees, among other bodies. In addition, research funding bodies, academic publication standards, and scientific and professional societies (i.e., self-regulation) also have a role to playin particular, the ISSCR and its role in the governance of pluripotent stem cell research and in addressing clinics offering unproven cell-based therapies. The National Academies of Sciences, Engineering, and Medicine played a critical role in the governance of ESC research, particularly from 2005 until 2010.

Affordability and reimbursement:While not strictly a matter of patient affordability, it is important to reiterate, as noted previously, that funding available for academic research has disproportionately benefited those with diseases such as cystic fibrosis and Duchenne muscular dystrophy, which are more common in White individuals of European descent, compared to SCD, which in the United States is more prevalent among non-Hispanic Black and Hispanic populations (Farooq et al., 2020; Demirci et al., 2019; Benjamin, 2011; Smith et al., 2006; Scott, 1970).

Private companies:Academicindustry research partnerships, including industry-funded clinical trials, are involved in this space; for example, the CRISPR-based clinical trial was funded by two biotechnology companies (Frangoul et al., 2021). Such partnerships are often predicated on exclusive intellectual property licenses to surrogate licensors (Contreras and Sherkow, 2017).

Social and ethical considerations:Extensive bioethics literature exists on the ethical, legal, and societal issues raised by human subjects research, first-in-human clinical trials, stem cell research, clinics offering unproven cell-based interventions, genome editing, health disparities, and structural racism. Much has also been written on the role of intellectual property and data and materials sharing in the context of human tissue research and genome editing.

Given the focus of CESTI on health and medicine, for the purpose of this case study, the primary actors within the nonprofit sector are those involved in health care, including hematopoietic stem and progenitor cell registries, health insurance companies, and medical profession associations.

Science and technology:HSPC transplants have been clinically available for decades, but research and improvement in this space continue.

Governance and enforcement:Today, the WMDA serves as the accrediting body for registries and promulgates regulations and standards to which the registries adhere on issues like the organization of a registry, the recruitment of volunteer donors, and the collection and transportation of HPCs (WMDA, 2022; Hurley et al., 2010). These standards represent the minimum guidelines for registries, which demonstrate their commitment to comply with WMDA Standards through the WMDA accreditation process (Hurley et al., 2010). Other groups involved in the governance of aspects of HSPC transplant are included inTable 1.

It is important to note that the nonprofit label in this context is somewhat fraught. Many (perhaps most) health care organizations are very much in the business of making money. One of these is the NMDP, which operates Be the Match, and which has diversified its portfolio over time, including the launch in 2016 of Be the Match BioTherapies, which partners with dozens of cell and gene therapy companies, supplying cells and services to advance the development of life-saving cell and gene therapies (Be the Match, 2021a,b).

The FDA generally has authority to regulate bone marrow transplantation through its oversight of bone marrow itself as a human cellular tissue product (HCT/P) and, therefore, a biologic (U.S. Code 262, n.d.). Typically, biologic products are required to submit to the FDAs premarket review process, including the filing of an investigative new drug application and clinical trials. With that said, the FDA has exempted certain types of bone marrow transplantation procedures from such review: namely, bone marrow products that are used in a same-day surgical procedure and those that are only minimally manipulated (FDA, 2020). Importantly, while the FDAs minimally manipulated exception broadly applies to autologous therapy, including the sort of therapy private cord blood banks are intended to plan for, it only applies to allogenic therapy if derived from a first-degree or second-degree blood relative; allogenic therapy using cells from more distant relatives requires the FDAs premarket review (FDA, 2020).

Cord blood matching and donor priority is controlled by the NMDP and regulated by the FDA (CFR, 2012). However, because cord blood therapy is almost always allogenic and usually from anonymized donors unrelated to the patient, cord blood HSPC transplant generally does not fulfill the FDAs minimal manipulation exemptions for HCT/P (FDA, 2020). As such, a total of eight public cord blood banks have applied for, and received, approval from the FDA for their cord blood products (FDA, 2022). Generally, public banks are held to transparent, rigorous storage and quality standards that do not apply to private banks, leading to lower overall quality of cord blood in private banks (Shearer et al., 2017; Sun et al., 2010; Committee on Obstetric Practice, 2008).

The American Academy of Pediatrics has taken a position on private versus public cord blood banks and supports public banking, as do the American Medical Association and the American Congress of Obstetricians and Gynecologists (AMA, n.d.; ACOG, 2019; Shearer et al., 2017).

Affordability and reimbursement:Both public and private insurers in the United States tend to distinguish autologous from allogenic bone marrow therapies, covering autologous transplantation for some indications and allogenic transplantation for others (CMS, 2016).

Leaving aside the broader issues of health insurance and health care affordability in the United States, annual and lifelong care costs for genetic hematologic diseases like SCD and thalassemia are considerablethe yearly cost of standard of care for a patient with SCD is more than $35,000 (Kauf et al., 2009). Novel therapiesboth pharmacologic and those based on HSPC transplantsare anticipated to be extraordinarily expensive, if proven safe and effective. For example, the drugs Oxbryta and Adakveo, approved in 2019 for treating SCD, are estimated to cost $84,000 and $88,000 per year, respectively (ICER, 2021; Sagonowsky, 2020). CART-T cell therapy, which as another novel, genetically modified cell-based therapy may be a reasonable bellwether for the cost of the SCD therapies described previously, costs at least $373,000 for a single infusion before hospital and other associated costs (Beasley, 2019). Many patients suffering from these diseases are from historically marginalized and underserved populations that tend to have lower levels of income. In addition, as therapies become more bespoke, scaling will increasingly become a challenge, from both a regulatory and delivery perspective. However, these delivery challenges may also open new business opportunities.

While donation of cord blood to a public bank is free to the donor, costs associated with private banking include a collection fee ($1,350$2,300) and annual storage fees ($100$175 a year), which are unlikely to be covered by health insurance (Shearer et al., 2017).

Private companies:Many private companies advertise private cord blood banking to new parents as a form of biological insurance; however, the costs of collection and storage are not generally covered by medical insurance (private companies offering unproven cell-based interventions are included under the private sector rather than health care).

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Regenerative Medicine: Case Study for Understanding and Anticipating ...

TORONTO and HAIFA, Israel, Feb. 14, 2025 (GLOBE NEWSWIRE) -- NurExone Biologic Inc. (TSXV: NRX) (OTCQB: NRXBF) (Germany: J90) (“NurExone” or the “Company”) is excited to announce that it will be presenting at the International Society for Cell & Gene Therapy (ISCT) 2025 Annual Meeting (“ISCT 2025”), a major global cell and gene therapy translation conference, taking place from May 7-10, 2025 in New Orleans, Louisiana, United States.

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NurExone Biologic Continues Expansion of U.S. Presence and Awareness with Prestigious Conference Presentation

Lille (France), Cambridge (Massachusetts, United States), Zurich (Switzerland), February 6, 2025 - GENFIT (Nasdaq and Euronext: GNFT), a biopharmaceutical company dedicated to improving the lives of patients with rare and life-threatening liver diseases, today outlines the design of new clinical trials within its Acute on-Chronic Liver Failure (ACLF) pipeline, with several clinical data readouts by end of 2025.

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GENFIT Outlines Anticipated New Clinical Trial Initiations, Development Milestones and Data Readouts in 2025

July 27, 2020, by NCI Staff

CRISPR is a highly precise gene editing tool that is changing cancer research and treatment.

Credit: Ernesto del Aguila III, National Human Genome Research Institute

Ever since scientists realized that changes in DNA cause cancer, they have been searching for an easy way to correct those changes by manipulating DNA. Although several methods of gene editing have been developed over the years, none has really fit the bill for a quick, easy, and cheap technology.

But a game-changer occurred in 2013, when several researchers showed that a gene-editing tool called CRISPR could alter the DNA of human cells like a very precise and easy-to-use pair of scissors.

The new tool has taken the research world by storm, markedly shifting the line between possible and impossible. As soon as CRISPR made its way onto the shelves and freezers of labs around the world, cancer researchers jumped at the chance to use it.

CRISPR is becoming a mainstream methodology used in many cancer biology studies because of the convenience of the technique, said Jerry Li, M.D., Ph.D., of NCIs Division of Cancer Biology.

Now CRISPR is moving out of lab dishes and into trials of people with cancer. In a small study, for example, researchers tested a cancer treatment involving immune cells that were CRISPR-edited to better hunt down and attack cancer.

Despite all the excitement, scientists have been proceeding cautiously, feeling out the tools strengths and pitfalls, setting best practices, and debating the social and ethical consequences of gene editing in humans.

Like many other advances in science and medicine, CRISPR was inspired by nature. In this case, the idea was borrowed from a simple defense mechanism found in some microbes, such as bacteria.

To protect themselves against invaders like viruses, these microbes capture snippets of the intruders DNA and store them away as segments called CRISPRs, or clustered regularly interspersed short palindromic repeats. If the same germ tries to attack again, those DNA segments (turned into short pieces of RNA) help an enzyme called Cas find and slice up the invaders DNA.

After this defense system was discovered, scientists realized that it had the makings of a versatile gene-editing tool. Within a handful of years, multiple groups had successfully adapted the system to edit virtually any section of DNA, first in the cells of other microbes, and then eventually in human cells.

CRISPR consists of a guide RNA (RNA-targeting device, purple) and the Cas enzyme (blue). When the guide RNA matches up with the target DNA (orange), Cas cuts the DNA. A new segment of DNA (green) can then be added.

Credit: National Institute of General Medical Sciences, National Institutes of Health

In the laboratory, the CRISPR tool consists of two main actors: a guide RNA and a DNA-cutting enzyme, most commonly one called Cas9. Scientists design the guide RNA to mirror the DNA of the gene to be edited (called the target). The guide RNA partners with Cas andtrue to its nameleads Cas to the target. When the guide RNA matches up with the target gene's DNA, Cas cuts the DNA.

What happens next depends on the type of CRISPR tool thats being used. In some cases, the target gene's DNA is scrambled while it's repaired, and the gene is inactivated. With other versions of CRISPR, scientists can manipulate genes in more precise ways such as adding a new segment of DNA or editing single DNA letters.

Scientists have also used CRISPR to detect specific targets, such as DNA from cancer-causing viruses and RNA from cancer cells. Most recently, CRISPR has been put to use as an experimental testto detect the novel coronavirus.

Scientists consider CRISPR to be a game-changer for a number of reasons. Perhaps the biggest is that CRISPR is easy to use, especially compared with older gene-editing tools.

Before, only a handful of labs in the world could make the proper tools [for gene editing]. Now, even a high school student can make a change in a complex genome using CRISPR, said Alejandro Chavez, M.D., Ph.D., an assistant professor at Columbia University who has developed several novel CRISPR tools.

CRISPR is also completely customizable. It can edit virtually any segment of DNA within the 3 billion letters of the human genome, and its more precise than other DNA-editing tools.

And gene editing with CRISPR is a lot faster. With older methods, it usually [took] a year or two to generate a genetically engineered mouse model, if youre lucky, said Dr. Li. But now with CRISPR, a scientist can create a complex mouse model within a few months, he said.

Another plus is that CRISPR can be easily scaled up. Researchers can use hundreds of guide RNAs to manipulate and evaluate hundreds or thousands of genes at a time. Cancer researchers often use this type of experiment to pick out genes that might make good drug targets.

And as an added bonus, its certainly cheaper than previous methods, Dr. Chavez noted.

With all of its advantages over other gene-editing tools, CRISPR has become a go-to for scientists studying cancer. Theres also hope that it will have a place in treating cancer, too. But CRISPR isnt perfect, and its downsides have made many scientists cautious about its use in people.

A major pitfall is that CRISPR sometimes cuts DNA outside of the target genewhats known as off-target editing. Scientists are worried that such unintended edits could be harmful and could even turn cells cancerous, as occurred in a 2002 study of a gene therapy.

If [CRISPR] starts breaking random parts of the genome, the cell can start stitching things together in really weird ways, and theres some concern about that becoming cancer, Dr. Chavez explained. But by tweaking the structures of Cas and the guide RNA, scientists have improved CRISPRs ability to cut only the intended target, he added.

Another potential roadblock is getting CRISPR components into cells. The most common way to do this is to co-opt a virus to do the job. Instead of ferrying genes that cause disease, the virus is modified to carry genes for the guide RNA and Cas.

Slipping CRISPR into lab-grown cells is one thing; but getting it into cells in a person's bodyis another story. Some viruses used to carry CRISPR can infect multiple types of cells, so, for instance, they may end up editing muscle cells when the goal was to edit liver cells.

Researchers are exploring different ways to fine-tune the delivery of CRISPR to specific organs or cells in the human body. Some are testing viruses that infect only one organ, like the liver or brain. Others have created tiny structures callednanocapsules that are designed to deliver CRISPR components to specific cells.

Because CRISPR is just beginning to be tested in humans, there are also concerns about how the bodyin particular, the immune systemwill react to viruses carrying CRISPR or to the CRISPR components themselves.

Some wonder whether the immune system could attack Cas (a bacterial enzyme that is foreign to human bodies) and destroy CRISPR-edited cells. Twenty years ago, a patient died after his immune system launched a massive attack against the viruses carrying a gene therapy he had received. However, newer CRISPR-based approaches rely on viruses that appear to be safer than those used for older gene therapies.

Another major concern is that editing cells inside the body could accidentally make changes to sperm or egg cells that can be passed on to future generations. But for almost all ongoing human studies involving CRISPR, patients cells are removed and edited outside of their bodies. This ex vivo approach is considered safer because it is more controlled than trying to edit cells inside the body, Dr. Chavez said.

However, one ongoing study is testing CRISPR gene editing directly in the eyes of people with a genetic disease that causes blindness, called Leber congenital amaurosis.

The first trial in the United States to test a CRISPR-made cancer therapy was launched in 2019 at the University of Pennsylvania. The study, funded in part by NCI, is testing a type of immunotherapy in which patients own immune cells are genetically modified to better see and kill their cancer.

The therapy involves making four genetic modifications to T cells, immune cells that can kill cancer. First, the addition of a synthetic gene gives the T cells a claw-like protein (called a receptor) that sees NY-ESO-1, a molecule on some cancer cells.

Then CRISPR is used to remove three genes: two that can interfere with the NY-ESO-1 receptor and another that limits the cells cancer-killing abilities. The finished product, dubbed NYCE T cells, were grown in large numbers and then infused into patients.

The first trial of CRISPR for patients with cancer tested T cells that were modified to better "see" and kill cancer.CRISPR was used to remove three genes: two that can interfere with the NY-ESO-1 receptor and another that limits the cells cancer-killing abilities.

Credit: National Cancer Institute

We had done a prior study of NY-ESO-1directed T cells and saw some evidence of improved response and low toxicity, said the trials leader, Edward Stadtmauer, M.D., of the University of Pennsylvania. He and his colleagues wanted to see if removing the three genes with CRISPR would make the T cells work even better, he said.

The goal of this study was to first find out if the CRISPR-made treatment was safe. It was tested in two patients with advanced multiple myeloma and one with metastatic sarcoma. All three had tumors that contained NY-ESO-1, the target of the T-cell therapy.

Initial findings suggest that the treatment is safe. Some side effects did occur, but they were likely caused by the chemotherapy patients received before the infusion of NYCE cells, the researchers reported. There was no evidence of an immune reaction to the CRISPR-edited cells.

Only about 10% of the T cells used for the therapy had all four of the desired genetic edits. And off-target edits were found in the modified cells of all three patients. However, none of the cells with off-target edits grew in a way that suggested they had become cancer, Dr. Stadtmauer noted.

The treatment had a small effect on the patients cancers. The tumors of two patients (one with multiple myeloma and one with sarcoma) stopped growing for a while but resumed growing later. The treatment didn't work at all for the third patient.

It'sexciting that the treatment initially worked for the sarcoma patientbecause solid tumors have been a much more difficult nut to crack with cellular therapy," Dr. Stadtmauer said. "Perhaps [CRISPR] techniques will enhance our ability to treat solid tumors with cell therapies.

Although the trial shows that CRISPR-edited cell therapy is possible, the long-term effects still need to be monitored, Dr. Stadtmauer continued. The NYCE cells are safe for as long as weve been watching [the study participants]. Our plan is to keep monitoring them for years, if not decades, he said.

While the study of NYCE T cells marked the first trial of a CRISPR-based cancer treatment, there are likely more to come.

This [trial] was really a proof-of-principle, feasibility, and safety thing that now opens up the whole world of CRISPR editing and other techniques of [gene] editing to hopefully make the next generation of therapies, Dr. Stadtmauer said.

Other clinical studies of CRISPR-made cancer treatments are already underway. A few trials are testing CRISPR-engineered CAR T-cell therapies, another type of immunotherapy. For example, one company is testing CRISPR-engineered CAR T cells in people with B cell cancers and people with multiple myeloma.

There are still a lot of questions about all the ways that CRISPR might be put to use in cancer research and treatment. But one thing is for certain: The field is moving incredibly fast and new applications of the technology are constantly popping up.

People are still improving CRISPR methods, Dr. Li said. Its quite an active area of research and development. Im sure that CRISPR will have even broader applications in the future.

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How CRISPR Is Changing Cancer Research and Treatment

Abstract

The convergence of artificial intelligence (AI) and precision medicine promises to revolutionize health care. Precision medicine methods identify phenotypes of patients with lesscommon responses to treatment or unique healthcare needs. AI leverages sophisticated computation and inference to generate insights, enables the system to reason and learn, and empowers clinician decision making through augmented intelligence. Recent literature suggests that translational research exploring this convergence will help solve the most difficult challenges facing precision medicine, especially those in which nongenomic and genomic determinants, combined with information from patient symptoms, clinical history, and lifestyles, will facilitate personalized diagnosis and prognostication.

In a recent National Academy of Medicine report about the current and future state of artificial intelligence (AI) in health care, the authors noted unprecedented opportunities to augment the care of specialists and the assistance that AI provides in combating the realities of being human (including fatigue and inattention) and the risks of machine error. Importantly, the report notes that whereas care must be taken with the use of these technologies, much promise exists.1 The digitization of healthrelated data and the rapid uptake in technology are fueling transformation and progress in the development and use of AI in healthcare.2, 3, 4 However, multimodal data integration, security, federated learning (which requires fundamental advances in areas, such as privacy, largescale machine learning, and distributed optimization), model performance, and bias may pose challenges to the use of AI in health care.5

Three main principles for successful adoption of AI in health care include data and security, analytics and insights, and shared expertise. Data and security equate to full transparency and trust in how AI systems are trained and in the data and knowledge used to train them. As humans and AI systems increasingly work together, it is essential that we trust the output of these systems.

Analytics and insights equate to purpose and people where augmented intelligence and actionable insights support what humans do, not replace them. AI can combine input from multiple structured and unstructured sources, reason at a semantic level, and use these abilities in computer vision, reading comprehension, conversational systems, and multimodal applications to help health professionals make more informed decisions (e.g., a physician making a diagnosis, a nurse creating a care plan, or a social services agency arranging services for an elderly citizen). Shared expertise equates to our complementary relationship with AI systems, which are trained by and are supporting human professionals, leading to workforce change, which leads to new skills. The ability to create cuttingedge AI models and build highquality business applications requires skilled experts with access to the latest hardware.

A vast amount of untapped data could have a great impact on our healthyet it exists outside medical systems.6 Our individual health is heavily influenced by our lifestyle, nutrition, our environment, and access to care. These behavioral and social determinants and other exogenous factors can now be tracked and measured by wearables and a range of medical devices. These factors account for about 60% of our determinants of health (behavioral, socioeconomical, physiological, and psychological data), our genes account for about 30%, and last our actual medical history accounts for a mere 10%.6 Over the course of our lifetimes, we will each generate the equivalent of over 300 million books of personal and healthrelated data that could unlock insights to a longer and healthier life.7

The phenomenon of big data can be described using the five Vs: volume, velocity, variety, veracity, and value. Volume refers to the vast amount of complex and heterogenous data, which makes data sets too large to store and analyze using traditional database technology. Velocity refers to the speed at which new data are generated and moves around. Variety refers to the different types of structured, semistructured, and unstructured data, such as social media conversations and voice recordings. Veracity refers to the certainty, accuracy, relevance, and predictive value of the data. Value refers to the conversion of data into business insights. Whereas the volume, variety, velocity, and veracity of data are contributing to the increasing complexity of data management and workloadscreating a greater need for advanced analytics to discover insightsmobile devices have made technology more consumable, creating user demand for interactive tools for visual analytics.

Big data analytics and AI are increasingly becoming omnipresent across the entire spectrum of health care, including the 5 Ps spanning: payer, provider, policy maker/government, patients, and product manufacturers. Up to 10% of global health care expenditure is due to fraud and abuse and AIbased tools help mitigate fraud, waste, and abuse in payer programs.8 Reliable identification of medical coding errors and incorrect claims positively impacts payers, providers, and governments by saving inordinate amounts of money, time, and efforts.9 As an example, IBM DataProbe, an AIbased business intelligence tool, was able to detect and recover US $41.5million in feeforservice payments over a 2year period in Medicaid fraud for Iowa Medicaid Enterprise.10 In the provider space, AI is used for evidencebased clinical decision support,11 detection of adverse events, and the usage of electronic health record (EHR) data to predict patients at risk for readmission.12 Healthcare policymakers and government use AIbased tools to control and predict infections and outbreaks. An example is FINDER, a machinelearned model for realtime detection of foodborne illness using anonymous and aggregated web search and location data.13 Another example is the integrated data hub and caremanagement solution using IBM Connect360 and IBM Watson Care Manager that Sonoma County, California government agencies used to transform health and healthcare for socially disadvantaged groups and other displaced individuals during a time of communitywide crisis.14 This solution enabled integration of siloed data and services into a unified citizen status view, identification of clinical and social determinants of health from structured and unstructured sources, construction of algorithms to match clients with services, and streamlining of care coordination during the 2017 and 2019 Sonoma County wildfires. With the advent of the global pandemic coronavirus disease 2019 (COVID19) in early 2020, such a model can be used to predict atrisk populations, and potentially provide additional risk information to clinicians caring for atrisk patients.15 The use of AI for patients and life sciences/healthcare products are addressed extensively in the sections that follow.

AI is not, however, the only datadriven field impacting health and health care. The field of precision medicine is providing an equal or even greater influence than AI on the direction of health care16 and has been doing so for more than a decade.17 Precision medicine aims to personalize care for every individual. This goal requires access to massive amounts of data, such as data collected through the United Kingdoms UK Biobank and the All of Us project, coupled with a receptive health care ecosystem willing to abandon the conventional approach to care in favor of a more highly individualized strategy. The convergence of these fields will likely accelerate the goals of personalized care and tightly couple AI to healthcare providers for the foreseeable future. In the sections that follow, we will briefly summarize the capabilities of existing AI technology, describe how precision medicine is evolving, and, through a series of examples, demonstrate the potentially transformative effect of AI on the rate and increasing breadth of application for precision medicine.

The past 10years have seen remarkable growth and acceptance of AI in a variety of domains and in particular by healthcare professionals. AI provides rich opportunities for designing intelligent products, creating novel services, and generating new business models. At the same time, the use of AI can introduce social and ethical challenges to security, privacy, and human rights.1

AI technologies in medicine exist in many forms, from the purely virtual (e.g., deeplearningbased health information management systems and active guidance of physicians in their treatment decisions) to cyberphysical (e.g., robots used to assist the attending surgeon and targeted nanorobots for drug delivery).18 The power of AI technologies to recognize sophisticated patterns and hidden structures has enabled many imagebased detection and diagnostic systems in healthcare to perform as well or better than clinicians, in some cases.19 AIenabled clinical decisionsupport systems may reduce diagnostic errors, augment intelligence to support decision making, and assist clinicians with EHR data extraction and documentation tasks.20 Emerging computational improvements in natural language processing (NLP), pattern identification, efficient search, prediction, and biasfree reasoning will lead to further capabilities in AI that address currently intractable problems.21, 22

Advances in the computational capability of AI have prompted concerns that AI technologies will eventually replace physicians. The term augmented intelligence, coined by W.R. Ashby in the 1950s,23 may be a more apt description of the future interplay among data, computation, and healthcare providers and perhaps a better definition for the abbreviation AI in healthcare. A version of augmented intelligence, described in the literature in Friedmans fundamental theorem of biomedical informatics,24 relates strongly to the role of AI in health care (depicted in Figure1). Consistent with Friedmans description of augmented intelligence, Langlotz at Stanford stated that Radiologists who use AI will replace radiologists who dont.25

A version of the Friedmans fundamental theorem of informatics describing the impact of augmented intelligence. The healthcare system with AI will be better than the healthcare system without it. AI, artificial intelligence.

An AI system exhibits four main characteristics that allow us to perceive it as cognitive: understanding, reasoning, learning, and empowering.26 An AI system understands by reading, processing, and interpreting the available structured and unstructured data at enormous scale and volume. An AI system reasons by understanding entities and relationships, drawing connections, proposing hypotheses, deriving inferences, and evaluating evidence that allows it to recognize and interpret the language of health and medicine. An AI system learns from human experts and realworld cases by collecting feedback, learning from outcomes at all levels and granularities of the system, and continuing to improve over time and experience. An AI system empowers and interacts clinicians and users by providing a more integrated experience in a variety of settings, combining dialog, visualization, collaboration, and delivering previously invisible data and knowledge into actionable insights. In contrast, humans excel at common sense, empathy, morals, and creativity.

Augmenting human capabilities with those provided by AI leads to actionable insights in areas such as oncology,27 imaging,28 and primary care.29 For example, a breast cancer predicting algorithm, trained on 38,444 mammogram images from 9,611 women, was the first to combine imaging and EHR data with associated health records. This algorithm was able to predict biopsy malignancy and differentiate between normal and abnormal screening results. The algorithm can be applied to assess breast cancer at a level comparable to radiologists, as well has having the potential to substantially reduce missed diagnoses of breast cancer.30 This combined machinelearning and deeplearning model trained on a dataset of linked mammograms and health records may assist radiologists in the detection of breast cancer as a second reader.

The field of precision medicine is similarly experiencing rapid growth. Precision medicine is perhaps best described as a health care movement involving what the National Research Council initially called the development of a New Taxonomy of human disease based on molecular biology, or a revolution in health care triggered by knowledge gained from sequencing the human genome.31 The field has since evolved to recognize how the intersection of multiomic data combined with medical history, social/behavioral determinants, and environmental knowledge precisely characterizes health states, disease states, and therapeutic options for affected individuals.32 For the remainder of this paper, we will use the term precision medicine to describe the health care philosophy and research agenda described above, and the term personalized care to reflect the impact of that philosophy on the individual receiving care.

Precision medicine offers healthcare providers the ability to discover and present information that either validates or alters the trajectory of a medical decision from one that is based on the evidence for the average patient, to one that is based upon individuals unique characteristics. It facilitates a clinicians delivery of care personalized for each patient. Precision medicine discovery empowers possibilities that would otherwise have been unrealized.

Advances in precision medicine manifest into tangible benefits, such as early detection of disease33 and designing personalized treatments are becoming more commonplace in health care.34 The power of precision medicine to personalize care is enabled by several data collection and analytics technologies. In particular, the convergence of highthroughput genotyping and global adoption of EHRs gives scientists an unprecedented opportunity to derive new phenotypes from realworld clinical and biomarker data. These phenotypes, combined with knowledge from the EHR, may validate the need for additional treatments or may improve diagnoses of disease variants.

Perhaps the most wellstudied impact of precision medicine on health care today is genotypeguided treatment. Clinicians have used genotype information as a guideline to help determine the correct dose of warfarin.35 The Clinical Pharmacogenetics Implementation Consortium published genotypebased drug guidelines to help clinicians optimize drug therapies with genetic test results.36 Genomic profiling of tumors can inform targeted therapy plans for patients with breast or lung cancer.34 Precision medicine, integrated into healthcare, has the potential to yield more precise diagnoses, predict disease risk before symptoms occur, and design customized treatment plans that maximize safety and efficiency. The trend toward enabling the use of precision medicine by establishing data repositories is not restricted to the United States; examples from Biobanks in many countries, such as the UK Biobank,37 BioBank Japan,38 and Australian Genomics Health Alliance39 demonstrate the power of changing attitudes toward precision medicine on a global scale.

Although there is much promise for AI and precision medicine, more work still needs to be done to test, validate, and change treatment practices. Researchers face challenges of adopting unified data formats (e.g., Fast Healthcare Interoperability Resources), obtaining sufficient and high quality labeled data for training algorithms, and addressing regulatory, privacy, and sociocultural requirements.

AI and precision medicine are converging to assist in solving the most complex problems in personalized care. Figure2 depicts five examples of personalized healthcare dogma that are inherently challenging but potentially amenable to progress using AI.40, 41, 42

Dimensions of synergy between AI and precision medicine. Both precision medicine and artificial intelligence (AI) techniques impact the goal of personalizing care in five ways: therapy planning using clincal, genomic or social and behavioral determinants of health, and risk prediction/diagnosis, using genomic or other variables.

Genomeinformed prescribing is perhaps one of the first areas to demonstrate the power of precision medicine at scale.43 However, the ability to make realtime recommendations hinges on developing machinelearning algorithms to predict which patients are likely to need a medication for which genomic information. The key to personalizing medications and dosages is to genotype those patients before that information is needed.44

This use case was among the earliest examples of the convergence between AI and precision medicine, as AI techniques have proven useful for efficient and highthroughput genome interpretation.45 As noted recently by Zou and colleagues,46 deep learning has been used to combine knowledge from the scientific literature with findings from sequencing to propose 3D protein configurations, identify transcription start sites, model regulatory elements, and predict gene expression from genotype data. These interpretations are foundational to identifying links among genomic variation and disease presentation, therapeutic success, and prognosis.

In medulloblastoma, the emergence of discrete molecular subgroups of the disease following AImediated analysis of hundreds of exomes, has facilitated the administration of the right treatment, at the right dosage, to the right cohort of pediatric patients.47 Although conventional treatment of this disease involved multimodal treatment, including surgery, chemotherapy, and whole brain radiation, precision genomics has enabled treatment of the wingless tumor subgroup, which is more common in children, with chemotherapy aloneobviating the need for radiation.48 Avoiding radiotherapy is particularly impactful for mitigating potential neurocognitive sequelae and secondary cancers from wholebrain radiation among disease survivors.49, 50

The initial successful paradigm of AI in imaging recognition has also given rise to radiogenomics. Radiogenomics, as a novel precision medicine research field, focuses on establishing associations between cancer imaging features and gene expression to predict a patients risk of developing toxicity following radiotherapy.51, 52, 53 For example, Chang et al. proposed a framework of multiple residual convolutional neural networks to noninvasively predict isocitrate dehydrogenase genotype in grades IIIV glioma using multiinstitutional magnetic resonance imaging datasets. Besides, AI has been used in discovering radiogenomic associations in breast cancer,52 liver cancer,54 and colorectal cancer.53 Currently, limited data availability remains the most formidable challenge for AI radiogenomics.51

Knowing the response to therapy can help clinicians choose the right treatment plan. AI demonstrates potential applications in this area. For example, McDonald et al. trained a support vector machine using patients gene expression data to predict their response to chemotherapy. Their data show encouraging outcomes across multiple drugs.55 Sadanandam et al. proposed approaches of discovering patterns in gene sequences or molecular signatures that are associated with better outcomes following nontraditional treatment. Their findings may assist clinicians in selecting a treatment that is most likely to be effective.56 Although tremendous progress has been made using AI techniques and genomics to predict treatment outcome, more prospective and retrospective clinical research and clinical studies still need to be conducted to generate the data that can then train the algorithms.

Incorporating environmental considerations into management plans require sufficient personal and environmental information, which may affect a patients risk for a poor outcome, knowledge about care alternatives, and conditions under which each alternative may be optimal.

One such example has been the challenge of identifying homelessness in some patients.57, 58, 59 These patients may require care in varying locations over a short period, requiring frequent reassessments of patient demographic data. Related issues, such as transportation, providing medications that require refrigeration, or using diagnostic modalities that require electricity (for monitoring), need to be modified accordingly.

Another environmental consideration is the availability of expertise in remote locations, including the availability of trained professionals at the point of need. AI has provided numerous examples of augmenting diagnostic capabilities in resourcepoor locations, which may translate into better patient classification and therefore more personalized therapy planning. Examples include the use of deep learning to identify patients with malaria60 and cervical cancer,61 as well as predicting infectious disease outbreaks,62 environmental toxin exposure,63 and allergen load.64

Finally, in addition to genomic considerations and social determinants of health, clinical factors are imperative to successful therapy planning. Age, comorbidities, and organ function in particular predicate treatment considerations and AI has emerged as a central pillar in stratifying patients for therapy. In one study, machine learning classifiers were used to analyze 30 comorbidities to identify critically ill patients who will require prolonged mechanical ventilation and tracheostomy placement.65 Other studies have used AI algorithms to analyze bedside monitored adverse events and other clinical parameters to predict organ dysfunction and failure.66, 67

Actress Angelina Jolies response to her inheritance of the BRCA gene illustrates the potential impact of more advanced genomic information on disease risk and prevention options.68 This case is not novel; the case of Woodie Guthrie and Huntingtons disease disclosed a similar conundrum for health care.69 Although the ethics of genetic testing without a clear cure continues to be debated, the broad availability of genetic information offered by nextgeneration sequencing and directtoconsumer testing renders personalized prevention and management of serious diseases a reality.70

Cardiovascular medicine is an area with a long history of embracing predictive modeling to assess patient risk.71 Recent work has uncovered methods to predict heart failure72 and other serious cardiac events in asymptomatic individuals.73 When combined with personalized prevention strategies,74, 75 these models may positively impact disease incidence and sequela. Complex diseases, such as cardiovascular disease, often involve the interplay among gender, genetic, lifestyle, and environmental factors. Integrating these attributes requires attention to the heterogeneity of the data.76 AI approaches that excel at discovering complex relationships among a large number of factors provide such opportunities. A study from Vanderbilt demonstrated early examples of combining EHR and genetic data, with positive results in cardiovascular disease prediction.77 AIenabled recognition of phenotype features through EHR or images and matching those features with genetic variants may allow faster genetic disease diagnosis.78 For example, accurate and fast diagnosis for seriously ill infants that have a suspected genetic disease can be attained by using rapid wholegenome sequencing and NLPenabled automated phenotyping.79

Automated speech analytics have benefited from improvements in the technical performance of NLP, understanding, and generation. Automated speech analytics may provide indicators for assessment and detection of earlystage dementia, minor cognitive impairment, Parkinsons disease, and other mental disorders.80, 81, 82, 83 Efforts also are underway to detect changes in mental health using smartphone sensors.84

AIassisted monitoring may also be used in realtime to assess the risk of intrapartum stress during labor, guiding the decision of cesarean section vs. normal vaginal deliveries, in an effort to decrease perinatal complications and stillbirths.85 This exemplifies realtime AIassisted monitoring of streaming data to reduce manual error associated with human interpretation of cardiotocography data during childbirth.

AI is also being used in the detection and characterization of polyps in colonoscopy.86 Wider adaptation of AI during endoscopy may lead to a higher rate of benign adenoma detection and reduction of cost and risk for unwarranted polypectomy.87 AImediated image analysis aimed at improving disease risk prediction and diagnosis will likely continue to increase in use for detection of diabetic retinopathy88 and metastasis in cancer,89 as well as for identification of benign melanoma.90 AIbased image analysis has become a part of a directtoconsumer diagnostic tool for anemia as well.91

The widespread use of home monitoring and wearable devices has long been accompanied by the expectation that collected data could help detect disease at an earlier state. Indeed, these advances have fueled new, noninvasive, wearable applications for monitoring and detecting specific health conditions, such as diabetes, epilepsy, pain management, Parkinsons disease, cardiovascular disease, sleep disorders, and obesity.92 Digital biomarkers are expected to facilitate remote disease monitoring outside of the physical confines of a hospital setting and can support decentralized clinical trials.93 Wearable tools that provide continuous multidimensional measurements of preselected biomarkers would enable the detection of minimum residual disease and monitor disease progression.94 In the field of cancer care, evolving technology using wearable devices continuously analyzes circulating tumor cells to screen for relapsed disease.95

We have observed increasing efforts to implement AI in precision medicine to perform tasks such as disease diagnosis, predicting risk, and treatment response. Although most of these studies showed promising experimental results, how AI improved health care is not fully demonstrated. In reality, the success of transforming an AI system to a realworld application not only depends on the accuracy but also relies on the capability of working accurately in a reliable, safe, and generalizable manner.5 For example, the difference among institutions in coding definitions, report formats, or cohort diversity, may result in a model trained using onesite data to not work well in another site (https://www.bmj.com/content/368/bmj.l6927). Here, we highlighted three main challenges that would impact the success of transitions to realworld healthcare.

Fairness and bias. The health data can be biased while building and processing the dataset (e.g., a lack of diverse sampling, missing values, and imputation methods; https://datasociety.net/library/fairnessinprecisionmedicine/). An AI model trained on the data might amplify the bias and make nonfavorable decisions toward a particular group of people characterized by age, gender, race, geographic, or economic level. Such unconscious bias may harm clinical applicability and health quality. Thus, it is crucial to detect and mitigate the bias in data and models. Some potential solutions include improving the diversity of the data, such as the All of Us program that aimed to recruit participants with diverse backgrounds. AI communities also proposed several techniques to fight against bias (https://arxiv.org/abs/1908.09635). IBM has developed an online toolkit (AI Fairness 360) that implemented a comprehensive set of fairness metrics to help researchers examine the bias among datasets and models, and algorithms to mitigate bias in classifiers (https://doi.org/10.1147/JRD.2019.2942287). However, fairness and protected attributes are closely related to the domain context and applications. More work is needed in biomedical research to define and explore the fairness and bias in AI models trained with historical patient data. To address the challenge, a collaborative effort that involves the AI and biomedical community is needed.

Socioenvironmental factors. The environmental factors and workflows where the AI model would be deployed may impact model performance and clinical efficacy. A recent prospective study carried out by Google Health evaluated an AI system for screening diabetic retinopathy in a real clinical environment. The AI system was developed to augment diabetic retinopathy screening by providing intime assessment, before this the process may take several weeks. Despite a specialistlevel accuracy (>90% sensitivity and specificity) achieved on retrospective patient data; however, the system has undergone unexpected challenges when applied to Thailand clinics (https://doi.org/10.1145/3313831.3376718). For example, the variety of conditions and workflows in clinics impaired the quality of the images that did not meet the system' high standards, resulting in a high rejection rate of images. The unstable internet connection restricted the processing speed of the AI models and caused a longer waiting time for the patients. Travel and travel costs may deter participants from remaining in the study. Such prospective studies highlighted the importance of validating the AI models in the clinical environment and considering an iteration loopthat collects users feedback as new input for learning and system improvement96 before applying the AI system widely. Of note, in healthcare, obtaining such feedback would take a long time at a high cost. It may take a longer time to evaluate a therapys effect and associated longterm health outcomes than what is required to validate whether a product is appealing to a customer. There is a need to explore other ways to facilitate creation of highperforming AI systems, for example, generating synthetic data that carries similar distributions and variances as the realworld data, or leveraging a simulated environment. Early examples by groups, such as Baowaly and colleagues,89 demonstrate much promise, but more AI research efforts are needed.

Data safety and privacy. Data is crucial to an AIdriven system. As AI and precision medicine are converging, data (e.g., genomics, medical history, behaviors, and social data that covers peoples daily lives) will be increasingly collected and integrated. Individuals concerns for data privacy are closely related to trust when they use AIenabled services. Building a safe and wellcontrolled ecosystem for data storage, management, and sharing is essential, requiring new technology adoptions, and collaborations, as well as the creation of new regulations and business models.

The training of AI methods and validation of AI models using large data sets prior to applying the methods to personal data may address many of the challenges facing precision medicine today. The cited examples reinforce the importance of another potential use of augmented intelligence, namely that of the role of technology in the hands of consumers to help communicate justintime risk or as an agent of behavior change. Although most studies to date are small and the data are limited, the ability to identify atrisk patients will translate into personalized care when identification is combined with strategies to notify and intervene. Researchers are actively pursuing the use of mobile apps, wearables, voice assistants, and other technology to create personspecific interfaces to intelligent systems. A review of these approaches is beyond the scope of this paper.

Active research in both AI and precision medicine is demonstrating a future where healthrelated tasks of both medical professionals and consumers are augmented with highly personalized medical diagnostic and therapeutic information. The synergy between these two forces and their impact on the healthcare system aligns with the ultimate goal of prevention and early detection of diseases affecting the individual, which could ultimately decrease the disease burden for the public at large, and, therefore, the cost of preventable health care for all.

This work was funded by a partnership between IBM Watson Health and Vanderbilt University Medical Center.

Drs. Weeraratne, Rhee, and Snowdon are employed by IBM Watson Health. All other authors declared no competing interests for this work.

The authors thank Karlis Draulis for his assistance with the figures.

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Precision Medicine, AI, and the Future of Personalized Health Care

When it comes to diseases that affect the eye, the earlier a condition is detected, the easier it is to manage. Accomplishing this takes medical expertise and the latest diagnostic tools. Youll find that and more at the NYU Langone Eye Center.

Diagnosis and Treatment of Glaucoma

Our physicians provide medical and surgical care for people with glaucoma.

Our ophthalmologists care for adults and children. We manage all conditions that affect the eye, including glaucoma, cataract, macular degeneration, and diabetic retinopathy. We also specialize in oculoplastic surgery, which is used to treat people with conditions that affect the eyelid and tear duct.

Our practice is led by Dr. Kathryn Colby, whose research has led to significant advances in treating complex diseases of the cornea and ocular surface.

Our team of ophthalmologists performs the latest surgical techniques, including minimally invasive glaucoma and vitreoretinal surgery, endoscopic orbital and skull base surgery, and cornea transplant. We also perform hyperspectral imaging, which allows us to look at the chemical makeup of the retina and detect any irregularities that could signal disease.

We are leaders in diagnosing and treating Fuchs dystrophy, a condition that ultimately leads to vision loss and is the most common reason for a corneal transplant in the United States. For people with this condition, the cells in the corneas endothelial layer gradually die off. These cells normally pump fluid out of the cornea to keep it clear. When these cells die, fluid builds up and the cornea thickens and swells, leading to cloudy or hazy vision, eye glare, and eye pain.

Our experts pioneered a minimally invasive surgical treatment for Fuchs dystrophy, called Descemet stripping only (DSO). This treatment involves removing the affected central corneal endothelial cells, which allows healthier peripheral cells to migrate in. Rather than relying on a donor cornea, DSO allows a persons own cells to rejuvenate their cornea. The selective removal of damaged tissue can effectively restore corneal function while eliminating the risks associated with corneal transplant, which include tissue rejection and blindness.

As leaders in this area, our team conducts research on DSO as an alternative to corneal replacement through a multicenter, multinational clinical trial.

Experts at the Eye Centers ocular imaging laboratory develop and study advanced diagnostic imaging technologies. At the Eye Centers Ophthalmic Imaging Research Laboratory, we study conditions that affect the retina, including macular degeneration. Our research is leading to new treatments for glaucoma, macular degeneration, diabetic retinopathy, and a host of other conditions.

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ABSTRACT

The ability to make site-specific modifications to the human genome has been an objective in medicine since the recognition of the gene as the basic unit of heredity. Thus, gene therapy is understood as the ability of genetic improvement through the correction of altered (mutated) genes or site-specific modifications that target therapeutic treatment. This therapy became possible through the advances of genetics and bioengineering that enabled manipulating vectors for delivery of extrachromosomal material to target cells. One of the main focuses of this technique is the optimization of delivery vehicles (vectors) that are mostly plasmids, nanostructured or viruses. The viruses are more often investigated due to their excellence of invading cells and inserting their genetic material. However, there is great concern regarding exacerbated immune responses and genome manipulation, especially in germ line cells. In vivo studies in in somatic cell showed satisfactory results with approved protocols in clinical trials. These trials have been conducted in the United States, Europe, Australia and China. Recent biotechnological advances, such as induced pluripotent stem cells in patients with liver diseases, chimeric antigen receptor T-cell immunotherapy, and genomic editing by CRISPR/Cas9, are addressed in this review.

Keywords: Gene therapy, Genetic Vectors, Gene transfer, horizontal, CRISPR-Cas9, CAR-T cell, Genetic therapy, Clustered regularly interspaced short palindromic repeats

A habilidade de fazer modificaes pontuais no genoma humano tem sido o objetivo da medicina desde o conhecimento do DNA como unidade bsica da hereditariedade. Entende-se terapia gnica como a capacidade do melhoramento gentico por meio da correo de genes alterados (mutados) ou modificaes stio-especficas, que tenham como alvo o tratamento teraputico. Este tipo de procedimento tornou-se possvel por conta dos avanos da gentica e da bioengenharia, que permitiram a manipulao de vetores para a entrega do material extracromossomal em clulas-alvo. Um dos principais focos desta tcnica a otimizao dos veculos de entrega (vetores) que, em sua maioria, so plasmdeos, nanoestruturados ou vrus sendo estes ltimos os mais estudados, devido sua excelncia em invadir as clulas e inserir seu material gentico. No entanto, existe grande preocupao referente s respostas imunes exacerbadas e manipulao do genoma, principalmente em linhagens germinativas. Estudos em clulas somticas in vivo apresentaram resultados satisfatrios, e j existem protocolos aprovados para uso clnico. Os principais trials tm sido conduzidos nos Estados Unidos, Europa, Austrlia e China. Recentes avanos biotecnolgicos empregados para o aprimoramento da terapia gnica, como clulas-tronco pluripotentes induzidas em pacientes portadores de doenas hepticas, imunoterapia com clulas T do receptor do antgeno quimera e edio genmica pelos sistema CRISPR/Cas9, so abordados nesta reviso.

Keywords: Terapia gnica, Vetores genticos, Transferncia gentica horizontal, CRISPR-Cas9, CAR-T cell, Terapia gentica, Repeties palindrmicas curtas agrupadas e regularmente espaadas

In 1991, James Watson declared that many people say they are worried about the changes in our genetic instructions. But these (genetic instructions) are merely a product of evolution, shaped so we can adapt to certain conditions which might no longer exist. We all know how imperfect we are. Why not become a little better apt to survive?.(1) Since the beginning, humans understand that the peculiar characteristics of the parents can be transmitted to their descendents. The first speculation originated from the ancient Greek students, and some of these theories continued for many centuries. Genetic-scientific studies initiated in the early 1850s, when the Austrian monk, Gregor Mendel, in a series of experiments with green peas, described the inheritance pattern by observing the traces that were inherited as separate units, which we know today as genes. Up until 1950, little was known as to the physical nature of genes, which was when the American biochemist, James Watson, and the British biophysicist, Francis Crick, developed the revolutionary model of the double strand DNA. In 1970, researchers discovered a series of enzymes that enabled the separation of the genes in predetermined sites along the DNA molecule and their reinsertion in a reproducible manner. These genetic advances prepared the scenario for the emergence of genetic engineering with the production of new drugs and antibodies, and as of 1980, gene therapy has been incorporated by scientists.(2,3)

In this review, we cover gene therapy, the different methodologies of genetic engineering used for this technique, its limitations, applications, and perspectives.

The ability to make local modificiations in the human genome has been the objective of Medicine since the knowledge of DNA as the basic unit of heredity. Gene therapy is understood as the capacity for gene improvement by means of the correction of altered (mutated) genes or site-specific modifications that have therapeutic treatment as target. Further on, diffrent strategies are described, which are often used for this purpose.(4)

Currently, gene therapy is an area that exists predominantly in research laboratories, and its application is still experimental.(5) Most trials are conducted in the United States, Europe, and Australia. The approach is broad, with potential treatment of diseases caused by recessive gene disorders (cystic fibrosis, hemophilia, muscular dystrophy, and sickle cell anemia), acquired genetic diseases such as cancer, and certain viral infections, such as AIDS.(3,6)

One of the most often used techniques consists of recombinant DNA technology, in which the gene of interest or healthy gene is inserted into a vector, which can be a plasmidial, nanoestrutured, or viral; the latter is the most often used due to its efficiency in invading cells and introducing its genetic material. On , a few gene therapy protocols are summarized, approved and published for clinical use, exemplifying the disease, the target, and the type of vector used.(3)

Gene therapy protocols

Although several protocols have been successful, the gene therapy process remains complex, and many techniques need new developments. The specific body cells that need treatment should be identified and accessible. A way to effectively distribute the gene copies to the cells must be available, and the diseases and their strict genetic bonds need to be completely understood.(3) There is also the important issue of the target cell type of gene therapy that currently is subdivided into two large groups: gene therapy of the germline(7) and gene therapy of somatic cells.(8) In germline gene therapy, the stem cells, e.g., with the sperm and egg, are modified by the introduction of functional genes, which are integrated into the genome. The modifications are hereditary and pass on to subsequent generations. In theory, this approach should be highly effective in the fight against genetic and hereditary diseases. Somatic cell gene therapy is when therapeutic genes are transferred to a patients somatic cells. Any modification and any effects are restricted only to that patient and are not inherited by future generations.

In gene therapy, a normal gene is inserted into the genome to replace an abnormal gene responsible for causing a certain disease. Of the various challenges involved in the process, one of the most significant is the difficulty in releasing the gene into the stem cell. Thus, a molecular carrier called a vector is used to release the gene, which needs to be very specific, display efficiency in the release of one or more genes of the sizes necessary for clinical applications, not be recognized by the immune system, and be purified in large quantities and high concentrations so that it can be produced and made available on a large scale. Once the vector is inserted into the patient, it cannot induce allergic reactions or inflammatory process; it should increase the normal functions, correct deficiencies, or inhibit deleterious activities. Furthermore, it should be safe not only for the patient, but also for the environment and for the professionals who manipulate it. Finally, the vector should be capable to express the gene, in general, for the patients entire life.(3,9)

Although the efficacy of viral vectors is confirmed, recently some studies demonstrated that the use of these carriers presented with several limitations. The presence of viral genetic material in the plasmid is a strong aggravating factor, since it can induce an acute immune response, besides a possible oncogenic transformation. Currently, there are two main approaches for genetic modifications of the cells, namely: virus-mediated () and via physical mechanisms, from preparations obtained by advanced nanotechnology techniques.(5) Within this context, included are polymers that form networks that capture a gene and release its load when they penetrate the cells, such as DNA microinjections,(10) cationic polymers,(11) cationic liposomes,(12,13) and particle bombardment.(14)

Viral vectors for gene therapy

Each exogenous material introduction technique differs from the other and depends on the type of application proposed. Some are more efficient, others more apt to carry large genes (>10kB) and integrate with the genome, allowing a permanent expression.(1)

Hematopoietic stem cells have become ideal targets for gene transfer due to the high potential for longevity and the capacity for self-renovation. One example of this combination of gene therapy and stem cells would be the production of gene transfer vectors for the creation of induced pluripotent stem cells (iPS), in order to generate the differentiation of the iPS and afford an additional phenotype from this differentiated derived cell. Patients with chronic liver disease and infection by the hepatitis virus (e.g., hepatitis B virus and hepatitis C virus), which require a liver transplant, may be likely to undergo the hepatic transplantation of mature hepatocytes or those derived from iPS.(15) Not only the transfer of genes might be needed to convert stem cells into hepatocytes; since the transplanted cells are susceptible to reinfection by the hepatitis virus, the transfer of a vector that encodes a short hairpin RNA directed against the virus would provide the transferred cells with resistance or immunity to reinfection. Resistant cells can repopulate the liver over time and restore normal hepatic function ().(15)

Combination of stem cells and gene therapy

shRA: short hairpin RNA; iPS: induced pluripotent stem cells.

Chimeric antigen recipient T (CAR-T) cell therapy is a type of immunotherapy that involves manipulation/reprogramming of immune cells (T lymphocytes) of the patients themselves, in order to recognize and attack the tumor T cells. Initial advancement in the design of the first CAR generation, by Eshhar et al.,(16) was marked by the fusion of a single chain fragment variable (scFv) to a transmembrane domain and an intracellular signaling unit: chain CD3 zeta.(17,18) This design combined the active element of a well-characterized monoclonal antibody with a signaling domain, increasing the recognition of the tumor-specific epitope and the activation of T cells, without depending on molecules from the histocompatibility complex.

An improvement in the first generation of CAR was made by means of integrating co-stimulating molecules necessary for signal transduction. The stimulatory recipient most commonly used in this CAR generation is CD28. This recipient acts as a second activating event of the route, enabling a marked proliferation of T cells along with an increased expression of cytokines.(19)

The most recent generation of CAR incorporated the addition of a co-stimulatory domain addition to increase the CAR function. Co-stimulatory molecules as recipients of the tumor necrosis factor (CD134 or CD137) are required for this methodology. In summary, the most recent forms of CAR include scFv, the initial chain of CD3-, along with the stimulatory chains of CD28 and CD134 or CD137.(20)

With the third CAR generation, Zhong et al., demonstrated an improvement in T cell activation of the Akt route (protein kinase B), which regulates the cell cycle. According to other studies, this last generation shows greater persistence of the T cells in comparison with the second generation of CAR.(21)

The most critical point of the adverse effects of CAR-T therapy is the identification of non-tumor cells that express the target epitope by CAR. Tumor antigens are molecules highly expressed in the tumor cells, but are not exclusive of these cells. For example, the CD19 antigen can be found in normal or malignant B cells, and the CAR design for the CD19 target in not capable of distinguishing them.(20,22) Other common toxicity for CAR-T therapy (and many other types of immunotherapy for cancer) is the cytokine release syndrome (CRS). Activation of the immune system after CAR-T infusion can induce a rapid increase in the levels of inflammatory cytokines.(20,23)

New developments in the design of vectors and trials with CAR-T provide balance and reinforcement in safety for amplification of the clinical application. The progressive improvement in the CAR trials has already advanced, as was observed from the first to the third generation. Knowledge and experience acquired in the assessment of CAR-T toxicity will increase the success of the progressive improvements for future trials.

During the 1980s, in the genome of Escherichia coli, a region was identified with an uncommon pattern, in which a highly variable sequence was intercalated by a repeated sequence with no known function. In 2005, it was assumed that the variable sequences were of extra-chromosomal origin, acting as an immune memory against phages and plasmids, starting the then unknown CRISPR system (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (Associated Proteins), that shines since 2012 as one of the primary biotechnological tools for gene edition.(24) Originating in the immune-adaptive system of procaryontes, this mechanism recognizes the invading genetic mateiral, cleaves it into small fragments, and integrates it into its own DNA. In a second infection by the same agent, the following sequence occurs: transcription of the CRISPR locus, RNAm processing, and creation of small fragments of RNA (crRNAs) that form complexes with the Cas proteins, and these recognize the alien nucleic acids and finally destroy them.(24)

Based on this natural mechanism, the CRIPSR technique was developed enabling editing of the target-specific DNA sequences of the genome of any organism by means of basically three molecules: nuclease (Cas9), responsible for cleavage of the double-strand DNA; an RNA guide, which guides the complex to the target; and the target DNA, as is shown in .(25,26)

CRISPR Cas-9 system. The technique involves basically three molecules: one nuclease (generally wild type Cas-9 of Streptococcus pyogenes), an RNA guide (known as single guide RNA), and the target (frequently the DNA)

Due to its simplicity and its precision when compared to other techniques (Zinc-Finger Nucleases, TALENs, and Gene Targeting), the CRISPR system arrives as a versitile tool that promotes the genetic editing by means of inactivation (knockout gene KO), integration of exogenous sequences (knock-in), and allele substitution, among others.(27,28)

The guide RNA hybridizes with the target DNA. Cas-9 recognizes this complex and should mediate cleavage of the DNA double strand and reparation in the presence of a (homologous) donor DNA. The result of this process is the integration of an exogenous sequence into the genome (knock-in) or allele substitution.

The rapid advancement of this new technology allowed the performance of translational trials in human somatic cells, using genetic editing by CRISPR. The first applications with a therapeutic focus already stood out in describing even the optimization steps of the delivery systems and specificity for the safety and efectiveness of the system.(28,29)

Researchers from the University of California and of Utah recently were successful in correcting the mutation of the hemoglobin gene, which originates sickle cell anemia. CD34+ cells from patients who are carriers of sickle cell anemia were isolated, edited by CRISPR-Cas9, and after 16 weeks, the results showed a reduction in the expression levels of the mutated gene and an increased gene expression of the wild type.(29)

The technology referred to is in use mainly in monogenic genetic pathologies, which, despite being rare, can reach about 10 thousand diseases already described.(4) Phase 1 clinical trials are foreseen for 2017, as well as the appearance of companies geared toward the clinical use of this system.

The possibility of genetically modifying germlines has been the object of heated discussion in the field of science for a long time. Bioethics is always present when new techniques are created, in order to assess the risks of the procedure and the moral implications involved.

A large part of the scientific community approves genetic therapy in somatic cells, especially in cases of severe disorders, such as cystic fibrosis and Duchenne muscular dystrophy.

In 2015, however, Chinese researchers went beyond the moral issues and announced, for the first time, the genetic modification of embryonic cells using the CRISPR-Cas9 technique. Next, another Chinese group also reported the conduction of the same process done with the intention of conferring resistance to HIV by insertion of the CCR5 gene mutation. The genetic analysis showed that 4 of the 26 embryos were successfully modified. The result clearly reveals the need for improving the technique, alerting that, possibly, such trials could be previously tested in animal models.(4,30)

These recent publications rekindled the debate regarding genetic editing. On one side, the Japanese Ethics Committee declared that the manner in which the experiment was conducted was correct, since there had been approval by the local Ethics Committee for the study conducted, as well as the consent of the egg donors. In the United Kingdom, the first project for healthy human embryo editing was approved. On the other hand, American research groups remained conservative, reiterating their position of not supporting this type of experiment and declaring that they await improvement in the techniques and of the definitions of ethical issues.(30)

Since the declaration of James Watson in 1991, in reference to the likely optimization of human genetics, gene therapy has advanced throughout the decades, whether by optimization of the types of vectors, by the introduction of new techniques, such as induced pluripotent stem cells in combination with current models of genetic editing (CRISPR-Cas9), and even by trials in germ cells, bringing with it the contradictory ethical and moral aspects that accompany the technique.

Local successes have already solidified the viability of treatments using gene therapy in clinical practice, as an alternative form for patients with congenital diseases or monogenic disorders and cancer, especially when the pharmacological or surgical interventions do not show good results.

The design of new experimental vectors, the increase in efficiency, the specificity of the delivery systems, and the greater understanding of the inflammatory response induction may balance the improvement of safety with the expansion of techniques in clinical applications. Yet the knowledge and experience acquired with the careful assessment of toxicity of these technologies also allow significant advances in the application of these methods.

Therefore, historically, gene therapy and the discovery of antibiotics and chemotherapy agents, or any new technology, need more clarifying preclinical studies. In the future, there is the promise of applying these techniques in several fields of Medicine and a greater percentage of clinical trials.

See the original post:
Gene therapy: advances, challenges and perspectives - PMC

Genes are small segments of DNA that instruct your cells to make certain proteins when specific conditions are met.

Mutated genes, on the other hand, may cause your cells to make too much or too little of the necessary protein. Even small changes can have a domino effect across your body just as tiny changes in computer code can affect an entire program.

Viral vectors

Scientists dont have tweezers small enough to edit your DNA by hand. Instead, they recruit a surprising ally to work on their behalf: viruses.

Typically, a virus would enter your cells and alter your DNA to create more copies of itself. But scientists can switch out this programming with their own, hijacking the virus to heal instead of harm. These vectors, as theyre called, dont have the parts they need to cause disease, so they cant make you sick the way a regular virus could.

There are two types of gene therapy:

Gene therapy is different from genetic engineering, which means changing otherwise healthy DNA for the purpose of enhancing specific traits. Hypothetically, genetic engineering could potentially reduce a childs risk of certain diseases or change the color of their eyes. But the practice remains highly controversial since it hovers very close to eugenics.

Gene therapy may be used to treat a variety of genetic conditions, including:

Inherited vision loss

When the RPE65 gene in your retinas doesnt work, your eyeballs cant convert light to electrical signals.

The gene therapy Luxturna, approved by the Food and Drug Administration (FDA) in 2017, can deliver a functional replacement of the RPE64 gene to your retinal cells.

Blood disorders

The FDA-approved Hemgenix can treat the bleeding disorder hemophilia B. The viral vector instructs your liver cells to create more of the factor IX protein, which helps your blood clot.

Meanwhile, the gene therapy Zynteglo, approved by the FDA in 2022, treats beta-thalassemia by giving your bone marrow stem cells correct instructions for creating hemoglobin.

This blood disorder can lower the oxygen in your body because it decreases your bodys hemoglobin production.

Spinal muscular atrophy (SMA)

In infantile-onset SMA, an infants body cant make enough of the survival of motor neuron (SMN) proteins necessary to build and repair motor neurons. Without these neurons, infants gradually lose their ability to move and breathe.

The gene therapy Zolgensma, approved by the FDA in 2019, replaces faulty SMN1 genes in an infants motor cells with genes that can create enough SMN proteins.

Cerebral adrenoleukodystrophy (CALD)

Your ABCD1 gene produces an enzyme that breaks down fatty acids in your brain. If you have cerebral adrenoleukodystrophy, this gene is either broken or missing.

Skysona, FDA approved as of 2022, delivers a functional ABCD1 gene so that fatty acids dont build up and cause brain damage.

Cancers

Most cancer gene therapies work indirectly by inserting new genes into a powerful antibody called a T cell. Your changed T cells can then latch on to cancerous cells and eliminate them, similar to how they attack viruses.

Some people considering gene therapy may feel uneasy about putting viruses in their body.

Keep in mind, though, that gene therapies undergo extensive testing before approval. The viruses in gene therapies are also fixed so they cant replicate similar to many vaccines.

That said, gene therapies may pose other risks:

Despite these issues, experts generally believe gene therapy offers more benefits than risks.

Most of the conditions treated with gene therapy are life threatening. The dangers of leaving them untreated often outweigh the risks of potential side effects.

Gene therapy does come with a few drawbacks that keep it from becoming a widespread treatment.

Limited targets

Gene therapy can only target certain mutations. This means it may not work for everyone with a specific condition.

For example, two people may have inherited vision loss. Currently, gene therapy can only treat vision loss caused by the RPE64 mutation.

Time to approval

Because gene therapy research is so new, experts do extensive safety testing before introducing their treatments to the public. It can take years to get FDA approval for each new therapy.

Expense

As you might imagine, gene therapies are expensive to manufacture and administer. This not only affects funding for clinical trials but also the price of the drug.

For example, the gene therapy Zolgensma is the most expensive drug in the United States at $2.1 million per dose. Even with insurance, that kind of price tag remains out of reach for the average American.

Scientists are trying to find ways to make the development process safer, cheaper, and more efficient so more people can access gene therapy.

Gene therapy works to treat several different genetic diseases by editing the mutations that cause them. As researchers further refine and expand this technology, they may find even more conditions that could be treated with it.

Experts are also continuing to explore options to make gene therapy more affordable so people who need these treatments have an easier time getting them.

Emily Swaim is a freelance health writer and editor who specializes in psychology. She has a BA in English from Kenyon College and an MFA in writing from California College of the Arts. In 2021, she received her Board of Editors in Life Sciences (BELS) certification. You can find more of her work on GoodTherapy, Verywell, Investopedia, Vox, and Insider. Find her on Twitter and LinkedIn.

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How Does Gene Therapy Work? Types, Uses, Safety - Healthline

Important:The authors, reviewers, and editors of this material have made extensive efforts to ensure that treatments, drugs, and discussions about medical practice are accurate and conform to the standards accepted at the time of publication. However, constant changes in information resulting from continuing research and clinical experience, reasonable differences in opinions among authorities, unique aspects of individual clinical situations, and the possibility of human error in preparing such an extensive text mean that other sources of medical information may differ from the information on this site. The information on this site is not intended to be professional advice and is not intended to replace personal consultation with a qualified physician, pharmacist, or other health care professional. The reader should not disregard medical advice or delay seeking it because of something found on this site.

Content in the Manuals reflects medical practice and information in the United States. Outside of the United States, clinical guidelines, practice standards, and professional opinion may differ and the reader is advised to also consult local medical sources.Please note, not all content that is available in English is available in every language.

Read the rest here:
Merck Veterinary Manual

Clin Transl Sci. 2020 May; 13(3): 440450.

1Division of Cardiac Surgery, University of Ottawa Heart Institute, OttawaOntario, Canada

2School of Human Kinetics, University of Ottawa, OttawaCanada

1Division of Cardiac Surgery, University of Ottawa Heart Institute, OttawaOntario, Canada

3Department of Cellular & Molecular Medicine, University of Ottawa, OttawaCanada

1Division of Cardiac Surgery, University of Ottawa Heart Institute, OttawaOntario, Canada

2School of Human Kinetics, University of Ottawa, OttawaCanada

3Department of Cellular & Molecular Medicine, University of Ottawa, OttawaCanada

Received 2019 Nov 6; Accepted 2019 Nov 7.

Despite regenerative medicine (RM) being one of the hottest topics in biotechnology for the past 3decades, it is generally acknowledged that the fields performance at the bedside has been somewhat disappointing. This may be linked to the novelty of these technologies and their disruptive nature, which has brought an increasing level of complexity to translation. Therefore, we look at how the historical development of the RM field has changed the translational strategy. Specifically, we explore how the pursuit of such novel regenerative therapies has changed the way experts aim to translate their ideas into clinical applications, and then identify areas that need to be corrected or reinforced in order for these therapies to eventually be incorporated into the standardofcare. This is then linked to a discussion of the preclinical and postclinical challenges remaining today, which offer insights that can contribute to the future progression of RM.

In 1954, Dr. Joseph Murray performed the first transplant in a human when he transferred a kidney from one identical twin to another.1 This successful procedure, which would go on to have a profound impact on medical history, was the culmination of >50years of transplantation and grafting research. In the following years, organ replacement became more widespread but also led to a plateau in terms of landmark successes.1 The technology was working, but limitations were already being encountered; the most prominent of them being the lack of organ availability and the increasing need from the aging population.2 During the same time period, chronic diseases were on the rise and the associated process of tissue degeneration was becoming evident. Additionally, the available clinical interventions were merely capable of treating symptoms, rather than curing the disease, and, therefore, once a loss of tissue function occurred, it was nearly impossible to regain.3 Overall, the coupling of all these factors that took place in the 1960s and 1970s created urgency for disruptive technologies and led to the creation of tissue engineering (TE).

TE can be described as a field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ.4 TE is considered to be under the umbrella of regenerative medicine (RM) and, according to Dr. Heather Greenwood et al., regenerative medicine is an emerging interdisciplinary field of research and clinical applications focused on the repair, replacement or regeneration of cells, tissues or organs to restore impaired function resulting from any cause, including congenital defects, diseases, trauma and aging.5 It uses a combination of technological approaches that moves it beyond traditional transplantation and replacement therapies. These approaches may include, but are not limited to, the use of soluble molecules, gene therapy, stem cell transplantation, tissue engineering, and the reprogramming of cell and tissue types.3, 6, 7 A summary of the recent history of RM is presented in Figure.

A summary timeline of the recent history of regenerative medicine (RM). Selected milestones in the development of RM are presented starting from the 1950s all the way up to the present day.

Although RM may have seemed novel, the principles of regeneration are as old as humanity and are found in its many cultures.8 A common example used is the tale of Prometheus that appeared in 8th century BCE. Prometheus, an immortal Titan in Greek mythology, stole fire and gave it to humanity for them to use, defying the gods in consequence. As punishment, Zeus decreed that he was to be bound to a rock where an eagle would feast on his liver every day and said liver would regenerate itself every night, leading to a continuous loop of torture.9 RM came about at the time it did, not only because of the combining factors mentioned above, but also because researchers had been successfully keeping tissue alive in vitro and understanding the biological processes involved in regeneration and degeneration. Consequently, possible therapeutic outcomes came into fruition. Since the arrival of TE and RM, strides made on the benchside have been ever increasing with now >280,000 search results on PubMed relating to regeneration. Discoveries and advances made by cell/molecular biologists, engineers, clinicians, and many more led to a paradigm shift from treatmentbased to curebased therapies.10 In addition to Greenwoods definition, RMs arsenal now contains controlled release matrices, scaffolds, and bioreactors.5, 8 Despite this impressive profile on the benchside, RM has so far underperformed in terms of clinical applications (i.e., poor therapy translation).8 Simply put, a disappointing number of discoveries are making it through clinical trials and onto the market.11 Although some experts say that the field is reaching a critical mass in terms of potential therapies and that we will soon see results, others, like Dr. Harper Jr. from the Mayo Clinic in Minnesota, say that the transformative power of RM is well recognized, but the complexity of translating isnt.7, 8, 12

This brings us to the subject matter of the present paper: RM and translation. The goals of this historical review are twofold. The first is to understand how RM, over the past 50years or so, has changed the way discoveries/new technologies are transferred to the clinic. How has the translational strategy changed in response to these new therapies? The second is to identify challenges that have led to RMs modest performance on the bedside. Some articles have already documented these but have focused on the clinical and postclinical factors, and whereas they will be briefly discussed here, the focus will be on preclinical factors.13 To accomplish these objectives, we will begin by summarizing the historical development of RM (which has been extensively documented by other works2, 3, 14, 15), followed by a detailed look at the definition of translational medicine (TM). With this background information established, we then look at the various preclinical and clinical impacts of RM on TM, as well as some of its effects on the private sector. Limiting factors of the field are then described, again focusing on those that are preclinical. This endeavor was initiated via a librarianassisted literature search for original research and historical documentation of the field of RM and other related subjects. The documents were then screened for relevance and the analyzed information was categorized into the themes discussed below. Conclusions were then drawn based on the interplay among these themes.

As mentioned, the idea of regeneration first started in myths and legends. This is logical because, as Drs. Himanshu Kaul and Yiannis Ventikos put it, myths shape ideas, and ideas then shape technologies.8 In addition to the tale of Prometheus, there are many others. For example, there is the Hindu myth of Raktabeej whose blood drops could each form a clone of himself, or the Indian story of the birth of the Kaurava brothers where pieces of flesh were grown in pots and treated with herbs to grow fullsized humans.8 The idea of regeneration has persisted throughout history and started to become a possibility in the early 1900s when scientists like Alexis Carrel (who invented the technique of cell culture) were finally able to keep cells and tissues alive outside of the body. This allowed them to study the mechanisms of cell renewal, regulation, and repair.8 In addition, studying regeneration goes handinhand with developmental biology. Seminal work in experimental embryology began in the 1820s with the detailed description of the differentiation of embryonic germ layers.16 An increased understanding of basic embryological mechanisms led to Hans Spemanns Nobel Prize for his theory of embryonic induction; a field that was further elaborated by his students and others, advancing it toward the possibility of cloning and demonstrating how development and regeneration are intimately linked.16 Before this era, the study of regeneration was done through the study of animals, with scientists studying the phenomena in serpents, snails, and crustaceans, for example.17, 18 However, the modern study of regeneration is said to have started with Abraham Trembleys study of the hydra, which showed that it was possible for an entire organism to regenerate from its cut appendage.19 The 18th century on through to the 19th century is also when scientists became intrigued by the amphibian newts and axolotls for their astonishing regenerative capabilities, which are still used today as the gold standard models for studying regeneration along with certain fish, such as the zebrafish.20

Now, although the term RM as we know it today would only be coined in 1999 by William Haseltine, the field itself started in the late 1970s in the form of TE (pioneered by Drs. Joseph Vacanti and Robert Langer) in the city of Boston.2, 14, 21 To address the need for novel therapies, biomedical engineers, material scientists, and biologists at Harvard and MIT started working on regenerating parts of the largest and simplest organ of the human body: the skin. In 1979, the first cellbased TE product appeared and was named Epicel.15 Developed by Dr. Howard Green et al., this technology consisted of isolating keratinocytes from a skin biopsy and having them proliferate outside of the body to make cell sheets that were then used as an autologous treatment for burn patients.15 Another famous product (this time allogeneic), developed in 1981, was Apligraf, a composite skin invention capable of rebuilding both the dermis and epidermis of skin wounds.15 With these two therapies and many more being created, TE in the 1980s was booming. At the time, researchers were also developing therapies for cartilage regeneration.

Once the 1990s came around, TE strategies were combined with stem cells (which had just been discovered) to create RM.3, 8 At that time, RM was a hot topic. After the first products for skin were commercialized, scientists became more enthused and started trying other tissues.15 Startup companies were popping up left and right, private funding was abnormally high, and public hype was gaining lots of traction. However, governments were not so quick to fund this research and took their time before making decisions, whereas private investors saw this field as very promising and thought it was their ticket to the top.14 Given that 90% of the funding of RM came from the private sector, this greatly influenced the direction of the research and its timeframe.14 People were simply trying to copy tissue formation rather than understanding it, so as to make the development process quicker.3 As a result, many of the technologies that initially looked promising failed in clinical trials or on the market.

These disappointing results coupled with the dot.com crash meant that by the end of 2002, the capital value of the industry was reduced by 90%, the workforce by 80%, and out of the 20 US Food and Drug Administration (FDA) products with clinical trials, only 4 were approved and none had any success.22 This phenomenon has been extensively studied and, according to Lysaght and Hazlehurst, five factors contributed to the industry crash22:

The products were not much better than the existing treatment options and so making the switch was not worth it for clinicians.

Even if the science was good, lowcost manufacturing procedures did not exist.

The approval process for these novel therapies was unrealistically challenging and the regulatory cost was too high.

Companies lacked the skill to market their new products.

The reimbursement strategies were unclear.

Despite these events, the industry had 89 firms survive the crash and stem cell research was not affected. In fact, from 2000 to 2004, the number of companies increased but the number of jobs decreased, which means investors were supporting research in basic and applied science with smaller firms that were lower risk, and by 2004, the field was dominated by startup companies.22 Before the crash, RM was primarily happening in the United States, but in 2004, other countries like the United Kingdom and Japan started catching up.22 The industry slowly started growing again. In 2006, the first engineered tissue (bladder) was implanted, and by 2008, commercial successes were being achieved.3, 10 As an example, hematopoietic stem cell transplants were approved and are now a curative treatment for blood disorders and other immunodeficiencies.7 Now, the RM field had ironically regenerated itself.3 It has gained increased governmental attention (federal funding has increased) and has been recognized as being at the forefront of health care.7, 22 There is once again intense media coverage that is raising public expectations.23 The number and variety of clinical trials is also increasing everywhere.23 According to allied market research, RM is predicted to be worth US $67.5 billion by 2020.10

Unfortunately, regardless of these seemingly cheerful notes, the fact remains that cell therapies remain experimental, except for the aforementioned hematopoietic stem cell treatments.13 The market for RM is still small and will remain so until RM proves that its therapies are better and cheaper than the existing ones.15 Yet, the pressure for clinical translation is increasing through the needs of the population, investors that are eager to make a return on their investments, and scientists who believe that these technologies are the future.23 Moreover, there has been a growing appreciation of the magnitude and complexity of the obstacles the field is facing, but it remains to be seen how they will be solved; although initial steps have already been taken, which will be discussed further below.

Now that we have established the background for RM, there needs to be a proper understanding of TM before conclusions on how the two are related can be drawn, which is the purpose of the following section.

The European Society for Translational Medicine (EUSTM) has defined TM as an interdisciplinary branch of the biomedical field supported by three main pillars: benchside, bedside, and community. The goals of TM are to combine disciplines, resources, expertise, and techniques within these pillars to promote enhancements in prevention, diagnosis, and therapies.24 TMs goals can be split into two categories: T1 and T2. T1 is to apply research from bench to bedside and back, whereas T2 is to help move successful new therapies from a research context to an everyday clinical context.25 In other words, TM is a medical practice explicitly devoted to helping basic research attain clinical application. Conceptual medical research, preclinical studies, clinical trials, and implementation of research findings are all included within TM.26

Between basic science and the clinic is an area that is popularly referred to as the valley of death.25 This gap is fraught with not only scientific obstacles (like an unknown molecular mechanism), but social and economic ones as well. This is where many novel ideas die and, consequently, companies are weary of going through this valley for fear of wasted financial resources.25 For these reasons, many of the approved drugs we get now are derivatives of others that have been previously approved.25 This is the area that TM seeks to impact, to be the bridge between idea and cure, and to act as a catalyst to increase the efficiency between laboratory and clinic.25, 26 The term bench to bedside and back is commonly used. The cost of development for a therapy is very high (estimated at US $800 million to $2.6 billion for a drug) because of increasing regulatory demands and the complexity of clinical trials, among others. TM aims to streamline the early development stages to reduce the time and cost of development.24

What will be important to note for the discussion below is that TM focuses more on the pathophysiological mechanisms of a disease and/or treatment and favors a more trialanderror method rather than an evidencebased method. Dr. Miriam Solomon argues in his book chapter entitled What is Translational Medicine? that most medical innovations proceed unpredictably with interdisciplinary teams and with shifts from laboratory to patient and back again, and that freedom of trialanderror is what will lead to more therapeutic translation.25 Furthermore, for years, TM did not have any technical suggestions for improving translation, only two broad categories that were claimed to be essential for translatability: improving research infrastructure and broadening the goals of inquiry. This discrepancy has since been identified and efforts have been made to address it. For example, the EUSTM provided a textbook called Translational Medicine: Tools and Techniques as an initiative to provide concise knowledge to the fields stakeholders.24

Presently, TM has attracted considerable attention with substantial funding and numerous institutions and journals committed to its cause.25, 27 But before this, its arrival had to be incited. TM emerged in the late 1990s to offer hope in response to the shortcomings of evidencebased medicine and basic science research, such as the unsatisfactory results from the Human Genome Project, for instance.25 There were growing concerns that the explosion of biomedical research was not being translated in a meaningful manner proportionate with the expenditures and growing needs of the patients.27 The research had ignored what it took to properly disseminate new ideas.25 The difficulties of translation from bench to bedside have always been known, but what is different with TM is the amount of emphasis that is now put on translation and the recognition on how difficult and multifaceted it is to translate technologies.25 Over the past 20years, the role, power, and research volume of the field has increased, and TM is now a top priority for the scientific community.26 TM is also often used as common justification for research funding and conveys the message to politicians and taxpayers that research activities ultimately serve the public, which is also why it appeals to todays generation of students who want to work on big, realworld problems and make a meaningful difference.28, 29

As already mentioned, RM therapies are proving difficult to translate to the clinic.11 Although the basic research discoveries are never ceasing (books such as NewPerspectives in Regeneration by Drs. HeberKatz and Stocum30, and articles such as "Tissue Engineering and Regenerative Medicine: Past, Present, and Future" by Dr. Antnio Salgado et al.,31 provide comprehensive summaries of these advancements), therapy approval is practically nonexistent.30, 31, 32 This may be due, in part, to a tendency for people to blame the lack of translation of their technologies on extrinsic factors, thus removing responsibility.11 Additionally, the failures are not being studied. For example, stem cell research looks good in small animals but often fails in larger ones and then does not progress beyond phase II or III clinical trials because no benefits are found, and historically we have not been exploring why.11, 32 Consequently, the next therapies that are developed are improved by guesses rather than through a better understanding of the disease in mind (Figure).11

The negative feedback cycle currently present in most discovery and development processes of regenerative medicine. This cycle obstructs progression of the field.

RM has the potential to impact not only the quality of healthcare but also the economy, because the costs that could be avoided with curative therapies are immense.33 For this reason, analyzing the impact of RM on the translational strategy over time can help identify aspects that should be encouraged or discouraged to drastically improve translation. Reflecting on this history cannot only help us to avoid past mistakes but can also aid in redirecting the field to a onceproductive path.34 In the following section, the preclinical impact of RM on TM will be discussed, focusing on the shift from evidencebased medicine to trialanderror, the role of the basic scientist, and the emergence of the multidisciplinary approach. Clinical impact is also covered, concentrating on regulatory modifications. Last, changes in the private sector are considered as the shift in business models is detailed.

Because the RM field is essentially comprised of new ideas on cell renewal and tissue healing, it is logical that most of its impact would be on the preclinical side, as this is where ideas are tested, finetuned, and developed. Coincidentally, it is also where the translational strategy begins. Considering certain aspects early in the developmental process, such as realistic applications and ease of use, can help facilitate translation. RMs influence on TM can thus be separated into the three themes below.

Before the late 20th century, the majority of medical research was done using evidencebased medicine. This is a systematic approach to solving a clinical problem that integrates the best available research evidence together with clinical signs, patient values, and individual clinical experience all to support scientific decision making and research progression.35 As such, evidencebased medicine favors clinical trials and does not allow for much tinkering and only that which possesses highquality clinical evidence is to be pursued. This has its limitations, as it devalues mechanistic reasoning, and both in vitro and animal studies. Therefore, evidencebased medicine may have played a role in RMs downfall in the early 2000s. TE in the 1990s was using evidencebased medicine and was simply trying to copy tissue formation rather than trying to understand it.3 That most of the funding was coming from the private sector probably did not help either. Investors saw TE as an opportunity for quick returns on their investments, so therapies were rushed to clinical trials, which led to inconsistent results.14, 25, 32

As well, evidencebased medicine obscured the need for different methods of discovery. After RMs decline and the idea of TM came about, a trialanderror method was adopted. This technique favors a team effort, mechanistic reasoning, and seeks to change the social structure of research.25 Although clinical trials are still deemed important, the trialanderror method identifies that an idea needs to first be explored and should not necessarily require the confirmation of a hypothesis.11, 25 This new method is based more so on facts and has stimulated a more informed dialogue among stakeholders (whereas the confirmation or refusal of a hypothesis cannot always be made relevant to people outside the field). This, in turn, can help the regulatory agencies reduce the burden on their review boards in the evaluation and acceptance of novel strategies.11 Therefore, the failures of RM had helped to highlight the boundaries of evidencebased medicine and, combined with the rising intensity put on TM in the 1990s, assisted in defining the trialanderror based method.

Another thing that is changed with the historical development of RM has been the role of the basic scientist. Please see Figure for a summary of the differences between the traditional and modern scientist discussed in this review. Traditionally, basic scientists have worked with a discovery mindset, but without a noticeable regard for potential therapeutic applications. It has been noted that RM has made us realize how important it is to take the practical and industrializing aspects (like cost, for example) into account even at the basic research level.7, 14 The needs of the end users need to be considered during the developmental phase if RM is to establish a proper foothold within the market.15 In view of this, over the past 2decades, medical philosophy has changed in that it encourages basic scientists to communicate more with clinicians and vice versa. Experts like Barry Coller, MD, Vice President for Medical Affairs and PhysicianinChief at the Rockefeller University Medical Center, have identified various skills that a basic scientist must possess if translational research is to be improved.26, 28 Additionally, other researchers have commented that more and more basic scientists are motivated to have an impact on global health and this passion can be a source of inspiration that can help fuel interdisciplinary cooperation.28 Efforts have also been made to familiarize basic scientists with regulatory requirements. For example, the FDA publishes guide documents with recommendations on how to address these requirements.36 Despite this, much remains to be done, as there is still a lack of TM professionals and the current research environment hampers cooperation between experts (e.g., specialization is still encouraged, and achievement awards are individualized).26, 28

A comparison between the traditional and modern scientist. Although traditional scientists are more hypothesisdriven and rigid in terms of research methodology, if the concepts shown above are used, it can generate the modern scientist who is better suited for the translation of regenerative therapies. RM, regenerative medicine.

An additional point that can be argued is that because RM got basic scientists more involved in the translational process, this has consequently made them more realistic.37 As already mentioned, early RM therapies were comprised of complex cell therapies that were not fully understood. From 2004 onward, the field diversified to include research into simpler acellular products.38 Other avenues, such as induced pluripotent stem cells, endogenous repair, nanotechnology, and regenerative pharmacology, are also being explored.37, 39, 40, 41 Increasingly, experts are trying to spread this message; for instance, in the field of cardiology, Dr. Mark Sussman, a world renowned cardiac researcher, and his colleague Dr. Kathleen Broughton at the San Diego State University Heart Institute and the Integrated Regenerative Research Institute, recently stated that After over a decade of myocardial regenerative research studies, the initial optimism and enthusiasm that fueled rapid and widespread adoption of cellular therapies for heart failure has given way to more pragmatic, realistic, and achievable goals.9

The last preclinical impact of RM to be discussed is the arrival of the multidisciplinary approach. This now widespread notion identifies that to improve translation and accelerate technology development, it is better to have a team composed of experts from multiple disciplines, because the various backgrounds and schools of thought can be combined with each contributing to a project in a different way.25, 39 What has surely incited its evolution is that RM inherently requires contributions from biologists, chemists, engineers, and medical professionals. This need has led to the formation of institutions that house all the required expertise under the same roof (such centers have increased in number since 2003), which promotes more teamwork between laboratories and clinics.28 Dr. Jennifer Hobin et al.28 states that bringing dissimilar research expertise together in close proximity is the key to creating an environment that facilitates collaboration. In addition, it could be said that these collaborative environments help minimize the flaws of medical specialization, which occurred in the second half of the 19th century; where the ideological basis that the human body can be categorized combined with the rapid arrival of new medical technologies led to the specialization of medical practice, which, in turn, led to the segregation of medical professionals from each other and the patient.42 Coincidentally, if one recalls the definition of TM, it, along with the trialanderror based method, suggests that improved research infrastructures and team efforts can facilitate the translation of therapies.

We now look at the influence that RM has had on the clinical side of therapy development. Before the subject is discussed, it is important to note that the reason clinical research has been affected is because of the uniqueness of RM therapies. Their novelty does not fit within the current regulatory process or use in clinical trials, and although the latter has yet to adapt, the regulatory sector has attempted over the years to facilitate the journey from bench to bedside.7, 43, 44

Initially, when RM was in its infancy, its therapies were regulated by the criteria originally developed for drugs; and as we have seen, this was identified as a factor that led to its downfall. Now, in 2019, several regulatory changes have been implemented to rectify this. What has helped has been the input from other countries. As mentioned above, RM started in the United States, but after the crash, other countries like the United Kingdom and Japan caught up, and their less stringent regulatory procedures have allowed them to better adapt the framework for these new therapies.22 In 2007, the European Union passed the Advanced Therapy Products Regulation law, which defined regenerative therapies, categorized them, and provided them with separate regulatory criteria for advanced approval.13, 43 In 2014, public pressure and researcher demands led Japan to enact three new laws: the Regenerative Medicine Promotion Act, the Pharmaceuticals, Medical Devices, and Other Therapeutic Products Act, and the Act on the Safety of Regenerative Medicine. These unprecedented national policies now help therapies gain accelerated and conditional approval to better conduct clinical trials and to better meet the demands of the patients.7, 13, 44, 45 During this time, the United States has not stood idle. In 2012, the US Congress passed the FDA Safety and Innovation Act (FDASIA), which expanded its existing Accelerated Approval Pathway to include breakthrough therapies, a category created for new emerging technologies, including regenerative strategies.13, 46 Drs. Celia Witten, Richard McFarland, and Stephanie Simek provide a wellwritten overview on the efforts of the FDA to accommodate RM.36 By and large, it is safe to say that RM has spurred a drastic change in traditional regulatory pathways to not only better manage these novel therapies but also put more weight on efficient translation.

It is also important to discuss changes in the private sector because manufacturing and marketing is and will remain one of the greatest obstacles facing RM, and, once again, the novelty of the field is responsible. Although the bulk of the problems remain, there has nonetheless been a change in business strategies that is worth appreciating.

Throughout its history, RM research has been carried out by academic research institutions or small and mediumsized enterprises.23, 47 With this in mind, the business model used in the health industry varies depending on the type of company. The royalty model is the one primarily used by biotech companies.8, 14 Here, businesses will develop a therapy up to the clinical stage and then hand it off to a company with more resources (usually a pharmaceutical one) who can carry out the larger scale studies. With this model, biotech companies make money simply through royalties and this carries both pros and cons (Figure).

A comparison of both the royalty and integrated business models used by private companies in the biomedical industry. The pros and cons are listed with the assumption that they are for a startup company in regenerative medicine.

Because the market for regenerative therapies currently is not big enough for the royalty model, startups have had to shift to an integrated model where the discovery, development, approval, and manufacturing of a new therapy are all done internally (which is unusual for small startups).8 Using this strategy, the companies can reap all the rewards but obviously also assume all the risk.

The market for regenerative therapies has so far been small enough that smaller firms do not have to manufacture large quantities of their products (like they do in the pharmaceutical industry) and they can start making money in a quicker fashion.8 Whether the business model will change again as the market grows or if the original startups will grow in proportion remains to be seen.14 What is to be highlighted here is that those who seek to commercialize regenerative therapies have had to shift to an integrated business model (that was not previously the norm for smaller ventures), which has affected translation by letting them have more influence in determining how their therapy is being developed, marketed, and manufactured.

Having detailed RMs relationship with the translation strategy and the aspects that changed in conjunction with the fields development, the remainder of the review will summarize the challenges that are contributing to RMs modest performance in the clinic.

With increased funding and a growing number of committed institutions, many countries have become increasingly invested in RMs success. For example, the US Department of Health and Human Services recognizes RM as being at the forefront of healthcare.7 As well, the UK government has identified RM as a field in which they can become global leaders and that will generate significant economic returns.44 The literature indicates that RM is reaching a critical mass and is on the verge of a significant clinical transition. The optimism is as high as it has ever been and the rush to succeed with clinical trials is equally felt.23 However, the bottom line is that the clinical and market performance is still very poor. Being that a gold standard for treatment in RM remains elusive, clinicians are often illinformed about current applications, and studies on safety and efficacy are lacking.23, 44, 48, 49 The National Institute of Health estimates that 8090% of potential therapies run into problems during the preclinical phase.28 Naturally, scientists have offered various explanations for these results, such as deficiencies in translational science and poor research practices in the clinical sciences.50 Shockingly, in a 2004 analysis, 101 articles by basic scientists were found that clearly promised a product with major clinical application, and yet 20years later, only 5 were licensed and only 1 had a major impact.50 Therefore, it is easily deducible that many challenges still lie ahead. The perceived riskbenefit ratio remains high and, as a consequence, clinical trials have been proceeding with caution.13, 23, 33 Numerous reviews have been published on these challenges but with an emphasis on those relating to the clinical phase.11, 13, 22, 51 Although these will be summarized below, the present study highlights the identification and analysis of the preclinical challenges. Please see Figure for a summary of the preclinical and clinical obstacles discussed herein.

Summary of the preclinical and postclinical challenges discussed. Even though preclinical obstacles to the translation of regenerative medicine therapies are more elusive, they are just as significant as their counterparts.

To begin, a possible explanation for the preclinical obstacles being underrepresented in the literature is because of the pliability of the phase itself. Although the clinical phase is composed of numerous subphases and strict protocols, the preclinical research is much less structured with less oversight. Whereas rigorous scientific method is applied to the experiments themselves, which usually consist of in vitro followed by in vivo experiments, the basic scientist has more flexibility regarding experimental organization, structure, and backtracking; thus, making explicit challenges possibly harder to recognize.

Some researchers have nevertheless attempted to do so. For example, Dr. Jennifer Hobin et al. have identified three major risks associated with RM technologies as being tumorigenicity, immunogenicity, and risks involved with the implantation procedure.13 The first two relate to arguably the largest preclinical challenge, which have been identified as needing a better understanding of the mechanism of action.12 Although the difficulties of identifying a mechanism are appreciated in the scientific community, it is imperative that improvements in this area are made as it will affect application and manufacturing decisions. Hence, greater emphasis on identifying the mechanism of action(s) will need to be adopted by basic scientists who are looking to develop a technology.

Another significant preclinical challenge is the lack of translation streamlining for basic scientists. Although basic scientists have become more involved in the translational process and more pragmatic over the years, there is, in general, still a lack of incentive and available resources to help a scientist translate their research. Academic faculty members are given tenure and promotion based on funding success (grants) and intellectual contributions (publications).28 Thus, researchers who have received money to conduct research and publish their work on a promising new therapy might stop short of translation as there may be no additional recognizable accomplishment or motivation for such an endeavor. For example, Jennifer Hobin et al. described the case of Dr. Daria MochlyRosen at Stanford Universitys Translational Research Program, who sought help for an interesting idea for a heart rate regulation therapy.13 She was turned down by numerous companies that found the clinical challenges too daunting and her colleagues offered no support but rather discouraged her from pursuing the idea saying that it would not be worthwhile for her career.

Last, a very important preclinical challenge that has gained recognition over the past few years is the lack of appropriate preclinical testing models. It is often reported that novel therapies that do well in the laboratory but then fail in larger animal studies or clinical trials. This is partly due to a lack of mechanistic insight, but also because of a shortage of appropriate in vitro, in vivo, and ex vivo models.9, 36 With properly validated preclinical models, we would be better able to gauge the performance of novel therapies and predict their future clinical success, but instead we are misidentifying the potential of therapies. Notably, the lack of appropriate models also contributes to the difficulty in obtaining reliable data on the underlying mechanism(s) of action of RM therapies, as differences may exist between the preclinical and clinical settings.

As far as clinical challenges go, they are numerous. Stem cell trials in particular have received criticism from a perceived lack of rigor and controlled trials.23 Related to this, a potent point that has arisen over the past few years is the absence of longterm followup studies for clinical trials, which is clearly necessary to establish the safety and efficacy of these interventions.13, 33 Unfortunately, they are costly and they are timeconsuming. Efforts are nonetheless being made to overcome these obstacles. For example, in 2015, the Mayo Clinic released an RM buildout perspective offering a blueprint for the discovery, translation and application of regenerative medicine therapies for accelerated adoption into standard of care.7 Institutions, such as Canadas Center for Commercialization of Regenerative Medicine, have been launched to help researchers mitigate the risks of cell therapy development by offering technical as well as business services.12, 51 Experts are also stepping up; for example, Drs. Arnold Caplan and Michael West proposed a new regulatory pathway that incorporates large postmarket studies into clinical trials.33

In terms of manufacturing, it is difficult to engage industry because the necessary technology to produce RM therapies at an industrial level does not exist yet. Scaleout and automated production methods for the manufacturing of regenerative therapies are needed.7, 10, 12, 23, 52, 53 This challenge stems from the complexity and natural intrinsic variation of the biological components, which makes longterm stability difficult to achieve and increases manufacturing costs.13, 44 Now, if RM therapies could establish their superiority over conventional treatments, then this would potentially alleviate costs and increase the likelihood of being reimbursed, but it remains to be seen.13 A hot topic at the moment is the choice between autologous or allogeneicbased products, which would entail either a centralized or decentralized manufacturing model, respectively (although hybrid models have been proposed).7, 23, 54 Autologous products, being patientspecific, have the advantage of having smaller startup costs, simpler regulations, and point of care processing.47 As for allogeneic products, they are more suitable for an off the shelf product, for a scaleout model and quality controls can be applied in bulk.47, 54 Dr. Yves Bayon et al.51 provided a thorough description of this topic while simultaneously indicating areas that have been identified for improvement.

As mentioned above, regulatory challenges are what have been most addressed thus far through scientific and public pressure. Moving forward, the goal identified by expert thinktank sessions is to harmonize RMspecific regulations across agencies and countries.7, 36 Reimbursement is the last of the regulatory challenges to be considered. In order for RM treatments to become broadly available, reimbursement is a necessity and both public and private healthcare need to determine how the regulations will be modified for disruptive therapies coming down the pipeline.13, 23, 44

RM has had an undeniable influence on the process of bench to bedside research. Preclinically, it has helped identify the limitations of evidencebased medicine and contributed to the paradigm shift to the trialanderror method. Likewise, the field has changed its mindset and the basic scientist is adopting new responsibilities becoming more motivated, pragmatic, and involved in TM, rivaling researchers in the applied sciences. The multidisciplinary approach has also been promoted by RM over the years and institutions dedicated to fostering collaborative research in RM have increased in numbers. Clinically, regulatory pathways that were developed for drugs and biomedical devices, and which have been in place for decades, have been adapted to aid RMs disruptive technologies, leading to new guidelines that favor translation. In the private sector, the novel nature of RM therapies has led to startup companies using an alternative business model that provides them toptobottom authority over the development of their products and it is yet to be seen if the business strategy in place will be sufficient as the industry grows.

If the translation of RM therapies is to be improved, many of the challenges to be overcome lie in the early stages of therapy development, such as identifying the mechanism(s) of action, validating preclinical experimental models, and incentivizing translational research for basic scientists. In later stages, regulatory changes have been made, but much still needs to be addressed. This includes the adoption of clinical trials that are more rigorous and include longterm followup studies, the development of appropriate manufacturing technology, the synchronization of regulatory agencies, and a clear plan for reimbursement strategies. Once again, these challenges have been discussed in greater detail in previous works.2, 3, 7, 12, 13, 15, 22, 23, 26, 31, 38, 44, 48, 51, 52 While it seems that the field may be at a tipping point with many challenges remaining, the fact that translation has been influenced in a positive way gives promise to the future progression of RM therapies.

This work was supported by a Collaborative Research Grant from the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council (NSERC; CPG158280 to E.J.S.), and the Hetenyi Memorial Studentship from the University of Ottawa (to E.J.).

All authors declared no competing interest for this work.

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The Progression of Regenerative Medicine and its Impact on Therapy ...

- Receipt of RMAT Designation is based on preliminary clinical evidence from the CANaspire Phase 1/2 clinical trial, which showed functional improvements in all dosed patients indicating that BBP-812 has potential to address the unmet needs of individuals with Canavan disease

- BridgeBio will leverage the benefits of RMAT designation, including early and more frequent interactions with the FDA, to establish an Accelerated Approval pathway for BBP-812

- If approved, BridgeBios gene therapy for Canavan disease could be the first therapeutic option for children born with this devastating and fatal neurodevelopmental disorder

PALO ALTO, Calif., Sept. 10, 2024 (GLOBE NEWSWIRE) -- BridgeBio Pharma, Inc. (Nasdaq: BBIO) (BridgeBio), a commercial-stage biopharmaceutical company focused on genetic diseases, today announced that the United States Food and Drug Administration (FDA) has granted Regenerative Medicine Advanced Therapy (RMAT) designation to BBP-812, an investigational intravenous (IV) adeno-associated virus serotype 9 (AAV9) gene therapy for the treatment of Canavan disease. RMAT designation was granted following the FDAs review of clinical data from the CANaspire Phase 1/2 clinical trial investigating BBP-812 as a potential therapy to address the unmet medical needs of individuals with Canavan disease.

RMAT is an expedited FDA program available to sponsors of regenerative medicine therapies intended to treat, modify, reverse, or cure serious conditions. Benefits of the RMAT designation include all the advantages of the Fast Track and Breakthrough Therapy Designation programs, including faster and more frequent interactions with the FDA to achieve early alignment on critical aspects of the program. FDA granted RMAT designation based on its review of 12 months of safety and efficacy data from the first eight patients with Canavan disease dosed with BBP-812 in the CANaspire Phase 1/2 clinical trial.

We are honored to be granted RMAT designation for BBP-812 and are eager to work closely with the FDA and the Canavan community with the goal of bringing our therapy to families living with Canavan disease as fast as possible, said Eric David, M.D., J.D., CEO at BridgeBio Gene Therapy. We are beyond grateful to the children and their families who are participating in CANaspire, as well as to the study investigators. RMAT will allow us to work more closely with FDA to ensure we are responding to the urgency that families feel.

To date, results from CANaspire show that all patients dosedwith at least one follow-up assessment havedemonstrated improvements in functional outcomes in key areas important to caregivers such as head control, sitting upright, reaching for and grasping objects, and visual tracking. All patients dosed with BBP-812 with at least one follow-up assessment have shownreductions in N-acetylaspartate (NAA), both in urine and in the central nervous system, to levels associated with mild disease. BBP-812 has been well-tolerated, with a safety profile generally consistent with that of other AAV9 gene therapy programs.

Canavan disease is an extremely rare and rapidly progressive neurodegenerative disease that prevents most children from meeting basic developmental milestones, such as crawling, walking, speaking, and even holding their heads up. It is a terminal diagnosis with no approved treatment to date. The news of the RMAT designation, coupled with the preliminary results seen in the clinical trial, provides hope to children worldwide living with Canavan disease and their families, said Kathleen Flynn,CEO of National Tay-Sachs & Allied Diseases Association, an advocacy organization dedicated to driving research, forging collaboration, and supporting families within the Tay-Sachs, Canavan, GM1, and Sandhoff disease communities.

In addition to RMAT designation, BBP-812 has been granted Orphan Drug, Rare Pediatric Disease (RPDD), and Fast Track Designations from the FDA, as well as Orphan Drug Designation from the European Medicines Agency. With RPDD, if approved, BridgeBio may qualify for a Priority Review Voucher.

About CANaspireCANaspire is a Phase 1/2 open-label study designed to evaluate the safety, tolerability, and pharmacodynamic activity of BridgeBios AAV9 gene therapy candidate, BBP-812, in pediatric patients with Canavan disease. Each eligible patient will receive a single IV infusion of BBP-812. The primary outcomes of the study are safety, as well as change from baseline of urine and central nervous system NAA levels. Motor function and development will also be assessed.

For more information about the CANaspire trial, visit TreatCanavan.com or ClinicalTrials.gov (NCT04998396).

About Canavan DiseaseAffecting approximately 1,000 children in the U.S. and European Union, Canavan disease is an ultra-rare, disabling and fatal disease with no approved therapy. Most children are not able to meet developmental milestones, are unable to crawl, walk, sit or talk, and die at a young age. The disease is caused by an inherited mutation of the ASPA gene that codes for aspartoacylase, a protein that breaks down a compound called NAA. Deficiency of aspartoacylase activity results in accumulation of NAA, and ultimately results in toxicity to myelin in ways that are not currently well understood. Myelin insulates neuronal axons, and without it, neurons are unable to send and receive messages as they should. The current standard of care for Canavan disease is limited to supportive therapy.

About BridgeBio Pharma, Inc.BridgeBio Pharma, Inc. (BridgeBio) is a commercial-stage biopharmaceutical company founded to discover, create, test and deliver transformative medicines to treat patients who suffer from genetic diseases. BridgeBios pipeline of development programs ranges from early science to advanced clinical trials. BridgeBio was founded in 2015 and its team of experienced drug discoverers, developers and innovators are committed to applying advances in genetic medicine to help patients as quickly as possible. For more information visitbridgebio.comand follow us onLinkedIn,Twitter and Facebook.

BridgeBio Pharma, Inc. Forward-Looking StatementsThis press release contains forward-looking statements. Statements BridgeBio makes in this press release may include statements that are not historical facts and are considered forward-looking within the meaning of Section 27A of the Securities Act of 1933, as amended (the Securities Act), and Section 21E of the Securities Exchange Act of 1934, as amended (the Exchange Act), which are usually identified by the use of words such as anticipates, believes, continues, estimates, expects, hopes, intends, may, plans, projects, remains, seeks, should, will, and variations of such words or similar expressions. BridgeBio intends these forward-looking statements to be covered by the safe harbor provisions for forward-looking statements contained in Section 27A of the Securities Act and Section 21E of the Exchange Act. These forward-looking statements, including statements relating to the timing and success of BridgeBios Phase 1/2 clinical trial of BBP-812 for the treatment of Canavan disease, expectations, plans and prospects regarding BridgeBios regulatory approval process for BBP-812, the ability of BBP-812 to be the first therapeutic treatment option for children born with Canavan disease, reflect BridgeBios current views about its plans, intentions, expectations, strategies and prospects, which are based on the information currently available to BridgeBio and on assumptions BridgeBio has made. Although BridgeBio believes that its plans, intentions, expectations, strategies and prospects as reflected in or suggested by those forward-looking statements are reasonable, BridgeBio can give no assurance that the plans, intentions, expectations or strategies will be attained or achieved. Furthermore, actual results may differ materially from those described in the forward-looking statements and will be affected by a number of risks, uncertainties and assumptions, including, but not limited to, BridgeBios ability to continue and complete its Phase 1/2 clinical trial of BBP-812 for the treatment of Canavan disease, BridgeBios ability to advance BBP-812 in clinical development according to its plans, the ability of BBP-812 to treat Canavan disease, the ability of BBP-812 to retain Fast Track Designation, Rare Pediatric Drug Designation, Regenerative Medicine Advanced Therapy Designation and Orphan Drug Designation from the U.S. Food and Drug Administration and Orphan Drug Designation from the European Medicines Agency, and potential adverse impacts due to global health emergencies, including delays in regulatory review, manufacturing and supply chain interruptions, adverse effects on healthcare systems and disruption of the global economy, the impacts of current macroeconomic and geopolitical events, including changing conditions from hostilities in Ukraine and in Israel and the Gaza Strip, increasing rates of inflation and rising interest rates, on our business operations and expectations as well as those risks set forth in the Risk Factors section of BridgeBios most recent Annual Report on Form 10-K, and BridgeBios other filings with the U.S. Securities and Exchange Commission. Moreover, BridgeBio operates in a very competitive and rapidly changing environment in which new risks emerge from time to time. These forward-looking statements are based upon the current expectations and beliefs of BridgeBios management as of the date of this press release and are subject to certain risks and uncertainties that could cause actual results to differ materially from those described in the forward-looking statements. Except as required by applicable law, we assume no obligation to update publicly any forward-looking statements, whether as a result of new information, future events or otherwise.

BridgeBio Contact:Vikram Balicontact@bridgebio.com(650)-789-8220

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BridgeBio Receives FDAs Regenerative Medicine Advanced Therapy (RMAT ...

Signal Transduct Target Ther. 2022; 7: 272.

1 Department of Research and Development, Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, Vietnam

2Department of Cellular Therapy, Vinmec High-Tech Center, Vinmec Healthcare System, Hanoi, Vietnam

1 Department of Research and Development, Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, Vietnam

2Department of Cellular Therapy, Vinmec High-Tech Center, Vinmec Healthcare System, Hanoi, Vietnam

1 Department of Research and Development, Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, Vietnam

1 Department of Research and Development, Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, Vietnam

1 Department of Research and Development, Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, Vietnam

1 Department of Research and Development, Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, Vietnam

1 Department of Research and Development, Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, Vietnam

3Institute for Science & Technology in Medicine, Keele University, Keele, UK

4Department of Biology, Stanford University, Stanford, CA USA

1 Department of Research and Development, Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, Vietnam

1 Department of Research and Development, Vinmec Research Institute of Stem Cell and Gene Technology, Vinmec Healthcare System, Hanoi, Vietnam

2Department of Cellular Therapy, Vinmec High-Tech Center, Vinmec Healthcare System, Hanoi, Vietnam

3Institute for Science & Technology in Medicine, Keele University, Keele, UK

4Department of Biology, Stanford University, Stanford, CA USA

Received 2022 Mar 15; Revised 2022 Jul 19; Accepted 2022 Jul 21.

Recent advancements in stem cell technology open a new door for patients suffering from diseases and disorders that have yet to be treated. Stem cell-based therapy, including human pluripotent stem cells (hPSCs) and multipotent mesenchymal stem cells (MSCs), has recently emerged as a key player in regenerative medicine. hPSCs are defined as self-renewable cell types conferring the ability to differentiate into various cellular phenotypes of the human body, including three germ layers. MSCs are multipotent progenitor cells possessing self-renewal ability (limited in vitro) and differentiation potential into mesenchymal lineages, according to the International Society for Cell and Gene Therapy (ISCT). This review provides an update on recent clinical applications using either hPSCs or MSCs derived from bone marrow (BM), adipose tissue (AT), or the umbilical cord (UC) for the treatment of human diseases, including neurological disorders, pulmonary dysfunctions, metabolic/endocrine-related diseases, reproductive disorders, skin burns, and cardiovascular conditions. Moreover, we discuss our own clinical trial experiences on targeted therapies using MSCs in a clinical setting, and we propose and discuss the MSC tissue origin concept and how MSC origin may contribute to the role of MSCs in downstream applications, with the ultimate objective of facilitating translational research in regenerative medicine into clinical applications. The mechanisms discussed here support the proposed hypothesis that BM-MSCs are potentially good candidates for brain and spinal cord injury treatment, AT-MSCs are potentially good candidates for reproductive disorder treatment and skin regeneration, and UC-MSCs are potentially good candidates for pulmonary disease and acute respiratory distress syndrome treatment.

Subject terms: Stem-cell research, Mesenchymal stem cells

The successful approval of cancer immunotherapies in the US and mesenchymal stem cell (MSC)-based therapies in Europe have turned the wheel of regenerative medicine to become prominent treatment modalities.13 Cell-based therapy, especially stem cells, provides new hope for patients suffering from incurable diseases where treatment approaches focus on management of the disease not treat it. Stem cell-based therapy is an important branch of regenerative medicine with the ultimate goal of enhancing the body repair machinery via stimulation, modulation, and regulation of the endogenous stem cell population and/or replenishing the cell pool toward tissue homeostasis and regeneration.4 Since the stem cell definition was introduced with their unique properties of self-renewal and differentiation, they have been subjected to numerous basic research and clinical studies and are defined as potential therapeutic agents. As the main agenda of regenerative medicine is related to tissue regeneration and cellular replacement and to achieve these targets, different types of stem cells have been used, including human pluripotent stem cells (hPSCs), multipotent stem cells and progenitor cells.5 However, the emergence of private and unproven clinics that claim the effectiveness of stem cell therapy as magic cells has raised highly publicized concerns about the safety of stem cell therapy. The most notable case involved the injection of a cell population derived from fractionated lipoaspirate into the eyes of three patients diagnosed with macular degeneration, resulting in the loss of vision for these patients.6 Thus, as regenerative medicine continues to progress and evolve and to clear the myth of the magic cells, this review provides a brief overview of stem cell-based therapy for the treatment of human diseases.

Stem cell therapy is a novel therapeutic approach that utilizes the unique properties of stem cells, including self-renewal and differentiation, to regenerate damaged cells and tissues in the human body or replace these cells with new, healthy and fully functional cells by delivering exogenous cells into a patient.7 Stem cells for cell-based therapy can be of (1) autologous, also known as self-to-self therapy, an approach using the patients own cells, and (2) allogeneic sources, which use cells from a healthy donor for the treatment.8 The term stem cell were first used by the eminent German biologist Ernst Haeckel to describe the properties of fertilized egg to give rise to all cells of the organism in 1868.9 The history of stem cell therapy started in 1888, when the definition of stem cell was first coined by two German zoologists Theodor Heinrich Boveri and Valentin Haecker,9 who set out to identify the distinct cell population in the embryo capable of differentiating to more specialized cells (Fig. ). In 1902, studies carried out by the histologist Franz Ernst Christian Neumann, who was working on bone marrow research, and Alexander Alexandrowitsch Maximov demonstrated the presence of common progenitor cells that give rise to mature blood cells, a process also known as haematopoiesis.10 From this study, Maximov proposed the concept of polyblasts, which later were named stem cells based on their proliferation and differentiation by Ernst Haeckel.11 Maximov described a hematopoietic population presented in the bone marrow. In 1939, the first case report described the transplantation of human bone marrow for a patient diagnosed with aplastic anemia. Twenty years later, in 1958, the first stem cell transplantation was performed by the French oncologist George Mathe to treat six nuclear researchers who were accidentally exposed to radioactive substances using bone marrow transplantation.12 Another study by George Mathe in 1963 shed light on the scientific community, as he successfully conducted bone marrow transplantation in a patient with leukemia. The first allogeneic hematopoietic stem cell transplantation (HSCT) was pioneered by Dr. E. Donnall Thomas in 1957.13 In this initial study, all six patients died, and only two patients showed evidence of transient engraftment due to the unknown quantities and potential hazards of bone marrow transplantation at that time. In 1969, Dr. E. Donnall Thomas conducted the first bone marrow transplantation in the US, although the success of the allogeneic treatment remained exclusive. In 1972, the year marked the discovery of cyclosporine (the immune suppressive drug),14 the first successes of allogeneic transplantation for aplastic anemia and acute myeloid leukemia were reported in a 16-year-old girl.15 From the 1960s to the 1970s, series of works conducted by Friendenstein and coworkers on bone marrow aspirates demonstrated the relationship between osteogenic differentiation and a minor subpopulation of cells derived from bone marrow.16 These cells were later proven to be distinguishable from the hematopoietic population and to be able to proliferate rapidly as adherent cells in tissue culture vessels. Another important breakthrough from Friendensteins team was the discovery that these cells could form the colony-forming unit when bone marrow was seeded as suspension culture following by differentiation into osteoblasts, adipocytes, and chondrocytes, suggesting that these cells confer the ability to proliferate and differentiate into different cell types.17 In 1991, combined with the discovery of human embryonic stem cells (hESCs), which will be discussed in the next section, the term mesenchymal stem cells, previously known as stromal stem cells or osteogenic stem cells, was first coined in Caplan and widely used to date.18 Starting with bone marrow transplantation 60 years ago, the journey of stem cell therapy has developed throughout the years to become a novel therapeutic agent of regenerative medicine to treat numerous incurable diseases, which will be reviewed and discussed in this review, including neurological disorders, pulmonary dysfunctions, metabolic/endocrine-related diseases, reproductive disorders, skin burns, and cardiovascular conditions).

Stem cell-based therapy: the history and cell source. a The timeline of major discoveries and advances in basic research and clinical applications of stem cell-based therapy. The term stem cells was first described in 1888, setting the first milestone in regenerative medicine. The hematopoietic progenitor cells were first discovered in 1902. In 1939, the first bone marrow transplantation was conducted in the treatment of aplasmic anemia. Since then, the translation of basic research to preclinical studies to clinical trials has driven the development of stem cell-based therapy by many discoveries and milestones. The isolations of mesenchymal stem cells in 1991 following by the discovery of human pluripotent stem cells have recently contributed to the progress of stem cell-based therapy in the treatment of human diseases. b Schematic of the different cell sources that can be used in stem cell-based therapy. (1) Human pluripotent stem cells, including embryonic stem cells (derived from inner cell mass of blastocyst) and induced pluripotent stem cells confer the ability to proliferate indefinitely in vitro and differentiate into numerous cell types of the human body, including three germ layers. (2) Mesenchymal stem cells are multipotent stem cells derived from mesoderm possessing self-renewal ability (limited in vitro) and differentiation potential into mesenchymal lineages. The differentiated/somatic cells can be reprogrammed back to the pluripotent stage using OSKM factors to generate induced pluripotent stem cells. It is important to note that stem cells show a relatively higher risk of tumor formation and lower risk of immune rejection (in the case of mesenchymal stem cells) when compared to that of somatic cells. The figure was created with BioRender.com

In this review, we described the different types of stem cell-based therapies (Fig. ), including hPSCs and MSCs, and provided an overview of their definition, history, and outstanding clinical applications. In addition, we further created the first literature portfolio for the targeted therapy of MSCs based on their origin, delineating their different tissue origins and downstream applications with an in-depth discussion of their mechanism of action. Finally, we provide our perspective on why the tissue origin of MSCs could contribute greatly to their downstream applications as a proposed hypothesis that needs to be proven or disproven in the future to further enhance the safety and effectiveness of stem cell-based therapy.

The clinical applications of stem cell-based therapies for heart diseases have been recently discussed comprehensively in the reviews19,20 and therefore will be elaborated in this study as the focus discussions related to hPSCs and MSCs in the following sections. In general, the safety profiles of stem cell-based therapies are supported by a large body of preclinical and clinical studies, especially adult stem cell therapy (such as MSC-based products). However, clinical trials have not yet yielded data supporting the efficacy of the treatment, as numerous studies have shown paradoxical results and no statistically significant differences in infarct size, cardiac function, or clinical outcomes, even in phase III trials.21 The results of a meta-analysis showed that stem cells derived from different sources did not exhibit any therapeutic effects on the improvement of myocardial contractility, cardiovascular remodeling, or clinical outcomes.22 The disappointing results obtained from the clinical trials thus far could be explained by the fact that the administered cells may exert their therapeutic effects via an immune modulation rather than regenerative function. Thus, well-designed, randomized and placebo-controlled phase III trials with appropriate cell-preparation methods, patient selection, follow-up schedules and suitable clinical measurements need to be conducted to determine the efficacy of the treatments. In addition, concerns related to optimum cell source and dose, delivery route and timing of administration, cell distribution post administration and the mechanism of action also need to be addressed. In the following section of this review, we present clinical trials related to MSC-based therapy in cardiovascular disease with the aim of discussing the contradictory results of these trials and analyzing the potential challenges underlying the current approaches.

Gastrointestinal diseases are among the most diagnosed conditions in the developed world, altering the life of one-third of individuals in Western countries. The gastrointestinal tract is protected from adverse substances in the gut environment by a single layer of epithelial cells that are known to have great regenerative ability in response to injuries and normal cell turnover.23 These epithelial cells have a rapid turnover rate of every 27 days under normal conditions and even more rapidly following tissue damage and inflammation. This rapid proliferation ability is possible owing to the presence of a specific stem cell population that is strictly compartmentalized in the intestinal crypts.24 The gastrointestinal tract is highly vulnerable to damage, tissue inflammation and diseases once the degradation of the mucosal lining layer occurs. The exposure of intestinal stem cells to the surrounding environment of the gut might result in the direct destruction of the stem cell layer or disruption of intestinal functions and lead to overt clinical symptoms.25 In addition, the accumulation of stem cell defects as well as the presence of chronic inflammation and stress also contributes to the reduction of intestinal stem cell quality.

In terms of digestive disorders, Crohns disease (CD) and ulcerative colitis are the two major forms of inflammatory bowel disease (IBD) and represent a significant burden on the healthcare system. The former is a chronic, uncontrolled inflammatory condition of the intestinal mucosa characterized by segmental transmural mucosal inflammation and granulomatous changes.26 The latter is a chronic inflammatory bowel disease affecting the colon and rectum, characterized by mucosal inflammation initiating in the rectum and extending proximal to the colon in a continuous fashion.27 Cellular therapy in the treatment of CD can be divided into haematopoietic stem cell-based therapy and MSC-based therapy. The indication and recommendation of using HSCs for the treatment of IBD were proposed in 1995 by an international committee with four important criteria: (1) refractory to immunosuppressive treatment; (2) persistence of the disease conditions indicated via endoscopy, colonoscopy or magnetic resonance enterography; (3) patients who underwent an imminent surgical procedure with a high risk of short bowel syndromes or refractory colonic disease; and (4) patients who refused to treat persistent perianal lesions using coloproctectomy with a definitive stroma implant.28 In the standard operation procedure, patents HSCs were recruited using cyclophosphamide, which is associated with granulocyte colony-stimulating factor (G-CSF), at two different administered concentrations (4g/m2 and 2g/m2). Recently, it was reported that high doses of cyclophosphamide do not improve the number of recruited HSCs but increase the risk of cardiac and bladder toxicity. An interest in using HSCTs in CD originated from case reports that autologous HSCTs can induce sustained disease remission in some29,30 but not all patients3133 with CD. The first phase I trial was conducted in Chicago and recruited 12 patients with active moderate to severe CD refractory to conventional therapies. Eleven of 12 patients demonstrated sustained remission after a median follow-up of 18.5 months, and one patient developed recurrence of active CD.31 The ASTIC trial (the Autologous Stem Cell Transplantation International Crohn Disease) was the first randomized clinical trial with the largest cohort of patients undergoing HSCT for refractory CD in 2015.34 The early report of the trial showed no statistically significant improvement in clinical outcomes of mobilization and autologous HSCT compared with mobilization followed by conventional therapy. In addition, the procedure was associated with significant toxicity, leading to the suggestion that HSCT for patients with refractory CD should not be widespread. Interestingly, by using conventional assessments for clinical trials for CD, a group reassessed the outcomes of patients enrolled in the ASTIC trial showing clinical and endoscopic benefits, although a high number of adverse events were also detected.35 A recent systematic review evaluated 18 human studies including 360 patients diagnosed with CD and showed that eleven studies confirmed the improvement of Crohns disease activity index between HSCT groups compared to the control group.36 Towards the cell sources, HSCs are the better sources as they afforded more stable outcomes when compared to that of MSC-based therapy.37 Moreover, autologous stem cells were better than their allogeneic counterparts.36 The safety of stem cell-based therapy in the treatment of CD has attracted our attention, as the risk of infection in patients with CD was relatively higher than that in those undergoing administration to treat cancer or other diseases. During the stem cell mobilization process, patient immunity is significantly compromised, leading to a high risk of infection, and requires carefully nursed and suitable antibiotic treatment to reduce the development of adverse events. Taken together, stem cell-based therapy for digestive disease reduced inflammation and improved the patients quality of life as well as bowel functions, although the high risk of adverse events needs to be carefully monitored to further improve patient safety and treatment outcomes.

The liver is the largest vital organ in the human body and performs essential biological functions, including detoxification of the organism, metabolism, supporting digestion, vitamin storage, and other functions.38 The disruption of liver homeostasis and function might lead to the development of pathological conditions such as liver failure, cirrhosis, cancer, alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), and autoimmune liver disease (ALD). Orthotropic liver transplantation is the only effective treatment for severe liver diseases, but the number of available and suitable donor organs is very limited. Currently, stem cell-based therapies in the treatment of liver disease are associated with HSCs, MSCs, hPSCs, and liver progenitor cells.

Liver failure is a critical condition characterized by severe liver dysfunctions or decompensation caused by numerous factors with a relatively high mortality rate. Stem cell-based therapy is a novel alternative approach in the treatment of liver failure, as it is believed to participate in the enhancement of liver regeneration and recovery. The results of a meta-analysis including four randomized controlled trials and six nonrandomized controlled trials in the treatment of acute-on-chronic liver failure (ACLF) demonstrated that clinical outcomes of stem cell therapy were achieved in the short term, requiring multiple doses of stem cells to prolong the therapeutic effects.39,40 Interestingly, although MSC-based therapies improved liver functions, including the model of end-stage liver disease score, albumin level, total bilirubin, and coagulation, beneficial effects on survival rate and aminotransferase level were not observed.41 A randomized controlled trial illustrated the improvement of liver functions and reduction of severe infections in patients with hepatitis B virus-related ACLF receiving allogeneic bone marrow-derived MSCs (BM-MSCs) via peripheral infusion.42 HSCs from peripheral blood after the G-CSF mobilization process were used in a phase I clinical trial and exhibited an improvement in serum bilirubin and albumin in patients with chronic liver failure without any specific adverse events related to the administration.43 Taken together, an overview of stem cell-based therapy in the treatment of liver failure indicates the potential therapeutic effects on liver functions with a strong safety profile, although larger randomized controlled trials are still needed to assure the conclusions.

Liver cirrhosis is one of the major causes of morbidity and mortality worldwide and is characterized by diffuse nodular regeneration with dense fibrotic septa and subsequent parenchymal extinction leading to the collapse of liver vascular structure.44 In fact, liver cirrhosis is considered the end-stage of liver disease that eventually leads to death unless liver transplantation is performed. Stem cell-based therapy, especially MSCs, currently emerges as a potential treatment with encouraging results for treating liver cirrhosis. In a clinical trial using umbilical cord-derived MSCs (UC-MSCs), 45 chronic hepatitis B patients with decompensated liver cirrhosis were divided into two groups: the MSC group (n=30) and the control group (n=15).45 The results showed a significant reduction in ascites volume in the MSC group compared with the control. Liver function was also significantly improved in the MSC groups, as indicated by the increase in serum albumin concentration, reduction in total serum bilirubin levels, and decrease in the sodium model for end-stage liver disease score.45 Similar results were also reported from a phase II trial using BM-MSCs in 25 patients with HCV-induced liver cirrhosis.46 Consistent with these studies, three other clinical trials targeting liver cirrhosis caused by hepatitis B and alcoholic cirrhosis were conducted and confirmed that MSC administration enhanced and recovered liver functions.4749 With the large cohort study as the clinical trial conducted by Fang, the safety and potential therapeutic effects of MSC-based therapies could be further strengthened and confirmed the feasibility of the treatment in virus-related liver cirrhosis.49 In terms of delivery route, a randomized controlled phase 2 trial suggested that systemic delivery of BM-MSCs does not show therapeutic effects on patients with liver cirrhosis.50 MSCs are not the only cell source for liver cirrhosis. Recently, an open-label clinical trial conducted in 19 children with liver cirrhosis due to biliary atresia after the Kasai operation illustrated the safety and feasibility of the approach by showing the improvement of liver function after bone marrow mononuclear cell (BMNC) administration assessed by biochemical tests and pediatric end-stage liver disease (PELD) scores.51 Another study using BMNCs in 32 decompensated liver cirrhosis patients illustrated the safety and effectiveness of BMNC administration in comparison with the control group.52 Recently, a long-term analysis of patients receiving peripheral blood-derived stem cells indicated a significant improvement in the long-term survival rate when compared to the control group, and the risk of hepatocellular carcinoma formation did not increase.53 CD133+ HSC infusion was performed in a multicentre, open, randomized controlled phase 2 trial in patients with liver cirrhosis; the results did not support the improvement of liver conditions, and cirrhosis persisted.54 Notably, these results are in line with a previous randomized controlled study, which also reported that G-CSF and bone marrow-derived stem cells delivered via the hepatic artery did not introduce therapeutic potential as expected.55 Thus, stem cell-based therapy for liver cirrhosis is still in its immature stage and requires larger trials with well-designed experiments to confirm the efficacy of the treatment.

Nonalcoholic fatty liver disease (NAFLD) is the most common medical condition caused by genetic and lifestyle factors and results in a severe liver condition and increased cardiovascular risk.56 NAFLD is the hidden enemy, as most patients are asymptomatic for a long time, and their routine life is unaffected. Thus, the detection, identification, and management of NAFLD conditions are challenging tasks, as patients diagnosed with NAFLD often develop nonalcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma.57 Although preclinical studies have shown that stem cell administration could enhance liver function in NAFLD models, a limited number of clinical trials were performed in human subjects. Recently, a multi-institutional clinical trial using freshly isolated autologous adipose tissue-derived regenerative cells was performed in Japan to treat seven NAFLD patients.58 The results illustrated the improvement in the serum albumin level of six patients and prothrombin activity of five patients, and no treatment-related adverse events or severe adverse events were observed. This study illustrates the therapeutic potential of stem cell-based therapy in the treatment of NAFLD.

Autoimmune liver disease (ALD) is a severe liver condition affecting children and adults worldwide, with a female predominance.59 The condition occurs in genetically predisposed patients when a stimulator, such as virus infection, leads to a T-cell-mediated autoimmune response directed against liver autoantigens. As a result, patients with ALD might develop liver cirrhosis, hepatocellular carcinoma, and, in severe cases, death. To date, HSCT and bone marrow transplantation are the two common stem cell-based therapies exhibiting therapeutic potential for ALD in clinical trials. An interesting report illustrated that haploidentical HSCTs could cure ALD in patients with sickle cells.60 This report is particularly important, as it illustrates the potential therapeutic approach of using haploidentical HSCTs to treat patients with both sickle cells and ALD. Another case report described a 19-year-old man with a 4-year history of ALD who developed acute lymphoblastic leukemia and required allogeneic bone marrow transplantation from this wholesome brother.61 The clinical data showed that immunosuppressive therapy for transplantation generated ALD remission in the patient.62 However, the data also provided valid information related to the sustained remission and the normalization of ASGPR-specific suppressor-inducer T-cell activity following bone marrow transplantation, suggesting that these suppressor functions originated from donor T cells.61 Thus, it was suggested that if standard immunosuppressive treatment fails, alternative cellular immunotherapy would be a viable option for patients with ALD. Primary biliary cholangitis (PBC), usually known as primary biliary cirrhosis, is a type of ALD characterized by a slow, progressive destruction of small bile ducts of the liver leading to the formation of cirrhosis and accumulation of bile and other toxins in the liver. A pilot, single-arm trial from China recruited seven patents with PBC who had a suboptimal response to ursodeoxycholic acid (UDCA) treatment.63 These patients received UDCA treatment in combination with three rounds of allogeneic UC-MSCs at 4-week intervals with a dose of 0.5106 cells/kg of patient body weight via the peripheral vein. No treatment-related adverse events or severe adverse events were observed throughout the course of the study. The clinical data indicated significant improvement in liver function, including reduction of serum ALP and gamma-glutamyltransferase (GGT) at 48 weeks post administration. The common symptoms of PBC, including fatigue, pruritus, and hypogastric ascites volume, were also reduced, supporting the feasibility of MSC-based therapy in the treatment of PBC, although major limitations of the study were nonrandomized, no control group and small sample size. Another study was conducted in China with ten PBC patients who underwent incompetent UDCA treatment for more than 1 year. These patients received a range of 35 allogeneic BM-MSCs/kg body weight by intravenous infusion.64 Although these early studies have several limitations, such as small sample size, nonrandomization, and no control group, their preliminary data related to safety and efficacy herald the prospects and support the feasibility of stem cell-based therapy in the treatment of ALD.

In summary, the current number of trials for liver disease using stem cell-based therapy has provided fundamental data supporting the safety and potential therapeutic effects in various liver diseases. Unfortunately, due to the small number of trials, several obstacles need to be overcome to prove the effectiveness of the treatments, including (1) stem cell source and dose, (2) administration route, (3) time of intervention, and (4) clinical assessments during the follow-up period. Only by addressing these challenges we will be able to prove, facilitate and promote stem cell-based therapy as a mainstream treatment for liver diseases.

Arthritis is a general term describing cartilage conditions that cause pain and inflammation of the joints. Osteoarthritis (OA) is the most common form of arthritis caused by persistent degeneration and poor recovery of articular cartilage.65 OA affects one or several diarthrodial joints, such as small joints at the hand and large joints at the knee and hips, leading to severe pain and subsequent reduction in the mobility of patients. There are two types of OA: (1) primary OA or idiopathic OA and secondary OA caused by causative factors such as trauma, surgery, and abnormal joint development at birth.66 As conventional treatments for OA are not consistent in their effectiveness and might cause unbearable pain as well as long-term rehabilitation (in the case of joint replacement), there is a need for a more reliable, less painful, and curative therapy targeting the root of OA.67 Thus, stem cell therapy has recently emerged as an alternative approach for OA and has drawn great attention in the regenerative field.

The administration of HSCs has been proven to reduce bone lesions, enhance bone regeneration and stimulate the vascularization process in degenerative cartilage. Attempts were made to evaluate the efficacy of peripheral blood stem cells in ten OA patients by three intraarticular injections. Post-administration analysis indicated a reduction in the WOMAC index with a significant reduction in all parameters. All patients completed 6-min walk tests with an increase of more than 54 meters. MRI analysis indicated an improvement in cartilage thickness, suggesting that cartilage degeneration was reduced post administration. To further enhance the therapeutic potential of HSCT, CD34+ stem cells were proposed to be used in combination with the rehabilitation algorithm, which included three stages: preoperative, hospitalization and outpatient periods.68 Currently, a large wave of studies has been directed to MSC-based therapy for the treatment of OA due to their immunoregulatory functions and anti-inflammatory characteristics. MSCs have been used as the main cell source in several multiple and small-scale trials, proving their safety profile and potential effectiveness in alleviating pain, reducing cartilage degeneration, and enhancing the regeneration of cartilage structure and morphology in some cases. However, the best source of MSCs, whether from bone marrow, adipose tissue, or umbilical cord, for the management of OA is still a great question to be answered. A systematic review investigating over sixty-one of 3172 articles with approximately 2390 OA patients supported the positive effects of MSC-based therapy on OA patients, although the study also pointed out the fact that these therapeutic potentials were based on limited high-quality evidence and long-term follow-up.69 Moreover, the study found no obvious evidence supporting the most effective source of MSCs for treating OA. Another systematic review covering 36 clinical trials, of which 14 studies were randomized trials, provides an interesting view in terms of the efficacy of autologous MSC-based therapy in the treatment of OA.70 In terms of BM-MSCs, 14 clinical trials reported the clinical outcomes at the 1-year follow-up, in which 57% of trials reported clinical outcomes that were significantly better in comparison with the control group. However, strength analysis of the data set showed that outcomes from six trials were low, whereas the outcomes of the remaining eight trials were extremely low. Moreover, the positive evidence obtained from MRI analysis was low to very low strength of evidence after 1-year post administration.70 Similar results were also found in the outcome analysis of autologous adipose tissue-derived MSCs (AT-MSCs). Thus, the review indicated low quality of evidence for the therapeutic potential of MSC therapy on clinical outcomes and MRI analysis. The low quality of clinical outcomes could be explained by the differences in interventions (including cell sources, cell doses, and administration routes), combination treatments (with hyaluronic acid,71 peripheral blood plasma,72 etc.), control treatments and clinical outcome measurements between randomized clinical trials.73 In addition, the data of the systematic analysis could not prove the better source of MSCs for OA treatment. Taken together, although stem cell-based therapy has been shown to be safe and feasible in the management of OA, the authors support the notion that stem cell-based therapy could be considered an alternative treatment for OA when first-line treatments, such as education, exercise, and body weight management, have failed.

Stem cell therapy in the treatment of cancer is a sensitive term and needs to be used and discussed with caution. Clinicians and researchers should protect patients with cancer from expensive and potentially dangerous or ineffective stem cell-based therapy and patients without a cancer diagnosis from the risk of malignancy development. In general, unproven stem cell clinics employed three cell-based therapies for cancer management, including autologous HSCTs, stromal vascular fraction (SVF), and multipotent stem cells, such as MSCs. Allogeneic HSCTs confer the ability to generate donor lymphocytes that contribute to the suppression and regression of hematological malignancies and select solid tumors, a specific condition known as graft-versus-tumor effects.74 However, stem cell clinics provide allogeneic cell-based therapy for the treatment of solid malignancies despite limited scientific evidence supporting the safety and efficacy of the treatment. High-quality evidence from the Cochrane library shows that marrow transplantation via autologous HSCTs in combination with high-dose chemotherapy does not improve the overall survival of women with metastatic breast cancer. In addition, a study including more than 41,000 breast cancer patients demonstrated no significant difference in survival benefits between patients who received HSCTs following high-dose chemotherapy and patients who underwent conventional treatment.75 Thus, the use of autologous T-cell transplants as monotherapy and advertising stem cell-based therapies as if they are medically approved or preferred treatment of solid tumors is considered untrue statements and needs to be alerted to cancer patients.76

Over the past decades, many preclinical studies have demonstrated the potential of MSC-based therapy in cancer treatment due to their unique properties. They confer the ability to migrate toward damaged sites via inherent tropism controlled by growth factors, chemokines, and cytokines. MSCs express specific CXC chemokine receptor type 4 (CXCR4) and other chemokine receptors (including CCR1, CCR2, CCR4, CCR7, etc.) that are essential to respond to the surrounding signals.77 In addition, specific adherent proteins, including CD49d, CD44, CD54, CD102, and CD106, are also expressed on the MSC surface, allowing them to attach, rotate, migrate, and penetrate the blood vessel lumen to infiltrate the damaged tissue.78 Similar to damaged tissues, tumors secrete a wide range of chemoattractant that also attract MSC migration via the CXCL12/CXCR4 axis. Previous studies also found that MSC migration toward the cancer site is tightly controlled by diffusible cytokines such as interleukin 8 (IL-8) and growth factors including transforming growth factor-beta 1 (TGF-1),79 platelet-derived growth factor (PDGF),80 fibroblast growth factor 2 (FGF-2),81 vascular endothelial growth factor (VEGF),81 and extracellular matrix molecules such as matrix metalloproteinase-2 (MMP-2).82 Once MSCs migrate successfully to cancerous tissue, accumulating evidence demonstrates the interaction between MSCs and cancer cells to exhibit their protumour and antitumour effects, which are the major concerns of MSC-based therapy. MSCs are well-known for their regenerative effects that regulate tissue repair and recovery. This unique ability is also attributed to the protumour functions of these cells. A previous study reported that breast cancer cells induce MSC secretion of chemokine (CC motif) ligand 5 (CCL-5), which regulates the tumor invasion process.83,84 Other studies also found that MSCs secrete a wide range of growth factors (VEGF, basic FGF, HGF, PDGF, etc.) that inhibits apoptosis of cancer cells.85 Moreover, MSCs also respond to signals released from cancer cells, such as TGF-,86 to transform into cancer-associated fibroblasts, a specific cell type residing within the tumor microenvironment capable of promoting tumorigenesis.87 Although MSCs have been proven to be involved in protumour activities, they also have potent tumor suppression abilities that have been used to develop cancer treatments. It has been suggested that MSCs exhibit their tumor inhibitory effects by inhibiting the Wnt and AKT signaling pathways,88 reducing the angiogenesis process,89 stimulating inflammatory cell infiltration,90 and inducing tumor cell cycle arrest and apoptosis.91 To date, the exact functions of MSCs in both protumour and antitumor activities are still a controversial issue across the stem cell field. Other approaches exploit gene editing and tissue engineering to convert MSCs into a Trojan horse that could exhibit antitumor functions. In addition, MSCs can also be modified to express specific anticancer miRNAs exhibiting tumor-suppressive behaviors.92 However, genetically modified MSCs are still underdeveloped and require intensive investigation in the clinical setting.

To date, ~25 clinical trials have been registered on ClinicalTrials.gov aimed at using MSCs as a therapeutic treatment for cancer.93 These trials are mostly phase 1 and 2 studies focusing on evaluating the safety and efficacy of the treatment. Studies exploiting MSC-based therapy have combined MSCs with an oncolytic virus approach. Oncolytic viruses are specific types of viruses that can be genetically engineered or naturally present, conferring the ability to selectively infect cancer cells and kill them without damaging the surrounding healthy cells.94 A completed phase I/II study using BM-MSCs infected with the oncolytic adenovirus ICOVIR5 in the treatment of metastatic and refractory solid tumors in children and adult patients demonstrated the safety of the treatment and provided preliminary data supporting their therapeutic potential.95 The same group also reported a complete disappearance of all signs of cancer in response to MSC-based therapy in one pediatric case three years post administration.96 A reported study in 2019 claimed that adipose-derived MSCs infected with vaccinia virus have the potential to eradicate resistant tumor cells via the combination of potent virus amplification and senitization of the tumor cells to virus infection.97 However, in a recently published review, a valid question was posed regarding the 2019 study that do these reported data merit inclusion in the publication record when they were collected by such groups using a dubious therapeutic that was eventually confiscated by US Marshals?76

Taken together, cancer research and therapy have entered an innovative and fascinating era with advancements in traditional therapies such as chemotherapy, radiotherapy, and surgery on one hand and stem cell-based therapy on the other hand. Although stem cell-based therapy has been considered a novel and attractive therapeutic approach for cancer treatment, it has been hampered by contradictory results describing the protumour and antitumour effects in preclinical studies. Despite this contradictory reality, the use of stem cell-based therapy, especially MSCs, offers new hope to cancer patients by providing a new and more effective tool in personalized medicine. The authors support the use of MSC-based therapy as a Trojan horse to deliver specific anticancer functions toward cancer cells to suppress their proliferation, eradicate cancer cells, or limit the vascularization process of cancerous tissue to improve the clinical safety and efficacy of the treatment.

The discovery of hPSCs, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), has revolutionized stem cell research and cell-based therapy.98 hESCs were first isolated from blastocyst-stage embryos in 1998,99 followed by breakthrough reprogramming research that converted somatic cells into hiPSCs using just four genetic factors.100,101 Methods have been developed to maintain these cells long-term in vitro and initiate their differentiation into a wide variety of cell types, opening a new era in regenerative medicine, particularly cell therapy to replace lost or damaged tissues.

hPSCs are defined as self-renewable cell types that confer the ability to differentiate into various cellular phenotypes of the human body, including three germ layers.102 Historically, the first pluripotent cell lines to be generated were embryonic carcinoma (EC) cell lines established from human germ cell tumors103 and murine undifferentiated compartments.104 Although EC cells are a powerful tool in vitro, these cells are not suitable for clinical applications due to their cancer-derived origin and aneuploidy genotype.105 The first murine ESCs were established in 1981 based on the culture techniques obtained from EC research.106 Murine ESCs are derived from the inner cell mass (ICM) of the pre-implantation blastocyst, a unique biological structure that contains outer trophoblast layers that give rise to the placenta and ICM.107 In vivo ESCs only exist for a short period during the embryos development, and they can be isolated and maintained indefinitely in vitro in an undifferentiated state. The discovery of murine ESCs has dramatically changed the field of biomedical research and regenerative medicine over the last 40 years. Since then, enormous investigations have been made to isolate and culture ESCs from other species, including hESCs, in 1998.99 The success of Thomson et al. in 1998 triggered the great controversy in media and ethical research boards across the globe, with particularly strong objections being raised to the use of human embryos for research purposes.108 Several studies using hESCs have been conducted demonstrating their therapeutic potential in the clinical setting. However, the use of hESCs is limited due to (1) the ethical barrier related to the destruction of human embryos and (2) the potential risk of immunological rejection, as hESCs are isolated from pre-implantation blastocysts, which are not autologous in origin. To overcome these two great obstacles, several research groups have been trying to develop technology to generate hESCs, including nuclear transfer technology, the well-known strategy that creates Dolly sheep, although the generation of human nuclear transfer ESCs remains technically challenging.109 Taking a different approach, in 2006, Yamanaka and Takahashi generated artificial PSCs from adult and embryonic mouse somatic cells using four transcription factors (Oct-3/4, Sox2, Klf4, and c-Myc, called OSKM) reduced from 24 factors.100 Thereafter, in 2007, Takahashi and colleagues successfully generated the first hiPSCs exhibiting molecular and biological features similar to those of hESCs using the same OSKM factors.101 Since then, hiPSCs have been widely studied to expand our knowledge of the pathogenesis of numerous diseases and aid in developing new cell-based therapies as well as personalized medicine.

Since its beginning 24 years ago, hPSC research has evolved momentously toward applications in regenerative medicine, disease modeling, drug screening and discovery, and stem cell-based therapy. In clinical trial settings, the uses of hESCs are restricted by ethical concerns and tight regulation, and the limited preclinical data support their therapeutic potential. However, it is important to acknowledge several successful outcomes of hESC-based therapies in treating human diseases. In 2012, Steven Schwartz and his team reported the first clinical evidence of using hESC-derived retinal pigment epithelium (RPE) in the treatment of Stargardts macular dystrophy, the most common pediatric macular degeneration, and an individual with dry age-related macular degeneration.110,111 With a differentiation efficiency of RPE greater than 99%, 5104 RPEs were injected into the subretinal space of one eye in each patient. As the hESC source of RPE differentiation was exposed to mouse embryonic stem cells, it was considered a xenotransplantation product and required a lower dose of immunosuppression treatment. This study showed that hESCs improved the vision of patients by differentiating into functional RPE without any severe adverse events. The trial was then expanded into two open-label, phase I/II studies with the published results in 2015 supporting the primary findings.112 In these trials, patients were divided into three groups receiving three different doses of hESC-derived RPE, including 10104, 15104 and 50104 RPE cells per eye. After 22 months of follow-up, 19 patients showed improvement in eyesight, seven patients exhibited no improvement, and one patient experienced a further loss of eyesight. The technical challenge of hESC-derived RPE engraftment was an unbalanced proliferation of RPE post administration, which was observed in 72% of treated patients. A similar approach was also conducted in two South Korean patients diagnosed with age-induced macular degeneration and two patients with Stargardt macular dystrophy.113 The results supported the safety of hESC-derived RPE cells and illustrated an improvement in visual acuity in three patients. Recently, clinical-graded hESC-derived RPE cells were also developed by Chinese researchers under xeno-free culture conditions to treat patients with wet age-related degeneration.114 As hESC development is still associated with ethical concerns and immunological complications related to allogeneic administration, hiPSC-derived RPE cells have emerged as a potential cell source for macular degeneration. Although RPE differentiation protocols have been developed and optimized to improve the efficacy of hiPSC-derived RPE cells, they are still insufficient, time-consuming and labor intensive.115,116 For clinical application, an efficient differentiation of primed to nave state hiPSCs toward the RPE was developed using feeder-free culture conditions utilizing the transient inhibition of the FGF/MAPK signaling pathway.117 Overexpression of specific transcription factors in hiPSCs throughout the differentiation process is also an interesting approach to generate a large number of RPE cells for clinical use. In a recent study, overexpression of three eye-field transcription factors, including OTX2, PAX6, and MITF, stimulated RPE differentiation in hiPSCs and generated functional RPE cells suitable for transplantation.118 To date, although reported data from phase I/II clinical trials have been produced enough to support the safety of hESC-derived RPE cells, the treatment is still in its immature stage. Thus, future studies should focus on the development of the cellular manufacturing process of RPE and the subretinal administration route to further improve the outcomes of RPE fabrication and engraftment into the patients retina (recommended review119).

Numerous studies have demonstrated that hESC-derived cardiomyocytes exhibit cardiac transcription factors and display a cardiomyocyte phenotype and immature electrical phenotype. In addition, using hPSC-derived cardiomyocytes could provide a large number of cells required for true remuscularization and transplantation. Thus, these cells can be a promising novel therapeutic approach for the treatment of human cardiovascular diseases. In a case report, hESC-derived cardiomyocytes showed potential therapeutic effects in patients with severe heart failure without any subsequent complications.120 This study was a phase I trial (ESCORT [Transplantation of Human Embryonic Stem Cell-derived Progenitors in Severe Heart Failure] trial) to evaluate the safety of cardiomyocyte progenitor cells derived from hESCs seeded in fibrin gel scaffolds for 10 patients with severe heart failure (NCT02057900). The encouraging results from this study demonstrated the feasibility of producing hESC-derived cardiomyocyte progenitor cells toward clinical-grade standards and combining them with a tissue-engineered scaffold to treat severe heart disease (the first patient of this trial has already reached the 7-year follow-up in October 2021).121 Currently, the two ongoing clinical trials using hPSC-derived cardiomyocytes have drawn great attention, as their results would pave the way to lift the bar for approving therapies for commercial use. The first trial was conducted by a team led by cardiac surgeon Yoshiki Sawa at Osaka University using hiPSC-derived cardiomyocytes embedded in a cell sheet for engraftment (jRCT2052190081). The trials started first with three patients followed by ten patients to assess the safety of the approach. Once safety is met, the treatment can be sold commercially under Japans fast-track system for regenerative medicine.122 Another trial used a collagen-based construct called BioVAT-HF to contain hiPSC-derived cardiomyocytes. The trial was divided into two parts to evaluate the cell dose: (Part A) recruiting 18 patients and (Part B) recruiting 35 patients to test a broad range of engineered human myocardium (EHM) doses. The expected results from this study will provide the proof-of-concept for the use of EHM in the stimulation of heart remuscularization in humans. To date, no adverse events or severe adverse events have been reported from these trials, supporting the safety of the procedure. However, as the number of treated patients was relatively small, limitations in drawing conclusions regarding efficacy are not yet possible.21,123

One of the first clinical trials using hPSC-based therapy was conducted by Geron Corporation in 2010 using hESC-derived oligodendrocyte progenitor cells (OPC1) to treat spinal cord injury (SCI). The results confirmed the safety one year post administration in five participants, and magnetic resonance imaging demonstrated improvement of spinal cord deterioration in four participants.124 Asterias Biotherapeutic (AST) continued the Geron study by conducting the SCiStar Phase I/IIa study to evaluate the therapeutic effects of AST-OPC1 (NCT02302157). The trials results published in clinicaltrials.gov demonstrated significant improvement in running speed, forelimb stride length, forelimb longitudinal deviations, and rear stride frequency. Interestingly, the recently published data of a phase 1, multicentre, nonrandomized, single-group assignment, interventional trial illustrated no evidence of neurological decline, enlarging masses, further spinal cord damage, or syrinx formation in patients 10 years post administration of the OPC1 product.125 This data set provides solid evidence supporting the safety of OPC1 with an event-free period of up to 10 years, which strengthens the safety profile of the SCiStar trial.

Analysis of the global trends in clinical trials using hPSC-based therapy showed that 77.1% of studies were observational (no cells were administered into patient), and only 22.9% of studies used hPSC-derived cells as interventional treatment.126 The number of studies using hiPSCs was relatively higher than that using hESCs, which was 74.8% compared to 25.2%, respectively. The majority of observational studies were performed in developed countries, including the USA (41.6%) and France (16.8%), whereas interventional studies were conducted in Asian countries, including China (36.7%), Japan (13.3%), and South Korea (10%). The trends in therapeutic studies were also clear in terms of targeted diseases. The three most studied diseases were ophthalmological conditions, circulatory disorders, and nervous systems.127 However, it is surprising that the clinical applications of hPSCs have achieved little progress since the first hESCs were discovered worldwide. The relatively low number of clinical trials focusing on using iPSCs as therapeutic agents to administer into patients could be ascribed to the unstable genome of hiPSCs,128 immunological rejection,129 and the potential for tumor formation.130

Approximately 55 years ago, fibroblast-like, plastic-adherent cells, later named mesenchymal stem cells (MSCs) by Arnold L. Caplan,18 were discovered for the first time in mouse bone marrow (BM) and were later demonstrated to be able to form colony-like structures, proliferate, and differentiate into bone/reticular tissue, cartilage, and fat.131 Protocols were subsequently established to directly culture this subpopulation of stromal cells from BM in vitro and to stimulate their differentiation into adipocytes, chondroblasts, and osteoblasts.132 Since then, MSCs have been found in and derived from different human tissue sources, including adipose tissue (AT), the umbilical cord (UC), UC blood, the placenta, dental pulp, amniotic fluid, etc.133 To standardize and define MSCs, the International Society for Cell and Gene Therapy (ISCT) set minimal identification criteria for MSCs derived from multiple tissue sources.134 Among them, MSCs derived from AT, BM, and UC are the most commonly studied MSCs in human clinical trials,135 and they constitute the three major tissue sources of MSCs that will be discussed in this review.

The discovery of MSCs opened an era during which preclinical studies and clinical trials have been performed to assess the safety and efficacy of MSCs in the treatment of various diseases. The major conclusion of these studies and trials is that MSC-based therapy is safe, although the outcomes have usually been either neutral or at best marginally positive in terms of the clinically relevant endpoints regardless of MSC tissue origin, route of infusion, dose, administration duration, and preconditioning.136 It is important to note that a solid background of knowledge has been generated from all these studies that has fueled the recent translational research in MSC-based therapy. As MSCs have been intensively studied over the last 55 years and have become the subject of multiple reviews, systematic reviews, and meta-analyses, the objective of this paper is not to duplicate these publications. Rather, we will discuss the questions that both clinicians and researchers are currently exploring with regard to MSC-based therapy, diligently seeking answers to the following:

With a solid body of data supporting their safety profiles derived from both preclinical and clinical studies, does the tissue origin of MSCs also play a role in their downstream clinical applications in the treatment of different human diseases?

Do MSCs derived from AT, BM, and UC exhibit similar efficacy in the treatment of neurological diseases, metabolic/endocrine-related disorders, reproductive dysfunction, skin burns, lung fibrosis, pulmonary disease, and cardiovascular conditions?

To answer these questions, we will first focus on the most recently published clinical data regarding these targeted conditions, including neurological disorders, pulmonary dysfunctions, metabolic/endocrine-related diseases, reproductive disorders, skin burns, and heart-related diseases, to analyze the potential efficacy of MSCs derived from AT, BM, and UC. Based on the level of clinical improvement observed in each trial, we analyzed the potential efficacy of MSCs derived from each source to visualize the correlation between patient improvement and MSC sources. We will then address recent trends in the exclusive use of MSC-based products, focusing on the efficacy of treatment with MSCs from each of the abovementioned sources, and we will analyze the relationship between the respective efficacies of MSCs from these sources in relation to the targeted disease conditions. Finally, we propose a hypothesis and mechanism to achieve the currently still unmet objective of evaluating the use of MSCs from AT, BM, and UC in regenerative medicine.

In general, MSCs are reported to be isolated from numerous tissue types, but all of these types can be organized into two major sources: adult137 and perinatal sources138 (Fig. ). Adult sources of MSCs are defined as tissues that can be harvested or obtained from an individual, such as dental pulp,139 BM, peripheral blood,140 AT,141 lungs,142 hair,143 or the heart.144 Adult MSCs usually reside in specialized structures called stem cell niches, which provide the microenvironment, growth factors, cell-to-cell contacts and external signals necessary for maintaining stemness and differentiation ability.145 BM was the first adult source of MSCs discovered by Friedenstein131 and has become one of the most documented and largely used MSC sources to date, followed by AT. BM-MSCs are isolated and cultured in vitro from BM aspirates using a Ficoll gradient-centrifugation method146 or a red blood cell lysate buffer to collect BM mononuclear cell populations, whereas AT-MSCs are obtained from stromal vascular fractions of enzymatically digested AT obtained through liposuction,141 lipoplasty, or lipectomy procedures.147 These tissue collection procedures are invasive and painful for the patient and are accompanied by a risk of infection, although BM aspiration and adipose liposuction are considered safe procedures for BM and AT biopsies. The number of MSCs that can be isolated from these adult tissues varies significantly in a tissue-dependent manner. The percentage of MSCs in BM mononuclear cells ranges from 0.001 to 0.01% following gradient centrifugation.132 The number of MSCs in AT is at least 500 times higher than that in BM, with approximately 5,000 MSCs per 1g of AT. Perinatal sources of MSCs consist of UC-derived components, such as UC, Whartons jelly, and UC blood, and placental structures, such as the placental membrane, amnion, chorion membrane, and amniotic fluid.138 The collection of perinatal MSCs, such as UC-MSCs, is noninvasive, as the placenta, UC, UC blood, and amnion are considered waste products that are usually discarded after birth (with no ethical barriers).148 Although MSCs represent only 107% the cells found in UC, their higher proliferation rate and rapid population doubling time allow these cells to rapidly replicate and increase in number during in vitro culture.149 Under standardized xeno-free and serum-free culture platforms, AT-MSCs show a faster proliferation rate and a higher number of colony-forming units than BM-MSCs.149 UC-MSCs have the fastest population doubling time compared to AT-MSCs and BM-MSCs in both conventional culture conditions and xeno- and serum-free environments.149 MSCs extracted from AT, BM and UC exhibit all minimal criteria listed by the ISCT, including morphology (plastic adherence and spindle shape), MSC surface markers (95% positive for CD73, CD90 and CD105; less than 2% negative for CD11, CD13, CD19, CD34, CD45, and HLR-DR) and differentiation ability into chondrocytes, osteocytes, and adipocytes.150

The two major sources of MSCs: adult and perinatal sources. The adult sources of MSCs are specific tissue in human body where MSCs could be isolated, including bone marrow, adipose tissue, dental pulp, peripheral blood, menstrual blood, muscle, etc. The perinatal sources of MSCs consist of umbilical cord-derived components, such as umbilical cord, Whartons jelly, umbilical cord blood, and placental structures, such as placental membrane, amnion, chorion membrane, amniotic fluid, etc. The figure was created with BioRender.com

In fact, although MSCs derived from either adult or perinatal sources exhibit similar morphology and the basic characteristics of MSCs, studies have demonstrated that these cells also differ from each other. Regarding immunophenotyping, AT-MSCs express high levels of CD49d and low levels of Stro-1. An analysis of the expression of CD49d and CD106 showed that the former is strongly expressed in AT-MSCs, in contrast to BM-MSCs, whereas CD106 is expressed in BM-MSCs but not in AT-MSCs.151 Increased expression of CD133, which is associated with stem cell regeneration, differentiation, and metabolic functions,152 was observed in BM-MSCs compared to MSCs from other sources.153 A recent study showed that CD146 expression in UC-MSCs was higher than that in AT- and BM-MSCs,153 supporting the observation that UC-MSCs have a stronger attachment and a higher proliferation rate than MSCs from other sources, as CD146 is a key cell adhesion protein in vascular and endothelial cell types.154 In terms of differentiation ability, donor-matched BM-MSCs exhibit a higher ability to differentiate into chondrogenic and osteogenic cell types than AT-MSCs, whereas AT-MSCs show a stronger capacity toward the adipogenic lineage.150 The findings from an in vitro differentiation study indicated that BM-MSCs are prone to osteogenic differentiation, whereas AT-MSCs possess stronger adipogenic differentiation ability, which can be explained by the fact that the epigenetic memory obtained from either BM or AT drives the favored MSC differentiation along an osteoblastic or adipocytic lineage.155 Interestingly, although UC-MSCs have the ability to differentiate into adipocytes, osteocytes, or chondrocytes, their osteogenic differentiation ability has been proven to be stronger than that of BM-MSCs.156 The most interesting characteristic of MSCs is their immunoregulatory functions, which are speculated to be related to either cell-to-cell contact or growth factor and cytokine secretion in response to environmental/microenvironmental stimuli. MSCs from different sources almost completely inhibit the proliferation of peripheral blood mononuclear cells (PBMCs) at PBMC:MSC ratios of 1:1 and 10:1.149 At a higher ratio, BM-MSCs showed a significantly higher inhibitory effect than AT- or UC-MSCs.153 Direct analysis of the immunosuppressive effects of BM- and UC-MSCs has revealed that these cells exert similar inhibitory effects in vitro with different mechanisms involved.157 With these conflicting data, the mechanism of action related to the immune response of MSCs from different sources is still poorly understood, and long-term investigations both in preclinical studies and in clinical trial settings are needed to shed light on this complex immunomodulation function.

The great concern in MSC-based therapy is the fate of these cells post administration, especially through different delivery routes, including systemic administration via an intravenous (IV) route or tissue-specific administration, such as dorsal pancreatic administration. It is important to understand the distribution of these cells after injection to expand our understanding of the underlying mechanisms of action of treatments; in addition, this knowledge is required by authorized bodies (the Food and Drug Administration (FDA) in the United States or the regulation of advanced-therapy medicinal products in Europe, No. 1394/2007) prior to using these cells in clinical trials. The preclinical data using various labeling techniques provide important information demonstrating that MSCs do not have unwanted homing that could lead to the incorrect differentiation of the cells or inappropriate tumor formation. In a mouse model, human BM-MSCs and AT-MSCs delivered via an IV route are rapidly trapped in the lungs and then recirculate through the body after the first embolization process, with a small number of infused cells found mainly in the liver after the second embolization.158 Using the technetium-99 m labeling method, intravenously infused human cells showed long-term persistence up to 13 months in the bone, BM compartment, spleen, muscle, and cartilage.159 A similar result was reported in baboons, confirming the long-term homing of human MSCs in various tissues post administration.160 Although the retainment of MSCs in the lungs might potentially reduce their systemic therapeutic effects,161 it provides a strong advantage when these cells are used in the treatment of respiratory diseases. Local injection of MSCs also revealed their tissue-specific homing, as an injection of MSCs via the renal artery route resulted in the majority of the injected cells being found in the renal cortex.162 Numerous studies have been conducted to track the migration of administered MSCs in human subjects. Henriksson and his team used MSCs labeled with iron sucrose in the treatment of intervertebral disc degeneration.163 Their study showed that chondrocytes differentiated from infused MSCs could be detected at the injured intervertebral discs at 8 months but not at 28 months. A study conducted in a patient with hemophilia A using In-oxine-labeled MSCs showed that the majority of the cells were trapped in the lungs and liver 1h post administration, followed by a reduction in the lungs and an increase in the number of cells in the liver after 6 days.164 Interestingly, a small proportion of infused MSCs were found in the hemarthrosis site at the right ankle after 24h, suggesting that MSCs are attracted and migrate to the injured site. The distribution of MSCs was also reported in the treatment of 21 patients diagnosed with type 2 diabetes using 18-FDG-tagged MSCs and visualized using positron emission tomography (PET).165 The results illustrated that local delivery of MSCs via an intraarterial route is more effective than delivery via an IV route, as MSCs home to the pancreatic head (pancreaticoduodenal artery) or body (splenic artery). Therefore, although the available data related to the biodistribution of infused MSCs are still limited, the results obtained from both preclinical and clinical studies illustrate a comparable set of data supporting results on homing, migration to the injured site, and the major organs where infused MSCs are located. The following comprehensive and interesting reviews are highly recommended.166168

To date, 1426 registered clinical trials spanning different trial phases have used MSCs for therapeutic purposes, which is four times the number reported in 2013.169,170 As supported by a large body of preclinical studies and advancements in conducting clinical trials, MSCs have been proven to be effective in the treatment of numerous diseases, including nervous system and brain disorders, pulmonary diseases,171 cardiovascular conditions,172 wound healing, etc. The outcomes of MSC-based therapy have been the subject of many intensive reviews and systematic analyses with the solid conclusion that these cells exhibit strong safety profiles and positive outcomes in most tested conditions.173175 In addition, the available data have revealed several potential mechanisms that could explain the beneficial effects of MSCs, including their homing efficiency, differentiation potential, production of trophic factors (including cytokines, chemokines, and growth factors), and immunomodulatory abilities. However, it is still not known which MSC types should be used for which diseases, as it seems to be that MSCs exhibit beneficial effects regardless of their sources.169

The theory that brain cells can never regenerate has been challenged by the discovery of newly formed neurons in the human adult hippocampus or the migration of stem cells in the brain in animal models.176 These observations have triggered hope for regeneration in the context of neuronal diseases by using exogenous stem cell sources to replenish or boost the stem cell population in the brain. Moreover, the limited regenerative capacity of the brain and spinal cord is an obstacle for traditional treatments of neurodegenerative diseases, such as autism, cerebral palsy, stroke, and spinal cord injury (SCI). As current treatments cannot halt the progression of these diseases, studies throughout the world have sought to exploit cell-based therapies to treat neurodegenerative diseases on the basis of advances in the understanding and development of stem cell technology, including the use of MSCs. Successful stem cell therapy for treating brain disease requires therapeutic cells to reach the injured sites, where they can repair, replace, or at least prevent the deteriorative effects of neuronal damage.177 Hence, the gold standard of cell-based therapy is to deliver the cells to the target site, stimulate the tissue repair machinery, and regulate immunological responses via either cell-to-cell contact or paracrine effects.178 Among 315 registered clinical trials using stem cells for the treatment of brain diseases, MSCs and hematopoietic stem cells (HSCs; CD34+ cells isolated from either BM aspirate or UC blood) are the two main cell types investigated, whereas approximately 102 clinical trials used MSCs and 62 trials used HSCs for the treatment of brain disease (main search data from clinicaltrial.gov). MSCs are widely used in almost all clinical trials targeting different neuronal diseases, including multiple sclerosis,179 stroke,180 SCI,181 cerebral palsy,182 hypoxic-ischemic encephalopathy,183 autism,184 Parkinsons disease,185 Alzheimers disease185 and ataxia. Among these trials in which MSCs were the major cells used, nearly two-thirds were for stroke, SCI, or multiple sclerosis. MSCs have been widely used in 29 registered clinical trials for stroke, with BM-MSCs being used in 16 of these trials. With 26 registered clinical trials, SCI is the second most common indication for using MSCs, with 16 of these trials using mainly expanded BM-MSCs. For multiple sclerosis, 15 trials employed BM-MSCs among a total of 23 trials conducted for the treatment of this disease. Hence, it is important to note that in neuronal diseases and disorders, BM-MSCs have emerged as the most commonly used therapeutic cells among other MSCs, such as AT-MSCs and UC-MSCs.

The outcomes of the use of BM-MSCs in the treatment of neuronal diseases have been widely reported in various clinical trial types. A review by Zheng et al. indicated that although the treatments appeared to be safe in patients diagnosed with stroke, there is a need for well-designed phase II multicentre studies to confirm the outcomes.173 One of the earliest trials using autologous BM-MSCs was conducted by Bang et al. in five patients diagnosed with stroke in 2005. The results supported the safety and showed an improved Barthel index (BI) in MSC-treated patients.186 In a 2-year follow-up clinical trial, 16 patients with stroke received BM-MSC infusions, and the results showed that the treatment was safe and improved clinical outcomes, such as motor impairment scale scores.187 A study conducted in 12 patients with ischemic stroke showed that autologous BM-MSCs expanded in vitro using autologous serum improved the patients modified Rankin Scale (mRS), with a mean lesion volume reduced by 20% at 1 week post cell infusion.188 In 2011, a modest increase in the Fugl Meyer and modified BI scores was observed after autologous administration of BM-MSCs in patients with chronic stroke.189 More recently, a prospective, open-label, randomized controlled trial with blinded outcome evaluation was conducted, with 39 patients and 15 patients in the BM-MSC administration and control groups, respectively. The results of this study indicated that autologous BM-MSCs with autologous serum administration were safe, but the treatment led to no improvements at 3 months in modified Rankin Scale (mRS) scores, although leg motor improvement was observed.180 Researchers explored whether the efficacy of BM-MSC administration was maintained over time in a 5-year follow-up clinical trial. Patients (85) were randomly assigned to either the MSC group or the control group, and follow-ups on safety and efficacy were performed for 5 years, with 52 patients being examined at the end of the study. The MSC group exhibited a significant improvement in terms of decreased mRS scores, whereas the number of patients with an mRS score increase of 03 was statistically significant.187 Although autologous BM-MSCs did not improve the Basel index, mRS, or National Institutes of Health Stroke Scale (NIHSS) score 2 years post infusion, patients who received BM-MSC therapy showed improvement in their motor function score.190 In addition, a prospective, open-label, randomized controlled trial by Lee et al. showed that autologous BM-MSCs primed with autologous ischemic serum significantly improved motor functions in the MSC-treated group. Neuroimaging analysis also illustrated a significant increase in interhemispheric connectivity and ipsilesional connectivity in the MSC group.191 Recently, a single intravenous infection of allogeneic BM-MSCs has been proven to be safe and feasible in patients with chronic stroke with a significant improvement in BI score and NIHSS score.192

In two systematic reviews using MSCs for the treatment of SCI, BM-MSCs (n=16) and UC-MSCs (n=5) were reported to be safe and well-tolerated.193,194 The results indicated significant improvements in the stem cell administration groups compared with the control groups in terms of a composite of the American Spinal Injury Association (ASIA) impairment scale (AIS) grade, AIS grade A, and ASIA sensory scores and bladder function (Table ). However, larger experimental groups with a randomized and multicentre design are needed for further confirmation of these findings. For multiple sclerosis, several early-phase (phase I/II) registered clinical studies have used BM-MSCs. A study compared the potential efficacy of BM-MSC and BM mononuclear cell (BMMNC) transplantation in 105 patients with spastic cerebral palsy.195 The results showed that the GMFM (gross motor function measure) and the FMFM (fine motor function measure) scores of the BM-MSC transplant group were higher than those of the BMNNC transplant group at 3, 6, and 12 months of assessment. In terms of autism spectrum disorder, a review of 254 children after BMMNC transplantation found that over 90% of patients ISAA (Indian Scale for Assessment of Autism) and CARS (Childhood Autism Rating Scale) scores improved. Young patients and those in whom autism spectrum disorder was detected early generally showed better improvement.196

The reported clinical trials using MSCs from AT, BM, and UC in the treatment of brain-related injuries and neurological disorders

One of the biggest limitations when using BM-MSCs is the bone marrow aspiration process, as it is an invasive procedure that can introduce a risk of complications, especially in pediatric and elderly patients.197 Therefore, UC-MSCs have been suggested as an alternative to BM-MSCs and are being studied in clinical trials for the treatment of neurological diseases in approximately 1550 patients throughout the world; however, only three studies have been completed, with data published recently.198 A recent study showed that UC-MSC administration improved both gross motor function and cognitive skills, assessed using the Activities of Daily Living (ADL), Comprehensive Function Assessment (CFA), and GMFM, in patients diagnosed with cerebral palsy. The improvements peaked 6 months post administration and lasted for 12 months after the first transplantation.199 In a single-targeted phase I/II clinical trial using UC-MSCs for the treatment of autism, Riordan et al. reported decreases in Autism Treatment Evaluation Checklist (ATEC) and CARS scores for eight patients, but the paper has been retracted due to a violation of the journals guidelines.200 In an open-label, phase I study, UC-MSCs were used as the main cells to treat 12 patients with autism spectrum disorder via IV infusions. It is important to note that five participants developed new class I anti-human leukocyte antigen in response to the specific lot of manufactured UC-MSCs, although these responses did not exhibit any immunological response or clinical manifestations. Only 50% of participants showed improvements in at least two autism-specific measurements.201 Although not as widely used as BM-MSCs, these trials have demonstrated the efficacy of using UC-MSCs in the treatment of SCIs. In a pilot clinical study, Yang et al. showed that the use of UC-MSCs has the potential to improve disease status through an increase in total ASIA and SCI Functional Rating Scale of the International Association of Neurorestoratology (IANR-SCIFRS) scores, as well as an improvement in pinprick, light touch, motor and sphincter scores.202 A study of 22 patients with SCIs showed a potential therapeutic effect in 13 patients post UC-MSC infusion.203 AT-MSCs were also used to treat SCI, with a single case report indicating an improvement in neurological and motor functions in a domestic ferret patient.204 However, a result obtained from another phase I trial using AT-MSCs showed mild improvements in neurological function in a small number of patients.205 A phase II, randomized, double-blind, placebo-controlled, single-center, pilot clinical trial using AT-MSCs in the treatment of acute ischemic stroke published a data set that supports the safety of the therapy, although patients who received AT-MSCs showed a nonsignificant improvement after 24 months of follow-up.206 In all of the above studies, the safety of using either AT-MSCs or UC-MSCs was evaluated, and no significant reactions were reported after infusion.

Therefore, based on the number of recovered patients post-transplantation and the number of recruited patients in large-scale trials using BM-MSCs, it seems that BM-MSCs are the prominent cells in regard to treating neurodegenerative disease with potentially good outcomes (Table ). It is important to note that we do not negate the fact that AT- and UC-MSCs also show positive outcomes in the treatment of neuronal diseases, with numerous ongoing large-scale, multicentre, randomized, and placebo-control trials,207,208 but we suggest alternative and thoughtful decisions regarding which sources of MSCs are best for the treatment of neuronal diseases and degenerative disorders.

In the last decade, significant increases in respiratory disease incidence due to air pollution, smoking behavior, population aging, and recently, respiratory virus infections such as coronavirus disease 2019 (COVID-19)209 have been observed, leading to substantial burdens on public health and healthcare systems worldwide. Respiratory inflammatory diseases, including bronchopulmonary dysplasia (BPD), chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS), have recently emerged as three prevalent pulmonary diseases in children and adults. These conditions are usually associated with inflammatory cell infiltration, a disruption of alveolar structural integrity, a reduction in alveolar fluid clearance ability, cytokine release and associated cytokine storms, airway remodeling, and the development of pulmonary fibrosis. Traditional treatments are focused on relieving symptoms and preventing disease progression using surfactants, artificial respiratory support, mechanical ventilation, and antibiotic/anti-inflammatory drugs, with limited effects on the damaged airway, alveolar fluid clearance, and other detrimental effects caused by the inflammatory response. MSCs are known for their immunomodulatory abilities, showing potential in injury reduction and aiding lung recovery after injury. According to ClinicalTrials.gov, from 2017 to date, there have been 159 studies testing the application of MSCs in the treatment of pulmonary diseases, including but not limited to BPD, COPD, and ARDS, suggesting a trend in the use of MSCs as an alternative approach for the treatment of respiratory diseases, especially MSCs from UC as an off-the-shelf and allogeneic source.

Extremely premature infants are born with arrested lung development at the canalicular-saccular phases prior to alveolarization and before pulmonary maturation occurs, which results in the development of BPD.210 These infants require intensive care during the first three months of life using postnatal interventions, including positive pressure mechanical ventilation, external oxygen support, and surfactant infusions, and the newborns have recurrent infections that further compromise normal lung development.211 To date, 13 clinical trials have been proposed to use UC-MSCs in the treatment of BPD, recruiting ~566 premature infants throughout the world, including Vietnam, Korea, the United States, Spain, Australia, and China. The majority of these trials use UC-derived stem cells for phases I and II, focusing on evaluating the safety and efficacy of stem cell-based therapy.212 Human UC tissue and its derivative components are considered the most attractive cell sources for MSCs in the treatment of BPD due to the ease of obtaining them, being readily available, with no ethical concerns, low antigenicity, a high cell proliferation rate, and superior regenerative potential. Chang et al. used MSCs derived from UC blood in a phase I dose-escalation clinical trial to treat 9 preterm infants via intratracheal administration to prevent the development of BPD.213 All 9 preterm infants survived, and only three developed BPD; these infants had significantly decreased BPD severity compared with the historically matched control group. A follow-up study of the same patients after 24 months indicated that only one infant had an E. cloacae infection after discharge at 4 months, with subsequent disseminated intravascular coagulation, which was later proven to be unrelated to the intervention. The remaining eight patients survived with normal pulmonary development and function, suggesting that the therapy was safe. MSCs from UC blood were also used for the treatment of 12 extremely low birthweight preterm patients using the same administration route, which further confirmed the safety of the therapy in the treatment of BPD, although ten of 12 infants still developed severe BPD at 36 weeks.214 Our group also reported the safety and potential efficacy of using UC-MSCs in the treatment of four preterm infants, and the results supported the safety of UC-MSCs and demonstrated that patients could be weaned from oxygen supply and develop normal lung structure and function.215 A phase II clinical trial of 66 infants born at 2328 weeks with a birthweight of 5001250g who were recruited and randomized into an MSC-administration group and a control group was conducted. Although the results supported the safety of MSC administration in preterm infants, the efficacy of the treatment was not supported by statistical analysis, potentially due to the small sample size. Subgroup analysis showed that patients with severe BPD born at 2324 weeks showed a significant improvement in BPD severity, but those born at 2528 weeks did not.216 Hence, it is important to conduct controlled phase II clinical trials with larger cohort sizes to further substantiate the efficacy of UC blood-derived MSCs in the treatment of infants with BPD.

With more than 65 million patients worldwide, COPD was the third-leading cause of death in 2020, according to World Health Organization records. COPD is classified as a chronic inflammatory and destructive pulmonary disease characterized by a progressive reduction in lung function. Averyanov et al. performed a randomized, placebo-controlled phase I/IIa study in 20 patients with mild to moderate idiopathic pulmonary fibrosis (IPF). Treatment group patients received two IV doses of allogeneic MSCs (2108cells) every 3 months, and the second group received a placebo.217 Evaluation tests were performed at weeks 13, 26, 39, and 52. The 6-min walking test distance (6MWTD) results showed that patient fitness improved from week 13 onwards and was maintained until up to the 52nd week. Pulmonary function indicators improved markedly before and after treatment in the treated group but did not change significantly in the placebo group. The goal of MSC therapy in the treatment of COPD is to promote the regeneration of parenchymal cells and alveolar structure and the restoration of lung function. Based on the results of a phase I trial of a commercial BM-MSC product, ProchymalTM, which led to improvements in pulmonary function in treated patients, a multicentre, double-blind, placebo-controlled phase II trial was conducted in 62 patients diagnosed with COPD to determine the safety and potential efficacy of the product. Although the results supported the safety of BM-MSCs, their effectiveness in the treatment of COPD was not assured. No statistically significant differences in FEV1 or FEV1%, total lung capacity, or carbon monoxide diffusing capacity were detected after 2 years of follow-up between the two treatment groups. To date, there have been five clinical trials using BM-MSCs as the main stem cells for the treatment of COPD, but the overall clinical outcomes did not demonstrate the potential therapeutic effects of the treatment.218222 In clinical trial NCT001110252, the results showed that there was an overall reduction in the process of COPD pathological development 3 years after the administration of BM-MSCs, although the trial had a phase I design, with no control group, and evaluated only a small cohort (four patients).219 To alleviate local inflammatory progression in COPD, Oliveira et al. studied the combination treatment of one-way endobronchial valve (EBV) and BM-MSC intubation.223 Ten GOLD (Global Initiative for Obstructive Lung Disease) stage C or D patients were equally divided into 2 groups: one group received a dose of 108 cells before valve insertion, and the other group received a normal saline infusion. The follow-up time was 90 days. Inflammation was significantly improved as assessed by the CRP (C-reactive protein) index at 30 and 90 days after infusion. In addition, improvements in St. Georges Respiratory Questionnaire (SGRQ) scores indicated improved patient quality of life. Furthermore, an investigation into the homing ability of MSCs in vivo was performed on 9 GOLD patients, from stage A to stage D. Each patient received two 2106 BM-MSC/kg IV infusions 1-week apart.224 The marking of MSCs with indium-111 showed that MSCs were retained in the pulmonary vasculature longer in patients with mild COPD and that the levels of inflammatory mediators improved after 7 days of treatment. The results of the evaluation survey conducted after 1 year showed that the number of COPD exacerbations decreased to six times/year compared to 11 times/year before treatment. In addition, AT-MSCs present in the stromal vascular fraction were used to treat patients with COPD, and no adverse events were observed after 12 months of follow-up, but the clinical improvements post administration were not clear.225 The results from a phase I clinical trial using AT-MSCs in eight patients with COPD also reported no significant change in pulmonary function test parameters.226 A study evaluating the use of AT-MSCs as adjunctive therapy for COPD in 12 patients was performed.227 AT was obtained using standard liposuction, MSCs were isolated, and 150300 million cells were intravenously infused. The patients showed improvements in quality of life, with improved SGRQ scores after 3 and 6 months of treatment. Recently, UC-MSCs have emerged as potential allogeneic stem cell candidates for the treatment of COPD.228 In a pilot clinical study, it was demonstrated that allogeneic administration of UC-MSCs in the treatment of COPD was safe and potentially effective.229 In one study, 20 patients, including 9 at stage C and 11 at stage D per the GOLD classification, with histories of smoking were recruited and received cell-based therapy. The patients who received UC-MSC treatment showed significant reductions in Modified Medical Research Council scores, COPD assessment test scores, and the number of pulmonary exacerbations 6 months post administration. The results of the second trial using UC-MSCs showed that the mean FEV1/FVC ratios were increased along with improvements in SGRQ scores and 6MWTDs at three months post administration.230 Although thorough assessments of the effectiveness of UC-MSCs are still in the early stages, the number of trials using UC-MSCs for the treatment of COPD is increasing steadily, with larger sample sizes and stronger designs (randomized or matched casecontrol studies), providing a data set strongly supporting the future applications of UC-MSCs.231

The ongoing pandemic of the 21st century, the COVID-19 pandemic, emerged as a major pulmonary health problem worldwide, with a relatively high mortality rate. Numerous studies, reviews, and systematic analyses have been conducted to discuss and expand our knowledge of the virus and propose different mechanisms by which the virus could alter the immune system.232 One of the most critical mechanisms is the generation of cytokine storms, which result from the initiation of hyperreactions of the adaptive immune response to viral infection.233 These cytokine storms are formed by the establishment of waves of hypercytokinaemia generated from overreactive immune cells, which enhance their expression of TNF-, IL-6, and IL-10, preventing T-lymphocyte recruitment and proliferation and culminating in T-lymphocyte apoptosis and T-cell exhaustion. In COVID-19, once a cytokine storm is formed, it spreads from an initial focal area through the body via circulation, which has been discussed in a comprehensive review by Jamilloux et al.234 At the time of writing this review, there were 74 clinical trials using MSCs from UC (29 trials; including WJ-derived MSCs (WJ-MSCs) and placenta-derived MSCs (PL-MSCs)), AT (15 trials), and BM (11 trials) (comprehensive review171,235). Hence, UC-MSCs have emerged as the most common MSCs for the treatment of COVID-19, with a total of 1047 patients participating in these trials. Among these trials, 15 completed trials using UC-MSCs (including WJ- and PL-MSCs) have been reported, with clinical data from approximately 600 recruited patients.232 Eight of these 15 studies used allogenic UC-MSC transplantation to treat critically ill patients.236 A list of case reports using UC-MSCs showed that the treatments were safe and well-tolerated in 14 patients with COVID-19, with the primary outcomes including increased percentages and numbers of T cells,237,238 improved respiratory and renal functions,239 reductions in inflammatory biomarker levels,240 and positive outcomes in the PaO2/FiO2 ratio.240 In a pilot study conducted in ten patients with severe COVID-19, a single dose of UC-MSCs was safe and improved clinical outcomes, although the study did not investigate whether multiple doses of UC-MSCs could further improve the outcomes.241 Two trials without a control group were conducted in 47 patients, and the results indicated that UC-MSCs were safe and feasible for the treatment of patients with COVID-19.235,242 A single-center, open-label, individually randomized, standard treatment-controlled trial was performed in 41 patients (12 patients assigned to the UC-MSC group), and the results showed that significant improvements in C-reactive protein levels, IL-6 levels, oxygen indices, and lymphocyte numbers were found in the MSC groups. Chest computed tomography (CT) illustrated significant reductions in lung inflammatory responses as reflected by CT findings, the number of lobes involved, and pulmonary consolidation.238 In a phase I trial conducted in 18 hospitalized patients with COVID-19, UC-MSCs were administered via an IV route in nine patients (five patients with moderate COVID-19 and 4 patients with severe COVID-19) at days 0, 3, and 6, with no treatment-related adverse events or severe adverse events.243 Only one patient in the UC-MSC group required mechanical ventilation, compared to four patients in the control group. However, the clinical outcomes, such as COVID-19 symptoms, laboratory test results, CT findings of lung damage, and pulmonary function test parameters, were improved in both groups. Interestingly, a 1-year follow-up of the same sample revealed that the patients who received UC-MSC administration improved in terms of whole-lung lesion volume compared to the control group.244 Moreover, chest CT at 12 months showed significant regeneration of lung tissue in the MSC-administered groups, whereas lung fibrosis was found in all patients in the control group. This finding is of interest because it indicates that a long time is needed to detect the regenerative functions of MSC-based therapy, as the biological process to enhance lung tissue regeneration occurs relatively slowly and requires multiple steps. The effects of UC-MSCs in the attenuation and prevention of the development of cytokine storms were illustrated in an interventional, prospective, three-parallel arm study with two control arms conducted in 30 patients in moderate and critical clinical conditions.245 The results indicated a significant decrease in proinflammatory cytokines (IFN, IL-6, IL-17A, IL-2, and IL-12) and an increase in anti-inflammatory cytokines (IL-10, IL-13, and IL-1ra), suggesting that UC-MSCs might participate in the prevention of cytokine storm development. Lanzoni et al. performed a double-blind, randomized, controlled trial and found that UC-MSC infusions significantly decreased cytokine levels at day 6 and improved survival in patients with COVID-19 with ARDS. In this trial, 24 patients were randomized and assigned 1:1 to receive either MSCs or placebo.246 MSC treatment was associated with a significant improvement in the survival rate without serious adverse events. To date, other trials conducted using UC-MSCs as the main MSCs provide a solid data set on their safety and efficacy in preventing the development of cytokine storms, reducing the inflammatory response, improving pulmonary function, reducing intensive care unit (ICU) stay duration, enhancing lung tissue regeneration, and reducing lung fibrosis progression.240,247249 In two large cohort studies (phase I with 210 patients and phase II with 100 patients), the volume of lung lesions and solid component injuries of patients lungs were reduced significantly after the administration of UC-MSCs,250 and clinical symptoms and inflammatory levels were improved.251 Of the 26 reported clinical trials for the treatment of COVID-19 with MSCs, 1 study used AT-MSCs as the main MSCs.236 Thirteen COVID-19 adult patients under invasive mechanical ventilation who had received previous antiviral and/or anti-inflammatory treatments (including steroids, lopinavir/ritonavir, hydroxychloroquine, and/or tocilizumab, among others) were treated with allogeneic AT-MSCs. With a mean follow-up time of 16 days after infusion, 9/13 patients clinical symptoms improved, and 7/13 patients were intubated. A decrease in inflammatory cytokines and an increase in immunoregulatory cells were also observed in patients, especially in the group of patients with overall clinical improvement. Although there is a lack of clinical efficacy data supporting the use of AT-MSCs in the treatment of patients with COVID-19, AT-MSCs are still potential candidates for inhibiting COVID-19 due to their high secretory activity, strong immune-modulatory effects, and homing ability.252254

For ARDS, in a phase IIa trial, 60 patients with moderate to severe disease were randomized into 2 groups. A group of 40 patients received a single infusion of BM-MSCs at a dose of 1106 cells/kg body weight, and another 20 patients received a placebo.255 After 6 and 24h of infusion, the decrease in plasma inflammatory cytokine levels in the MSC group was significantly greater than that in the placebo group. For severe pulmonary hypertension (PH) associated with BPD (BPD-PH), in a small trial, two preterm infants born at 2627 weeks of age were intravenously administered heterologous BM-MSCs at a dose of 5106 cells per kg of body weight; the treatment reduced oxygen requirements and supported respiration in the infants.256 The administration of allogeneic AT-MSCs in the treatment of ARDS appeared to be safe and well-tolerated in 12 adult patients, but clinical outcomes were not observed.257 The results of two patients who received BM-MSCs showed that both patients had improved respiratory function and hemodynamic function and a reduction in multiorgan failure.258 Although the safety of BM-MSCs was confirmed in a multicentre, open-label, dose-escalation, phase I clinical trial (The Stem cells for ARDS treatmentSTART trial),259 no significant improvements were found in a phase II trial, including in respiratory function and ARDS conditions.260 The safety profile of UC-MSCs is also supported by the findings of a previous phase I clinical trial conducted in 9 patients, which showed that a single IV administration of UC-MSCs was safe and led to positive outcomes in terms of respiratory function and a reduction in the inflammatory response.261 The findings of this study were also supported by those of the REALIST (Repair of Acute Respiratory Distress with Stromal Cell Administration) trial, which further confirmed the maximum tolerated dose of allogeneic UC-MSCs in patients with moderate to severe ARDS.262

Although AT- and BM-MSCs have demonstrated therapeutic potential with similar mechanisms of action, UC-MSCs have emerged as potential candidates in the treatment of pulmonary diseases due to their ease of production as off-the-shelf products, rapid proliferation, noninvasive isolation methods, and supreme immunological regulation as well as anti-inflammatory effects.263 However, it is important to note that there is a need to conduct phase III clinical trials with larger cohorts and trials with at least two sources of MSCs in the treatment of pulmonary conditions to further confirm this speculation.264 Table summarizes several clinical trials with published results discussed in this review.

The reported clinical trials using MSCs from AT, BM, and UC in the treatment of respiratory diseases

The human body maintains function and homeostatic regulation via a complex network of endocrine glands that synthesize and release a wide range of hormones. The endocrine system regulates body functions, including heartbeat, bone regeneration, sexual function, and metabolic activity. Endocrine system dysregulation plays a vital role in the development of diabetes, thyroid disease, growth disorder, sexual dysfunction, reproductive malfunction, and other metabolic disorders. The central dogma of regenerative medicine is the use of adult stem cells as a footprint for tissue regeneration and organ renewal. The functions of these stem cells are tightly regulated by microenvironmental stimuli from the nervous system (rapid response) and endocrine signals via hormones, growth factors, and cytokines. This harmonized and orchestrated system creates a symphony of signals that directly regulate tissue homeostasis and repair after injury. The disruption of these complex networks results in an imbalance of tissue homeostasis and regeneration that can lead to the development of endocrine disorders in humans, such as diabetes, sexual hormone deficiency, premature ovarian failure (POF), and Asherman syndrome.

In recent years, obesity and diabetes (type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM)) have been the two biggest challenges in endocrinology research, and the application of MSCs has emerged as a novel approach for therapeutic consideration. T1DM is characterized by the autoimmune destruction of pancreatic -cells, whereas T2DM is defined as a combination of insulin resistance and pancreatic insulin-producing cell dysfunction. Regenerative medicine seeks to provide an exogenous cell source for replacing damaged or lost -cells to achieve the goal of stabilizing patients blood glucose levels. To date, there are 28 clinical trials using MSCs in the treatment of T1DM (http://www.clinicaltrials.gov, searched in October 2021), among which three trials were completed using autologous BM-MSCs (NCT01068951), allogeneic BM-MSCs (NCT00690066), and allogeneic AT-MSCs (NCT03920397). Interestingly, UC-MSCs were the most favored MSCs for the remaining trials. All published studies confirmed the safety of MSC therapy in the treatment of T1DM with no adverse events. The first study using autologous BM-MSCs showed that patients who were randomized into the MSC-administration group showed an increase in C-peptide levels in response to a mixed-meal tolerance test (MMTT) in comparison to the control group.265 Unfortunately, there was no significant improvement in C-peptide levels, HbA1C or insulin requirements. The use of autologous AT-MSCs in combination with vitamin D was safe and improved HbA1C levels 6 months post administration.266 WJ-MSCs were used as the main MSCs for the treatment of new-onset T1DM, which showed a significant improvement in both HbA1C and C-peptide levels when compared to those of the control group at three and six months post administration.267,268 The combination of allogeneic WJ-MSCs with autologous BM-derived mononuclear cells improved insulin secretion and reduced insulin requirements in patients with T1DM.269 In terms of T2DM, 23 studies were registered on clinicaltrials.gov (searched in October 2021), with six completed studies (three studies used BM-MSCs and three studies used allogeneic UC-MSCs). Although the number of studies using MSCs for the treatment of T2DM is small, their findings support the safety of MSCs, with no severe adverse events observed during the course of these studies.270 It was confirmed that MSC therapy potentially reduced fasting blood glucose and HbA1C levels and increased C-peptide levels. However, these effects were short-term, and multiple doses were required to maintain the MSC effects. Interestingly, the autologous MSC approach in the treatment of patients with diabetes in general is hampered, as both BM-MSCs and AT-MSCs isolated from patients with diabetes showed reduced stemness and functional characteristics.271,272 In addition, the durations of diabetes and obesity are strongly associated with autologous BM-MSC metabolic function, especially mitochondrial respiration, and the accumulation of mitochondrial DNA, which directly interfere with the functions of BM-MSCs and reduce the effectiveness of the therapy.271 Therefore, the allogeneic approach using MSCs from healthy donors provides an alternative approach for stem cell therapy in the treatment of patients with diabetes.

Modern society is increasingly facing the problem of infertility, which is defined as the inability to become pregnant after more than 1 year of unprotected intercourse.273 This problem has emerged as an important worldwide health issue and social burden. Assisted reproductive techniques and in vitro fertilization technology have recently become the most effective methods for the treatment of infertility in humans, but the use of these approaches is limited, as they cannot be applied in patients with no sperm or those who are unable to support implantation during pregnancy, they are associated with complications, they are time-consuming and expensive, and they are associated with ethical issues in certain territories.274 Numerous conditions are related to infertility, including POF, nonobstructive azoospermia, endometrial dysfunction, and Asherman syndrome. Recent progress has been illustrated in preclinical studies for the potential applications of stem cell-based therapy for reproductive function recovery, especially recent studies in the field of MSCs, which provide new hope for patients with infertility and reproductive disorders.275

POF is characterized by a loss of ovarian activity during middle age (before 40 years old) and affects 12% of women of reproductive age.276 Patients diagnosed with POF exhibit oligo-/amenorrhea for at least 4 months, with increased levels of follicle-stimulating hormone (FSH) (>25IU/L) on two occasions more than 1 month apart.277 Diverse factors, such as genetic backgrounds, autoimmune disorders, environmental conditions, and iatrogenic and idiopathic situations, have been reported to be the cause of POF.278 POF can be treated with limited effectiveness via psychosocial support, hormone replacement intervention, and fertility management.279 MSCs from AT, BM, and UC have been used in the treatment of POF, with improvements in ovarian function in preclinical studies using chemotherapy-induced POF animal models. The early published POF study using BM-MSCs as the main cell source is a single case report in which a perimenopausal woman showed an improvement in follicular regeneration, and increased AMH levels resulted in a successful pregnancy followed by delivery of a healthy infant.280 A report using autologous BM-MSCs in two women with POF illustrated an increase in baseline estrogen levels and the volume of the treated ovaries along with amelioration of menopausal symptoms.281 The clinical procedures used in this early trial were invasive, as patients underwent two operations: (1) BM aspiration and (2) laparoscopy. A similar approach was used in two trials conducted in 10 women with POF (age range from 2633 years old) and 30 patients (age from 18 to 40 years old).282 A later study investigated two different routes of cell delivery, including laparoscopy and the ovarian artery, but the results have not been reported at this time.282 Based on the positive outcomes of the mouse model, an autologous stem cell ovarian transplantation (ASCOT) trial was deployed using BM-derived stem cells with encouraging observations of improved ovarian function, as determined by elevated levels of AMH and AFC in 81.3% of participants, six pregnancies, and the successful delivery of three healthy babies.283 A randomized trial (NCT03535480) was conducted in 20 patients with POF aged less than 39 years to further elaborate on the results of the ASCOT trial.284 To date, there are no completed trials using AT-MSCs or UC-MSCs in the treatment of patients with POF, limiting the evaluation of these MSCs in the treatment of POF. The speculated reason is that POF is a rare disease, affecting 1% of women younger than 40 years, and with improvements in assisted productive technology, patients have several alternative options to enhance the recovery of reproductive function.285

Burns are the fourth most common injury worldwide, affecting ~11 million people, and are a major cause of death (180,000 patients annually). The severity of burns is defined based on the percentage of surface area burned, burn depth, burn location and patient age, and burns are usually classified into first-, second-, third-, and fourth-degree burns on the basis of their severity.286 Postburn recovery depends on the severity of the burn and the effectiveness of treatment. Rapid healing may occur over weeks, while alternatively, healing can take months, with the ultimate result being scar formation and disability in patients with severe burns. Different from mechanical injury, burn injury is an invasive progression of damage to tissue at the burn site, including both mechanical damage to the skin surface and biological damage caused by natural apoptosis that prolongs excessive inflammation, oxidative stress, and impaired tissue perfusion.287 To date, completely reversing the devastating damage of severe burns remains unachievable in medicine, and stem cell therapy provides an alternative option for patients with burn injury. The first case report of the use of BM-MSCs to treat a 45-year-old patient with burns on 40% of their body demonstrated the safety of the therapy and showed partial improvements in vascularization at the wound site and reduced coarse cicatrices.288,289 Later, patients with second- and third-degree burns as well as deep burns were treated using either autologous BM-MSCs or allogeneic BM-MSCs by spraying the MSCs onto the burn sites or adding MSCs over a dermal matrix sheet to cover the wound. The results in these case reports revealed the potential efficacy of MSC-based therapy, which not only enhanced the speed of wound recovery but also reduced pain and improved blood supply without introducing infection.288,290,291 In 2017, a study conducted in 60 patients with 1025% of their total body surface areas burned treated with either autologous BM-MSCs or UC-MSCs showed that both MSC types improved the rate of healing and reduced the hospitalization period.292 The drawback of BM-MSCs in the treatment of burns is the invasive harvesting method, which causes pain and possible complications in patients. Hence, treatment with allogeneic MSCs obtained from healthy donors is the method of choice, and AT- and UC-MSCs are two suitable candidates for this option. To date, a limited number of clinical trials have been conducted using MSC therapy. These trials have several limitations in trial design, such as a lack of a negative control group and blinding, small sample sizes, and the use of standardized measurement tools for burn injury and wound healing. Currently, AT-MSCs are being used in seven ongoing phase I and II trials in the treatment of burns. Hence, it is important to note that among the most widely studied MSCs, AT-MSCs have advantages over BM-MSCs when obtained from an allogeneic source, while their abilities in burn treatment remain to be determined. The main MSCs that should be used in the regeneration of burn tissue remain undefined (Table ), and we observed the trend that AT-MSCs are more suitable candidates due to their biological nature, which contributes to the generation of keratinocytes and secretion profiles that strongly enhance the skin regeneration process.293296

The reported clinical trials using MSCs from AT, BM, and UC in the treatment of the endocrinological disorder, reproductive disease, and skin healing

In the last two decades, great advancements have been achieved in the development of novel regenerative medicine and cardiovascular research, especially stem cell technology.297 The discovery of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs) opened a new door for basic research and therapeutic investigation of the use of these cells to treat different diseases.298 However, the clinical path of hiPSCs and hiPSC-derived cardiomyocytes in the treatment of cardiovascular diseases is limited due to the potential for teratoma formation with hiPSCs and the immaturity of hiPSC-derived cardiomyocytes, which might pose a risk of cancer formation,299 arrhythmia, and cardiac arrest to patients.300 A recently emerged stem cell type is adult stem cells/progenitor cells, including MSCs, which can stimulate myocardial repair post administration due to their paracrine effects. Promising results of MSC-based therapy obtained from preclinical studies of cardiac diseases enhance the knowledge and strengthen the clinical research to investigate the safety and efficacy in a clinical trial setting. There are papers that discuss the importance of MSC therapy in the treatment of cardiovascular diseases, with the following references being highly recommended.301306 To date, 36 trials have evaluated the therapeutic potential of MSCs in different pathological conditions, with the most prevalent types being BM-MSCs (25 trials), followed by UC-MSCs (7 trials) and AT-MSCs (4 trials).303 However, the reported results are contradictory and create controversy about the efficacy of the treatments.

One of the first trials using MSCs in the treatment of chronic heart failure was the Cardiopoietic Stem Cell Therapy in Heart Failure (C-CURE) trial, a multicentre, randomized clinical trial that recruited 47 patients. The trial findings supported the safety of BM-MSC therapy and provided a data set that demonstrated improvements in cardiovascular scores along with New York Heart Association functional class, quality of life, and general physical health.307 Despite these encouraging results in the phase I trial, the treatment failed to achieve the primary outcomes in the phase II/III trial (CHART-1 trial), including no significant improvements in cardiac structure or function or patient quality of life.308 A positive outcome was also found in a phase I/II, randomized pilot study called the POSEIDON trial, which was the first trial to demonstrate the superior effectiveness of the administration of allogeneic BM-MSCs compared to allogeneic MSCs from other sources.309,310 Published results from the MSC-HF study, with 4 years of follow-up results,311,312 and the TRIDENT study313 illustrated the positive outcomes of BM-MSCs in the treatment of heart failure. However, a contradictory result from the recently published CONCERT-HF trial demonstrated that the administration of autologous BM-MSCs to patients diagnosed with chronic ischemic heart failure did not improve left ventricular function or reduce scar size at 12 months post administration, but the patients quality of life was improved.314 This observation is similar to that of the TAC-HFT trial315 but completely different from the reported results of the MSC-HF trial. A comprehensive investigation is still needed to determine the reasons behind these contradictory results. The largest clinical trial to date using BM-MSCs is the DREAM-HF study, which was a randomized, double-blind, placebo-controlled, phase III trial that was conducted at 55 sites across North America and recruited a total of 565 patients with ischemic and nonischaemic heart failure.172 Although recent reports from the sponsor confirmed that the trial missed its primary endpoint (a reduction in recurrent heart failure-related hospitalization), other prespecified endpoints were met, such as a reduction in overall major adverse cardiac events (including death, myocardial infarction, and stroke).306 Thus, a complete report from the DREAM-HF trial will provide pivotal data supporting the therapeutic potential of BM-MSCs in the treatment of heart failure and open a new path for the FDA to approve cell-based therapy for cardiovascular diseases.

The early trial using AT-derived cells was the PRECISE trial, which was a phase I, randomized, placebo-controlled, double-blind study that examined the safety and efficacy of adipose-derived regenerative cells (ADRCs) in the treatment of chronic ischemic cardiomyopathy.316 ADRCs are a homogenous population of cells obtained from the vascular stromal fraction of AT, which contains a small proportion of AT-MSCs.317 Although the study supported the safety of ADRC administration and illustrated a preserved functional capacity (peak VO2) in the treated group and improvements in heart wall motion, neither poor left ventricle (LV) volume nor poor left ventricular ejection fraction (LVEF) was ameliorated. The follow-up trial of the PRECISE trial, called the ATHENA trial, was conducted in 31 patients, although the study was terminated prematurely because two cerebrovascular events occurred, which were not related to the cell product itself.318 The results of the study illustrated increases in functional capacity, hospitalization rate, and MLHFQ scores, but the LV volume and LVEF were not significantly different between the two groups. Kastrup and colleagues conducted the first in vitro expanded AT-MSC trial in ten patients with ischemic heart disease and ischemic heart failure in 2017. The results confirmed that ready-to-use AT-MSCs were well-tolerated and potentially effective in the treatment of ischemic heart disease and heart failure.319 Comparable results of AT-MSCs were also reported from the MyStromalCell Trial, which was a randomized placebo-controlled study. In this trial, 61 patients were randomized at a 2:1 ratio into two groups, with the results showing no significant difference in the primary endpoint, which was a change in the maximal bicycle exercise tolerance test (ETT) score from baseline to 6 months post administration.320 A 3-year follow-up report from the MyStromalCell Trial confirmed that patients who received AT-MSC administration maintained their preserved exercise capacity and their cardiac symptoms improved, whereas the control group experienced a significant reduction in exercise performance and a worsened cardiovascular condition.321

UC-MSCs are potential allogeneic cells for the treatment of cardiovascular disease, as they are ready to use and easy to isolate, they rapidly proliferate, and they secrete hepatocyte growth factors,322 which are involved in cardioprotection and cardiovascular regeneration.323 The pilot study using UC-MSCs in 30 patients with heart failure, called the RIMECARD trial, was the first reported trial for which the results supported the effectiveness of UC-MSCs, as seen in the improved ejection fraction, left ventricular function, functional status, and quality of life in patients administered UC-MSCs.324 Encouraging results reported from a phase I/II HUC-HEART trial325 showed improvements in LVEF and reductions in the size of the injured area of the myocardium. However, the opposite observations were also reported from a recently published phase I randomized trial using a combination of UC-MSCs and a collagen scaffold in patients with ischemic heart conditions, in which the size of fibrotic scar tissue was not significantly reduced.326

Although MSCs from AT, BM, and UC have proven to be safe and feasible in the treatment of cardiovascular diseases, the correlation between the MSC types and their therapeutic potentials is still uncertain because different results have been reported from different clinical trials (Table ). The mechanisms by which MSCs participate in recovery and enhance myocardial regeneration have been discussed comprehensively in a recently published review;305,327 therefore, they will not be discussed in this review. In fact, the challenges of MSC-based therapy in cardiovascular diseases have been clearly described previously,328 including (1) the lack of an in vitro evaluation of the transdifferentiation potential of MSCs to functional cardiac and endothelial cells,329 (2) the uncontrollable differentiation of MSCs to undesirable cell types post administration,330 and (3) the undistinguishable nature of MSCs derived from different sources with various levels of differentiation potential.331 Therefore, the applications of MSC-based therapy in cardiovascular disease are still in their immature stage, with potential benefits to patients. Thus, there is a need to conduct large-scale, well-designed randomized clinical trials not only to confirm the therapeutic potential of MSCs from various sources but also to enhance our knowledge of cardiovascular regeneration post administration.

The reported clinical trials using MSCs from AT, BM, and UC in the treatment of cardiovascular diseases

Bones are complex structures constituting a part of the vertebrate skeleton, and they play a vital role in the production of blood cells from HSCs. Similar to the functions of most vertebrate organs, bone function is tightly regulated by its constituents and by long-range signaling from AT and the adrenal glands, parathyroid glands, and nervous system.332 The central nervous system (CNS) orchestrates the voluntary and involuntary input transmitted by a network of peripheral nerves, which act as the bridge between the nervous system and target organs. The CNS controls involuntary responses via the autonomic nervous system (ANS), consisting of the sympathetic nervous system and the parasympathetic nervous system, and voluntary responses via the somatic nervous system. The ANS penetrates deep into the BM cavity, reaching the regions of hematopoietic activity to deliver neurotransmitters that tightly regulate BM stem cell niches.333 The BM microenvironment consists of various cell types that participate in the maintenance of HSC niches, which are composed of specialized cells, including BM-MSCs (Fig. ). The release of a specific neurotransmitter, circadian norepinephrine, from the sympathetic nervous system at nerve terminals leads to a reduction in the circadian expression of CX-C chemokine ligand 12 (CXCL12, which is also known as stromal cell-derived factor-1 (SDF-1)) by Nestin+/NG22+ BM-MSCs, resulting in the secretion of HSCs into the peripheral bloodstream.334,335 In fact, BM-MSCs play a significant role in the regulation of HSC quiescence and are closely associated with arterioles and sympathetic nervous system nerve fibers. Nestin-expressing BM-MSCs have been shown to express high levels of SDF-1, stem cell factor (SCF), angiopoietin-1 (Ang-1), interleukin-7, vascular cell adhesion molecule 1 (VCAM-1), and osteopontin (OPN), which are directly involved in the regulation and maintenance of HSC quiescence.336 The depletion of BM-MSCs in BM leads to the mobilization of HSCs into the peripheral bloodstream and spleen. The findings from a previous study demonstrated that reduced SDF-1 expression in norepinephrine-treated BM-MSCs resulted in the mobilization of CXCR4+ HSCs into circulation.337 The ability of BM-MSCs to produce SDF-1 is tightly related to their neuronal protective functions.338 SDF-1 is a member of a chemokine subfamily that orchestrates an enormous diversity of pathways and functions in the CNS, such as neuronal survival and proliferation. The chemokine has two receptors, CXCR4 and CXCR7, that are involved in the pathogenic development of neurodegenerative and neuroinflammatory diseases.339 In the damaged brain, SDF-1 functions as a stem cell homing signal, and in acquired immune deficiency syndrome (AIDS), SDF-1 has been reported to be involved in the protection of damaged neurons by preventing apoptosis. In a traumatic brain injury model, SDF-1 was found to function as an inhibitor of the caspase-3 pathway by upregulating the Bcl-2/Bax ratio, which in turn protects neurons from apoptosis.340 Moreover, the release of SDF-1 also facilitates cell recruitment, cell migration, and the homing of neuronal precursor cells in the adult CNS by activating the CXCR4 receptor.341,342 Existing data support that SDF-1 acts as the guiding signal for the regeneration of axon growth in damaged neurons and enhances spinal nerve regeneration.343,344 Hence, the ability of BM-MSCs to express SDF-1 in response to the neuronal environment provides a unique neuronal protective effect that could explain the potential therapeutic efficacy of BM-MSCs in the treatment of neurodegenerative diseases (Fig. ).

The nature of the stem niche of bone marrow-derived mesenchymal stem cells (BM-MSCs) supports their therapeutic potential in neuron-related diseases. a Bone marrow is a complex stem cell niche regulated directly by the central nervous system to maintain bone marrow homeostasis and haematopoietic stem cell (HSC) functions. MSCs in bone marrow respond to the environmental changes through the release of norepinephrine (NE) from the sympathetic nerves that regulate the synthesis of SDF-1 and the migration of HSCs through the sinusoids. The secretion of stem cell factors (SCFs), VCAM-1 and angiotensin-1 from MSCs also plays a significant role in the maintenance of HSCs. b BM-MSCs have the ability to produce and release SDF-1, which directly contributes to neuroprotective functions at the damaged site through interaction with its receptors CXCR4/7, located on the neuronal membrane. c Neuronal protection and the functional remyelination induced by BM-MSCs are also modulated by the release of a wide range of growth factors, including VEGF, BDNF, and NGF, by the BM-MSCs. d BM-MSCs also have the ability to regulate neuronal immune responses by direct interaction or paracrine communication with microglia. Figure was created with BioRender.com

The migration of exogenous MSCs after systemic administration to the brain is limited by the physical bloodbrain barrier (BBB), which is a selective barrier formed by CNS endothelial cells to restrict the passage of molecules and cells. The mechanism of molecular movement across the BBB is well established, but how stem cells can bypass the BBB and home to the brain remains unclear. Recent studies have reported that MSCs are able to migrate through endothelial cell sheets by paracellular or transcellular transport followed by migration to the injured or inflammatory site of the brain.345,346 During certain injuries or ischemic events, such as brain injury, stroke, or cerebral palsy, the integrity and efficiency of BBB protection is compromised, which allows MSC migration across the BBB via paracellular transport through the transient formation of interendothelial gaps.347 CD24 expression has been detected in human BM-MSCs, which are regulated by TGF-3,348 allowing them to interact with activated endothelial cells via P-selectin and initiate the tethering and rolling steps of MSCs.349 Additionally, BM-MSCs express high levels of CXCR4 or CXCR7,350,351 which bind to integrin receptors, such as VLA-4, to activate the integrin-binding process and allow the cells to anchor to endothelial cells, followed by the migration of MSCs through the endothelial cell layer and basement membrane in a process called transmigration.352 This process is facilitated by the secretion of matrix metalloproteinases (MMPs), which degrade the endothelial basement membrane, allowing BM-MSCs to enter the brain environment.353,354 BM-MSCs can also regulate the integrity of the BBB via the secretion of tissue inhibitor of matrix metalloproteinase-3 (TIMP3), which has been shown to ameliorate the effects of a compromised BBB in traumatic brain injury.355 The secretion of TIMP3 from MSCs directly blocked vascular endothelial growth factor a (VEGF-a)-induced breakdown of endothelial cell adherent junctions, demonstrating the potential mechanism of BM-MSCs in the regulation of BBB integrity.

The therapeutic applications of BM-MSCs in neurodegenerative conditions have been significantly increased by the demonstration of BM-MSC involvement in axonal and functional remyelination processes. Remyelination is a spontaneous regenerative process occurring in the human CNS to protect oligodendrocytes, neurons, and myelin sheaths from neuronal degenerative diseases.356 Remyelination is considered a neuroprotective process that limits axonal degeneration by demyelination and neuronal damage. The first mechanism of action of BM-MSCs related to remyelination is the activation of the JAK/STAT3 pathway to regulate dorsal root ganglia development.357 It was reported that BM-MSCs secrete vascular endothelial growth factor-A (VEGF-A),358 brain-derived neurotrophic factor (BDNF), interleukin-6, and leukemia inhibitor factor (LIF), which directly function in neurogenesis and neurite growth.357 VEGF-A is a key regulator of hemangiogenesis during development and bone homeostasis. Postnatally, osteoblast- and MSC-derived VEGF plays a critical role in maintaining and regulating bone homeostasis by stimulating MSC differentiation into osteoblasts and suppressing their adipogenic differentiation.359361 To balance osteoblast and adipogenic differentiation, VEGF forms a functional link with the nuclear envelope protein laminin A, which in turn directly regulates the osteoblast and adipocyte transcription factors Runx2 and PPAR, respectively.361,362 In the brain, VEGF is a potent growth factor mediating angiogenesis, neural migration, and neuroprotection. VEGF-A, secreted from BM-MSCs under in vitro xeno- and serum-free culture conditions, is the most studied member of the VEGF family and is suggested to play a protective role against cognitive impairment, such as in the context of Alzheimers disease pathology or stroke.363365 Recently, it was reported that the neurotrophic and neuroprotective function of VEGF is mediated through VEGFR2/Flk-1 receptors, which are expressed in the neuroproliferative zones and extend to astroglia and endothelial cells.366 In animal models of intracerebral hemorrhage and cerebral ischemia, the transfusion of Flk-1-positive BM-MSCs promotes behavioral recovery and anti-inflammatory and angiogenic effects.367,368 Moreover, supplementation with VEGF-A in neuronal disorders enhances intraneural angiogenesis, improves nerve regeneration, and promotes neurotrophic capacities, which in turn increase myelin thickness via the activation of the prosurvival transcription factor nuclear factor-kappa B (NF-kB). This activation, together with the downregulation of Mdm2 and increased expression of the pro-apoptotic transcription factor p53, is considered to be the neuroprotective process associated with an increased VEGF-A level.369371 An analysis of microRNA (miRNA) in extracellular vesicles (EVs) secreted from BM-MSCs revealed that BM-MSCs release substantial amounts of miRNA133b, which suppresses the expression of connective tissue growth factor (CTGF) and protects hippocampal neurons from apoptosis and inflammatory injury372374 (Fig. ).

In terms of immunoregulatory functions, the administration of human BM-MSCs into immunocompetent mice subjected to SCI or brain ischemia showed that BM-MSCs exhibited a short-term neuronal protective function against neurological damage (Fig. ). Further investigation demonstrated the ability of BM-MSCs to directly communicate with host microglia/macrophages and convert them from phenotypic polarization into alternative activated microglia/macrophages (AAMs), which are key players in axonal extension and the reconstruction of neuronal networks.375 Other studies have also illustrated that the administration of AAMs directly to the injured spinal cord induced axonal regrowth and functional improvement.376 The mechanism by which BM-MSCs activate the conversion of microglia/macrophages occurs through two representative macrophage-related chemokine axes, CCL2/CCR2 and CCL-5/CCR5, both of which exhibit acute or chronic elevation following brain injury or SCI.377 The CCL2/CCR2 axis contributed to the enhancement of inflammatory function, and BM-MSC-mediated induction of CCL2 did not alter the total granulocyte number (Fig. ). Although the chemokine-mediated mechanism of BM-MSCs in the activation of AAMs and enhanced axonal regeneration at the damage sites is evident, the direct mechanism by which the communication between BM-MSCs and the target cells results in these phenomena remains unclear, and further investigation is needed.

BM-MSCs also confer the ability to regulate the inflammatory regulation of the immune cells present in the brain by (1) promoting the polarization of macrophages toward the M2 type, (2) suppressing T-lymphocyte activities, (3) stimulating the proliferation and differentiation of regulatory T cells (Tregs), and (4) inhibiting the activation of natural killer (NK) cells. BM-MSCs secrete glial cell line-derived neurotrophic factor (GDNF), a specific growth factor that contributes directly to the transition of the microglial destructive M1 phenotype into the regenerative M2 phenotype during the neuroinflammatory process.378 A similar result was also found in AT-379 and UC-MSCs380 under neuroinflammation-associated conditions, suggesting that AT-, BM-, and UC-MSCs share the same mechanism in promoting macrophage polarization. In terms of T-lymphocyte suppression, compared to MSCs from AT and BM, UC-MSCs show the strongest potential to inhibit the proliferation of T-lymphocytes by promoting cell cycle arrest (G0/G1 phase) and apoptosis.381 In addition, UC-MSCs have been proven to be more effective in promoting the proliferation of Tregs382 and inhibiting NK activation.383 Although MSCs are well-known for their inflammatory regulatory ability, the mechanism is not exclusive to BM-MSCs, especially in neurological disorders.384

In contrast to AT-MSCs and BM-MSCs, UC-MSCs have lower expression of major histocompatibility complex I (MHC I) and no expression of MHC II, which prevents the complications of immune rejection.385 Moreover, as UC is considered a waste product after birth, with the option of noninvasive collection, UC-MSCs are easier to obtain and culture than AD- and BM-MSCs.386 These advantages of UC-MSCs have contributed to their use in the treatment of pulmonary diseases, especially during the rampant COVID-19 pandemic, as off-the-shelf products. Numerous pulmonary diseases have been the subject of applications of UC-MSCs, including BPD, COPD, ARDS, and COVID-19-induced ARDS. In BPD, premature infants are born before the alveolarization process, resulting in arrested lung development and alveolar maturation. Upon administration via an IV route, the majority of exogenous UC-MSCs reach the immature lung and directly interact with immune cells to exert their immunomodulatory properties via cell-to-cell interaction mechanisms (Fig. ). UC-MSCs interact with T cells via the PD-L1 ligand, which binds to the PD-1 inhibitory molecule on T cells, resulting in the suppression of CD3+ T-cell proliferation and effector T-cell responses.387 In addition, UC-MSCs also express CD54 (ICAM-1), which plays a crucial role in the immunomodulatory functions of T cells.388 Direct contact between UC-MSCs and macrophages via CD54 expression on UC-MSCs promotes the immune regulation of UC-MSCs via the regulation of phagocytosis by monocytes.389 Moreover, the contact of UC-MSCs with macrophages during proinflammatory responses increases the secretion of TSG-6 by UC-MSCs, which in turn promotes the inhibitory regulation of CD3+ T cells, macrophages, and monocytes by MSCs.390 Recently, upregulation of SDF-1 was described in neonatal lung injury, especially in layers of the respiratory epithelium.391 SDF-1 has been shown to participate in the migration and initiation of the homing process of MSCs via the CXCR4 receptors on their surface.392 It was reported that UC-MSCs express low levels of CXCR4, allowing them to induce SDF-1-associated migration processes via the Akt, ERK, and p38 signal transduction pathways.393 Hence, in BPD, the upregulation of SDF-1 together with the homing ability of UC-MSCs strongly supports the therapeutic effects of UC-MSCs in the treatment of BPD. Furthermore, UC-MSCs have the ability to communicate with immune cells via cell-to-cell contact to reduce proinflammatory responses and the production of proinflammatory cytokines (such as TGF-, INF-, macrophage MIF, and TNF-). The modulation of the human innate immune system by UC-MSCs is mediated by cellcell interactions via CD54-LFA-1 that switch macrophage polarization processes, promoting the proliferation of M2 macrophages, which in turn reduce inflammatory responses in the immature lung.394 Moreover, UC-MSCs also have the ability to produce VEGF and hepatocyte growth factors (HGFs), promoting angiogenesis and enhancing lung maturation.395

Adipose tissue-derived mesenchymal stem cells (AT-MSCs) and the nature of their tissue of origin support their use in therapeutic applications. a Adipose tissue is considered an endocrine organ, supporting and regulating various functions, including appetite regulation, immune regulation, sex hormone and glucocorticoid metabolism, energy production, the orchestration of reproduction, the control of vascularization, and blood flow, the regulation of coagulation, and angiogenesis and skin regeneration. b In terms of metabolic disorders, such as type 2 diabetes mellitus (T2DM), as adipose tissue is directly involved in the metabolism of glucose and lipids and the regulation of appetite, the detrimental effects of T2DM also alter the functions of AT-MSCs, which in turn, hampers their therapeutic effects. Hence, the use of autologous AT-MSCs is not recommended for the treatment of metabolic disorders, including T2DM, suggesting that allogeneic AT-MSCs from healthy donors could be a better alternative approach. c AT-MSCs are suitable for the treatment of reproductive disorders due to their unique ability to mobilize and home to the thecal layer of the injured ovary, enhance the regeneration and maturation of thecal cells, increase the structure and function of damaged ovaries via exosome-activated SMAD, decrease oxidative stress and autophagy, and increase the proliferation of granulosa cells via PI3K/AKT pathways. These functions are regulated specifically by growth hormones produced by AT-MSCs in response to the surrounding environment, including HGF, TGF-, IGF-1, and EGF. d AT-MSCs are also good candidates for skin healing and regeneration as their growth factors strongly support neovascularization and angiogenesis by reducing PLL4, increase anti-apoptosis via the activation of PI3K/AKT pathways, regulate inflammation by downregulating NADPH oxidase isoform 1, and increase immunoregulation through the inhibition of NF-B activation. The figure was created with BioRender.com

COPD is characterized by an increase in hyperinflammatory reactions in the lung, compromising lung function and increasing the development of lung fibrosis. The mechanism by which UC-MSCs contribute to the response to COPD is inflammatory regulation (Fig. ). The administration of UC-MSCs prevented the infiltration of inflammatory cells in peribronchiolar, perivascular, and alveolar septa and switched macrophage polarization to M2.396 A significant reduction in proinflammatory cytokines, including IL-1, TNF-, and IL-8, was also observed following UC-MSC administration.224 MSCs, including UC-MSCs, have been reported to trigger the production of secretory leukocyte protease inhibitors in epithelial cells through the secretion of HGF and epidermal growth factor (EGF), which is believed to have beneficial effects on COPD.397,398 In addition to their inflammatory regulation ability, UC-MSCs exhibit antimicrobial effects through the inhibition of bacterial growth and the alleviation of antibiotic resistance during Pseudomonas aeruginosa infection.399 The combination of the regulation of the host immune response and the antimicrobial effects of UC-MSCs may be relevant for the prevention and treatment of COPD exacerbations, as inflammation and bacterial infections are important risk factors that significantly contribute to the morbidity and mortality of patients with COPD. In terms of regenerative functions, UC-MSCs were reported to be able to differentiate into type 2 alveolar epithelial cells in vitro and alleviate the development of pulmonary fibrosis via -catenin-regulated cell apoptosis.400 Furthermore, UC-MSCs enhanced alveolar epithelial cell migration and proliferation by increasing matrix metalloproteinase-2 levels and reduced their endogenous inhibitors, tissue inhibitors of matrix metalloproteinases, providing a potential mechanism underlying their anti-pulmonary-fibrosis effects.401,402

In ARDS, especially that associated with COVID-19, the proinflammatory state is initiated by increases in plasma concentrations of proinflammatory cytokines, such as IL-1 beta, IL-7, IL-8, IL-9, IL-10, bFGF, granulocyte colony-stimulating factor (G-CSF), GM-CSF, IFN-, and TNF-. The significant increases in the concentrations of these cytokines in patient plasma suggest the development of a cytokine storm, which is a leading cause of COVID-induced mortality. In addition to the immunomodulatory functions regulated via cell-to-cell interactions between UC-MSCs and immune cells, such as macrophages, monocytes, and T cells, UC-MSCs exert their functions via paracrine effects through the secretion of growth factors, cytokines, and exosomes (Fig. ). The most relevant immunomodulatory function of UC-MSCs is considered to be their inhibition of effector T cells via the induction of T-cell apoptosis and cell cycle arrest by the production of indoleamine 2,3- dioxygenase (IDO), prostaglandin E2 (PGE-2), and TGF-. Elevated levels of PGE-2 in patients with COVID-19 are reported to be a crucial factor in the initiation of inflammatory regulation by UC-MSCs post administration and prevent the development of cytokine storms by direct inhibition of T- and B lymphocytes.403 UC-MSCs exert these inhibitory activities through a PGE-2-dependent mechanism.404 It was reported that UC-MSCs confer the ability to secrete tolerogenic mediators, including TGF-1, PGE-2, nitric oxide (NO), and TNF-, which are directly involved in their immunoregulatory mechanism. The secretion of NO from UC-MSCs is reported to be associated with the desensitization of T cells via the IFN-inducible nitric oxide synthase (iNOS) pathways and to stimulate the migration of T cells in close proximity to MSCs that subsequently suppress T-cell sensitivities via NO.405 Lung infection with viruses usually leads to impairments in alveolar fluid clearance and protein permeability. The administration of UC-MSCs enhances alveolar protection and restores fluid clearance in patients with COVID-19. UC-MSCs secrete growth factors associated with angiogenesis and the regeneration of pulmonary blood vessels and micronetworks, including angiotensin-1, VEGF, and HGF, which also reduce oxidative stress and prevent fibrosis formation in the lungs. These trophic factors have been identified as key players in the modulation of the microenvironment and promote pulmonary repair. Additionally, UC-MSCs are more effective than BM-MSCs in the restoration of impaired alveolar fluid clearance and the permeability of airways in vitro, supporting the use of UC-MSCs in the treatment of patients with pulmonary pneumonia.406 In the context of pulmonary regeneration, UC-MSCs were shown to inhibit apoptosis and fibrosis in pulmonary tissue by activating the PI3K/AKT/mTOR pathways via the secretion of HGF, which also acts as an inhibitory stimulus that blocks alveolar epithelial-to-mesenchymal transition.407,408 Moreover, UC-MSCs can reverse the process of fibrosis via enhanced expression of macrophage matrix-metallopeptidase-9 for collagen degradation and facilitate alveolar regeneration via Toll-like receptor-4 signaling pathways.409 UC-MSCs were shown to communicate with CD4+ T cells through HGF induction not only to inhibit their differentiation into Th17 cells, reducing the secretion of IL-17 and IL-22 but also to switch their differentiation into regulatory T cells.410,411 In addition, UC-MSCs conferred the ability to facilitate the number of M2 macrophages and reduce M1 cells via the control of the macrophage polarization process.412

There are several potential mechanisms of UC-MSCs in the treatment of patients with pulmonary diseases and pneumonia, including the regulation of immune cell function, immunomodulation, the enhancement of alveolar fluid clearance and protein permeability, the modulation of endoplasmic reticulum stress, and the attenuation of pulmonary fibrosis. Hence, based on these discussions, UC-MSCs are recommended as suitable candidates for the treatment of pulmonary disease both in pediatric and adult patients.

Human AT was first viewed as a passive reservoir for energy storage and later as a major site for sex hormone metabolism, the production of endocrine factors (such as adipsin and leptin), and a secretion source of bioactive peptides known as adipokines.413 It is now clear that AT functions as a complex and highly active metabolic and endocrine organ, orchestrating numerous different biological features414 (Fig. ). In addition to adipocytes, AT contains hematopoietic-derived progenitor cells, connective tissue, nerve tissue, stromal cells, endothelial cells, MSCs, and pericytes. AT-MSCs and pericytes mobilize from their perivascular locations to aid in healing and tissue regeneration throughout the body. As AT is involved directly in energy storage and metabolism, AT-MSCs are also mediated and regulated by growth factors related to these pathways. In particular, interleukin-6 (IL-6), IL-33, and leptin regulate the maintenance of metabolic activities by increasing insulin sensitivity and preserving homeostasis related to AT. Nevertheless, in the development of obesity and diabetes, omental and subcutaneous AT maintains a low-grade state of inflammation, resulting in the impairment of glucose metabolism and potentially contributing to the development of insulin resistance.415 In normal AT, direct regulation of Pre-B-cell leukemia homeobox (Pbx)-regulating protein-1 (PREP1) by leptin and thyroid growth factor-beta 1 (TGF-1) in AT-MSCs and mature adipocytes is involved in the protective function and maintenance of AT homeostasis. However, under diabetic conditions, the balance between the expression of leptin and the secretion of TGF-1 is compromised, resulting in the malfunction of AT-MSC metabolic activity and the proliferation, differentiation, and maturation of adipocytes. Therefore, the use of autologous AT-MSCs in the treatment of diabetic conditions is not a suitable option, as the functions of AT-MSCs are directly altered by diabetic conditions, which reduces their effectiveness in cell-based therapy (Fig. ).

Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are good candidates for the treatment of pulmonary diseases. a Lung immaturity and fibrosis are the major problems of patients with bronchopulmonary dysplasia and lead to increased levels of SDF-1, the development of fibrosis, the induction of the inflammatory response, and the impairment of alveolarization. UC-MSCs are attracted to the damaged lung via the chemoattractant SDF-1, which is constantly released from the immature lung via SDF-1 and CXCR4 communication. Moreover, UC-MSCs reduce the level of proinflammatory cytokines (TGF-, INF-, macrophage MIF, and TNF-) via a cell-to-cell contact mechanism. The ability of UC-MSCs to produce and secrete VEGF also involves in the regeneration of the immature lung through enhanced angiogenesis. b Upon an exacerbation of chronic obstructive pulmonary disease (COPD), UC-MSCs respond to the surrounding stimuli by reducing IL-8 and TNF- levels, resulting in the inhibition of the inflammatory response but an increase in the secretion of growth factors participating in the protection of alveoli, fluid clearance and reduced oxidative stress and lung fibrosis, including HGF, TGF-, IGF-1, and exosomes. c In a similar manner, UC-MSCs prevent the formation of cytokine storms in coronavirus disease 2019 (COVID-19) by inhibiting CD34+ T-cell differentiation into Th17 cells and enhancing the number of regulatory T cells. Moreover, UC-MSCs also have antibacterial activity by secreting LL-3717 and lipocalin. Figure was created with BioRender.com

Preclinical studies and clinical trials have revealed the therapeutic effects of MSCs, in general, and AT-MSCs, in particular, in the management of POF, with relatively high efficacy and enhanced regeneration of the ovaries. Understanding the molecular and cellular mechanisms underlying these effects is the first step in the development of suitable MSC-based therapies for POF. One of the mechanisms by which MSCs exert their therapeutic effects is their ability to migrate to sites of injury, a process known as homing. Studies have shown that MSCs from different sources have the ability to migrate to different compartments of the injured ovary. For example, BM-MSCs administered through IV routes migrated mostly to the ovarian hilum and medulla,416 whereas a significant number of UC-MSCs were found in the medulla.417 Interestingly, AT-MSCs were found to be engrafted in the theca layers of the ovary but not in the follicles, where they acted as supportive cells to promote follicular growth and the regeneration of thecal layers.418 The structure and function of the thecal layer have a great impact on fertility, which has been reviewed elsewhere.419 In brief, the thecal layer consists of two distinct parts, the theca interna, which contains endocrine cells, and the theca externa, which is an outer fibrous layer. The thecal layer contains not only endocrine-derived cells but also vascular- and immune-derived cells, whose functions are to maintain the structural integrity of the follicles, transport nutrients to the inner compartment of the ovary and produce key reproductive hormones such as androgens (testosterone and dihydrotestosterone) and growth factors (morphogenic proteins, e.g., BMPs and TGF-).420 As AT-MSCs originate from an endocrine organ, their ability to sense signals and migrate to the thecal layer is anticipated. Additionally, secretome analysis of AT-MSCs showed a wide range of growth factors, including HGF, TBG-, VEGF, insulin-like growth factor-1 (IGF-1), and EGF,421 that are directly involved in the restoration of the structure and function of damaged ovaries by stimulating cell proliferation and reducing the aging process of oocytes via the activation of the SIRT1/FOXO1 pathway, a key regulator of vascular endothelial homeostasis.422,423 In POF pathology, autophagy and its correlated oxidative stress contribute to the development of POF throughout a patients life. Recently, AT-MSCs were shown to be able to improve the structure and function of mouse ovaries by reducing oxidative stress and inflammation, providing essential data supporting the mechanism of AT-MSCs in the treatment of POF.424 Several studies have illustrated that AT-MSCs secrete biologically active EVs that regulate the proliferation of ovarian granulosa cells via the PI3K/AKT pathway, resulting in the enhancement of ovarian function.425 Direct regulation of ovarian cell proliferation modulates the state of these cells, which in turn restores the ovarian reserve.426 Other mechanisms supporting the effectiveness of MSCs have been carefully reviewed, confirming the therapeutic potential of MSCs derived from different sources426 (Fig. ).

In the last decade, the number of clinical trials using AT-MSCs in the treatment of chronic skin wounds and skin regeneration has exponentially increased, with data supporting the enhancement of the skin healing processes, the reduction of scar formation, and improvements in skin structure and quality. Several mechanisms are directly linked to the origin of AT-MSCs, including differentiation ability, neovascularization, anti-apoptosis, and immunological regulation. AT is a connective and supportive tissue positioned just beneath the skin layers. AT-MSCs have a strong ability to differentiate into adipocytes, endothelial cells,427 epithelial cells428 and muscle cells.429 The adipogenic differentiation of AT-MSCs is one of the three mesoderm lineages that defines MSC features, and AT-MSCs are likely to be the best MSC type harboring this ability compared to BM- and UC-MSCs. Recent reports detailed that AT-MSCs accelerated diabetic wound tissue closure through the recruitment and differentiation of endothelial cell progenitor cells into endothelial cells mediated by the VEGF-PLC-ERK1/ERK2 pathway.430 Upon injury, the skin must be healed as quickly as possible to prevent inflammation and excessive blood loss. The reparation process occurs through distinct overlapping phases and involves various cell types and processes, including endothelial cells, keratinocyte proliferation, stem cell differentiation, and the restoration of skin homeostasis.431 Hence, the differentiation ability of AT-MSCs plays a critical role in their therapeutic effect on skin wound regeneration and healing processes. AT-MSCs accelerate wound healing via the production of exosomes that serve as paracrine factors. It was reported that AT-MSCs responded to skin wound injury stimuli by increasing their expression of the lncRNA H19 exosome, which upregulated SOX9 expression via miR-19b, resulting in the acceleration of human skin fibroblast proliferation, migration, and invasion.432 In addition, the engraftment of AT-MSCs supported wound bed blood flow and epithelialization processes.433 Anti-apoptosis plays a critical role in AT-MSC-based therapy, as without a microvascular supply network established within 4 days post injury, adipocytes undergo apoptosis and degenerate. Exogenous sources of AT-MSCs mediate anti-apoptosis via IGF-1 and exosome secretion by triggering the activation of PI3K signaling pathways.434 Another mechanism supporting the therapeutic potential of AT-MSCs is their anti-inflammatory function, which results in the reduction of proinflammatory factors, such as tumor necrosis factor (TNF) and interferon- (IFN-), and increases the production of the anti-inflammatory factors IL-10 and IL-4. Exosomes from AT-MSCs in response to a wound environment were found to contain high levels of Nrf2, which downregulated wound NADPH oxidase isoform 1 (NOX1), NADPH oxidase isoform 4 (NOX4), IL-1, IL-6, and TNF- expression. The anti-inflammatory functions of AT-MSCs are also regulated by their immunomodulatory ability, partially through the inhibition of NF-B activation in T cells via the PD-L1/PD-1 and Gal-9/TIM-3 pathways, providing a novel target for the acceleration of wound healing435 (Fig. ).

Therefore, as an endocrine organ in the human body, AT and its derivative stem cells, including AT-MSCs, have shown great potential in the treatment of reproductive disorders and skin diseases. Their potential is supported by mechanisms that are directly related to the nature of AT-MSCs in the maintenance of tissue homeostasis, angiogenesis, anti-apoptosis, and the regulation of inflammatory responses.

Over the past decades, MSC-based research and therapy have made tremendous advancements due to their advantages, including immune evasion, diverse tissue sources for harvesting, ease of isolation, rapid expansion, and cryopreservation as off-the-shelf products. However, several important challenges have to be addressed to further enhance the safety profile and efficacy of MSC-based therapy. In our opinion, the most important challenge of MSC-based therapy is the fate of these cells post administration, especially the long-term survival of allogeneic cells in the treatment of certain diseases. Although reported data confirm that the majority of MSCs are trapped in the lung and rapidly removed from the circulation, caution has been raised related to the occurrence of embolism events post infusion, which was proven to be related to MSC-induced innate immune attack (called instant blood-mediated inflammatory reaction).436 Another related challenge is the homing ability of infused cells, as successful homing at targeted tissue might result in long-term benefits to patients. Other concerns related to MSC-based therapy are the number of dead cells infused into the patients. An interesting study reported that dead MSCs alone still exerted the same immunomodulatory property as live MSCs by releasing phosphatidylserine.437 This is an interesting observation, as there is always a certain number of dead cells present in the cell-based product, and concerns are always raised related to their effects on the patients health. Finally, the hypothesis presented in this review is also a great challenge of the field, which has been proposed for future studies to answer the question: What is the impact of MSC sources on their downstream application?. Tables and illustrate the comparative studies that were conducted in preclinical and clinical settings to address the MSC source challenge. Other challenges of MSC-based therapies have been discussed in several reviews and systematic studies,135,185,438,439 which are highly recommended.

Comparative analysis of the effectiveness of MSC sources in a preclinical setting

Increase BDNF levels in the injured spinal cord, reduce lesion cavity volume and microglia/macrophage infiltration

Induce angiogenesis, axonal regeneration

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Stem cell-based therapy for human diseases - PMC