StemCell Therapy

Over the last several decades, research has greatly increased our understanding of the immune system, genetics, and biology. This research is now showing results in several areas important to rheumatoid arthritis. Scientists are thinking about RA in exciting ways that were not possible years ago.

NIAMS-supported researchers have identified several genetic factors that may make some people more likely to develop rheumatoid arthritis, as well as factors that affect disease severity. Scientists have learned that dozens of genes determine whether a person develops rheumatoid arthritis and how severe the disease will become. Researchers are studying these findings to help identify new treatment approaches for the disease.

Researchers are also investigating the potential connection between health, disease, and the human microbiome, which are microorganisms that inhabit the human body, such as the intestines and the mouth. One study found that the presence of a specific type of gut bacteria correlated with rheumatoid arthritis in newly diagnosed, untreated people. Another study has found that bacteria in the mouth can contribute to the autoimmune RA process, indicating that good dental hygiene is important. More work is being done to understand how bacteria interacts with the immune system in the disease.

NIAMS intramural researchers are studying the natural history of rheumatoid arthritis in children and adults to understand how the disease progresses and affects patient symptoms and functional status.

Investigators are also exploring whether patients with rheumatoid arthritis in remission while taking tumor necrosis factor (TNF) inhibitors can remain in remission after tapering the dose of these medications. Most studies so far suggest that taking them away completely leads to flares. However, researchers are studying and identifying what factors predict who will relapse when treatments are reduced.

Scientists are continuing to understand what happens at the molecular level in rheumatoid arthritis and are working to develop tests that could help diagnose RA earlier and identify patients who would benefit most from specific treatments.

Joint inflammation. NIAMS-funded researchers have determined that joint inflammation can continue in rheumatoid arthritis even after clinical symptoms have eased. This finding may help doctors determine when a patient is truly in remission and can safely stop treatment.

To date, there is still no cure for RA. Researchers continue to identify genes and molecules that contribute to the development and worsening of rheumatoid arthritis and thus are potential targets for new treatments. The path between identifying the molecule and developing a drug that targets it is long and difficult. Fortunately, several new medications for RA have emerged over the past decades that substantially reduce symptoms and damage in rheumatoid arthritis. However, over time, medications may stop working for some people, creating a need for new advanced therapies. Researchers continue to identify more candidate drugs, with hopes that these will have fewer side effects or will cure more patients.

National Institutes of Health Accelerating Medicines Partnership.NIAMS and the National Institute of Allergy and Infectious Diseases are leading the Accelerating Medicines Partnership Autoimmune and Immune-Mediated Diseases (AMP AIM) Program. AMP AIM is a unique publicprivate partnership that aims to characterize the cellular interactions and biological pathways that cause inflammation, injury, abnormal function, and clinical disease in autoimmune diseases such as rheumatoid arthritis.

See the article here:
Rheumatoid Arthritis: Research & Resources - National Institute of ...

The most common symptom of atopic dermatitis is itching, which can be severe. Other common symptoms include:

The symptoms can flare in multiple areas of the body at the same time and can appear in the same locations and in new locations. The appearance and location of the rash vary depending on age; however, the rash can appear anywhere on the body.Patients with darker skin tones often experience darkening or lightening of the skin in areas of skin inflammation.

During infancy and up to 2 years of age, it is most common for a red rash, which may ooze when scratched, to appear on the:

Some parents worry that the infant has atopic dermatitis in the diaper area; however, the condition rarely appears in this area.

During childhood, usually 2 years of age to puberty, it is most common for a red thickened rash, which may ooze or bleed when scratched, to appear on the:

During the teenage and adult years, it is most common for a red to dark brown scaly rash, which may bleed and crust when scratched, to appear on the:

Other common skin features of atopic dermatitis include:

In addition, people with atopic dermatitis often have other conditions, such as:

Researchers continue to study why having atopic dermatitis as a child can lead to the development of asthma and hay fever later in life.

Complications of atopic dermatitis can happen. They include:

Read more:
Atopic Dermatitis Treatment, Symptoms & Causes | NIAMS

Abstract

Personalized medicine (PM) is about tailoring a treatment as individualized as the disease. The approach relies on identifying genetic, epigenomic, and clinical information that allows the breakthroughs in our understanding of how a person's unique genomic portfolio makes them vulnerable to certain diseases. PM approach is a complete extension of traditional approach (One-Size-Fits-All) to increasing our ability to predict which medical treatments will be safe and effective for individual patient, and which ones will not be, based on the patient's unique genetic profile. Implementation of PM has the potential to reduce financial and time expenditure, and increase quality of life and life extension of patients. Knowledge of PM facilitates earlier disease detection via enhanced use of existing biomarkers and detection of early genomic and epigenomic events in disease development, particularly carcinogenesis. The PM approach predominantly focuses on preventative medicine and favours taking pro-active actions rather than just reactive. This approach delays or prevents the need to apply more severe treatments which are usually less tolerated and with increased quality of life and financial considerations. Increasing healthcare costs have placed additional pressure on government funded healthcare systems globally, especially regarding end of life care. PM may increase the effectiveness of existing treatments and negate the inherent problems associated with non-PM approaches. PM is a young but rapidly expanding field of healthcare where a physician can select a treatment based on a patient's genetic profile that may not only minimize harmful side effects and guarantee a more successful result, but can be less cost effective compared with a trial-and-error approach to disease treatment. The less efficient non-PM (trial-and-error) approach, which can lead to drug toxicity, severe side effects, reactive treatment and misdiagnosis continue to contribute to increasing healthcare costs. Increased patient stratification will allow for the enhanced application of PM and pro-active treatment regimens, resulting in reduced costs and quality of life enhancement.

Keywords: personalized medicine, target therapy, tailored therapy

Personalized medicine (PM) is currently a particularly novel and exciting topic in the medicine and healthcare industries. It is a concept that has the potential to transform medical interventions by providing effective, tailored therapeutic strategies based on the genomic, epigenomic and proteomic profile of an individual, whilst also remaining mindful of a patient's personal situation. The power of PM lies not only in treatment, but in prevention. Increased utilisation of molecular stratification of patients, for example assessing for mutations that give rise to resistance to certain treatments, will provide medical professionals with clear evidence upon which to base treatment strategies for individual patients. With this development, there will no longer be a dependence on the adverse outcomes of trial and error prescribing methods (1,2). Currently, when prescribed medication is not effective, the patient may switch to a different medication. This trial and error approach leads to poorer outcomes for patients, in terms of adverse side effects, drug interaction, potential disease progression whilst effective treatment is delayed and patient dissatisfaction (3).

The 21st century vision of PM is to provide the right drug, with the right dose at the right time to the right patient (4). Effective application of PM relies heavily on the availability of rigorous diagnostic tools which allow for the optimal selection of therapeutic product to improve patient outcomes. The products are fully regulated by manufacturers and Food and Drug Administration (FDA) bodies (5). According to the FDA, the aim of PM is to elevate benefits and reduce risks to patients by targeting prevention and treatment more effectively. PM does not seek to establish novel medication for patients, but to stratify individuals into subpopulations that vary in their response to a therapeutic agent for their specific disease. For example, Herceptin is an extremely useful drug for around 2030% of breast cancer patients who have elevated expression of HER2. However, some patients with elevated HER2 are inherently resistant to Herceptin due to mutations to the HER2 gene. Therefore, intelligent molecular characterisation of breast cancer patients at both a genetic and epigenetic level allows for the optimal use of Herceptin through stratification of patients (6).

The revolution of PM has created a lucrative opportunity for pharmaceutical companies developing molecular-targeted therapeutics, but also through the optimised use and repurposing of existing drugs and combination therapies. Adopting PM will alter the approach to diagnosis and treatment, and will lead to increased participation of the patient during and after treatment. For example, active surveillance in prostate cancer gives patients the choice on if they would like curative treatment immediately, with potential complications and discomfort, or wait until there is signs of disease progression (7). This aspect of PM incorporates the circumstances of the patient as an influence on the appropriate treatment strategy for them as a person, not just as a patient.

The advanced commercialization of molecular medicine has produced the novel concept of pharmacogenetics, the application of which is now acknowledged as PM. Molecular targeted therapies include monoclonal antibody (MAb) based therapeutics like herceptin which targets HER2 in breast cancer but MAb therapies are also used clinically to molecularly target therapies for rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis and inflammatory bowel disease as well as several types of cancer (8). Currently, we are seeing the translation of immunotherapy research into clinical practice, including highly PM centric ex vivo modification of immune cells. These treatments, such as Sipuleucel (Provenge) extract dendritic cells from a patient's own blood, incubate and mature them in the presence of prostatic acid phosphatase (PAP), which is expressed on the surface of around 95% of prostate cancer cells. When these modified dendritic cells are re-infused to the patient, they are able to present PAP to the patient's immune system, directing the body to attack its own tumour (9). With the advent of CRISPR-Cas9, scientists now have even greater ability to engineer cells at a genetic level (10). Ex vivo modifications using CRISPR-Cas9 to gene edit specific oncogenes is already underway (11) and could be used to create treatments based on the unique tumour evolution path of each specific patient. Clinical trials using CRISPR-Cas9 gene editing have also begun in the USA (12).

To fully realise the potential of PM, pharmaceutical companies will need to invest in development of new diagnostic techniques, that will help to stratify patients at a higher resolution, allowing for optimised therapeutic selection and timing. PM requires coordinated adjustments to each aspect of the value chain, from discovery to development and from commercialization to lifecycle management (13).

PM is considered to be an innovation in the healthcare system; it is preventive, synchronized and proven (1,2). In the current healthcare system, stakeholders and consumers do not yet fully recognise the benefits of PM. Recent studies demonstrate the following challenges to the development of PM: Scientific challenges (wherein genetic markers are the most clinically significant, with a poor understanding of the molecular mechanisms of certain diseases) (14); economic challenges; operational issues (difficulty identifying technology and operational systems that will save costs); and protection of private information during the investigation and development stages (3). Furthermore, there are policy challenges regarding the association between government research and regulatory agencies (15).

PM has the potential to offer improved medication selection and targeted therapy, reduce adverse effects, increase patient compliance, shift the goal of medicine from reaction to prevention, improve cost effectiveness, and increase patient confidence post-marketing by approving novel therapeutic strategies and altering the perception of medicine in the healthcare system (15).

For the development and rapid adoption of PM it is vital that pharmaceutical companies invest in these new technologies and show willingness to work collaboratively with academic research teams. Identification of more stringent biomarkers and are necessary to inform a pro-active approach to PM. One example is the recent development of liquid biopsies, which can be used to detect DNA circulating in the blood. This type of biopsy is non-invasive, much lower risk than traditional biopsy and has been used to detect disease extremely early. One of the first uses of liquid biopsy was a test for Down syndrome is pregnant mothers (16). Now studies such as TRACERx are using ctDNA (circulating tumour DNA) to analyse and predict the tumour evolution of lung cancer (17). Approaches like this will allow medical professionals to apply PM pro-actively by pre-empting the course of tumour evolution and switching patients onto different therapeutics as soon as signs of drug resistance are detected. This may be able to delay the onset of resistance For long-term purposes, pharmaceutical companies must educate themselves in order to be profitable using the novel diagnostic and treatment methods in markedly reduced volumes. Novel drug development is prohibitively expensive and pharmaceutical companies are increasingly keen to repurpose existing drugs. PM allows for optimisation of treatment regimens and therefore increases the utility of existing products.

All pharmaceutical companies implement PM according to their own methods as follows: i) Transitioning from traditional drug methods to PM is not an option, but a necessity. Accept that each molecule in the pipeline will be personalized to precise patient populations, rather than the mass market. ii) PM is an innovative approach towards delivering improved healthcare and reducing overall healthcare costs. This will be achieved by implementing the digitalization of healthcare, by improving the healthcare IT system and with innovative technologies, such as developing single-cell omics, which permits the investigation of different cells in a high throughput manner (18). iii) Embedding PM skills into the existing healthcare system. The implementation of PM requires a united effort of a broad community of stakeholders, all working towards a primary goal of exploiting breakthrough in science to improve patient care (19,20). iv) Biomarkers (an indicator of biological state) are facilitating the support of research and design (R&D) in healthcare industries. R&D is improved by decreasing trial sizes and increasing the speed to market. Support smaller-market therapeutic agents that are more likely to succeed. v) Access novel capabilities by forming partnerships; for example, contact with world-class diagnostics by corporations with assay developers and different industries. vi) Intelligent sales forces with the most up-to-date expertise. Sales teams will be required to have knowledge of patient history, as well as diagnostic and treatment methods. Furthermore, sales teams must understand molecular analysis and disease pathways. vii) Post market surveillance is particularly important in PM to allow more focused clinical trials of pharmaceutical products (21).

In addition, in the coming decades, the demand for PM will increase, as consumers will become more educated about this novel treatment approach. This will encourage the shift from the current medicine module to the novel methods of diagnosis and treatment. Furthermore, currently, clinical trials are time-consuming and require significant manpower; however, in future the concept of clinical trials will be more advanced and easy to accommodate PM with the help of regulatory approval. The development of PM R&D map by improving public/private sector. Establish a simple method of identifying and prioritising the disease, which may benefit from the application of novel technology. Additionally, development of joint venture programmes for validation of study designs and biomarker standardisation (13).

PM has the potential to fulfil the requirement to improve health outcomes by reducing healthcare costs, drug-development costs and time. This revolution in the healthcare system will only be possible to achieve by equal contribution of patient and consumers in participating in clinical trials, entrepreneurs and innovators to develop smart tools and analyze the genetic information, regulators by educating consumers and providers, and support essential revolutions in policy and regulation, physicians to understand the disease at the molecular level, academic researchers by accompanying innovative research to uncover new insights at the molecular basis of disease and supporting target-based drug development, IT sector by creating unique electronic tools to collect and secure patient information, stakeholders, payer and policy makers by exploring new business models, novel diagnostics tools, target therapy and other personalized treatment protocols. PM has the potential to have a positive effect on the healthcare system. In future, with use of the personalized approach, each individual, on the day of their birth, will receive their full genomic information to place into an individual medical record. This information would allow physicians and clinicians to implement more effective healthcare approaches based on patient exposure to different diseases.

Articles from Biomedical Reports are provided here courtesy of Spandidos Publications

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Personalized medicine could transform healthcare - PMC

Abstract

Significant progress has been made in regenerative medicine for skin repair and rejuvenation. This review examines core technologies including stem cell therapy, bioengineered skin substitutes, platelet-rich plasma (PRP), exosome-based therapies, and gene editing techniques like CRISPR. These methods hold promise for treating a range of conditions, from chronic wounds and burns to age-related skin changes and genetic disorders. Challenges remain in optimizing these therapies for broader accessibility and ensuring long-term safety and efficacy.

Keywords: skin regeneration, rejuvenation, regenerative medicine, stem cells, bioengineered skin substitutes, wound healing, chronic wounds, burns

This review systematically analyzed articles published in English from 2015 to 2024 focusing on regenerative medicine approaches for skin regeneration and rejuvenation. Searches were conducted using PubMed, Scopus, and Web of Science databases. Keywords included combinations of skin regeneration, skin rejuvenation, regenerative medicine, and specific treatment modalities (e.g., stem cell therapy, platelet-rich plasma, exosomes). Studies were included if they presented original research or comprehensive reviews related to the specified topic. Exclusion criteria included studies not published in English, those focusing solely on animal models without human relevance, and those not meeting minimum methodological quality standards such as lack of adequate controls, small sample sizes.

Regenerative medicine is a developing field focused on the repair, rejuvenation, replacement, or regeneration of tissues and organs to reestablish normal function (Mao and Mooney, 2015; Sampogna et al., 2015; Jafarzadeh et al., 2024). In the context of skin, regenerative medicine offers innovative approaches to healing (Shimizu et al., 2022; Mahajan et al., 2024) and rejuvenating the skin (Jo et al., 2021; Taub, 2024), the bodys largest organ and serves as the first line of defense.

The skin is a complex, multi-layered organ composed of the epidermis (outer layer), dermis (middle layer), and hypodermis (subcutaneous tissue). It possesses several critical functions such as a) barrier protection against pathogens, ultraviolet radiation (UV), and physical injury; b) temperature regulation, maintaining body temperature through sweat production and blood vessel dilation or constriction, c) sensory perception, since it contains nerve endings and d) immune defense, where host immune cells protect against infections and participate in wound healing (Yousef et al., 2024).

Skin can be damaged by several factors, including injury, diseases, aging, and environmental stressors (Parrado et al., 2019; Arabpour et al., 2024). Traditional treatments often focus on managing symptoms rather than focusing on the underlying damage. On the other hand, regenerative medicine for skin focuses on repairing or replacement of damaged skin tissue, restoring the skin normal function and appearance by promoting the regeneration of the tissue (Fadilah et al., 2022). Innovative techniques, including stem cell therapy, tissue engineering, and growth factors, have been developed to address conditions such as chronic wounds, burns, scars, chronic ulcers, and aged skin (Shimizu et al., 2022). These approaches aim to accelerate healing, minimize scarring, and restore skin integrity (Fadilah et al., 2022).

Specifically, chronic wounds, such as diabetic foot ulcers (DFU), venous leg (VLU) or pressure ulcers, can be difficult to treat and often fail to heal with conventional methods (Frykberg and Banks, 2015; OuYang et al., 2023). Regenerative approaches such as stem cell therapy and bioengineered skin can promote faster and effective healing in DFU (Chiu et al., 2023). Platelet-rich plasma (PRP), which is rich in growth factors, can be used to enhance healing and regeneration in chronic skin conditions where skin healing is impaired (Xu et al., 2020).

Also, severe burns can result in significant tissue loss and scarring. Traditional burn treatments often involve skin grafts, which can be painful and leave significant scarring (Chogan et al., 2023). On the other hand, regenerative medicine, requires advanced therapies like bioengineered skin grafts, stem cell treatments, exosome therapy, topical application of growth factors, such as Epidermal Growth Factor (EGF) and Platelet Derived Growth Factor (PDGF) directly to burn wounds, to promote faster and more effective healing (Chogan et al., 2023), reducing scarring, and improving functionality, elasticity, and sensitivity, as well as aesthetic outcomes. Additionally, advanced dressings and scaffolds are being proposed such as hydrogel dressings, which provide a moist environment that promotes healing and reduces pain. They can also be infused with growth factors, stem cells, or other regenerative agents to further enhance wound healing (Surowiecka et al., 2022). As far as burned skin, fractional CO2 laser therapy can be applied to remodel scar tissue and improve the texture and elasticity of the skin. This therapy can also be combined with stem cells or growth factors, as treatments to enhance skin regeneration (Roohaninasab et al., 2023). In case of burns, many regenerative treatments, such as stem cell therapy can also be tailored to the individual patient, reducing the risk of immune rejection and improving outcomes (Lukomskyj et al., 2022).

Chronic skin conditions like psoriasis, eczema, and vitiligo often present significant challenges in terms of management and treatment. Regenerative medicine offers new perspectives by targeting the underlying causes of these diseases and promoting long-term regeneration of skin (Paganelli et al., 2020; Daltro et al., 2020; Park et al., 2019; Bellei et al., 2022). It has been shown, for example, that mesenchymal stem cells (MSCs) can reduce the hyperproliferation of skin cells and modulate the immune response, potentially leading to remission of symptoms in autoimmune-related skin conditions like psoriasis and eczema (Shin et al., 2017; Daltro et al., 2020; Diotallevi et al., 2022). Also, exosomes can be topically applied or injected into affected areas to improve skin health and reduce the symptoms of chronic skin conditions (Wang et al., 2019; Farabi et al., 2024). In vitiligo, exosomes may help restore pigmentation by stimulating melanogenesis and melanocyte-stimulating factors (Wong et al., 2020). Gene therapy is another modality that offers a promising approach to correcting the genetic mutations underlying chronic skin diseases. In conditions like epidermolysis bullosa, a severe blistering disorder, gene therapy can be used to restore functional genes in skin cells, potentially improving skin integrity and improving skin integrity and reducing blister formation (Bischof et al., 2024). In addition, CRISPR/Cas9, a gene-editing technology has the potential to correct mutations at the DNA level, a potential solution for certain genetic skin diseases (Abdelnour et al., 2021).

Finally, skin aging, a complex interplay of internal and external factors, can now be targeted with regenerative medicine (He et al., 2023). Since skin health is considered one of the main factors associated with welfare and the perception of health in humans, numerous anti-aging strategies have been developed and proposed (Ganceviciene et al., 2012). In the context of regenerative medicine and skin rejuvenation, anti-aging therapies focus on reversing or slowing down the signs of skin aging, which includes wrinkles, loss of elasticity, pigmentation, and thinning. The aim is to repair damaged skin, promote and/or stimulate collagen production, and restore young skin features (Wong et al., 2020; Ribaudo and Gianoncelli, 2023).

Given stem cells remarkable potential to differentiate into various cell types, they can be used to rejuvenate tissues and organs, enhancing their regenerative capacity (Jin et al., 2023). Mesenchymal stem cells (MSCs) are not only capable of differentiating into skin cells but also are able to release growth factors and cytokines that enhance collagen synthesis and skin tissue regeneration. In this way, stem cell-based treatments are being explored for reducing wrinkles, improving skin elasticity, and treating scars (Jo et al., 2021). Also, adipose-derived stem cells (ADSCs) can be harvested from fat tissue and used in skin rejuvenation procedures, specifically ADSCs injected into areas of the face to restore volume and improve skin texture (Surowiecka and Struyna, 2022). Exosomes from stem cells have been shown to be particularly effective in promoting healing after procedures like laser therapy or microneedling, as they can accelerate skin regeneration (Prasai et al., 2022). In skin care, topical growth factors such as EGF and transforming growth factor-beta (TGF-) or injected formulations can stimulate collagen synthesis, accelerate wound healing, and reduce the appearance of fine lines (He et al., 2023). In the same line, studies suggest that PRP can improve fine lines, wrinkles, and overall skin texture by promoting cellular repair and enhancing skin regeneration (Phoebe et al., 2024). Lately, gene editing tools such as CRISPR are being explored to repair age-related genetic damage (Yu et al., 2022) as well as some therapies targeting genes associated with aging, like telomerase activation, have been proposed to extend the lifespan of cells (Tenchov et al., 2024). Finally, advances in 3D bioprinting and tissue scaffolds are enabling the development of engineered skin substitutes for cosmetic applications (Pleguezuelos-Beltrn et al., 2024). These skin substitutes can provide a platform for delivering stem cells or growth factors to damaged skin, promoting regeneration and rejuvenation.

Skin regenerative medicine generally encompasses two key approaches: cell-based and cell-free therapies. These include stem cell therapy, platelet-rich plasma (PRP), growth factors, cytokines, wound dressings, gene therapy, and tissue engineering, encompassing the use of biomaterials, skin grafts, bioengineered skin substitutes, and 3D bioprinting.

Stem cells are undifferentiated cells with the ability to self-replicate, self-renewal, homing and plasticity potential. They can differentiate into various cell types, such as nerve cells, cardiomyocytes, liver cells and skin cells (Jin et al., 2023). They possess anti-inflammatory properties, promote epithelial cell proliferation, inhibit wound scarring, maintain homeostasis, repair tissue injuries, and accelerate healing (Jin et al., 2023; Khandpur et al., 2021; Zhang and Huang, 2023). Stem cells also secrete bioactive molecules, including growth factors, cytokines, chemokines and exosomes, which are responsible for the paracrine effects of these cells in bone tissue and nervous system regeneration, as well as in wound healing and endothelial cells (Tran et al., 2023; Wu et al., 2024).

There are different sources for using stem cells in regenerative medicine classified according to their differentiation potential (Khandpur et al., 2021), including embryonic stem cells (ESCs), umbilical cord mesenchymal stem cells (UCMSCs), induced pluripotent stem cells (iPSCs), MSCs, ADSCs and bone marrow mesenchymal stem cells (BMMSC) (Jin et al., 2023; Semsarzadeh and Khetarpal, 2022; Tran et al., 2023).

MSCs are originated from the mesoderm and ectoderm, and can differentiate into various cell types, such as osteocytes, chondrocytes, adipocytes, neurons and endothelial cells (Ma et al., 2023; Tran et al., 2023). Additionally, MSCs can be found in bone marrow, adipose tissue, synovial tissue, muscle, lung, liver, and umbilical cord blood (Khandpur et al., 2021; Huynh et al., 2022; Jin et al., 2023). MSCs play a pivotal role in wound healing and exhibit additional functions including immunomodulation, anti-inflammatory and anti-apoptotic effects, and pro-angiogenic activity (Khandpur et al., 2021; Wu et al., 2024).

Currently, there are several therapies using MSCs for the treatment of graft-versus-host disease, bone defects, ischemic heart failure, burns, autoimmune-related skin conditions, and diabetic foot (Jin et al., 2023; Wu et al., 2024; Farabi et al., 2024). Since MSCs can differentiate into skin cells such as keratinocytes, fibroblasts, and endothelial cells, they can be used to enhance the healing process. This is not only due to their cell differentiation capabilities but also because of their crosstalk with macrophages, which play a role in the wound repair process (Wu et al., 2024).

MSCs can be applied to burn injuries by inhibiting cellular inflammation through the release of anti-inflammatory cytokines. Additionally, MSCs exert paracrine effects that polarize macrophages from an inflammatory phenotype (M1) to a wound-healing phenotype (M2), promoting tissue repair and clearance (Ma et al., 2023; Wu et al., 2024; Zhang and Huang, 2023). Moreover, they stimulate the formation of new blood vessels, which results in increased blood perfusion and the delivery of nutrients to affected areas, thereby accelerating the healing process (Jin et al., 2023; Semsarzadeh and Khetarpal, 2022). On top of that, MSCs improve the wound structure by secreting proteins that make up the extracellular matrix (ECM), such as collagen, elastin and fibronectin, enhancing the reconstruction of the dermis (Mazini et al., 2020; Tran et al., 2023; Wu et al., 2024).

Beyond that, bone marrow-derived MSCs can be used for scars reduction, anti-aging, and systemic sclerosis (Tran et al., 2023). Conget et al. (2010) conducted a study in which they used these cells to treat two patients with severe generalized recessive dystrophic epidermolysis bullosa (EB). After 12 weeks of intradermal treatment, the ulcers healed completely, however this effect only lasted 4months.

Another type of stem cells are the iPSCs. They are capable of differentiating into more cell types compared to MSCs, and they have the ability for unlimited self-renewal (Zhang and Huang, 2023). Studies show that these cells promote angiogenesis, perfusion, collagen deposition, and accelerate the natural healing process in murine models (Clayton et al., 2018; Farabi et al., 2024).

Besides MSCs and IPSCs, ADSCs are another promising cell type for regenerative therapy. ADSCs have shown potential applications in dermatology and aesthetics, including scar reduction, anti-aging, wrinkle reduction, and hair loss treatment (Khandpur et al., 2021; Suh et al., 2019; Tran et al., 2023). Anderi et al. (2018) injected ADSCs, derived from liposuction, into 20 patients with alopecia areata, and after 6months of follow-up, they observed a statistically significant difference in hair growth rates among all treated patients. Indeed, Zhang et al. (2014) evaluated the antioxidant effects of ADSCs in a mouse model and found that, after 28 days of injection, ADSCs reversed the aging phenotype, increased dermal thickness and collagen content, and enhanced skin vascular density. In addition, Chen et al. (2020) showed that ADSCs were effective in improving the appearance of the skin, particularly in reducing wrinkles in UV-damaged skin. These results highlight the paracrine effects of ADSCs in promoting skin rejuvenation.

Interestingly, Li et al. (2016) used the conditioned medium from ADSCs (ADSC-CM) to evaluate its effect on hypertrophic scars ex vivo, injecting them into rats. Through Western blotting, they analyzed key proteins involved in healing, such as collagen I, collagen III, and -SMA (alpha-smooth muscle actin). The results showed that the use of ADSC-CM reduced collagen deposition in hypertrophic scar tissues and improved fibrosis in these tissues.

Not only ADSC-CM, but also conditioned medium from MSCs (MSC-CM) have been investigated to be used on regenerative medicine because it contains anti-apoptotic, anti-inflammatory and anti-aging substances. Because of that, there are pre-clinical studies using MSC-CM to show whether it influence on lung progenitor cell development or its paracrine effect influences tissue regeneration (Smolinsk et al., 2023).

Yet another ADSCs derivative, nano-fat, obtained through a multi-step process involving mechanical digestion and filtration of fat tissue, has demonstrated several benefits, including reduced scar size, improved skin color, and enhanced overall skin quality (Suh et al., 2019; Hajimortezayi et al., 2024).

In addition, UCMSCs can also be used in regenerative medicine, having advantages over other types of stem cells because they are abundant, easy to collect, cause no harm to donors, have low immunogenicity, and high differentiation capacity (Jin et al., 2023; Li et al., 2024). They can be used for the treatment of cardiovascular diseases, liver diseases, degenerative muscle diseases, and autoimmune diseases (Wu et al., 2024). They can also be used for treating burns and psoriasis vulgaris (Tran et al., 2023) and for treating chronic wounds, facial and body rejuvenation, even combination therapies with other biomaterials (Li et al., 2024).

The Stromal Vascular Fraction (SVF) is isolated from adipose tissue and contains various cell types, such as MSCs, endothelial cells, stromal cells, and immune cells (Surowiecka and Struyna, 2022). For example, in a pilot study, SVF was used for the treatment of alopecia, where a significant increase in hair growth in patients was observed (Semsarzadeh and Khetarpal, 2022).

While various stem cell types exist, extensive preclinical and clinical research suggests that mesenchymal stem cells (MSCs) and MSC-based products offer the most promising balance of safety and efficacy, with a low risk of tumor formation and minimal immune rejection (Hoang et al., 2022; Farabi et al., 2024). Despite their widespread use, MSCs have limitations, such as a decline in viability and activity with age (Wu et al., 2024).

PRP was first used in 1954 to improve wound healing in dentistry (Kingsley, 1954). Since its inception, the application of PRP has grown significantly, becoming a valuable tool in tissue repair and regeneration across various medical fields. (Cecerska-Hery et al., 2022). The treatment is based on injections of the patients own platelets highly concentrated in plasma and separated from other blood components by centrifugation cycles (Samadi et al., 2019; Pixley et al., 2023).

The peripheral blood contains at least five times fewer platelets than PRP. This increased platelet concentration in PRP enhances its potential for wound healing (Davies and Miron, 2024). Platelets are involved in a wide range of growth factors, proteins, cytokines and other biological agents that have effects in processes like cellular migration, proliferation, differentiation, angiogenesis, tissue regeneration and collagen synthesis (Pincelli et al., 2024). The variety of molecules derived from PRP enables efficient wound healing, as the healing process involves multiple molecules and complex pathways (Samadi et al., 2019; Davies and Miron, 2024).

Platelet-derived molecules could be secreted by -granules, dense granules and lysosomes. Platelet derived growth factor (PDGF), insulin-like growth factor 1 (IGF-1), EGF, TGF-, fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) are secreted from -granules, in addition to adhesive proteins, coagulation factors, angiogenic regulators, cytokines and exosomes. They are the more abundant secretory granules and are responsible for releasing the greatest number of molecules with a direct effect on wound healing (Cecerska-Hery et al., 2022; Pincelli et al., 2024). On the other hand, dense granules contribute to platelet activation and subsequent release of -granules constituents, in which it has been shown that PRP lysosomes functions participate in antimicrobial activity and the degradation of extracellular matrices (Everts et al., 2024).

The composition of PRP can vary depending on the preparation. It can be categorized as pure (P-PRP), leukocyte-poor (LP-PRP), leukocyte-rich (LR-PRP) or platelet-rich fibrin (PRF), considering the centrifugation time, speed and the presence or absence of non-autologous anticoagulants (Karimi and Rockwall, 2019; Everts et al., 2024). PRF specifically has gained prominence in regenerative medicine in recent years due to its benefits, such as the release of platelet-related therapeutic granules over a longer period and at a slower rate than PRP (Narayanaswamy et al., 2023). In this way, PRF also offers a valuable adjunct to both surgical and non-surgical interventions, demonstrating great potential for enhancing treatment outcomes.

PRP treatment offers several advantages, including low cost, ease of preparation, versatility, and safety. However, further research is necessary to standardize preparation procedures and establish regulations regarding the composition of PRP injectables. Additionally, the efficacy of PRP and its various categories remains uncertain in relation to specific diseases and clinical conditions (Everts et al., 2020; Cecerska-Hery et al., 2022; Pincelli et al., 2024).

Micro-vesicles, currently known as exosomes, were first identified in 1983. Initially, exosomes were considered just a cell waste, however, later research revealed that exosomes play important roles in cell communication and signal transduction. As an alternative for cell-based therapies, exosomes emerged as potential tools for treating skins conditions, such as improving wound healing and even skin rejuvenation. Since exosomes are small vesicles secreted by different cells, there are many possibilities for their isolation and use as an effective carrier for bioactive compounds or genetic material. Exosomes not only transport and protect molecules from degradation, but also exhibit biocompatibility, reducing the risk of immune reactions and tumorigenesis. In this way, clinical application of exosomes is a promising avenue for free-cell therapies (Hajialiasgary Najafabadi et al., 2024; Pea and Martin, 2024; Quan, 2023; Sonbhadra and Pandey, 2023; Zhou et al., 2023).

Exosomes are extracellular vesicles, approximately 30200nm in size, which can be obtained from cell culture and that function as membrane-bound carriers of biomolecules and metabolites, reflecting their cellular origin. These vesicles, spherical in solution, exhibit a lipid bilayer structure, which decreases the risks of immune responses and provides protection of their cargo from degradation. Furthermore, exosomes pose a low risk of uncontrolled cell proliferation and differentiation, minimizing concerns related to tumorigenicity. Their ability to carry and deliver bioactive substances to cells makes exosome-based therapies a promising possibility for therapeutic applications like various skin conditions, including psoriasis, atopic dermatitis and vitiligo, as well as for promoting skin regeneration, such as in diabetic wound healing, hypertrophic scarring and keloid formation, and even for addressing skin aging (Gurung et al., 2021; Hajialiasgary Najafabadi et al., 2024; Yu et al., 2024).

Exosomes are ubiquitous in various body fluids, including serum, saliva, milk, cerebrospinal fluid, urine and semen. Among these, stem cell-derived exosomes have been extensively investigated for their potential role in mediating the biological effects of paracrine factors, particularly in wound healing (Zhou et al., 2023).

Tissue repair is a complex process involving initial clot formation, followed by inflammatory cell signaling, cell proliferation and remodeling. These phases overlap, each with its own purpose and time before the next starts. Mesenchymal stem cells exosomes (MSC-exos) derived from different tissues, like ADSC, hold a promise for cutaneous repair due to their ability to stimulate fibroblast activity. In the dermis, fibroblasts play a pivotal role in wound healing through collagen synthesis, making MSC-exos, which enhance fibroblast function, valuable contributors to the healing microenvironment and the promotion of wound repair. ADSC-derived exosomes demonstrate significant potential for treating diabetic wounds due to their ability to induce collagen I and III production in the early stages of healing, which can help prevent scar and keloid formation (Song et al., 2023; Zhou et al., 2023; Pea and Martin, 2024).

Data from an ECM loaded with ADSC-exosomes indicate that this combination is also a possible therapeutic approach for both normal and pathological wound healing. The results demonstrated that ECM-loaded exosomes promoted increased cell growth, cell migration, collagen deposition, and decreased inflammation in vivo (Song et al., 2023).

Moreover, given their role in wound healing, stem cell-derived exosomes could potentially be used to treat skin photoaging, a condition marked by uneven pigmentation and wrinkles (Hajialiasgary Najafabadi et al., 2024).

Park et al. (2023) demonstrated that exosomes derived from human foreskin fibroblasts (BJ-5ta Exo) can mitigate oxidative stress by upregulating the expression of antioxidant genes CAT, SOD-1, SOD-2, and GPX. Additionally, BJ-5ta Exo promoted a decrease in programmed cell death and cell cycle arrest.

In addition, it was suggested that exosomes obtained from 3D culture of human dermal fibroblasts (HDFs) may be able to promote collagen synthesis and reduce skin inflammation. Data also revealed that 3D-HDF-exos, through tumor necrosis factor alpha (TNF-) downregulation and TGF- upregulation, promoted a procollagen type I increase while reducing matrix metalloproteinase-1 (MMP-1) expression. Given the age-related decline in HDF collagen production and repair, coupled with increased MMP-mediated ECM degradation, these findings suggest that exosomes may possess anti-aging properties (Xiong et al., 2021).

Given the abundance of exosomes in bovine milk, recent studies have explored the potential of milk-derived exosomes (MK-exo) as novel anti-aging compounds. These investigations have revealed that MK-exo can stimulate the expression of filaggrin and CD44 in keratinocytes, as well as hyaluronidase levels in fibroblasts. Furthermore, MK-exo protected collagen biosynthesis from UV-induced damage. Notably, these exosomes also stimulated increased cell migration rates in fibroblasts (Lu et al., 2024).

However, while exosomes demonstrate significant promise as therapeutic agents, several challenges must be addressed for their clinical application. These include standardizing isolation and analysis, ensuring safety and purity, and preserving exosome activity during preparation and storage (Lu et al., 2024; Rezaie et al., 2022).

Despite above stated challenges, exosomes, being versatile carriers of biomolecules, offer promise for clinical applications. Their biocompatibility, low risk of immune responses and tumorigenicity make them attractive candidates for treating diseases. In the context of skin, their potential to influence collagen synthesis and inflammation suggests their value for wound healing and skin rejuvenation. Future research should focus on exploring these possibilities and ensuring the safety of exosomes for medical use.

Dermal fillers, also known as facial fillers, are soft, gel-like substances injected beneath the skin. Over time, the loss of soft tissue volume, fat redistribution, reduced skin elasticity, and thickness contribute to the formation of wrinkles and folds characteristic of aging. In this context, injectable fillers are an option for treating wrinkles, scars, folds, and areas under the skin lacking volume (Callan et al., 2013; Maio, 2018; Colon et al., 2023; Faris, 2024).

The principle behind the use of these products is based on strengthening the ECM in the dermal layer. They are made of various low-crosslinking polymeric ingredients with different effects on the skin (Yi et al., 2024). Among the most used ingredients are hyaluronic acid, poly-L-lactic acid, polymethylmethacrylate, and calcium hydroxyapatite (CaHA) (Colon et al., 2023).

Hyaluronic acid is the most commonly used dermal filler, naturally found in the skin, being safe and effective for use (Yi et al., 2024). Poly-L-lactic acid is synthetic, acting as a collagen stimulator, recommended for softening lines and treating wrinkles (Ao et al., 2024). Similarly, according to the American Board of Cosmetic Surgery (2022), polymethylmethacrylate comprises synthetic microspheres that remain under the skin indefinitely, providing continuous support and containing collagen in their composition. In addition, CaHA is a natural substance found in bones that helps stimulate the skins natural collagen production and is recommended for treating deeper lines.

Focusing on hyaluronic acid fillers due to their widespread use, it is the most abundant glycosaminoglycan found in the human dermis, contributing to tissue hydration and volume, as well as providing structural support (Ballin et al., 2015; Wongprasert et al., 2022). A recent study by Chen et al. (2023) demonstrated the anti-aging ability of hyaluronic acid fillers by inhibiting the expression of MMP-1, promoting collagen accumulation, and increasing the expression of dermo-epidermal junction proteins.

One of the most significant advantages of hyaluronic acid fillers is that they can be easily removed by injecting hyaluronidase (Wongprasert et al., 2022). However, some limitations persist in their use, which can sometimes extend to other types of fillers, such as the duration of the compounds, discomfort caused by the injections, and the ability to achieve precise delivery of the fillers to the intended location and skin layer (Colon et al., 2023).

Growth factors are polypeptides or proteins that regulate physiological processes in and between cells. They are naturally found in the skin, secreted by various cell types in this tissue. Many growth factors are involved in wound healing, both acute and chronic, and are some of the most important signalers during this process (Pamela, 2018; Yamakawa and Hayashida, 2019; Vaidyanathan, 2021). Furthermore, the processes of aging and wound healing naturally stimulate the release of growth factors, which affect critical biochemical repair pathways in the dermal matrix and have inspired the use of these factors to improve skin appearance (Kremer and Burkemper, 2024).

Some of the growth factors used in skin treatment are EGF, FGF, hepatocyte growth factor (HGF), and IGF-1, which can be used in combination (Vaidyanathan, 2021; Yamakawa and Hayashida, 2019). They can come from various sources, including human and non-human cell cultures and recombinant sources (Quinlan et al., 2023).

Treatments involving the use of different growth factors have been reported to improve wrinkles, skin texture, photo-damage, and the overall appearance of facial skin (Quinlan et al., 2023). In addition to cosmetic use, human EGF (hEGF) is also applied in regenerative medicine for the treatment of alopecia, dermatitis following chemotherapy, burns, diabetic foot ulcers, and post-surgical ulcers (Kong and Hong, 2013; Kim et al., 2017; Esquirol-Caussa and Herrero-Vila, 2019; Jeon et al., 2019; Kahraman et al., 2019; Lou, 2021; Vaidyanathan, 2021).

The use of EGF for aesthetic purposes also shows broad applications, presenting a regenerative effect in the aging skin process by promoting the migration of aged fibroblasts and increasing the synthesis of hyaluronic acid and collagen in this tissue (Kim et al., 2015; Miller-Kobisher et al., 2021; Vaidyanathan, 2021). Growth factors can also be combined with other treatments for apparent skin rejuvenation, such as lasers, microneedling, radiofrequency, or chemical peels, to amplify results or improve side effects from these treatments (Quinlan et al., 2023). However, in recent years, the development of cell-penetrating peptides associated with growth factors has improved the topical application of these factors, increasing their ability to penetrate the skin and epithelial cells (Chen et al., 2017; Choi et al., 2018; Jeon et al., 2019, Lee et al., 2020, Xie et al., 2020.

Aiming to correct genetic defects and thereby prevent or cure genetic disorders, gene therapy has been applied to skin regenerative medicine. All types of skin diseases are candidates for gene therapy, from inflammatory diseases to skin cancers and genodermatoses (Ain et al., 2021), as well as more aesthetic aspects like skin regeneration and scar and keloid treatment (Hosseinkhani et al., 2023; Luo et al., 2023).

Gene therapy technologies include the use of messenger RNA, silencing RNA, antisense oligonucleotides (AON), plasmid DNA, minicircle DNA, mini-string DNA, and CRISPR/Cas9 technology (Wan et al., 2021; Guri-Lamce et al., 2024; Tenchov et al., 2024), which must have specific action on the desired area of the skin, representing one of the current challenges in this type of therapy.

Currently, there are three main methods of delivering gene therapies to the skin: viral delivery, nanoparticles and physical methods. In this perspective, viral delivery is the most used and effective method (Picano-Castro et al., 2020), though it presents challenges such as limitations in efficacy due to pre-existing immunity, the inability to redose, and genome integration capacity, which increases the risk of unwanted insertional mutagenesis. Additionally, there are size limitations for the genetic cargo (Anzalone et al., 2019; Ain et al., 2021). Lipid-based nanoparticles, a more recent and promising method (Guri-Lamce et al., 2024); polymeric nanoparticles, capable of associating with negatively charged genetic cargoes and forming spherical complexes, but which still present high toxicity related to particle size (Blakney et al., 2020); and physical methods, such as electroporation, ultrasound application, or microneedles (Wan et al., 2021), which have limitations for clinical application, such as limited cargo capacity and challenges with whole-body administration (Ain et al., 2021).

Other substances can be incorporated into skin care products, playing an active role in tissue regeneration or inflammation by improving ECM synthesis or inhibiting its degradation, neutralizing free reactive species, or reducing proinflammatory factors (Makpol et al., 2013; Wang H. et al., 2021; Torres et al., 2023). Among these substances, zinc-based compounds stand out. This metal has been shown to reduce inflammation and the risk of infections, being involved in cell proliferation and migration and collagen synthesis, thus promoting epithelialization (Lin et al., 2017; Chen et al., 2022; Pino et al., 2023). Other substances primarily act as moisturizers, provided by ingredients that increase the synthesis of structural skin lipids, directly restoring the skin barrier, such as vitamin A, or hygroscopic substances like dexpanthenol, which bind to and retain water in the stratum corneum (Liu, 2022). Other substances are lipids, oils, and fatty acids, known as emollients, such as petrolatum derivatives, which also promote hydration by directly replacing missing fatty acids in the tissue (Elias, 2022; Torres et al., 2023).

Smart dressings were developed to enhance wound care management based on the injury type and patient conditions (Raju et al., 2022). Multifunctional wound dressings promote wound healing by encapsulating bioactive substances, sustaining the release of medicines by stimuli-responsive technology (Raju et al., 2022). Biopolymers such as alginate, chitosan, polyvinyl alcohol (PVA), and collagen are increasingly used to create innovative wound dressings due to their cost-effectiveness and eco-friendliness. Among these, alginate dressings are the most popular because they promote skin regeneration, accelerate wound closure, minimize scarring, absorb exudates effectively, and are biocompatible. Alginates gelling properties and stability in warm environments make it ideal for various applications, including hydrogels, nanofibers, dermal patches, films, and foams (Nqoro et al., 2022).

Other than that, hydrocolloids are effective in wound care because they can absorb significant amounts of wound fluid and are impermeable to water vapor, creating a moist healing environment. They also block oxygen, which speeds up epithelialization and collagen synthesis while lowering the pH to reduce bacterial growth (Nguyen et al., 2023). Nonetheless, new smart hydrogel wound dressings with embedded sensors have been rapidly developed to monitor wound conditions. Notable examples include flexible pH-sensing alginate-based hydrogel fibers for skin wounds and PVA/xyloglucan (PVA/XG) hydrogel membranes that absorb exudate and release biological factors (Tamayol et al., 2016; Ajovalasit et al., 2018; Tavakoli and Klar, 2020).

Although high-end wound dressings have been developed in recent years, the products face several limitations, including a complicated production process, inadequate quality assurance for biological materials and questions about the effectiveness of their components for widespread use. In addition to that, more trials and experiments are needed to assess the true effectiveness of these advanced dressings in wound healing (Nguyen et al., 2023).

Tissue engineering has become a multidisciplinary research field that involves the use of techniques to replicate biological prototypes, such as skin, to study the regeneration of physiological tissue on repairing or replacement of damaged skin (Berthiaume et al., 2011; Deepa and Bhatt, 2024; Wei et al., 2024). Indeed, it is possible to introduce biomaterials and hydrogels, which can be used as scaffolds to facilitate wound healing, combined with the knowledge of cell culture, for the improvement of techniques such as the use of nanomaterials and 3D bioprinting (Kondej et al., 2024; Wei et al., 2024; Loukelis et al., 2024; Bian et al., 2024).

Studies in the last 20years have involved the use of different skin cell cultures (mainly keratinocytes), umbilical cord mesenchymal stem cells differentiated to keratinocytes, or co-culture of skin cells with other cell types, including immune cells and dermal fibroblasts, in a two-dimensional (2D) monolayer (Abaci et al., 2017; Santos et al., 2023), to study the signaling pathways of skin diseases such as psoriasis or melanoma, wound healing, also to test the efficacy of safety treatments (Karras and Kunz, 2024). However, 2D models do not often represent a sufficient level of complexity to assess the various cell-cell and cell-extracellular matrix interactions, as well as oxygen and nutrient gradients (Loke et al., 2021; Santos et al., 2023).

On the other hand, it is possible to better understand most complex skin diseases in the three-dimensional (3D) microenvironment by involving additional cell lineages, such as immune cells or skin appendages as innervation type, to generate more effective in vitro skin models, using spheroids or skin constructs, for example, to improve skin replacement therapy (Abaci et al., 2017; Karras and Kunz, 2024; Loke et al., 2021).

The in vivo model can be better mimicked in spheroid cultures compared to 2D models, representing a more complex tissue architecture, with increased cell-cell contacts and heterogeneous cell growth. In addition, spheroids have been used for semi-high-throughput drug screening, as well as being used in co-culture models to evaluate different tissue responses under paracrine stimulation (Raghavan et al., 2016; Loke et al., 2021; Karras and Kunz, 2024).

The application of spheroid cultures in skin models can be generated from cell aggregates of fibroblast or keratinocyte lineages, for example, under non-adherent conditions, by so-called spinner culture, hanging drop, magnetic levitation or gel incorporation (Abaci et al., 2017; Klicks et al., 2019; Schfer et al., 2021; Ohguro et al., 2024). To improve the formation of the spheroids, it is important to choose an ECM based on biomaterials, such as hydrogels or collagens, for the architecture of the dermal matrix (Enyedi et al., 2023; Santos et al., 2023).

Some methods for evaluating the different cell configurations in spheroids still remain limited, but the main ones based on microscopy, such as phase contrast microscopy, are used to analyze the size and shape of spheroids. Other methods, such as cell surface staining and flow cytometry, are used to analyze the presence of specific molecules. Cryosectioning, after fixation in formalin and embedding in paraffin, is used for a deeper view of the sectioned spheroid (Filipiak-Duliban et al., 2022; Habanjar et al., 2021; Karras and Kunz, 2024).

Due to the cell aggregate structure, spheroid nuclei are exposed to low oxygen conditions, as well as limited access to nutrients and metabolites, which can lead to an increase in apoptotic cells (Karras and Kunz, 2024; Loke et al., 2021). On the other hand, the cells on the periphery are proliferative, due to the availability of oxygen and nutrients. Interestingly, the middle layer contains quiescent and senescent cells, and as a result, this spheroid configuration becomes a suitable model for testing pathophysiological conditions (Karras and Kunz, 2024; Loke et al., 2021; Ohguro et al., 2023).

Another model of organotypic cultures are the so-called raft cultures, also known as skin reconstructs. Cells are established in a manner as to allow the stratified epithelium and the dermal component of the skin, to be reconstituted in a tissue culture environment. Keratinocytes, for example, are seeded in a dermal equivalent containing fibroblast and, when raised to the air-liquid interface, reproduce the process of stratification and terminal differentiation of keratinocyte (Klicks et al., 2019; Santos et al., 2023). Histological analysis of these skin reconstructs shows the similarity and tissue organization to human skin, with a cornified epidermal-equivalent appearing on top of a dermal, containing human fibroblasts (Klicks et al., 2019; Loke et al., 2021; Santos et al., 2023).

This skin reconstruct is a useful system for testing pharmacological dynamics, efficacy tests, analysis of absorption by different forms of administration, or for preclinical screening of drugs and cosmetics (Torre et al., 2020; Portugal-Cohen et al., 2023; Suthar et al., 2024). This model takes less time to obtain results and is less expensive than performing experiments using animals (Abaci et al., 2017; Karras and Kunz, 2024). Therefore, it has been emphasized in recent years that organotypic cultures for skin reconstructions can also be obtained using the bioprinting technique, in order to construct highly reproducible dermal equivalents, with architecture similar to the in vivo (Cubo et al., 2016; Fernandes et al., 2022; Bian et al., 2024), to be widely used in regenerative medicine or in strategies for testing immunotherapy (Ao et al., 2022; Santos et al., 2023).

Tissue bioengineering has been expanding as a new strategy by employing advanced techniques of bioprinting, biopolymer engineering, stem cell research and nanomedicine (Augustine, 2018; Pasierb et al., 2022; Wei et al., 2024). Bioprinting has attracted attention as a promising technique, in which the technology aims to generate, precisely, a controlled and organized complex with similar architectures of native tissues (Loukelis et al., 2024; Bian et al., 2024). Bioprinting has been used to generate tissues and transplants, including skin and its multilayers, tracheal splints, cardiac tissue and cartilaginous textures (Kaur et al., 2019; Miguel et al., 2019; Bian et al., 2024).

In fact, bioprinting technique has the potential to revolutionize contemporary regenerative medicine, considering that by taking advantage of tissue regeneration techniques. Using approaches that facilitates the production of skin and consequently its use in cases of wound closure, it is also possible within this model to mimic characteristic inflammatory profiles, in order to study drug-related toxicity or investigate the pathological mechanism of some skin diseases, including psoriasis and atopic dermatitis (Randall et al., 2018; Lorthois et al., 2019; Derr et al., 2019; Liu et al., 2020; Deepa and Bhatt, 2024).

The simultaneous incorporation of different cells into the bioprint, including fibroblasts and melanocytes in dermal equivalents, makes it possible to study the impact of UV radiation (Pasierb et al., 2022). Compared to machining prototypes, bioprinting makes skin production cheaper and faster, as the prototype can be finished in hours, allowing the process to be efficient, even with design modifications in production. The manufacturing process can also reduce material costs, as it uses only the amount of material needed for the prototype itself, minimizing or eliminating waste (Wang H. et al., 2021; Wang Z. et al., 2021).

Other advantages of using the bioprinting technique include: 1) customization of the skin to be used. Depending on the shape and depth of the wound surface, imaging technology using computer digitization can quickly print the skin graft compatible with the wound. Indeed, this technique confers the characteristics of punctuality, high flow and high repeatability (Weng et al., 2021). 2) the use of bioinks, which can be deposited flexibly and precisely with different biological agents, including living cells, nucleic acids, growth factors, among others, is usually required to help build skin structures (Zhu et al., 2016; Wei et al., 2024; Loukelis et al., 2024). According to the different printing materials, three different techniques of bioprinting can be mentioned, such as: droplet-based bioprinting (DBB), laser-assisted bioprinting (LAB) and extrusion-based bioprinting (EBB) (Gudapati et al., 2016; Weng et al., 2021; Kang et al., 2022).

DBB technique includes the drop-by-drop mode. In this model, drops of biomaterial on a substrate are deposited when necessary. DBB-based bioprinters are suitable for deposition and patterning of materials, due to their high precision and minimal biomaterial waste. In addition, DBB mainly uses piezoelectric, thermal or electrostatic forces to generate droplets, which can precisely deposit the biomaterial to make a spatially heterogeneous tissue structure (Gudapati et al., 2016; Matai et al., 2020; Kang et al., 2022). Its non-contact printing mode is more suitable for biological printing directly onto a wound, for example,. Some studies have used the DBB technique to print human keratinocytes and fibroblasts directly onto dermal wounds on the backs of mice. Compared to the control group without any biological dressing, the skin grafts in the experimental group promoted wound healing (Gudapati et al., 2016; Matai et al., 2020; Wang Z. et al., 2021). On the other hand, DBB has some limitations. Its inkjet injector is small, measuring up to 150m, which can be easily blocked by biomaterials. Only low-viscosity hydrogels or other low-concentration biological agents can be used (Wang H. et al., 2021).

LAB technique consists of the emission of laser light, that is focused on the metal film on the back of the silicate glass and heated locally, so that the bioink deposited on the equipment evaporates and is sprayed onto the substrate in the form of liquid drops (Matai et al., 2020; Wang Z. et al., 2021). The LAB technique mainly uses a nanosecond laser with ultraviolet wavelengths as an energy source, and its printing resolution can reach the picogram level, performing bioprinting without direct contact with the substrate and can print cells with high resolution. (Zhang et al., 2023). However, LAB does not have a suitable rapid gelling mechanism yet, which limits its ability to produce high-performance prints (Wang H. et al., 2021; Zhang et al., 2023).

EBB technique makes controllable impressions using fluid distribution systems and automated machines. Under the control of a computer, the biomaterial passes through a catheter, using pneumatic, piston or screw approaches (Zhang et al., 2023). Hydrogels better perform in bioprinting by pneumatic extrusion, because it is kept in this material the profile of printed filaments, after extrusion. The screw-driven structure can bioprint biomaterial at high viscosity, which is conducive to producing a more stable bioprinted tissue (Zieliski et al., 2023). In addition, extrusion bioprinting can print a porous grid structure, promote the circulation of nutrients and metabolites, which allows better control over porosity, shape and distribution of cells in the printed prototype (Pasierb et al., 2022; Zhang et al., 2023). Compared to DBB and LAB models, the advantages of EBB include faster bioprinting speed, more usable bioink types (including cell clusters, high-viscosity hydrogels, microcarriers and cell matrix components) (Pasierb et al., 2022; Zhang et al., 2023), more versatility and suitability for manufacturing prosthetic implants for tissue bioengineering. However, the limitation of this technique is that it has a lower resolution of at least 100m (Zhang et al., 2023; Zieliski et al., 2023).

In general, different approaches in bioprinting techniques are used, in order to allow specialists to acquire more precision and high resolution in the regeneration of the skin and its appendages, including hair follicles, sebaceous and sweat glands. The same approaches might be used on selecting between the different biomaterials, which make the skin cell lineages remain highly viable and metabolically active, to keep the accuracy in replicating the tissue layers and to not compromise functionality (Weng et al., 2021).

Biomaterials can be of natural, synthetic, or a combination of both origins and are of great interest in tissue engineering due to their properties of biocompatibility, biodegradability, promoting cell adhesion and migration in scaffolds, potential resemblance the extracellular matrix, and presenting controllable properties and architecture (Chaudhari et al., 2016; Liu et al., 2023).

Natural biomaterials are primarily derived from proteins, such as collagen and spider silk, for example, (Liu et al., 2023), but can also originate from carbohydrates, such as alginate (Farshidfar et al., 2023).

Firstly, collagen is a protein that contains triple helices capable of forming strong and stable fibers through cross-linking. This stability can be used to form scaffolds that resemble the ECM of living tissues (Chattopadhyay and Raines, 2014; Amirrah et al., 2022; Zhu et al., 2022). Thus, in regenerative medicine, collagen as a biomaterial can be used as a wound dressing, promoting healing, or as a supplement for the skin, improving aspects such as elasticity and hydration (Ghomi et al., 2021).

Spider silk, as a natural biomaterial, is explored for its biocompatibility and low density. Because it is difficult to cultivate directly from arachnids, this protein has been produced recombinantly for the construction of scaffolds for cell culture. Using these supports, the regeneration of bone, cartilage, muscle, nerve, and epidermal tissues, especially in burn patients, has been studied. (Salehi et al., 2020).

It is important to highlight that some materials have limited properties, such as alginate, which has low stability due to its chemical properties, and polydopamine, which has low hydrogel-forming capacity. However, they can be used in combination with other materials, such as hydroxyapatite, chitosan, gelatin, and collagen, to achieve results and applications in tissue engineering. Thus, in combination, they can be used for bone tissue repair, corneal reconstruction, wound healing and covering, and even in drug delivery systems (Farshidfar et al., 2023; Yazdi et al., 2022).

Another way to apply the biomaterials, in general, is using as hydrogels, which provide a moist environment with the ability to retain proteins, growth factors, and nutrients within the gel structure and release these molecules into the medium (Berthiaume et al., 2011; Lei et al., 2022). Due to technological advancements in tissue engineering, it has become possible to work on an increasingly smaller scale of these gels, creating nanogels which can reach smaller and more internal wounds than hydrogels, ensuring drug release in the region, facilitating wound healing and tissue regeneration (Grimaudo et al., 2019; Brianna et al., 2024). In addition to hydrogels, there are also nanomaterials, which can be made of a single chemical element, such as silver or gold, that possess antimicrobial characteristics, can stimulate cell growth and can be delivered in systems along with nanogels (Bellu et al., 2021). The field of nanostructures in regenerative medicine and tissue engineering is still a novelty and shows extreme promise with ongoing research advancements.

As mentioned previously, the field of regenerative medicine for skin regeneration and rejuvenation, while promising, faces several significant limitations as described:

1. High costs and limited accessibility:many regenerative therapies, especially those involving stem cells or bioengineered tissues, are expensive and require specialized equipment and expertise, limiting their accessibility to many patients.

2. Variability in treatment outcomes: the effectiveness of regenerative therapies can vary significantly depending on factors like disease severity, individual genetic background, and overall health. This makes it challenging to predict outcomes and standardize treatment protocols.

3. Long development times and regulatory hurdles: developing and gaining regulatory approval for new regenerative therapies is time-consuming and costly, which slows down the introduction of promising treatments to the market.

4. Technical challenges in production and application: producing sufficient quantities of high-quality regenerative materials (stem cells, bioengineered tissues, exosomes) consistently and reliably for clinical applications remains a significant technological hurdle. The delivery and distribution of these materials in the body can also be complex and may not always be effective.

5. Uncertain long-term efficacy and safety: while many therapies show promise in short-term studies, the long-term efficacy and safety of regenerative treatments are often not fully established. Potential risks such as tumorigenicity or immune responses need further investigation.

6. Ethical considerations: the use of stem cells and gene editing technologies raises ethical concerns, including issues related to stem cell sources (embryonic vs. adult), the potential for off-target effects in gene editing, and the ethical considerations surrounding the use of human tissue and data. Animal testing used in preclinical research may also raise ethical questions.

7. Standardization and quality control: there is a lack of standardization in the preparation and quality control of several regenerative materials and treatments (e.g., PRP, exosomes). This affects the reproducibility and reliability of treatment outcomes.

8. Complex biological systems: the skin is a complex organ with multiple interacting cell types and signaling pathways. Fully understanding these complexities is crucial for developing truly effective regenerative therapies. Current therapies often target only a subset of these mechanisms, limiting their overall impact.

9. Limited understanding of underlying mechanisms: while many regenerative therapies show promise, a complete understanding of their precise mechanisms of action is often lacking. This limits the ability to further improve and refine treatments.

Table 1 summarizes various techniques and approaches used in skin regeneration and treatment, categorized into in vitro and in vivo methods, as well as clinical applications. For each technique, it details specific findings, strengths, and limitations or challenges encountered. The in vitro methods include organotypic cultures (2D and 3D), spheroids, skin reconstructs, cell cultures, exosome studies, and ADSC injections. In vivo techniques cover PRP injections, growth factor application, exosome application, bioprinting, animal models, stem cell and gene therapy. Finally, clinical applications include PRP therapy, growth factor therapy, and dermal fillers. The table provides a comprehensive overview of the current state of skin regeneration research and its therapeutic potential, highlighting both the advantages and drawbacks of different methodologies.

In vitro, in vivo and clinical approaches to skin repair: strengths, limitations, and specific findings.

Table 2 was designed in order to compare various techniques used in skin regeneration, categorized into cell-based and cell-free methods, providing a concise overview of advantages and disadvantages. The cell-based methods include cell therapy, platelet-rich plasma, and growth factors and cytokines, while the cell-free methods encompass exosomes, wound dressings, and gene therapy. Additionally, there is a section for bioengineered skin including biomaterials and nanodevices.

Comparison of cell-based and cell-free approaches for skin regeneration.

Basically, cell-based therapies rely on the biological activity of living cells (e.g., growth factors, immunomodulation), while cell-free methods utilize components derived from cells or synthetic materials to stimulate tissue repair. In general, cell-based therapies offer potential for multifaceted benefits, nevertheless they are often more complex and costly to produce than cell-free options. Also, it is possible to assume that while some cell-based therapies (like PRP) have gained clinical traction, others are still in earlier stages of development. Similarly, the table indicates cell-free options like dermal fillers are established clinically whereas gene therapy, for example, remains limited due to distinct factors such as cost, scalability, and ethical issues. In addition, the comparison sheds light on the potential safety concerns associated with each technique. While cell-based methods carry risks such as immune rejection, cell-free methods have limitations regarding long-term stability and potential off-target effects (e.g., gene therapy). This is vital for evaluating the risk-benefit profile of each approach. By highlighting the limitations of current technologies, the comparison stimulates innovation and the development of new techniques. For instance, challenges in exosome isolation and standardization may boost research into improved purification and delivery methods.

In summary, comparing cell-based and cell-free methods provides a framework for assessing the strengths and weaknesses of different regenerative approaches, contributing to decisions about research direction, clinical translation, and resource allocation within the field of skin regeneration. Addressing these limitations requires continued research, development of standardized protocols, improved manufacturing processes, robust clinical trials, and careful ethical consideration of the technologies making them more effective, affordable, and accessible.

Since the cave age, man has been healing his wounds, treating burns and preventing bleeding. Nowadays, the importance of aesthetics and the growth of the geriatric population is propelling the demand for skin regeneration and rejuvenation products and services. In this context, the interest in maintaining a skin with youthful appearance, the demand for treatment of disorders/disease and superficial or full-thickness skin injuries, has led to the development of regenerative medicine-based approaches, with the aim of repair, replace, regenerate, and rejuvenate (the four Rs) the skin. In recent years we have seen rapid growth in the field of regenerative medicine-based approaches for skin.

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Advances in regenerative medicine-based approaches for skin ...

Angelman syndrome is a rare genetic disorder caused by a mutation on chromosome 15, which hinders the production of a protein crucial for brain function. As a result, people living with Angelman syndrome experience severe developmental and intellectual disabilities.

For more than a decade, February 15 has been designated as International Angelman Syndrome Day, a significant date as February is Rare Disease Month, and the day symbolizes chromosome 15. Angelman syndrome affects approximately one in 15,000 individuals in the United Statesor about 500,000 globallyand like most rare diseases, there is currently no cure. Among the 10,000 known rare diseases, there are fewer than 900 FDA-approved treatments.

But researchers are hopeful that cures for Angelman syndrome and other rare diseases, such as the neurodevelopmental disorder known as H1-4 syndrome, are within reach. Yong-Hui Jiang, MD, PhD, professor and chief of medical genetics, and Jiangbing Zhou, PhD, Nixdorff-German Professor of Neurosurgery, are among researchers at Yale School of Medicine dedicated to the development of new gene-editing treatments that aim to correct genetic alterations underlying rare neurogenetic disorders. Jiang is also the director of the Yale National Organization for Rare Disorders (NORD) Center of Excellence.

We spoke to Jiang and Zhou about exciting new rare disease research and therapies on the horizon.

Jiang: As a clinical geneticist, working with rare diseases is part of my job. As a researcher, it was my choice to focus on rare diseases. The patients I see while working in the clinic motivate me to understand their conditions from the scientific perspective and figure out how to help them.

For most genetic diseases, there are almost no treatments that specifically target the genetic defects. With the application of new genome technology in clinics, we are successfully identifying the genetic cause of these diseases and diagnosing them, but we are often not able to actually offer the next step of treatment or intervention. A better understanding of how to develop treatments that target the genetic defect is our ultimate goal.

Zhou: Im a biomedical engineer who mainly works on developing non-viral, gene-based therapies. Ive been working in this area for over 16 years. I think gene therapy could be widely used for a lot of major diseases in the future. But at this stage, I feel that gene therapy is particularly suitable for rare diseases such as Angelman syndrome, because they often have a very defined genetic cause.

This is one of the reasons I have focused on rare diseases over the past few years. Ive been collaborating with Dr. Jiang, who sees patients with genetic disorders every day. Its a benefit for scientists like me to work with physicians or physician-scientists such as Dr. Jiang because they see the great clinical need for new treatments.

Jiang: There are a few aspects. One is that we want people to know that although rare diseases are rare individually, we estimate that there are about 10,000 rare diseases in total. So altogether, they actually are not rare. Almost one in 10 Americans is affected by a rare disease.

Second, although we know of 10,000 rare diseases, we only understand the cause of about half of them. For the other 5,000, we dont even have names. As geneticists, we do a lot of rare disease research because almost 80% of rare diseases are genetic. But not every rare disease is geneticthat is also a common misperception.

The third is that there is a great unmet clinical need for families contending with a rare disease. But rare disease researchers have very limited resources. Almost 10,000 diseases are in need of research to better understand how to treat them, but not every disease gets resources from the government or any other sort of funding source. This can be very frustrating for a lot of families who have spent years trying to find a diagnosis, but in the end find very little information or help because there is so little knowledge.

Jiang: Yale has been one of the leading institutions for rare disease research for almost half a century. Our history started with Leon Rosenberg, MD, who was the founding chair of the department of human genetics back in 1972, and he led the first clinical genetics division at Yale New Haven Hospital. During his tenure at Yale, he was a pioneer in the rare disease field, particularly for what we call metabolic diseases, such as methylmalonic acidemia and homocystinuria.

Following Leon Rosenberg, Richard Lifton, MD, PhD, another former chair of the department of genetics, and many other faculty and clinicians at Yale also dedicated their research to rare diseases. Yale investigators have discovered genetic bases for several hundred rare genetic diseases. Those efforts helped lead to the creation of the Centers for Mendelian Genomics, supported by the National Institutes of Health (NIH), as well as the creation of the Yale Center for Genome Analysis.

Jiang: Weve accomplished quite a bit, mostly in patient care. For example, we organize a rare disease event every year to promote public awareness of these diseasesespecially rare genetic diseasesand educate attendees on how to recognize them. The event brings together leading experts and patient advocates who lead lectures and roundtables on new insights and ways to support the rare disease community. We were also awarded NIH Undiagnosed Diseases Network (UDN) Phase III funding to join UDN as a new Diagnostic Center of Excellence.

Jiang: The majority of rare diseases are genetic. Over the last 20 yearsdue to a new generation of genome technology in clinics, such as an exome sequencing method that was pioneered at Yalewe have diagnosed genetic rare diseases much more rapidly. However, the challenge is the treatment or intervention; almost 95% of rare genetic diseases have no available treatment options.

For all genetic diseases, the best treatment would be to correct the genetic mistake, which could potentially slow down or stop the disease progression and offer a cure. CRISPR-mediated genome editing technology [which is designed to modify an individuals DNA] offers promise and hope. Two-thirds of all rare genetic diseases affect the brain, which is the most challenging organ for gene therapy.

Thats where we step in. We hope that the STEP platform can eventually apply to rare genetic diseases that affect the brain. Were currently focusing on neurodevelopmental disorders like Angelman and H1-4 syndrome because of our expertise. But this technology could also eventually be helpful for many brain disorders, including neurodegenerative conditions such as Alzheimers or Parkinsons disease. It will have very broad applications.

Zhou: STEP technology is a non-viral, chemical-based delivery system developed here at Yale. I have not seen anyone else working on this type of delivery system. Its unique in that it uses chemicals instead of other vectors such as viral vectors or nanoparticlesthe two most commonly used vectors in the fieldto deliver genome editors to the brain.

It seems that the STEP technology works well for many neurogenetic diseases. We have applied the delivery system to a few diseases, including Angelman syndrome and H1-4 syndrome, and our findings have been very exciting. We have been working together with the NIH to translate this technology to clinical use. Hopefully, we can achieve that in the next few years.

Zhou: We have an array of new technologies under development or under evaluation for correcting genes through either gene correction or epigenetic regulation. This will allow us to potentially treat many rare genetic diseases. Dr. Jiang and I are working on ways to treat Rett syndrome, ALS, and Alzheimers disease, among others. The advances in our technologies open the door to study many genetic, neurodevelopmental, and neurodegenerative diseases.

Jiang: Our goal is to be able to treat or cure every rare genetic disease. The FDA approved CRISPR genome editing treatment for sickle cell therapy in 2023which is quite impressive since CRISPR technology is relatively new. So I have a lot of hope that our work will move fast over the next decade because of the success of the sickle cell program. But we do expect challenges. For instance, many of the critical steps required to navigate CRISPR technology from the bench to the clinic are new to the FDA, the NIH, the research community, and pharmaceutical companies.

However, we remain optimistic that society will address these issues accordingly. We hope that we will be able to deliver therapy for a few dozen of these diseases over the next five years in the clinic.

Zhou: I think that with advances in gene-editing technology, it will now be possible to treat many rare diseases, even through a one-time administration. Along with FDA regulation, there will be challenges in how to engineer our system for efficiency, specificity, and delivery. But we have seen a lot of progress in the field, and we are definitely optimistic about the treatment options that will be available in the next decade.

Jiang: CRISPR editing aims to correct the genetic mistakeoften there is only one mistake in the entire genome. But the technology itself may cause what we call off-target events [in which the technology edits DNA at sites other than the intended target] that might cause harm in an individuals genome. The question is first, how can we maximize the safety of our technology from the design perspective? And second, how do we assess off-target events in the clinical sense?

Another major gap is in resources. From beginning to end, the development of each biological drug requires tens of millions of dollars. And then, we have 5,000 rare genetic diseases now that are eligible for CRISPR gene-editing technology. But because the individual diseases are rare overall, pharmaceutical companies may not be interested in investing due to financial reasons.

Jiang: We have our Rare Disease Day celebration scheduled for February 21 on Yale School of Medicines campus. It will include families dealing with rare diseases, physicians from Yale School of Medicine and Yale New Haven Hospital who treat patients, and rare disease researchers from across campus. Rare diseases can affect any organ system. We want to raise awareness across the academic hospital community because we hope that other specialties will take interest in investing more in rare diseases in terms of clinical care and research.

Zhou: Were lucky to have support from the NIH for our work on Angelman syndrome. Its an exciting time, but hopefully there will be support and investments from other sources so that we can continue these programs and help families. Over the last couple of years, we have been supported by multiple philanthropy efforts that have helped offset some of these limitations.

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The Future of Gene-Editing Treatments for Rare Diseases

Biotechnology is a field of life science that uses living organisms and biological systems to create modified or new organisms or useful products. A major component of biotechnology is _genetic engineering_.

The popular concept of biotechnology is one of experiments happening in laboratories and cutting-edge industrial advances, but biotechnology is much more integrated into most people's everyday lives than it seems.

The vaccines you get, the soy sauce, cheese and bread you buy at the grocery store, the plastics in your daily environment, your wrinkle-resistant cotton clothing, the cleanup after news of oil spills and more are all examples of biotechnology. They all "employ" living microbes to create a product.

Even a Lyme disease blood test, a breast cancer chemotherapy treatment or an insulin injection might be the result of biotechnology.

Biotechnology relies on the field of genetic engineering, which modifies DNA to alter the function or other traits of living organisms.

Early examples of this are selective breeding of plants and animals thousands of years ago. Today, scientists edit or transfer DNA from one species to another. Biotechnology harnesses these processes for a wide variety of industries, including medicine, food and agriculture, manufacturing and biofuels.

Biotechnology would not be possible without genetic engineering. In modern terms, this process manipulates cells' genetic information using laboratory techniques in order to change the traits of living organisms.

Scientists may use genetic engineering in order to change the way an organism looks, behaves, functions, or interacts with specific materials or stimuli in its environment. Genetic engineering is possible in all living cells; this includes micro-organisms such as bacteria and individual cells of multicellular organisms, such as plants and animals. Even the human genome can be edited using these techniques.

Sometimes, scientists alter genetic information in a cell by directly altering its genes. In other cases, pieces of DNA from one organism are implanted into the cells of another organism. The new hybrid cells are called transgenic.

Genetic engineering may seem like an ultra-modern technological advance, but it has been in use for decades, in numerous fields. In fact, modern genetic engineering has its roots in ancient human practices that were first defined by Charles Darwin as _artificial selection_.

Artificial selection, which is also called selective breeding, is a method for deliberately choosing mating pairs for plants, animals or other organisms based on desired traits. The reason to do this is to create offspring with those traits, and to repeat the process with future generations to gradually strengthen the traits in the population.

Although artificial selection does not require microscopy or other advanced lab equipment, it is an effective form of genetic engineering. Although it began as an ancient technique, humans still use it today.

Common examples include:

Breeding livestock. Creating flower varieties. Breeding animals, such as rodents or primates, with specific desired traits like susceptibility for diseases for research studies.

The first known example of humans engaging in the artificial selection of an organism is the rise of Canis lupus familiaris, or as it is more commonly known, the dog. About 32,000 years ago, humans in a an area of East Asia that is now China, lived in hunter-gatherer groups. Wild wolves followed the human groups and scavenged on carcasses that hunters left behind.

Scientists think it is most likely that humans only allowed the docile wolves that were not a threat to live. In this way, the branching off of dogs from wolves began by self-selection, as the individuals with the trait that allowed them to tolerate the presence of humans became the domesticated companions to the hunter-gatherers.

Eventually, humans began to intentionally domesticate and then breed generations of dogs for desired traits, especially docility. Dogs became loyal and protective companions to humans. Over thousands of years, humans selectively bred them for specific traits such as coat length and color, eye size and snout length, body size, disposition and more.

The wild wolves of East Asia of 32,000 years ago that split off 32,000 years ago into dogs comprise almost 350 different dog breeds. Those early dogs are most closely genetically related to the modern dogs called Chinese native dogs.

Artificial selection manifested in other ways in ancient human cultures, as well. As humans moved toward agricultural societies, they utilized artificial selection with an increasing number of plant and animal species.

They domesticated animals by breeding them generation after generation, only mating the offspring that exhibited desired traits. These traits depended on the purpose of the animal. For example, modern domesticated horses are commonly used in many cultures as transportation and as pack animals, part of a group of animals commonly called beasts of burden.

Therefore, traits that horse breeders might have looked for are docility and strength, as well as robustness in cold or heat, and an ability to breed in captivity.

Ancient societies utilized genetic engineering in ways other than artificial selection, too. 6,000 years ago, Egyptians used yeast to leaven bread and fermented yeast to make wine and beer.

Modern genetic engineering happens in a laboratory instead of by selective breeding, since genes are copied and moved from one piece of DNA to another, or from one organism's cell to another organism's DNA. This relies on a ring of DNA called a plasmid.

**Plasmids** are present in bacterial and yeast cells, and are separate from chromosomes. Although both contain DNA, plasmids are typically not necessary for the cell to survive. While bacterial chromosomes contain thousands of genes, plasmids contain only as many genes as you would count on one hand. This makes them much simpler to manipulate and analyze.

The discovery in the 1960s of restriction endonucleases, also known as restriction enzymes, led to a breakthrough in gene editing. These enzymes cut DNA at specific locations in the chain of base pairs.

Base pairs are the bonded nucleotides that form the DNA strand. Depending on the species of bacteria, the restriction enzyme will be specialized to recognize and cut different sequences of base pairs.

_Related content: The Definition of Molecular Biology_

Scientists discovered that they were able to use the restriction enzymes to cut out pieces of the plasmid rings. They were then able to introduce DNA from a different source.

Another enzyme called DNA ligase attaches the foreign DNA to the original plasmid in the empty gap left by the missing DNA sequence. The end result of this process is a plasmid with a foreign gene segment, which is called a vector.

If the DNA source was a different species, the new plasmid is called recombinant DNA, or a chimera. Once the plasmid is reintroduced into the bacterial cell, the new genes are expressed as if the bacterium had always possessed that genetic makeup. As the bacterium replicates and multiplies, the gene will also be copied.

If the goal is to introduce the new DNA into the cell of an organism that is not bacteria, different techniques are required. One of these is a gene gun, which blasts very tiny particles of heavy-metal elements coated with the recombinant DNA at plant or animal tissue.

Two other techniques require harnessing the power of infectious disease processes. A bacterial strain called Agrobacterium tumefaciens infects plants, causing tumors to grow in the plant. Scientists remove the disease-causing genes from the plasmid responsible for the tumors, called the Ti, or tumor-inducing plasmid. They replace these genes with whatever genes they want to transfer into the plant so that the plant will become "infected" with the desirable DNA.

_Related content: Cell Biology: An Overview of Prokaryotic & Eukaryotic Cells_

Viruses often invade other cells, from bacteria to human cells, and insert their own DNA. A viral vector is used by scientists to transfer DNA into a plant or animal cell. The disease-causing genes are removed and replaced with the desired genes, which may include marker genes to signal that the transfer occurred.

The first instance of modern genetic modification was in 1973, when Herbert Boyer and Stanley Cohen transferred a gene from one strain of bacteria into another. The gene coded for antibiotic resistance.

The following year, scientists created the first instance of a genetically modified animal, when Rudolf Jaenisch and Beatrice Mintz successfully inserted foreign DNA into mouse embryos.

Scientists began applying genetic engineering to a wide field of organisms, for a burgeoning number of new technologies. For example, they developed plants with herbicide resistance so that farmers could spray for weeds without damaging their crops.

They also modified foods, especially vegetables and fruits, so that they would grow much larger and last longer than their unmodified cousins.

Genetic engineering is the foundation of biotechnology, since the biotechnology industry is, in a general sense, a vast field that involves making use of other living species for humans' needs.

Your ancestors from thousands of years ago who were selectively breeding dogs or certain crops were making use of biotechnology. So too are modern-day farmers and dog breeders, and so is any bakery or winery.

_Related content: How to Contact Your Representative About Climate Change_

Industrial biotechnology is used for fuel sources; this is where the term "biofuels" originates. Micro-organisms consume fats and turn them into ethanol, which is a consumable fuel source.

Enzymes are used to produce chemicals with less waste and cost than traditional methods, or to clean up manufacturing processes by breaking down chemical byproducts.

From stem cell treatments to improved blood tests to a variety of pharmaceuticals, the face of healthcare has been changed by biotechnology. Medical biotechnology companies use microbes to create new medications, such as monoclonal antibodies (these drugs are used to treat a variety of conditions, including cancer), antibiotics, vaccines and hormones.

A significant medical advance was the development of a process to create synthetic insulin with the help of genetic engineering and microbes. DNA for human insulin is inserted into bacteria, which replicate and grow and produce the insulin, until the insulin can be collected and purified.

In 1991, Ingo Potrykus used agricultural biotechnology research to develop a kind of rice that is fortified with beta carotene, which the body converts to vitamin A, and is ideal to be grown in Asian countries, where childhood blindness from vitamin A deficiency is a particular problem.

The miscommunication between the science community and the public have led to great controversy over genetically modified organisms, or GMOs. There was such fear and outcry over a genetically modified food product such as Golden Rice, as it is called, that despite having the plants ready for distribution to Asian farmers in 1999, that distribution has not yet occurred.

E., Rebecca. "Biotechnology & Genetic Engineering: An Overview" sciencing.com, https://www.sciencing.com/biotechnology-genetic-engineering-an-overview-13718445/. 28 May 2019.

E., Rebecca. (2019, May 28). Biotechnology & Genetic Engineering: An Overview. sciencing.com. Retrieved from https://www.sciencing.com/biotechnology-genetic-engineering-an-overview-13718445/

E., Rebecca. Biotechnology & Genetic Engineering: An Overview last modified August 30, 2022. https://www.sciencing.com/biotechnology-genetic-engineering-an-overview-13718445/

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Biotechnology & Genetic Engineering: An Overview - Sciencing

• Findings support the development of a novel peptide vaccine targeting the immunosuppressive effects of Transforming Growth Beta (TGF-?) in solid tumors

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IO Biotech Announces New Preclinical Data for Additional Pipeline Candidate Presented at the AACR-IO Conference

Abstract

There is a great deal of hype surrounding the concept of personalized medicine. Personalized medicine is rooted in the belief that since individuals possess nuanced and unique characteristics at the molecular, physiological, environmental exposure and behavioral levels, they may need to have interventions provided to them for diseases they possess that are tailored to these nuanced and unique characteristics. This belief has been verified to some degree through the application of emerging technologies such as DNA sequencing, proteomics, imaging protocols, and wireless health monitoring devices, which have revealed great inter-individual variation in disease processes. In this review, we consider the motivation for personalized medicine, its historical precedents, the emerging technologies that are enabling it, some recent experiences including successes and setbacks, ways of vetting and deploying personalized medicines, and future directions, including potential ways of treating individuals with fertility and sterility issues. We also consider current limitations of personalized medicine. We ultimately argue that since aspects of personalized medicine are rooted in biological realities, personalized medicine practices in certain contexts are likely to be an inevitability, especially as relevant assays and deployment strategies become more efficient and cost-effective.

Keywords: Precision medicine, biomarkers, patient monitoring, genomics

The application of emerging, high-throughput, data-intensive biomedical assays, such as DNA sequencing, proteomics, imaging protocols, and wireless monitoring devices, has revealed a great deal of inter-individual variation with respect to the effects of, and mechanisms and factors that contribute to, disease processes. This has raised questions about the degree to which this inter-individual variation should impact decisions about the optimal way to treat, monitor, or prevent a disease for an individual. In fact, it is now widely believed that the underlying heterogeneity of many disease processes suggests that strategies for treating an individual with a disease, and possibly monitoring or preventing that disease, must be tailored or personalized to that individual's unique biochemical, physiological, environmental exposure, and behavioral profile. A number of excellent reviews on personalized medicine have been written, including a growing number of textbooks on the subject meant for medical students and clinicians. It should be noted that although many use the term personalized medicine interchangeably with the terms individualized and precision medicine (as we do here), many have argued that there are some important, though often subtle, distinctions between them.(1, 2))

There are a number of challenges associated with personalized medicines, especially with respect to obtaining their approval for routine use from various regulatory agencies. In addition, there have many issues associated with the broad acceptance of personalized medicines on the part of different health care stakeholders, such as physicians, health care executives, insurance companies, and, ultimately, patients. Almost all of these challenges revolve around a need to prove that personalized medicine strategies simply outperform traditional medicine strategies, especially since many tailored or personalized therapies, such as autologous CAR-T cell transplant therapies for certain types of cancer(3) and mutation-specific medicines such as ivacaftor to treat cystic fibrosis (4, 5), can be very expensive(6). In this review we consider the history and motivation of personalized medicine and provide some context on what personalized medicines strategies have emerged in the last few decades, what limitations are slowing their advance, and what is on the horizon. We also consider strategies for proving that personalized medicine protocols and strategies can outperform traditional medicine protocols and strategies. Importantly, we distinguish examples and challenges associated with personalized disease prevention, personalized health monitoring, and personalized treatment of overt disease.

There is much in the history of western medicine that anticipates the emergence of personalized medicine. For reasons of brevity, we will not focus on all of these events, but rather only a few that we feel encompass the most basic themes behind personalized medicine. More than a century ago Archibald Garrod, an English physician, began studying in earnest diseases that would later become known as inborn errors of metabolism. Garrod studied a number of rare diseases with overt, visible phenotypic manifestations including alkaptonuria, albinism, cystinuria and pentosuria. Of these, his focused work on alkaptonuria led to some notoriety when he observed that some members of families exhibiting alkaptonuria showed measurably outlying values for certain basic biochemical assays, e.g., from urine, relative to the values of family members who did not possess alkaptonuria. This led him to conclude that alkaptonuria was due to a specific altered course of metabolism among affected individuals, which was subsequently proven correct.(7) Further, in considering other rare diseases like alkaptonuria, Garrod argued that the thought naturally presents itself that these [conditions] are merely extreme examples of variation of chemical behavior which are probably everywhere present in minor degrees and that just as no two individuals of a species are absolutely identical in bodily structure neither are their chemical processes carried out on exactly the same lines. This more than hints at his belief that, at least with respect to metabolism, humans vary widely and that these differences in metabolism could help explain overt phenotypic differences between individuals, such as their varying susceptibilities to diseases and the ways in which they manifest diseases.(8, 9)

Garrod was working in the backdrop of a great deal of debate about the emerging field of genetics. Although the specific entities we now routinely refer to as genes (i.e., stretches of DNA sequence that code for a protein and related regulatory elements), were unknown to Garrod and his contemporaries, he and others often referred to factors influencing disease possessed by certain individuals that were consistent with the modern notion of genes. Claims about the very presence of such factors were born out of discussions rooted in the findings of Mendel (later, it would be shown that many of the metabolic outliers Garrod observed in people with diseases like alkaptonuria were due to defects in genes possessed by people with those diseases). Mendel observed consistent connections between the emergence of very specific phenotypes only when certain breeding protocols were followed in peas that anticipated the modern field of genetics.(10) Essentially, as discussed in an excellent book by William Provine,(11) many in the research community at the time debated how genes or factors of the type Garrod and others were considering could explain the broad variation in phenotypic expression observed in nature. One group of academics and researchers, referred to as the Mendelians in the historical literature, which included William Bateson and Hugo de Vries, focused on the discrete nature of the factors likely to be responsible for many observable inheritance patterns (such as those of focus in Mendel's studies and observations like Garrod's in the context of rare disease). In opposition to the Mendelians were the Biometricians, represented most notably by Karl Pearson, whose focus on continuous or graded phenotypes, like height, gave them concerns about how to reconcile such continuous variation with the overtly discrete (either/or) factors and inheritance patterns considered by the Mendelians and researchers like Garrod.

The Mendelian vs. Biometrician debate was resolved to a great extent by the statistician Ronald Fisher in a series of seminal papers. Fisher argued that one could reconcile continuous phenotypic variation with discrete, heritable factors that contribute to this variation by suggesting that many factors (i.e., genes) might contribute in a small way to a particular phenotype. The collective effect, or sum total, of these factors could then create variation in phenotypes that give the appearance of continuity in the population at large (e.g., an individual who inherited only 1 of 25 genetic variants known to increase height would be shorter on average than someone who inherited 10 or 12, and much shorter, relatively speaking, than an individual who inherited 22 or 25).(12) The belief that there might be many genes that contribute to phenotypic expression broadly, some with more pronounced effects and some with less pronounced effects, that interact and collectively contribute to a phenotype in a myriad of ways, has been validated through the application of modern high-throughput genetic technologies such as genotyping chips and DNA sequencing. As a result, much of the contemporary focus on personalized medicine is rooted in the findings of genetic studies, as it has been shown that individuals do in fact vary widely as each individual possesses subsets of literally many millions of genetic variants that exist in the human population as a whole. In addition, subsets of these genetic variants may have arisen as de novo mutations and hence may be unique to an individual. These extreme genetic variation explains, in part, why individuals vary so much with respect to phenotypes, in particular their susceptibilities to disease and their responses to interventions.(13) It should be emphasized that although personalized medicine has its roots in the results of genetic studies, it is widely accepted that other factors, e.g., environmental exposures, developmental phenomena and epigenetic changes, and behaviors, all need to be taken into account when determining the optimal way to treat an individual patient (see Figure 1).(14-16)

Graphical depiction of elements in need of integration and assessment in pursuing truly personalized medicine. Access to health care is important since some individuals may not be able to access expertise and technologies due to geographic or economic barriers and therefore interventions might need to be crafted for those individuals with this in mind. Inherited genetic information is really only predictive or diagnostic in nature however somatic changes to DNA can provide valuable insight into pathogenic processes. Tissue biomarkers (e.g., routine blood-based clinical chemistry panels) are useful for detecting changes in health status, as are imaging and radiology exams as well as data collected routinely via wireless monitors. Environmental exposures and behaviors can really impact the success of an intervention and exhibit great inter-individual variability. Epigenetic phenomena reshape gene function based on exposures and developmental or stochastic phenomena and should be monitored as well as indicators of a health status change.

Another, sadly more obscure, publication was also prescient for personalized medicine, although this publication bears more on the need for clinical practices that are consistent with personalized medicine rather than a scientific justification of personalized medicine. More than 60 years ago Hogben and Sim considered how clinical practice needs to pay attention to nuanced characteristics of patients in order to determine an appropriate intervention for them.(17-19) Although more will be discussed about their paper in the section on Testing Personalized Medicines, suffice it to say that the authors believed that in order to determine an optimal course of action for an individual patient in the absence of any a priori understanding of how best to treat that patient given his or her characteristics or profile, a number of items would need to be obtained. Thus, greater information about that patient would have to be gathered, a plan to vet the utility of an intervention chosen on the basis of that information would have to be pursued, and a strategy for incorporating the results of the patient-oriented study into future care would have to be crafted. Although simple in theory, the practical issues surrounding gathering more information about a patient and pursuing an the empirical assessment of a personalized intervention can be daunting. For example, questions surrounding how one can know that a chosen intervention works unless meticulous patient follow-up information is kept, how one would know if a patient satisfied with what they are experiencing with the intervention, and how one could assess the difference between other interventions that could have been chosen and the chosen personalized intervention, would all need to be addressed. In fact, practical issues surrounding the implementation of personalized medicine that Hogben and Sim considered are often overlooked in contemporary discussions about personalized medicine, especially since different technologies for profiling patients are constantly being developed and refined, and more and more evidence for inter-individual variation in factors associated with diseases (from technologies such as DNA sequencing, proteomics, sophisticated imaging protocols, etc.) is emerging.

There have been a great many examples of interventions tailored to individual patient profiles, virtually all of them based on genetic profiles. Before providing a few classic examples, it should be emphasized that personalized medicine can be practiced not only for the treatment of disease, but also for the early detection and prevention of disease. We provide some historical examples of personalized disease treatments here and consider early detection and prevention in the next section, as developments in personalized disease detection and prevention are much more recent.

The human body deals with traditional pharmacotherapies (i.e., drugs) to treat disease in two general ways. Initially, the body must respond to a drug. This response occurs in steps, with the first step involving the absorption of the drug by the body. The drug must then be distributed throughout the body (during this process the drug might be biotransformed or metabolized into useful components) and then begin to elicit effects. Finally, any remaining drug or drug components are excreted. These processes are often lumped under the heading of pharmacokinetics and collectively referred to as the ADME of a drug (Absorption, Distribution, Metabolism and Excretion). Pharmacokinetic activity is often under the control of a unique set of genes (e.g., drug metabolizing enzymes) that could harbor naturally-occurring genetic variants (or polymorphisms) that influence their function and hence how the body ultimately deals with a particular drug. Once a drug is within the body, how it interacts with its target (typically a gene or protein encoded by a gene) to elicit an effect is known as its pharmacodynamic properties. These properties include the affinity the drug has for its target(s), the drug's ability to modulate the target(s) (or its efficacy), and the potency of the drug, or how much of the drug is needed to induce a certain change in its target. Pharmacodynamic properties of a drug are also under genetic control.

Many early examples of personalized medicines were associated with genetically-mediated pharmacokinetic aspects of drugs. This was due in part to the biomedical science community's understanding of drug metabolizing enzymes and the role they play in the body's response to drugs. An excellent introduction to pharmacogenetic properties of drugs, as well genetic variants in genes that influence the efficacy and side effects of drugs (especially with respect to genetic variants in drug metabolizing enzymes) is the book by Weber.(20) Warfarin is a widely used blood thinning medication that, if not dosed properly, could cause a potentially life-threatening adverse drug reaction. Warfarin targets a particular gene, VKORC1, and is metabolized in part by the gene CYP2C9. Naturally-occurring genetic variation in both the VKORC1 and CYP2C9 genes leads to variation in the pharmacodynamic and pharmacokinetic properties of Warfarin across individuals, ultimately creating variation in individuals' responses to warfarin. The US Food and Drug Administration has therefore recommended that dosing for warfarin take into consideration an individual's genotype (i.e., the dose must be personalized to an individual based on the specific genetic variants they possess in the VKORC1 and CYP2C9 genes).(21)

Another classic example of a drug that should only be provided to individuals with a certain genetic profile is primaquine (PQ). PQ has been used to manage malaria with some success in parts of the world where malaria is endemic. However, military doctors working in the past observed that some of the soldiers they treated for malaria that were provided the drug became jaundiced and anemic, and ultimately exhibited symptoms of what would later be termed acute haemolytic anaemia (AHA). It was later shown that the individuals exhibiting AHA after PQ administration carried variants in the G6PD gene.(22) Current clinical practice with PQ therefore calls for the genotyping of individual patients to see if they carry relevant variants in the G6PD gene that might discourage PQ use for them.

A final, often-cited example of a personalized medicine is the drug imatinib.(23) Imatinib is used to treat chronic myelogenous leukemia (CML). Imatinib inhibits an enzyme, tyrosine kinase, that is increased by the formation of a fusion of two genomic regions, one encompassing the Abelson proto-oncogene (abl) and the other the breakpoint cluster region (bcr). This fusion event arises in many tumors contributing to the development of CML and is referred to as the bcr-abl fusion or Philadelphia chromosome. However, not all individuals with CML have tumors harboring the bcr-abl fusion mutation. Therefore, imatinib is typically given only to individual CML patients with this fusion event.

Drugs like warfarin, PQ and imatinib that appear to only work or only work without side effects when a patient possesses a certain genetic profile, have generated tremendous interest in identifying factors, such as genetic variants, that influence an individual patient's response to any number of drugs and interventions. This interest in crafting personalized medicines to treat diseases has expanded into personalized disease surveillance (i.e., early detection protocols) and personalized disease prevention strategies as well. We briefly describe a few very recent examples of this activity.

Instead of developing a drug and then identifying factors that mitigate its efficacy or side effects through observational studies on individuals provided the drug, as with warfarin, PQ and imatinib, there are now attempts to identify, e.g., genetic profiles possessed by patients and then craft therapies that uniquely target those profiles. For example, the drug ivacaftor mentioned earlier was designed to treat individuals with cystic fibrosis (CF) that have very specific pathogenic mutations in the gene CFTR.(4) The CFTR gene has many functions, but one set of functions is dictated by a gate-like structure in the CFTR gene's encoded protein that can open and close to control the movement of salts in and out of cells. If the CFTR gene is dysfunctional, then the gate is closed, causing a build-up of mucus and other material in the lungs. Different mutations in the CFTR gene cause different types of dysfunction. For example, some mutations simply cause the CFTR gene to not produce anything, whether the gate is open or not. Other mutations cause the gate mechanism to dysfunction. Ivacaftor is designed to open the gate for longer periods of time in the presence of certain mutations that tend to cause the gate to be closed. Therefore, ivacaftor is only useful for the small subset of CF patients whose CFTR mutations lead to this specific gating problem. Connections between genetic variants and drug efficacy and side effects are growing in number, and in fact the US FDA provides a list of approved drug-based interventions that require a test to determine their appropriateness for an individual: https://www.fda.gov/Drugs/ScienceResearch/ucm572698.htm. Other publications consider the practical implications of approved personalized medicine interventions, such as the report produced by the Personalized Medicine Coalition (PMC).(24)

A second example involves the emerging set of cancer treatments known as immunotherapies.(25) Although there are many types of immunotherapies, all of them seek to prime or trigger an individual's own immune system to attack a cancer. One type of immunotherapy exploits potentially unique sets of genetic alterations that arise in a cancer patient's tumor cells, known as neo-antigens, which are often capable of raising an immune response if recognized properly by the host's immune cells. Essentially, this type of immunotherapy works by harvesting cells from a patient that mediate that patient's immune reactions, such as T cells, then modifying those cells to specifically recognize and target the neo-antigens found to be present in the patient's tumor. These modified cells are then put back in the patient's body so these cells can attack the tumor cells giving off the neo-antigen signals. Cytotoxic T cell therapies like this, as well as immunotherapies in general, have had notable successes, but can be very patient-specific for two reasons. First, the neo-antigen profile of a patient might be very unique, such that cytotoxic T cells made to recognize and attack a specific set of neo-antigens will not work in someone whose tumor does not have those neo-antigens. Second, if autologous constructs are used, then the patient's own T cells are modified, and hence not likely to work as well in another patient, although attempts to make allogeneic constructs in which one individual's T cells are modified and introduced into another patient's body are being pursued aggressively.(25)

If an individual is susceptible to a disease, or susceptible to recurrence of a disease, then that individual should be monitored. It is now believed that such monitoring should be pursued with use of personal thresholds, as opposed to population thresholds, to make claims about evidence or signs of disease or a pathogenic process.(26) Population thresholds are derived from epidemiologic data and population surveys and include, for example, cholesterol levels > 200 being an indicator for risk of heart disease, or systolic blood pressure > 140 being an indicator of hypertension, risk of stroke or heart disease. Personal thresholds are established from legacy values of a measure collected on an individual over time that used to gauge how deviant future values of that measure might be for that individual. Significant deviations from historical or average legacy values are taken as a sign of a health status change, irrespective of whether or not those values are beyond a population threshold.(27) As an example, Drescher et al.(26) explored the utility of personal thresholds applied to longitudinal CA125 levels collected on a number of women, a subset of whom developed ovarian cancer. The authors found that in all but one instance, the application of personal thresholds would have captured the presence of ovarian cancer at the same time as, or importantly earlier than, the application of population thresholds. Further, the authors showed that the use of personal thresholds could have captured the ovarian cancer almost a year earlier, on average, then the use of population thresholds. As the costs and convenience associated with monitoring assays and technologies improves (i.e., they become cheap and non-intrusive, if not transparent, to an individual user, say through an easily implantable wireless device), then the use of personal thresholds will likely become the rule rather than the exception in health monitoring protocols.

The use of genetic information to develop personalized disease prevention strategies is now well established in the scientific community, but not yet widely adopted in clinical practice. There are many excellent examples of how the use of genetic information can lead to both a decreased risk of disease development as well as decreased complications from standard treatment and screening strategies. A prime example relates to colorectal cancer, which remains the third leading cause of cancer deaths despite being a highly preventable illness. In 2012 Liao et al. reported an improvement in overall survival and a decreased risk for cancer-specific deaths in patients taking postoperative aspirin if they exhibited a somatic mutation in the PIK3CA gene in their colorectal cancers compared with patients whose cancers had the wild-type PIK3CA gene.(28) In 2015, Nan et al. reported varying effects of aspirin use on risk for development of colorectal cancer depending on an individual's genotype, with individuals possessing different genotypes having either lower, higher or no change in their risk of colorectal cancer development with aspirin use.(29) Given that aspirin use can have serious side effects associated with intestinal and intracranial bleeding, it would be ideal to limit the use of this medication for those individuals predicted to have a side effect, based on genotype.

As another example, in 2018, Jeon et al. reported the use of expanded risk prediction models for determining when to begin colorectal cancer screening. Currently the guidelines use only age and family history as variables. Jeon et al. showed that by using information about an individual's environmental exposure and genetic profile, specifically the presence of colorectal cancer associated genetic variants, recommendations for when to start screening could change by 12 years for men and 14 years for women.(30) The accuracy of relevant predictions about an individual's risk for colorectal cancer has been studied and suggests that the area under the curve (AUC) value for a model including environmental and genetic factors, where an AUC of 1.0 would suggest a model with perfect predictive accuracy, was 0.63 for men and 0.62 for women. This is compared to an AUC value of 0.53 (men) 0.54 (women) when only family history information was considered. Although there is still room for improvement given the AUCs were only 0.62 for the model with patient environmental exposure and genetic information, the considerable improvement over models that did not include genetic or environmental information justifies their use.

Although we have argued that personalized medicine is rooted in a great number of legacy insights and historical precedents, mostly related to genetics and rare diseases, its recognition as a paradigm that should be embraced broadly by the biomedical research and clinical communities is relatively recent. This suggests that not enough time has elapsed since the time of this recognition for researchers to show that personalized medicine actually works in a wide enough variety of settings to motivate its broad adoption. In this light, questions of how the community can vet or test the utility of personalized medicine arise. We describe three emerging strategies for vetting personalized medicines below, including N-of-1 clinical trials, intervention-matching trials, and adaptive clinical trials, and argue that although these strategies borrow elements from traditional randomized clinical trials (RCTs), they deviate significantly from historical population-based RCTs that were prominent in the past.

If there is no reason to believe that any one of a set of different interventions matches an individual's profile (e.g., genomic, behavioral, etc.) better than others, then there is equipoise among those interventions. In this case it becomes an empirical question as to which intervention might be optimal for the individual in question. Trials focusing on an individual's response to different interventions to determine an optimal intervention are referred to as N-of-1 or single subject trials. N-of-1 trials often exploit a simple cross-over design or even a repeated crossover designs, such as ABABAB designs, where A and B refer to different interventions, and the sequence ABABAB refers to the order in which the interventions are provided to a patient. Alternating interventions, and collecting data on the individual's response to those interventions, allows comparisons of those interventions (for example between a test intervention and a comparator, or placebo, intervention. Randomization, blinding, washout periods, multiple endpoints, and many other design elements can be used in N-of-1 trials.(27, 31, 32)

N-of-1 trials involving the provision of different interventions in sequence to an individual and evaluating outcomes for each, need to accommodate serial correlation between the observations, as well as possible carry-over effects from one intervention to another, but these issues can largely be overcome with appropriate analytical methods and study design.(32) Cross-over based N-of-1 trials are impractical, if not unethical, in settings where an individual is suffering from an acute or life-threatening condition, since switching from one intervention to another may exacerbate that individual's condition. However, sequential N-of-1 designs, in which measures are continuously monitored in real time to determine if an intervention is causing harm or working, have been proposed for these situations.(27) Given that the focus of an N-of-1 trial is on the identification of an optimal intervention for an individual, rather than on the average response to an intervention in the population at large (which is often the focus of traditional RCTs), they may be most appropriate to conduct in actual clinical practice when a physician is faced with equipoise, as considered by Hogen and Sim.(33, 34)

If evidence is found that certain features in individual patients' profiles can be used to identify interventions that might work for each of them, then a question arises as to how to test that the hypothesis that providing interventions to those individuals based on these matches leads to better outcomes than providing those individuals interventions based on some other scheme or strategy. One could test each individual match, but this may require pursuing many small clinical trials, which may be logistically complicated and hard to find financial support and infrastructure to implement. As an alternative, one could test an entire matching strategy against an alternative way of providing interventions (e.g., giving everyone the same intervention). This is more or less the motivation behind basket and umbrella trials currently in use, primarily in oncology settings.(35, 36) In oncology contexts, basket and umbrella trials enroll multiple individual patients into a trial knowing that they each might have unique features in their profile that could indicate that different interventions are appropriate. Basket trials enroll individuals without regard to the specific tissue affected by cancer (e.g., lung, breast and colorectal cancer patients can be enrolled) whereas umbrella trials only consider a single tissue (only lung cancer patients are enrolled). Each patient's tumor is profiled, usually via DNA sequencing. The tumor genome is analyzed to see if there are actionable driver perturbations in the tumor, such as mutations affecting particular genes, that are likely contributing to the growth of the tumor. If the mechanisms of action of a group of interventions (i.e., cancer drugs) are understood well enough, it may be possible to match those drugs to the perturbations in the tumor (e.g., if the EGFR gene is mutated and overexpressed in the tumor, then using a drug like cetuximab, which inhibits the EGFR gene, would be logical). Thus, each patient is steered towards a particular intervention basket (e.g., the EGFR inhibitor basket). The trial then seeks to test the hypothesis that the use of the different intervention baskets based on the matching scheme results in better outcomes than interventions provided to individual patients based on some other scheme that does not involve tumor profiling and matching.

If the trial is a failure (i.e., the matching scheme doesn't lead to better outcomes than something else), then an argument could be made that the matching scheme was flawed and not necessarily that the interventions considered in the trial are flawed. It would also be wrong to assume that the concept of personalized medicine is flawed as a result of a failure of a basket or bucket trial if in fact the matching scheme was found to be flawed. Some basket trials only have a single basket and no comparison group, but rely on determining which patient profiles appear to be associated with better outcomes for the intervention being tested.(37) Intervention matching schemes are likely to become the rule rather than the exception in medicine, especially since the introduction of computational environments like IBM's Watson system. Essentially, Watson is system that includes a very large database extracted in part from the vast medical literature, providing links between information about a patient (e.g., genetic profiles, age, sex, etc.) to outcomes (such as drug response). These links have been enhanced by leveraging statistical methods to further assess relationships between patient profiles and outcomes. For example, Watson has been trained to identify and establish links about perturbations often observed in a tumor and how those perturbations might be combatted by available drugs and interventions generally. Thus, if Watson was provided a patient profile, it could look up the best possible intervention given the current state of the science reflected in the literature and Watson's methods for establishing links between profiles and outcomes. The use of IBM's Watson system in actual clinical settings has led to discussions about how best to test and deploy such as a system as a way of supporting, as opposed to replacing, physicians' decisions about an intervention choice for individual patients.(38)

Adaptive and sequential clinical trials have been used for decades but their consideration and use in personalized medicine contexts is much more recent.(35) Essentially, adaptive trials have as one of their focal points a desire to minimize the amount of time a patient is on what is likely to be an inferior therapy. In the context of personalized medicine, if there is equipoise with respect to available interventions or between an untested and a conventional intervention for an individual patient, then the evaluation of the effects of each intervention on an individual to determine the best one for that individual (as in a very elaborate N-of-1 study) might be impractical or cause more harm than good. This is the case because some, if not all, of the interventions might not actually benefit that individual. In this light, it makes sense to implement studies in which biomarkers reflecting response or adverse effects are collected on an individual trial participant and monitoring of those biomarkers is pursued to determine if there are signs an intervention is not working. If there are, e.g., signs that an intervention is not working, the individual could cross-over to a new intervention. Although adaptive designs can be difficult to implement given their real-time evaluation and updating components, and can also produce data that might be more complicated to analyze than data from fixed, non-adaptive trials, they are often seen as more ethical. In addition, adding adaptive components to N-of-1 and aggregated N-of-1 trials as well as intervention-matching trials is possible. Although there are a growing number of papers describing adaptive trials, the work of Murphy and colleagues has received a great deal of attention because of its focus on minimizing the amount of time a patient is on an inferior treatment.(39-41)

There are a number of very recent research and clinical activities that are charting new territory for personalized medicine. We focus on four of these activities in the following, providing a brief overview of each. These activities include the use of patient-derived cell and organoid avatars for determining the best therapies for that patient, the use of intense individualized diagnostic and monitoring protocols to detect signs of disease, the development of personalized digital therapeutics, and the use of personalized medicine approaches in treating patients with fertility issues.

It is now possible to harvest cells from individuals and use pluripotency induction (i.e., induced pluripotent stem cell or iPSC) methods on those cells to generate additional cell types of relevance to a patient's condition without having to directly biopsy the affected tissue. This allows researchers to essentially develop a disease in a dish cellular model of a patient's condition.(42-44) These in vitro cellular avatars can be studied to identify key molecular pathologies that might give an indication as to how best to treat an individual patient of interest. The use of iPSC technologies in this manner can be extended with a few additional, very recently developed, technologies to create even better models of an individual's condition. For example, if the patient has a known mutation causing his or her condition, it is possible to use assays based on, e.g., Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and related constructs to create isogenic cells in which some cells have the mutation in question and some do not. Comparison of these cells allows direct insight into the effects of the mutation while controlling for all relevant genetic background effects associated with the patient's genome.(45, 46) In addition, it is possible to create partial organs or organoids from cells obtained from an individual.(47) Organoids can provide greater insight into molecular pathologies associated with an individual patient's condition since they can model cell:cell interactions and more global tissue function.(48)

To achieve truly personalized medical care, the use of patient avatars derived from their own cells could be integrated with other pieces of information about a patient, as well as protocols for acting on that information. Schork and Nazor describe the motivation and integration of different aspects of patient diagnosis, intervention choice, and monitoring, using, among other things, patient avatars.(49) One important aspect of the use of cell-based patient avatars in personalized medicine is that they can accommodate personalized drug screening: literally testing thousands of drugs and compounds against a patient's cells (or organoids, possibly modified with CRISPR technologies) to identify drugs or compounds that uniquely correct the patient's molecular defects. If the drug or compound has actually been approved for use, possibly for another condition, then it could be tested for efficacy with the patient in question under an approved drug repurposing protocol. The use of patient-derived cells in personalized drug screening initiatives has shown some success in cancer settings, as tumor biopsies can yield appropriate material for drug screening.(48, 50) The biggest concern with this approach revolves around the question of whether or not the in vitro models capture relevant in vivo pathobiology and drug response information that may impact a patient's response to a chosen drug. A more direct strategy for in vivo experimental cancer intervention choice could involve implanting a device into a patient's tumor in vivo and then delivering different drugs through that device to see which ones have an effect.(51, 52)

The availability of inexpensive genotyping and sequencing technologies is allowing individuals and their health care providers to assess their genetically-mediated risk for disease and/or make a genetic diagnosis if they are already diseased. In addition, given the availability of health monitoring devices, online-ordered blood-based clinical assays, inexpensive imaging devices, etc. it is possible to continuously, or near continuously, monitor aspects of an individual's health (see Figure 1 and see the articles associated with the quantified self movement: http://quantifiedself.com)(53, 54). With this in mind, combining genetic risk or diagnostic assessment with intense health monitoring makes sense. A number of individuals with unique diseases and conditions have benefitted from a genetic diagnosis, as it uncovered potential genetically-mediated pathogenic mechanisms or revealed potential targets for pharmacotherapies for them.(49) In addition, a number of individuals have monitored their health intensely for the express purpose of identifying signs of a health status changes, some of which might be attributable to genetic susceptibilities.(55) Table 1 lists examples of published studies exploring the utility of genetic assays in generating a diagnosis for individuals with idiopathic conditions (or what have been referred to as diagnostic odysseys) as well as published studies exploring the utility of near continuous monitoring to identify evidence for a health status change in an individual. Such diagnoses and monitoring are highly personalized by definition.(15, 16, 56)

Monitoring individuals for health status changes is not trivial, however, if the measures being collected have not been evaluated in a population. This is because there will be no established norms that can be contrasted to the measures collected on an individual to know if those measures are abnormal. However, the community is quickly recognizing the utility of establishing personal thresholds for measures as opposed to population thresholds, as discussed in the Personalizing Early Detection Strategies section above (26, 27) As noted, population thresholds are established from epidemiologic and population survey data and include often-used thresholds for determining disease status such as a cholesterol level greater than 200 for heart disease or a systolic blood pressure greater than 140 mmHg for hypertension. Personal thresholds are established from longitudinal or legacy values of a measure collected on an individual and may be unique to the individual in question and their use in some settings suggests that they work better than population thresholds.(26)

The ubiquity of smart phones has attracted the interest of many researchers in the health professions as a vehicle for not only collecting health data through various apps but also to provide advice, feedback, coaching, imagery, music, text-messages, or connections with other resources, that could benefit an individual with a particular condition or disease. This has led to the emergence of the concept of a digital therapeutic: a smart phone app designed to treat and bring relief to an individual affected by a medical or psychological condition.(57) The content provided by a digital therapeutic app to an individual could vary depending on what is learned about that individual and his or her response to content provided in the app. In this way, the app can be personalized.(58) Many digital therapeutics have undergone evaluation for their ability to engage users and benefit them.(59) The US Food and Drug Administration (FDA) has created guidelines for registering digital therapeutics as bona-fide, insurance-reimbursable, approved health technologies, and has begun evaluating and approving many of them. The first approved digital therapeutic an app for substance abuse was approved by the FDA in 2017.(60) How easily digital therapeutics will be assimilated into the care stream is an open question.(61)

Personalized medicine strategies and approaches can be applied to treatments for fertility, as many researchers have proposed. For example, it has been suggested that one could leverage real world data of the type collected routinely on patients visiting reproductive medicine and fertility clinics (from, e.g., Electronic Medical Record (EMR) systems established at many hospitals and clinics), and use these data to in analyses exploring patterns, patient subgroups and individual patient profiles that could shed light on variation in fertility rates, responses to interventions to enhance fertility, etc. The results of these analyses could then guide future care for patients with fertility issues.(62) In the context of the use of digital medicine, proposals to develop smart phone apps that could provide personalized coaching content to enhance pregnancy have been put forth.(63). Genetic variants known to influence fertility have also been identified and could be used to support diagnoses or personalized intervention plans.(64)(65) Finally, adaptive trial designs have been proposed that could be used to assess the utility of personalized approaches to raising awareness about time to conception and fertility.(66)

In addition to these more traditional approaches to personalizing fertility interventions, there are a number of emerging strategies to enhance fertility in women that go beyond traditional ways of stimulating ovaries.(67) For example, it is now possible to cryopreserve a set oocytes and ovarian tissue samples from a woman and then implant them in her at a later time that may suit her desire to become pregnant.(68) Such a procedure would be highly personalized, since it would work with an individual's own cells and accommodate her preferences for becoming pregnant. However, this procedure would only work if the preserved tissues were viable and not damaged, although relevant cells in those tissues could, in theory, be corrected for genetic defects using gene editing techniques.(69) A more futuristic and controversial personalized fertility intervention, involves the concept that one could use cell reprogramming technologies to generate sperm and egg cells from other cells obtained from an individual (e.g., skin cells) that could be edited to generate de novo gametes for fertilization a concept known as in vitro gametogenesis.(70)

Personalized Medicine, or the practice of characterizing an individual patient on a number of levels (e.g., genomic, biochemical, behavioral, etc.) that might shed light on their response to an intervention, and then treating them accordingly, is a necessity given the fact that clinically meaningful inter-individual variation has, and will continue to be, identified. The availability of modern biomedical technologies such as DNA sequencing, proteomics, and wireless monitoring devices, has enabled the identification of this variation, essentially exposing the need for the personalization of medicine at some level. The future challenges associated with this reality will be to not only improve the efficiency in the way in which individuals are characterized, but also in the way in personalized medicines are crafted and vetted to show their utility. This is not to say that interventions that work ubiquitously (i.e., the traditional single agent block buster drugs) should be ignored if identified, but rather that they might be very hard to identify going forward.

There are a few other issues associated with personalized medicine that may hard to overcome in the near term. For example, the need for large data collections in order to identify factors that discriminate groups of individuals that might benefit more from one or another intervention, could create concerns about privacy and the data about those individuals possibly being used for nefarious purposes.(71-73) Fortunately, this issue is not necessarily unique to health care settings, whether current or future, as it has plagued many other industries including the banking, marketing, and social media industries. Strategies exploited in these other industries could be used in health care settings as well. In addition, developing more efficient ways of developing personalized medicines (for example, with respect to cell replacement therapies or mutation-specific drugs that work for a small fraction of patients) is crucial to meet the demands of all patients. Also, paying for personalized medicine practices in the future may be complicated given that they might be initially more expensive.(74) Finally, in order for various stakeholders to embrace personalized medicine, better strategies to educate and train health care professionals about personalized medicine must be developed and implemented.

Dr. Schork and his lab are funded in part by US National Institutes of Health Grants UL1TR001442 (CTSA), U24AG051129, U19G023122, as well as a contract from the Allen Institute for Brain Science (note that the content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the NIH).

LHG and NJS have no conflicts to declare with respect to this article.

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The Department of Genetic Medicine maintains several large centers funded in part by the National Institutes of Health. These research resources have a long history at Johns Hopkins and provide the foundation for innovative research, as well as providing services and information to scientists around the world.

OMIM, is an encyclopedia of genetic disorders, their clinical features and the genes that contribute to them. The database contains information on thousands ofMendelian conditions, disorders caused by errors in a single gene. The database was developed 55years ago by Victor McKusick and is now maintained byAda Hamosh, MD, MPH, and her team. OMIM is used by 2.7 million unique users per year around the world.

GRCFprovides year-roundresearch expertise, products, and services for the study of the human genome. At the leading edge of technology, the GRCF provides sophisticated tools and equipment oftentimes not available in individual research labs. The mission of the GRCF is to provide high quality, cost effective research services and products to investigators throughout the Johns Hopkins scientific community. Accordingly, GRCF services cover a broad segment of genetic research including:

JHG provides research and clinical genotyping and sequencing together with extensive analytic expertise. A partnership between the Departments of Genetic Medicineand Pathology, JHG opened its doors in 2017, co-localizing four existing labs:

CIDR,is a national resource, offering sequencing, genotyping and epigenetic services to scientists looking to discover genes and variants that contribute to human disease. As part of Johns Hopkins Genomics, CIDR researchers focus on the genetic architecture of complex traits, looking at conditions that result from many genetic variants and how these variants accumulate to manifest disease. This includes conditions such as all types of cancer risk, eye diseases, cleft lip and palate, oral health, environmental influences on child health outcomes, ADHD, structural brain disorders, obesity, alcoholism and aging. Most recent studies are focused on minority populations or extremely well-phenotyped populations. CIDR facilitates data cleaning and data sharing. The 140 CIDR studies posted in dbGaP are heavily utilized with > 7,600 data requests. Since opening its doors in 1996, CIDR has been continuously funded by contracts from a consortium of ten National Institutes of Health institutes (the CIDR Program) as well as through funding from many other genomic consortia, including most recently the national precision medicine initiative, the All of Us Research Program. As of January 2024, CIDR has completed 1,508 studies, consisting of > 1.7 million DNA samples and encompassing over 200 different phenotypes for 421 principal investigators world-wide.

BHCMGaccepts samples from thousands of peoplewith rare disorders submitted by a worldwide network of rare-disease experts. A collaboration between Baylor College of Medicine and Johns Hopkins, the goal of the center is to sequence the genomes of people with these conditions as well as appropriate family members to identify the genes and variants responsible for disorders whose molecular basis was previously unknown. In particular, the center seeks families with known or novel conditions for which the culprit gene is unknown. Successful identification of the responsible gene connects a particular gene with a particular set of clinical features, thereby enabling precise molecular diagnosis and prognosis.It alsoinforms research on the development of rational treatment and providing families with information about recurrence risk.

Focused on Kabuki syndrome and related Mendelian disorders of the epigenetic machinery. These rare disorders result from mutations in single genes encoding components of the systems that add, interpret or delete epigenetic marks with the result that sets of genes are mis-regulated. Currently we know of more than 40 such epigenetic disorders, most of which have intellectual disability and growth abnormalities as prominent clinical consequences. By understanding the features and pathogenesis of these precise abnormalities of the epigenetic system IGM investigators expect to understand not only each disorder but also to how the whole epigenetic systems functions and the pathophysiological consequences that accrue when the system malfunctions. This research complements the clinical services offered in the IGM Epigenetics and Chromatin Clinic where patients with these disorders are diagnosed, characterized and treated.

Focused on understanding the molecular pathophysiology of the vascular form of Ehlers-Danlos syndrome (vascular EDS) with the aim of providing informed management of these patients as well as developing new forms of therapy. The Center will utilize advanced genetic and molecular methods to discover the sequence of events that contribute to structural weakening of the arterial wall and internal tissues over time, ultimately leading to tear or rupture and the potential for early death. The research team has developed two mouse models of vascular EDS that demonstrate most of the important physical findings seen in patients with the disorder. As in people with vascular EDS, we observe tremendous variation in the timing of onset and severity of vascular disease in our mouse colonies. Our strong belief is that both genetic and environmental factors have the capacity to afford strong protection in vascular EDS. Once identified, we will attempt to mimic the mechanism of protection using medications or other strategies. The Center also aims to coordinate expert clinical care of individuals with vascular EDS, and to promote research in the clinical sciences that will improve both the length and quality of life for affected individuals. The Center for Vascular Ehlers-Danlos Syndrome Research has received generous and visionary funding from a variety of sources including the EDS Network CARES Foundation, the EDS Today Advocates, the DEFY Foundation, the Aldredge Family Foundation, and the Daskal Family Foundation.

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Research Centers | Johns Hopkins Institute of Genetic Medicine

The Evolution of Cell & Gene Therapy: Development and Manufacturing Insights and the Role of CDMOs  Pharmaceutical Technology Magazine

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The Evolution of Cell & Gene Therapy: Development and Manufacturing Insights and the Role of CDMOs - Pharmaceutical Technology Magazine

Abstract

Stem-cell therapy is a revolutionary frontier in modern medicine, offering enormous capacity to transform the treatment landscape of numerous debilitating illnesses and injuries. This review examines the revolutionary frontier of treatments utilizing stem cells, highlighting the distinctive abilities of stem cells to undergo regeneration and specialized cell differentiation into a wide variety of phenotypes. This paper aims to guide researchers, physicians, and stakeholders through the intricate terrain of stem-cell therapy, examining the processes, applications, and challenges inherent in utilizing stem cells across diverse medical disciplines. The historical journey from foundational contributions in the late 19th and early 20th centuries to recent breakthroughs, including ESC isolation and iPSC discovery, has set the stage for monumental leaps in medical science. Stem cells regenerative potential spans embryonic, adult, induced pluripotent, and perinatal stages, offering unprecedented therapeutic opportunities in cancer, neurodegenerative disorders, cardiovascular ailments, spinal cord injuries, diabetes, and tissue damage. However, difficulties, such as immunological rejection, tumorigenesis, and precise manipulation of stem-cell behavior, necessitate comprehensive exploration and innovative solutions. This manuscript summarizes recent biotechnological advancements, critical trial evaluations, and emerging technologies, providing a nuanced understanding of the triumphs, difficulties, and future trajectories in stem cell-based regenerative medicine. Future directions, including precision medicine integration, immune modulation strategies, advancements in gene-editing technologies, and bioengineering synergy, offer a roadmap in stem cell treatment. The focus on stem-cell therapys potential highlights its significant influence on contemporary medicine and points to a future in which individualized regenerative therapies will alleviate various medical disorders.

Keywords: biotechnology advancements, clinical trials, medical revolution, stem-cell therapy

Stem cell therapy represents a groundbreaking frontier in modern medicine, offering unprecedented potential to address a wide range of debilitating diseases and injuries.

Stem cells possess unique properties, including self-renewal and differentiation into specialized cell types, making them indispensable for regenerative medicine applications.

The historical journey of stem cell research, from foundational contributions in the late 19th and early 20th centuries to recent breakthroughs like the isolation of embryonic stem cells and induced pluripotent stem cells, highlights the monumental progress in medical science.

Stem cell therapy holds promise for treating various conditions, including cancer, neurodegenerative disorders, cardiovascular diseases, spinal cord injuries, diabetes, and tissue damage.

Despite the immense potential, stem cell therapy faces challenges such as immune rejection, tumorigenesis, and the precise manipulation of stem cell behaviors, necessitating innovative solutions for clinical translation.

Recent biotechnological advancements, such as exosome-based therapeutics, single-cell RNA sequencing, and CRISPR technology, have revolutionized stem cell research, offering new opportunities for precise genome editing and therapeutic interventions.

Regulatory considerations are paramount in the clinical translation of stem cell therapies, requiring adherence to strict guidelines and directives to ensure safety and efficacy.

The future of stem cell therapy lies in precision medicine integration, immune modulation strategies, advancements in gene editing technologies, and synergies with bioengineering, paving the way for continued evolution and personalized regenerative therapies.

Stem-cell therapy signifies a pioneering frontier in modern medicine that uses the extraordinary power of stem cells and their revolutionary potential to treat diverse illnesses. Stem cells play a crucial role in regenerative medicine and exhibit the extraordinary ability to differentiate into various cell types and to renew themselves. Their intrinsic capacity to repair and regenerate tissues holds immense promise for revolutionizing therapeutic interventions1,2. The historical journey of stem-cell investigation can be traced to pivotal contributions from visionaries such as Boveri, Hcker, Maximow, and Cohnheim during the late 19th and early 20th centuries3. Their foundational work placed the groundwork for comprehension of the fundamental principles of stem cells and for shedding light on their roles in developmental processes and tissue repair. These early insights have laid the foundation for contemporary stem-cell investigations, fueling a deeper exploration of their biological significance3,4. Important turning points in the history of this field include the identification of ESCs in 1981 by Kaufman and Evans57 and Thomsons discovery of iPSCs in 20078. Although stem-cell therapies have vast and promising potential, several challenges and complexities loom in their clinical translation9. Issues like immunological rejection, tumorigenesis, and precise manipulation of stem-cell behavior for optimal therapeutic outcomes are critical hurdles that necessitate comprehensive exploration and innovative solutions1,1012. Advances in biotechnology, especially the revolution in exosome-based therapeutics, single-cell RNA sequencing (scRNA-Seq), and CRISPR technology1315, one of the major developments in genetic engineering, has made precise and effective genome editing possible, which opens new avenues for modified genetic material, leading to advances in a variety of fields such as biotechnology and medicine16,17. Regenerative medicine represents a novel and promising therapeutic approach for individuals with exhausted or nonexistent options for managing their medical condition. Research studies, such as identification, clinical trials, and therapeutic applications on stem-cell have been extensive in recent years because of promising results from preclinical research (Fig. 1). The process of bringing these novel medicinal items from laboratories to the market is governed by strict guidelines and directives issued by qualified regulatory bodies18. Stem cells can be obtained for tissue engineering and cell treatments from four primary sources. The stem cells primary sources are embryonic and fetal tissues, comprising the placenta (including the chorion and amnion), umbilical cord (Wharton jelly), and particular tissues inside the adult, such as blood, skin, skeletal muscle, fat, and bone marrow, and somatic cells that have undergone genetic reprogramming to become distinct from their original state, such as iPSCs19.

A timeline depicting the introduction of mesenchymal stem cells (MSCs), their early research, and their substantial application in clinical trials, immunoregulation, and disease treatment.

Through an extensive synthesis of recent biotechnological advancements, critical evaluations, and emerging technologies, this review offers a nuanced comprehension of the advantages, difficulties, and future trajectories of stem cell-based regenerative therapy. By examining the historical foundations, current landscape, and prospects, this study endeavors to serve as a guide for researchers, clinicians, and stakeholders in navigating the intricate terrain of stem-cell therapy.

An extensive examination of existing literature was performed using the Embase, Web of Science, PubMed, and Scopus databases. The terms stem cell therapy, medical revolution, biotechnology advancements, and clinical trial were used in the search. Only articles published in English were included in the search. We assessed the abstracts of each article to determine the relevance of the retrieved papers to the topic. Subsequently, every relevant paper (in vivo, in vitro, and human-based research) was selected as part of the study.

ESCs exhibit characteristics that distinguish them from each other in stem cell biology. Notably, their pluripotency, which is defined by distinct features to differentiate into any human body cell, makes them highly adaptable and has great therapeutic promise20. Additionally, ESCs have a notably high self-renewal capacity, which contributes to their sustained presence and functionality over extended periods21. Potential ESC sources include mice, nonhuman primates, and humans. They are isolated from the blastocysts inner cell mass before implantation22,23. Because they are pluripotent cells, they can produce various kinds of cells from fetuses and adults in vivo and in vitro2426. Two methods were employed to separate ESCs from blastocysts inner cell masses. Microsurgery is the most commonly used surgical approach. Mechanical dissection in the microscopic direction is used to isolate cells of the trophoblastic lineage from the rest of the cell mass. The second approach entails employing an antibody to target trophoblast lineage cells27,28.

Regarding potential applications, the pluripotent nature of ESCs opens avenues for significant contributions to tissue regeneration and repair. Their capacity to undergo differentiation into many cell lineages holds promise for treating degenerative conditions and injuries, making them pivotal players in regenerative medicine. Furthermore, ESCs serve as invaluable tools in disease modeling for research purposes29. By replicating specific cellular environments, researchers can discover more about the workings of various disorders, providing a framework for cellular disease research and aiding in the creation of focused therapies. The unique properties of ESCs are relevant to drug testing and development30. Because of their pluripotency, a variety of cell populations can be created to provide a more complete picture of human cellular responses. This capability is particularly valuable for evaluating drug efficacy and safety and provides a sophisticated model for preclinical testing. Consequently, the multifaceted potential of ESCs dramatically enhances our comprehension of biology, fostering medical research and shaping the landscape of therapeutic innovation31,32.

ASCs stand out in the realm of regenerative biology because of their distinctive properties and vital roles in maintaining tissue homeostasis33. Multipotency is the ability of cells to possess various potential fates or abilities to develop into a restricted, diverse array of cellular phenotypes34. ASCs are endogenous stem cells that are crucial for preserving the tissues structural integrity, like bone, skin, and blood. They are located in specific niches or tissue sections35. ASCs have been discovered in several tissues, including blood, stomach, muscle, skin, brain, and heart36. They are less potent than ESCs; however, they have demonstrated efficacy in disease treatment. They can be extracted and harvested from individuals and used for tissue regeneration through autologous or allogeneic transplantation37. ASCs have a more specialized differentiation capability than pluripotent cells, such as ESCs, and can help generate particular cell lineages within their original tissue34.

Stem cells function in repairing damaged tissues and maintenance is essential throughout an individuals lifespan38. The unique ability of ASCs to maintain tissue and exhibit multipotency lends itself to a variety of possible uses within the regenerative medicine field39. Tissue-specific regeneration and repair are among the most promising approaches. ASCs can be utilized to regenerate damaged or deteriorated tissues due to their presence in diverse tissues, including the bone marrow, skin, and muscle40. Their capacity to undergo cell type-specific differentiation that is relevant to their native tissues places them at the forefront of tailored regeneration techniques, offering potential treatment options for ailments ranging from degenerative illnesses unique to certain organs to musculoskeletal injuries41.

ASCs are an appealing therapeutic choice for degenerative diseases. Because of their functions in tissue repair and regeneration, they are desirable targets for therapies aimed at slowing the advancement of illnesses marked by cellular degeneration42. Through the utilization of the regenerative capacity of these cells, scientists and medical professionals have investigated ways to create novel treatments that target the root causes of degenerative illnesses with the aim of enhancing patient outcomes and quality of life43. Within the class of ASCs, hematopoietic stem cells are a specific subset essential for bone marrow transplantation44. The immune system and blood regeneration rely on hematopoietic stem cells (HSCs), which are essential due to their versatility in cell differentiation into various blood cell types45. The utilization of these cells in bone marrow transplants represents a cornerstone in hematological therapies, offering a curative approach for conditions like leukemia and other disorders affecting the blood and immune systems46,47. Transplantation of hematopoietic stem cells is a life-saving intervention that reinstates functional blood and immune cell populations in individuals with hematopoietic disorders48.

Embryonic stem cells are derived from the amniotic fluid, placenta, and umbilical cord and represent a unique category within the spectrum of stem cell types49. Fetal cells possess multipotent capabilities and can differentiate into a restricted type of cells50. These cells are distinctively derived from tissues associated with the prenatal and perinatal stages of development, indicating their specialized origin49. Notably, perinatal stem cells exhibit a hybrid nature, sharing characteristics analogous to those of adults and ESCs. Their dual features make them adaptable and potentially useful for various regenerative medicine applications51. Perinatal stem cells offer a noncontroversial and ethically sound reservoir for therapeutic purposes49. Their properties, which are reminiscent of those of ESCs and ASCs, contribute to their unique regenerative potential. Since these cells undergo cell differentiation into a wide variety of cells, tailored approaches for tissue regeneration and repair are possible52. Perinatal stem cells show promise in furthering regenerative medicine across a range of tissues in terms of prospective uses. They are important components in targeted tissue renewal because of their capacity to specialize in particular cell lineages52.

Moreover, its therapeutic potential can be extended to other conditions, such as cerebral palsy and diabetes. Perinatal stem cells offer a novel and innovative approach to the development of medicines tailored to address the complexities of these disorders by exploiting their regenerative properties and versatile differentiation capabilities53. One notable advantage of perinatal stem cells is their potential for allogeneic transplantation without eliciting immune rejection. The immunomodulatory characteristics of these cells make them well-suited for transplantation across different individuals, eliminating the need for a perfect match between the donor and recipient54. This opens new possibilities for allogeneic stem-cell therapies, providing a feasible and practical approach to transplantation procedures without the intricate challenges associated with immune compatibility.

In summary, perinatal stem cells signify a distinct and highly promising category of stem cells with hybrid properties. Their application in regenerative medicine, therapeutic interventions for specific conditions, and allogeneic transplantation underscore their potential to reshape the landscape of stem cell-based therapies.

The iPSCs represent a revolutionary category in stem-cell studies and are characterized by properties that mirror those of ESCs55. Several human and mouse investigations have utilized fibroblasts and skin cells as the primary sources of adult cells. It has been discovered that adult brain stem cells have been identified as the primary cell type in investigations of reprograming cells56. Another study reported that murine bone marrow mononuclear cells can be reprogrammed more effectively than mouse embryonic fibroblasts57. Notably, iPSCs and their embryonic counterparts possess the capacity to undergo pluripotency to differentiate into distinct kinds of specialized cells58. One important way to iPSCs is to distinguish them from ESCs by their source, in which in order to create iPSCs, adult cells are reprogrammed. This methodology provides a novel means of addressing ethical concerns regarding the use of ESCs in scientific investigation59. Personalized medicine could undergo significant transformations if adult cells are reprogrammed to become iPSCs. The advancement of individualized cellular therapeutics involves the process of cellular reprogramming for individual patients is one of the main uses of iPSCs60. The iPSCs have the remarkable ability to transform into a wide variety of disease-specific cell types during cell reprogramming. This personalized approach improves the integrity and efficiency of cell-based treatments and offers a potential path in order to treat numerous illnesses and traumas.

Furthermore, iPSCs play a pivotal role in disease modeling in personalized medicine61. The capacity to generate iPSCs from individuals with particular genetic conditions has enabled researchers to create in vitro disease models. These models are extremely invaluable tools for understanding disease mechanisms at the cellular level and enable the exploration of targeted therapeutic interventions62. iPSC-based disease modeling advances the field of personalized medicine by enabling a more accurate and customized approach to medical research, thus opening the door for customized treatments. Beyond illness modeling and customized treatments, iPSCs have a major impact on toxicity assessments and drug development63. The pluripotent characteristics of iPSCs allow the generation of diverse cellular phenotypes, providing a flexible platform for evaluating the safety and effectiveness of pharmaceuticals. iPSC-based assays offer a more thorough understanding of how pharmaceuticals interact with various cell types, which helps identify possible side effects and directs advancements in the creation of remedies that are both safer and more effective64.

In conclusion, iPSCs offer a revolutionary approach to stem-cell investigation, owing to their pluripotent characteristics and the origin of adult cell reprogramming. Their applications in patient-specific cell therapies, disease modeling for personalized medicine, and drug discovery underscore their potential to revolutionize medical treatment and contribute to advancements in personalized healthcare.

Stem cells secrete numerous factors and exosomes that are responsible for immunomodulatory, antiapoptotic, antibacterial, and microbial properties. In addition to the ability for repair, communication, and regeneration (Fig. 2).

The schematic diagram represents the mesenchymal stem cells mechanism of action and their interaction with immune cells, including differentiation, immunomodulation, antiapoptotic effects, exosome and microvesicle release, migration and homing, and matrix remodeling.

Stem cells immunomodulatory actions have undergone extensive research when contrasted with other stem cell types65,66. Stem cells have a role in suppressing acute-phase responses by suppressing excessive activation of macrophages and T cells and initiating the secretion of inflammatory cytokines. This could decrease the likelihood of a cytokine storm67. Toll-like receptors (TLRs) present in MSCs detect injury signals and initiate immunomodulatory responses68. MSCs exhibit immunomodulatory properties via paracrine activity and direct intercellular communication facilitated by several bioactive compounds like cytokines, chemokines, and growth factors. These molecules affect both adaptive and innate immunity. MSCs can prevent the activation of T-cells via several immunomodulatory substances, such as TGF-1, PGE2, and HLA-G5. They also utilize molecules that are linked to a membrane, such as VCAM-1, PD-L1, and Gal-169,70. MSCs regulate NK cell cytotoxicity by reducing the expression of IFN-71. Cytokines are crucial for preserving the ability of ESCs to reproduce. This is achieved through the action of a specific cytokine called leukemia inhibitory factor (LIF), which belongs to the class of cytokines known as interleukin-672. The iPSCs can modulate the immune system, as demonstrated through their capacity to suppress the rapid increase of responder T cells in modified combined leukocyte reactions in vitro73.

In addition, apoptosis serves as a protective process within the immunological response of the host to combat pathogens and has a crucial function in interactions between the host and pathogens71. MSCs can inhibit apoptosis, which may occur due to pathogens, low oxygen levels, mechanical stress, or radiation. For instance, the ability of MSCs to avoid cell death (antiapoptotic effects) has been investigated in cardiac ischemia, neurological conditions, and respiratory ailments74. In addition, during apoptosis caused by hypoxia, MSCs stimulate the expression of certain proteins, including HGF, VEGF, and TGF-1, with the potential to prevent endothelial cell death75. Additional variables contribute to the antiapoptotic effect of MSCs, such as IL-6 and IGF-1, which results in enhanced secretion of SFRP2 protein76.

Stem cells exert their antimicrobial activity by secreting molecules and direct cell-to-cell interactions, namely by releasing antimicrobial peptides (AMPs). The antimicrobial activities are carried out by specific AMPs like the family of lipocalins (Lcn2), hepcidin, and b-defensins (hBD-1, hBD-2, and hBD-3)77,78. Stem cells boost their antimicrobial activity by upregulating LL-37, a peptide that is stimulated by bacteria and inhibits bacterial growth79.

Regeneration and restoration of damaged tissues rely heavily on stem cells because of their distinctive ability to suppress aberrant immune responses, their capacity to transform into specific tissues, and produce certain substances that stimulate the hosts reparative and regenerative systems80. Furthermore, the micro-vesicles and exosomes generated from stem cells are important for stem-cell communication and regeneration. Lipids, proteins, nucleic acids, including RNA and micro RNA, and signaling molecules are among the many bioactive compounds that are transported within the extracellular vesicles (EVs) emitted by stem cells of the body81. Compounds secreted by stem cells facilitate tissue regeneration by promoting the growth and specialization of stem/progenitor cells in the immediate vicinity. In addition, they control the placement of molecules in the extracellular matrix, activate pathways that prevent scarring, and promote the development of new blood vessels82,83. MSCs release soluble paracrine factors, including ANGPT1, HGF, EGF, VEGF, KGF, PGE2, and interleukin-10 (IL10). These factors can improve the restoration of epithelial and endothelial cells84,85.

Recent years have seen remarkable progress in stem-cell research that has greatly expanded our comprehension of stem-cell biology86. One notable milestone was the elucidation of novel mechanisms governing stem cell fate decisions. Researchers have uncovered key signaling pathways and transcription factors that play pivotal roles in directing stem-cell differentiation87,88. A cellular communication system known as the Notch signaling pathway is vital for various physiological and developmental functions89. Researchers have demonstrated the significance of the Notch pathway in determining the outcome of cells by either promoting the renewal of cells or their differentiation into various types of stem cells, including ESCs90, PSCs91, HSCs92, NSCs93, and ISCs94. Other instances of the signaling pathways are the PI3k/AKT signaling95 and TGF- signaling96. A transcription factor known as NF-B controls the diverse functions of NF-B in stem cells and developmental processes97. These findings enhance stem cell manipulation capabilities for specific therapeutic purposes, offering unprecedented opportunities for targeted cell-based interventions98. Recent studies have explored the nuances of lineage commitment and cellular specialization within the framework of stem-cell development. Scientists have identified regulatory networks that govern stem cell differentiation into distinct cell types, shedding light on the molecular events that dictate cell fate99,100.

Researchers have also unveiled insights into the epigenetic modifications associated with reprogramming, enhancing our comprehension of the molecular mechanisms by which somatic cells transform into pluripotent states101. For example, studies proved that gene expression and cellular identity are influenced by changes in DNA methylation patterns during the formation of iPSCs102. Modification of histones through acetylation and methylation, which affect chromatin structure and gene regulation, also play significant roles in reprogramming. This new understanding of epigenetic pathways helps clarify the complex processes involved in pluripotency induction and cellular reprogramming.

These advancements have contributed to improvements in iPSC-based methods for pharmaceutical innovation, disease modeling, and customized regenerative medicine62. Another significant stride in stem-cell research pertains to the tissue regeneration field103.

Transplantation of stem cells has great potential as a medicine applied to numerous illnesses. In neurology clinical trials, scientists are presently investigating stem cell therapys feasibility for the purpose of alleviating neurological disorders, such as Alzheimers and Parkinsons104. Additionally, investigations are being conducted on stem-cell therapy for cardiovascular illnesses, orthopedic conditions, hematological conditions, and diabetes. The adaptability of stem cells, coupled with advancements in delivery techniques, positions them as potential game-changers in regenerative medicine105. Emerging applications include the use of stem cells in immunotherapy, where they are engineered to target and treat certain cancers106. Furthermore, continuous investigations have investigated the possibility of using stem cells to regulate the immune system in disorders like autoimmune illnesses107. As these clinical applications progress from research to practice, the landscape of healthcare is poised to undergo significant transformation.

Neural stem-cell transplants have been administered to patients with PD in a clinical trial. In addition to improving motor system function, the data demonstrated a slowing of the diseases progression and suggested the prospects of stem cells for neurological regeneration108,109. Individuals with heart failure participated in a cardiac stem-cell clinical trial. The outcomes showed less scar tissue, increased angiogenesis, and improved heart function, indicating the effectiveness of stem-cell treatment in promoting the regrowth of cardiac tissue110,111. Additionally, bone marrow-derived MSCs (BM-MSCs) have been utilized in a clinical study of osteoarthritis. Patients experience decreased pain, improved joint function, and evidence of cartilage regeneration, demonstrating the therapeutic prospects of stem cells in orthopedic applications112114.

Treating leukemia with HSC transplantation (HSCT) has proven beneficial. Patients undergoing this procedure achieve complete remission and hematopoietic system reconstitution, leading to prolonged survival and improved quality of life46,115. Furthermore, clinical trials utilizing iPSCs to generate pancreatic progenitor cells have demonstrated promise for the treatment of diabetes. Patients exhibit restored insulin production and improved glycemic control, suggesting a regenerative approach to diabetes management116,117 (Table 1) (Fig. 3).

The advancement in stem-cell therapies in various diseases.

MSC sources, such as bone marrow, adipose tissue, and placenta, and their role in the therapy of different diseases. MSCs improve and combat diseases including pneumonia, leukemia, neuron diseases, osteoarthritis hear diseases, and the two types of diabetes. MSCs have immunoregulator and anti-inflammatory properties.

In combating the COVID-19 pandemic, universal vaccination remains the primary strategy; however, uncertainties persist regarding the duration of vaccine protection and the inability of any vaccine to provide absolute immunity137. Stem-cell therapy has arisen as a potential substitute, building on successes observed in severe H7N9 avian influenza138,139. Stem cells, particularly those derived from human umbilical cord stem cells (hUCMSCs), are effective and safe for treating severe COVID-19, demonstrating their potential in over 100 international clinical trials140. Allogeneic MSCs, notably hUCMSCs, contribute to anti-inflammatory responses, tissue repair, and the modulation of immune functions, showcasing their therapeutic promise141. Challenges include difficulties in recruitment due to the evolving clinical landscape, lack of preclinical data, and variations in stem-cell properties. Despite these hurdles, stem-cell therapy, especially considering advancements in organoid technology for better modeling of viral effects, has significant clinical potential142. Despite current limitations and technological challenges, the continuous advancement of stem-cell treatment offers optimism in the fight to preserve lives and improve treatment results for individuals with severe COVID-19 infection (Fig. 4).

Potential and mechanism of action of mesenchymal stem cell treatment for COVID-19 pneumonia using MSCs, which have immunoregulatory characteristics, can help control the cytokine storm and COVID-19 lung injury. Mesenchymal stromal cells (MSCs) play an important role in a number of processes, including preventing neutrophil infiltration and transforming hyperactivated T cells into regulatory T cells (Tregs). They also promote the production of anti-inflammatory cytokines, such as prostaglandin E2 (PGE2), transforming growth factor beta (TGF), indoleamine 2,3-dioxygenase (IDO), and interleukin 10 (IL-10). Nevertheless, MSCs play a crucial function by stimulating the synthesis of growth factors by endothelial and epithelial cells, which in turn inhibits fibrosis and boosts the infusion of alveolar fluid.

Regenerative medicine with stem cells has investigated significant capacity across diverse medical specialties, offering innovative solutions for previously challenging conditions143. Patients stem cells are harvested for autologous stem-cell treatment. Autologous stem cells that have been cultured are cultivated in the lab before transplantation. These cells have the potential to be categorized into modified and unmodified expanded autologous stem cells. Allogeneic stem cells are classified similarly to autologous stem cells, but they come from healthy donors18. Autologous stem cells can be readily acquired and do not cause immunological rejection after infusion. Allogeneic stem cells provide multiple benefits, including the ability to select a donor, availability from different sources, minimal likelihood of causing an immune response, and the convenience of being readily available. Allogeneic MSCs are also immunogenic, indicating that they can trigger an immunological response. These cells can generate a memory response in the immune system under specific circumstances144146.

Regenerative medicine can restore, repair, or regenerate impaired tissues or organs by harnessing the unique characteristics of stem cells147. This topic includes a range of approaches that seek to leverage the extraordinary capacity of stem cells for medical applications. Although stem cells possess the capacity to undergo self-renewal and differentiate into various distinct cell types, they hold great promise as therapeutic agents against various illnesses and wounds148. Regenerative medicine aims to create novel methods to repair damaged tissues caused by disease, injury, or aging using stem cells to restore normal function and structure to damaged organs or tissues149. These therapies have great potential to revolutionize medical treatments, particularly in areas where conventional medicine falls short of providing effective remedies or cures150.

This emerging field presents a promising avenue for personalized cancer treatments, as researchers have delved into harnessing the unique attributes of stem cells to create innovative strategies for cancer management and potential cures. These investigations signify a significant paradigm shift in oncology, offering a progressive outlook for tailored therapies and potential breakthroughs in cancer treatment151,152. Stem cell-based cancer treatments are becoming increasingly promising. Because stem cells can locate and target primary and metastatic tumors, and serve as innovative delivery approaches. In preclinical animal models, stem cells modified to express different cytotoxic chemicals consistently reduced tumor size and increased survival153,154. They have also been used to reduce side effects and improve primary medicinal efficacy by acting as carriers of viruses and nanoparticles. Additionally, stem cells have the potential for utilization in immunotherapy, anticancer drug screening, regenerative medicine, and cancer stem cell-targeted therapy for diverse forms of malignancies, including lung cancer, breast cancer, and osteosarcoma155.

Regenerative strategies in orthopedics include advanced osteonecrosis of the hip joint, intervertebral hernias, osteoporosis, targeted joint injuries, cartilage restoration, and bone healing through stem-cell and tissue-engineering methodologies156,157. Recent investigations have shown innovative approaches, like MSC therapy, platelet-rich plasma (PRP) injections, and biocompatible scaffolds infused with growth factors158. These methods aim to optimize cartilage repair and bone regeneration, offering promising outcomes under musculoskeletal conditions159,160. Research has focused on refining MSC isolation techniques, deciphering the crucial signaling pathways involved in tissue regeneration, and developing bioactive materials that enhance healing161.

In the cardiology field, innovative approaches, including stem-cell therapy and bioengineered cardiac patches, are being explored to mend and regenerate impaired heart tissues after cardiac events such as myocardial infarctions162. Current research has been focused on different stem-cell types, including iPSCs and cardiac progenitor cells, to regenerate impaired heart muscles and restore cardiac function. Furthermore, research has focused on creating bioengineered cardiac patches using cell-based structures and biomaterials that resemble genuine heart tissue163.

In the field of neurology, ongoing investigations have delved into the domain of medicines based on stem cells developed to fight diseases affecting the nervous system, including Parkinsons and Alzheimers164,165. Studies have focused on using stem cell-derived neurons to replace and regenerate impaired nerve cells166. Recent studies have shown that there are numerous varieties of stem cells, including neural stem cells and iPSCs, with the aim of producing functional neurons capable of integrating into damaged neural networks167,168.

Regenerative medicine in dermatology represents a dynamic frontier of research, particularly concerning stem-cell applications in the skin169. Stem cells residing in the skin tissues offer promising avenues for innovative therapeutic strategies that target various dermatological conditions and injuries170. Their remarkable regenerative potential holds immense promise for advancing wound healing, addressing burns, and managing skin disorders such as psoriasis and vitiligo171,172. Additionally, stem cell use in cosmetic dermatology for antiaging treatments and improving skin quality underscores their diverse clinical utility173. Researchers have actively explored methods to harness the inherent regenerative abilities of stem cells with the aim of developing tailored and effective therapies for combating skin-related diseases and facilitating cosmetic enhancements. This transformative approach involves tissue engineering techniques utilizing stem cells, biomaterials, and growth factors to create skin substitutes that promote tissue regeneration and repair174177.

Due to their potential function, an enormous amount of curiosity about stem cells has persisted in rejuvenating the retina and addressing corneal damage, particularly in diseases such as macular degeneration178. Noteworthy studies featured in journals such as Investigative Ophthalmology and Visual Science and British Journal of Ophthalmology delve into the strides made in utilizing stem cells for ocular regeneration179,180. Studies have employed stem-cell therapies to restore retinal cells and heal corneal injuries, presenting encouraging pathways for managing vision-related ailments181. These studies signify a burgeoning field of ophthalmology research, offering promising prospects for innovative treatments aimed at addressing ocular disorders and enhancing vision182.

Stem-cell utilization in oncology, regenerative medicine, and disease therapeutics is an expanding field of research and innovation151. Research has focused on leveraging stem cells for targeted cancer therapies and exploring their potential for cellular reprogramming and immune cell modulation to combat tumors183. The immunomodulatory potential of stem cells presents a compelling avenue in biomedical research, particularly in addressing autoimmune disorders and graft-versus-host disease (GVHD) and improving transplantation outcomes184. Stem cells show a remarkable ability to influence immune cell behavior and function, offering promising prospects for novel therapeutic interventions185. This intersection of immunology and stem-cell biology promises not only innovative treatments but also deeper insights into the complex mechanisms governing immune system regulation and dysregulation. This rapidly expanding field has an enormous potential to improve our knowledge of immune-related disorders and provide efficient treatment plans186.

Stem-cell utilization in hematology is a dynamic area of scientific inquiry and clinical application in the regenerative medicine field and therapeutic interventions for diseases187. Leveraging the potential of stem cells to regenerate is the main goal of research, particularly in HSCs, for transplanting bone marrow and exploring its role in immune cell therapies to combat various blood-related ailments188. This growing field represents a promising avenue for innovative treatments, emphasizing the pivotal role of stem cells in revolutionizing hematology by offering potential cures and personalized therapeutic solutions for blood disorders, thereby marking a transformative shift in disease management189,190.

Stem-cell research offers the potential for addressing illnesses such as inflammatory bowel disease (IBD)191,192 and managing various gastrointestinal disorders193. Researchers are investigating stem cell-based approaches to repair gastrointestinal tract injuries, manage ulcers, and alleviate the symptoms of chronic conditions like ulcerative colitis and Crohns disease194,195. Despite ongoing investigations, the clinical application of stem-cell therapies in gastroenterology remains the subject of clinical trials and extensive research, emphasizing the need for further exploration and understanding of their efficacy and safety in treating many immunopathological diseases (Fig. 5)196.

MSCs inhibit many immunopathological disease conditions, including skin infection, inflammatory bowel disease, and endocrine hormone disorders; they also suppress tumor cells, the aging process, and reproductive infertility.

Ongoing investigations explore the potential of stem cells in restoring lung tissue damaged by diseases like serious respiratory disease or chronic obstructive pulmonary disease (COPD)197. Researchers have investigated the capacity of stem cells to restore impaired lung tissue, alleviate COPD symptoms, and target conditions such as idiopathic pulmonary fibrosis198. Despite extensive research, the use of stem-cell therapies in pulmonology requires further examination to establish their safety, effectiveness, and long-term effects on respiratory illnesses199. Although this emerging field shows promise for future treatment, it requires thorough comprehension and robust clinical validation200.

Stem-cell research in reproductive medicine opens new avenues for treating infertility and addressing various reproductive system disorders201. Stem cells, whether derived from embryonic, adult, or induced pluripotent sources, hold promise for regenerating and repairing damaged reproductive tissues202. This area of study covers various aspects of reproductive health, including the restoration of ovarian function, addressing endometrial issues, and potentially aiding fertility preservation. Research endeavors detailed in publications such as the Journal of Assisted Reproduction and Genetics and Fertility and Sterility, explore the potential of interventions utilizing stem cells to revolutionize infertility treatments and offer new hope to individuals facing reproductive health challenges. These advancements represent a burgeoning field that may reshape the landscape of reproductive medicine and provide innovative solutions for the treatment of infertility and related disorders201,203.

Stem-cell research in endocrinology presents a promising avenue for managing endocrine disorders such as diabetes by focusing on the generation of insulin-producing cells and regenerating pancreatic tissues204. Through various studies documented in journals like Diabetes and Endocrine Reviews, researchers work to create functional beta cells or islet-like structures that can secrete insulin by utilizing the regeneration ability of stem cells205. This pioneering field aims to address deficiencies observed in traditional diabetes management by offering cell-based therapies that can potentially restore insulin production and regulate glucose levels206. The exploration of stem-cell therapies in endocrinology has heralded a new era of diabetes treatment, offering hope for more effective and sustainable management strategies for this chronic condition207.

In dentistry, cutting-edge research has focused on the innovative utilization of stem cells to regenerate crucial dental tissues, including tooth enamel, dentin, and dental pulp208. This revolutionary exploration seeks to redefine conventional approaches to dental care by offering transformative treatments for prevalent conditions such as cavities, gum diseases, and dental trauma209. Utilizing their unique regenerative stem-cell capacities, scientists aim to generate interventions that induce the natural regeneration and repair of diseased or impaired dental tissues, potentially revolutionizing the oral healthcare landscape210,211. This promising field of study in dentistry holds the potential to pave the way for novel therapeutic strategies that offer patients improved outcomes and enhanced oral health212.

In the domains of trauma and wound healing, intensive research efforts have focused on uncovering the regenerative processes of stem cells to address the complexities of chronic wounds, burns, and traumatic injuries213. Stem cells exhibit promising capabilities in fostering tissue regeneration and mitigating scarring by influencing cell differentiation and supporting repair mechanisms in damaged tissues214,215. This exploration of stem cell-based interventions aims to revolutionize conventional wound care approaches by fostering natural tissue regeneration, accelerating healing processes, and minimizing scarring, thereby offering renewed hope to patients with challenging wounds and traumatic injuries216. In the quest for more potent treatment approaches to enhance patient outcomes and accelerate recovery, the potential of stem cells in trauma and wound healing serves as a ray of hope217,218. Stem-cell regenerative medicine is a dynamic and expansive field, continuously expanding its applications across various medical disciplines to address a wide spectrum of health conditions and diseases219 (Table 2).

Stem-cell therapy is utilized in specific medical fields.

In addition, various types of stimulation have been utilized during stem-cell therapy to enhance differentiation proliferation and improve healing, such as shock wave stimulation242. MSCs are increasingly being acknowledged as valuable resources for various orthopedic applications, and radial shock waves have been shown to substantially enhance the development and regrowth of MSCs in a laboratory setting. Furthermore, this type of stimulation safely accelerates cartilage repair in living organisms, suggesting positive results for clinical applications243. IR is a type of high-energy radiation that has enough energy to dislodge firmly bound electrons from atoms, leading to the creation of ions. In addition to being a carcinogen, IR is also used as a therapeutic option for patients with cancer. However, there is increasing data showing that extranuclear components, such as mitochondria, play a significant role in the cellular response to IR, and the mitochondrial function of MSCs was observed to be considerably increased after 4h of exposure to ionizing radiation, as determined by measuring mitochondrial oxygen consumption244. Cell proliferation has been induced in many in vitro trials using a modest amount of laser therapy. Osteoblasts, lymphocytes, keratinocytes, and fibroblasts exhibit enhanced proliferation when exposed to laser irradiation245. Other types of stimulation include electrical stimulation to enhance stem-cell therapy in nerve regeneration242, electrical stimulation to promote cell differentiation and proliferation of fatal neuronal stem cells into neuronal stem cells246, and nonpeptide small molecules247, in addition to mechanical stimuli such as cyclic stretch, three forces, laminar shear stress, cyclic pressure248, and gamma radiation249.

Stem-cell studies have been significantly promoted by cutting-edge technologies that have revolutionized our understanding and utilization of these versatile cells. This discussion focuses on some of the most impactful biotechnological advancements in stem-cell studies, with a specific focus on exosome-based therapeutics, scRNA-Seq, and the revolutionary CRISPR-Cas9 gene-editing technology250252.

The new frontier of exosomes produced from stem cell-based therapeutics represents a promising avenue for the field of regenerative medicine253. RNAs, signaling molecules, and proteins are bioactive substances encapsulated in exosomes and small vessels secreted by stem cells. These nanovesicles are essential for intercellular interactions and can control a number of cellular functions254. Stem cell-derived exosomes exhibit unique properties that modulate immune responses, promote tissue regeneration, and foster repair mechanisms255. Harnessing the therapeutic potential of these exosomes holds considerable promise for developing innovative treatments for diverse medical conditions, including inflammatory disorders, neurodegenerative diseases, and tissue injuries253,256258. Stem cell-derived exosome-based therapies represent a burgeoning frontier in regenerative medicine, providing new opportunities for targeted, minimally invasive therapeutic interventions259.

Advances in scRNA-seq have allowed investigators to examine stem-cell transcriptomes individually, providing unprecedented insights into cellular heterogeneity and gene expression patterns13. This technology has played an essential role in comprehending the dynamics of stem-cell populations during differentiation and disease progression260,261.

With the advent of CRISPR-Cas9, a new era in gene editing has begun, which enables the precise modifications of stem-cell DNA14. Researchers can now edit or introduce specific genes with unprecedented accuracy, facilitating cancer and disease modeling, studying gene function, and developing potential therapeutic interventions262,263.

CRISPR-based technologies have enabled large-scale functional genomic studies and high-throughput screening of stem cells. That allows researchers to systematically interrogate gene function on a genome-wide scale, uncovering novel regulators of stem-cell fate, pluripotency, and differentiation264,265.

Beyond traditional CRISPR-Cas9, recent innovations, such as base editing and prime editing, offer enhanced precision in gene editing266. These techniques allow the modification of specific nucleotides without causing double-strand breaks, minimizing off-target effects and expanding the possibilities for therapeutic genome editing in stem cells267 (Fig. 6).

Immunotherapy chimeric antigen receptor (CAR) T-cell therapy can be filled with the help of recent developments in genome editing using CRISPR-Cas9. To enable robust, accurate, and controllable genetic alteration, genome editing techniques are used, such as base and prime editing. In both hematopoietic and non-hematopoietic cancers, T-cells can be circumvented through CRISPR-Cas9-induced multiplex deletion of inhibitory molecules, which enhances CAR T-cell growth and persistence. The use of targeted knock-in techniques during CAR T-cell engineering offers the possibility of producing highly effective and potent cell products. Lentivirus is viral particles modified to carry CRISPR components in T cells, CRISPR-Cas9 based on the precise insertion of CAR genes, more and strong CAR T-cells product engineered using CRISPR-Cas9 to overcome specific histocompatibility hurdles and with improved persistence/antitumor function could greatly improve the production of cellular immunotherapies and the therapeutic durability.

Overall, CRISPR-based gene editing shows great promise for therapeutic applications in stem cell-based regenerative medicine. This opens new avenues for correcting genetic mutations underlying various diseases, generating genetically modified cells for transplantation, and developing personalized cell therapies.

Stem cells are integral to the advancement of personalized medicine, aligned with the goal of tailoring healthcare to individual characteristics and encompassing genetic, environmental, and lifestyle factors16. From a patients cells, iPSCs provide a potent platform for building disease models that accurately reflect the persons genetic background268. This capability facilitates in-depth studies of disease mechanisms at the cellular and molecular levels, enabling more precise diagnosis and the establishment of targeted therapeutic strategies60. Moreover, modern gene-editing techniques, including CRISPR-Cas9, enable accurate alterations in stem-cell genomes269.

This breakthrough allowed the correction of genetic mutations associated with diseases, laying the groundwork for personalized therapies addressing specific genetic alterations in individual cells270. In pharmacogenomics, stem cells significantly contribute to the assessment of individual drug responses. Leveraging patient-derived stem cells in pharmacogenomic studies enables researchers to understand the impact of an individuals genetic composition on their reaction to various medications271. This knowledge serves as a guide for formulating personalized treatment plans, minimizing adverse reactions, and enhancing the overall therapeutic outcomes. Moreover, stem cells actively contribute to the identification of personalized biomarkers associated with specific diseases272. Differentiating patient-derived stem cells into cell types relevant to the disease makes it easier to identify molecular signatures that can be used as diagnostic indicators. These personalized biomarkers substantially improve the accuracy of disease detection and monitoring, marking a significant step toward more individualized and effective healthcare strategies273,274.

Stem-cell therapy is witnessing a surge in clinical trials, reflecting a growing interest in translating laboratory findings into viable treatments275. Clinical trials involving various stem-cell types are currently underway and include a wide range of health issues276. The goal of ongoing trials is to determine whether stem-cell therapies are effective in alleviating symptoms of neurological diseases such as Alzheimers, Parkinsons, and spinal cord injuries277 (Table 3). Researchers are investigating how stem cells might be able to repair damaged neurons, encourage brain regeneration, and lessen the symptoms of these crippling conditions288.

Examples of clinical trials with results involved in neurological diseases, cancer, cardiovascular, and Orthopedics, from http://clinicaltrials.gov/.

Clinical trials in cardiovascular medicine aim to evaluate the use of stem cells, such as progenitor cells and MSCs, for treating conditions like heart failure and ischemic heart disease. These trials explored the regenerative potential of stem cells in repairing impaired cardiac tissues and improving overall cardiac function289.

Research is now being conducted on stem cell-based therapeutics for cancer treatment, including studies focusing on HSC transplantation (SCT) to treat hematological malignancies290. In addition, researchers have explored potential applications for stem cells in conjunction with traditional cancer therapies in order to enhance therapeutic results and minimize negative consequences291. Additionally, clinical trials in orthopedics and musculoskeletal disorders involve the use of stem cells to treat conditions like osteoarthritis and bone defects. MSCs, which are known for their capacity to differentiate into bone and cartilage, are being studied for their regenerative potential in restoring joint and bone health292. Furthermore, stem-cell therapies are now under investigation for their potential applications to treat diabetes by replenishing pancreatic beta cells. Clinical trials have investigated the use of stem cell-derived insulin-producing cells as transplants to regulate blood glucose levels in patients with diabetes293.

Stem-cell therapy, although showing great promise, faces multiple obstacles and constraints that need to be carefully considered. One prominent challenge is the potential for tumorigenesis, wherein the number of transplanted stem cells may increase uncontrollably, leading to tumor formation294,295. The security of stem cells can only be ensured by thorough preclinical examinations before they can be used in clinical settings. Additionally, the immune response poses a challenge due to the recipients immune system perceiving the transplanted cells as alien, leading to rejection296,297. The development of strategies to mitigate immune rejection and improve engraftment remains an ongoing challenge.

Furthermore, precisely controlling stem cell development into the desired cell types is a significant challenge298. The variability in differentiation protocols and the possibility of off-target consequences raise concerns regarding the reliability and safety of the therapeutic outcomes. Additionally, scalability and cost-effectiveness in the production of sufficient quantities of quality-controlled stem cells for widespread clinical use remain logistic obstacles that must be overcome for the field to attain its full potential110,299,300.

Ethical considerations are central to the discourse surrounding stem-cell therapy, particularly the use of ESCs301. Discussions over the moral standing of the early human embryo arose because of the killing of embryos during the extraction of ESCs. Because of these concerns, scientists are looking at alternative sources of pluripotent stem cells, such as iPSCs, which are reprogrammed from adult cells and do not have the same ethical concerns as ESCs. Regulatory frameworks are essential for negotiating the moral challenges presented by different stem cell therapies302.

Countries have varying regulations governing the clinical utilization of stem cells, ranging from permissive to restrictive. Achieving a balance between promoting innovation and ensuring patient safety remains a challenge for regulatory bodies303. The evolving nature of stem-cell research and therapies necessitates dynamic regulatory frameworks that can be adapted for scientific advancement. Ongoing debates persist in this field, particularly regarding the commercialization of stem-cell therapies. Issues of accessibility, affordability, and equitable distribution of these therapies raise ethical questions.

Moreover, concerns regarding the premature marketing of unproven stem-cell therapies and the need for transparent communication regarding the state of scientific evidence contribute to the ethical complexity of this field304. In conclusion, addressing the difficulties and ethical considerations of stem-cell therapy requires a multidisciplinary approach that encompasses rigorous scientific research, transparent communication, and dynamic regulatory frameworks. Realizing the full promise of stem-cell therapies will require a careful balance between ethical responsibility and innovation as the field develops.

With the help of new technologies and the results of continuing research, stem-cell treatment might potentially transform many different areas of medicine. One key direction involves the integration of stem-cell therapy into precision medicine approaches, opening a new chapter in medical history, where customized care based on a persons genetic composition promises enhanced therapeutic outcomes and reduced side effects. Advances in genomics and the application of patient-specific stem cells are expected to drive this integration. Additionally, future research should focus on refining the immune modulation strategies associated with stem-cell therapies and addressing challenges such as immune rejection and graft-versus-host responses. Innovative approaches, including engineered stem cells and immunomodulatory molecules, aim to enhance compatibility with stem-cell treatment.

The continued evolution of gene-editing tools, including CRISPR-Cas9, will perform a key function in ensuring the precision and safety of stem-cell therapies. This technology enables the modification of specific genes in stem cells, offering avenues for targeted therapeutic interventions and correction of genetic disorders at the cellular level. The synergy between stem-cell therapy and bioengineering has emerged as a significant area of exploration. The integration of stem cells with advanced biomaterials can potentially create functional tissues and organs with improved structural and functional properties. Bioengineered constructs provide innovative solutions for tissue-specific regeneration and transplantation. These key directions underscore the multidimensional nature of future advancements in stem-cell therapy, bringing together precision medicine, immune modulation, gene editing, and bioengineering to propel the field toward transformative developments.

Recent developments in stem-cell therapy have illuminated a path of immense promise and transformative potential for revolutionizing modern medicine. The exploration of stem cells across diverse medical disciplines guided by advancements in science, biotechnology, and clinical trial applications has positioned this field at the forefront of biomedical research. The historical journey from foundational concepts laid by pioneering scientists in the late 19th and early 20th centuries to groundbreaking milestones such as the isolation of ESCs and the discovery of iPSCs underscores a monumental leap in medical science.

The regenerative processes of stem cells, categorized into embryonic, adult, induced pluripotent, and perinatal stem cells, offer unprecedented opportunities for therapeutic interventions. Development, tissue repair, and regeneration are all intricately linked to stem cells due to their remarkable capacity to differentiate into different cell types and self-renew. Their diverse applications include neurodegenerative disorders, cardiovascular ailments, spinal cord injuries, diabetes, and tissue damage, opening novel avenues for treating debilitating conditions. However, as the field advances, the critical challenges and complexities must be addressed. Problems like immunological rejection, tumorigenesis, and the precise manipulation of stem-cell behavior pose hurdles that demand comprehensive exploration and innovative solutions. The landscape of stem-cell therapy is intricate and requires a nuanced understanding of its historical foundations, current realities, and future trajectories.

In collating recent biotechnology advancements, critical trial evaluations, and emerging technologies, this review provides a comprehensive compass for clinicians, researchers, and stakeholders navigating the intricate terrain of stem-cell therapy. Future directions, marked by precision medicine integration, immune modulation strategies, advancements in gene-editing technologies, and synergy with bioengineering, offer a roadmap for the continued evolution of stem-cell therapies.

Resonating with the revolutionary promise of stem-cell therapy not only in the realms of science and medicine but also in the lives of individuals with debilitating diseases and injuries. The journey from conceptualization to practical utilization represents a testament to human ingenuity and the relentless pursuit of improving healthcare. As stem-cell research continues, it holds the promise of reshaping the landscape of medicine, bringing forth a new era in which personalized regenerative therapies can mitigate the impact of a spectrum of medical challenges.

Not applicable.

Not applicable.

No funding was received for this study.

B.M.H.: study design and data analysis; R.K.Y.: writing the paper; G.H.A.: data collection; S.R.A. and R.K.K.: data analysis and interpretation; S.A.M.: study design and writing the paper.

The authors declare no conflicts of interest.

Not applicable.

Bashdar Mahmud Hussen and Suhad A. Mustafa.

All the data are available in the manuscript.

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Revolutionizing medicine: recent developments and future prospects in ...

Abstract

In recent years, there has been an explosion of interest in stem cells, not just within the scientific and medical communities but also among politicians, religious groups and ethicists. Here, we summarize the different types of stem cells that have been described: their origins in embryonic and adult tissues and their differentiation potential in vivo and in culture. We review some current clinical applications of stem cells, highlighting the problems encountered when going from proof-of-principle in the laboratory to widespread clinical practice. While some of the key genetic and epigenetic factors that determine stem cell properties have been identified, there is still much to be learned about how these factors interact. There is a growing realization of the importance of environmental factors in regulating stem cell behaviour and this is being explored by imaging stem cells in vivo and recreating artificial niches in vitro. New therapies, based on stem cell transplantation or endogenous stem cells, are emerging areas, as is drug discovery based on patient-specific pluripotent cells and cancer stem cells. What makes stem cell research so exciting is its tremendous potential to benefit human health and the opportunities for interdisciplinary research that it presents.

Keywords: adult stem cells, ES cells, iPS cells, cell-based therapies, drug discovery

The human body comprises over 200 different cell types that are organized into tissues and organs to provide all the functions required for viability and reproduction. Historically, biologists have been interested primarily in the events that occur prior to birth. The second half of the twentieth century was a golden era for developmental biology, since the key regulatory pathways that control specification and morphogenesis of tissues were defined at the molecular level (Arias 2008). The origins of stem cell research lie in a desire to understand how tissues are maintained in adult life, rather than how different cell types arise in the embryo. An interest in adult tissues fell, historically, within the remit of pathologists and thus tended to be considered in the context of disease, particularly cancer.

It was appreciated long ago that within a given tissue there is cellular heterogeneity: in some tissues, such as the blood, skin and intestinal epithelium, the differentiated cells have a short lifespan and are unable to self-renew. This led to the concept that such tissues are maintained by stem cells, defined as cells with extensive renewal capacity and the ability to generate daughter cells that undergo further differentiation (Lajtha 1979). Such cells generate only the differentiated lineages appropriate for the tissue in which they reside and are thus referred to as multipotent or unipotent (figure1).

Origin of stem cells. Cells are described as pluripotent if they can form all the cell types of the adult organism. If, in addition, they can form the extraembryonic tissues of the embryo, they are described as totipotent. Multipotent stem cells have the ability to form all the differentiated cell types of a given tissue. In some cases, a tissue contains only one differentiated lineage and the stem cells that maintain the lineage are described as unipotent. Postnatal spermatogonial stem cells, which are unipotent in vivo but pluripotent in culture, are not shown (Jaenisch & Young 2008). CNS, central nervous system; ICM, inner cell mass.

In the early days of stem cell research, a distinction was generally made between three types of tissue: those, such as epidermis, with rapid turnover of differentiated cells; those, such as brain, in which there appeared to be no self-renewal; and those, such as liver, in which cells divided to give two daughter cells that were functionally equivalent (Leblond 1964; Hall & Watt 1989). While it remains true that different adult tissues differ in terms of the proportion of proliferative cells and the nature of the differentiation compartment, in recent years it has become apparent that some tissues that appeared to lack self-renewal ability do indeed contain stem cells (Zhao et al. 2008) and others contain a previously unrecognized cellular heterogeneity (Zaret & Grompe 2008). That is not to say that all tissues are maintained by stem cells; for example, in the pancreas, there is evidence against the existence of a distinct stem cell compartment (Dor et al. 2004).

One reason why it took so long for stem cells to become a well-established research field is that in the early years too much time and energy were expended in trying to define stem cells and in arguing about whether or not a particular cell was truly a stem cell (Watt 1999). Additional putative characteristics of stem cells, such as rarity, capacity for asymmetric division or tendency to divide infrequently, were incorporated into the definition, so that if a cell did not exhibit these additional properties it tended to be excluded from the stem cell list. Some researchers still remain anxious about the definitions and try to hedge their bets by describing a cell as a stem/progenitor cell. However, this is not useful. The use of the term progenitor, or transit amplifying, cell should be reserved for a cell that has left the stem cell compartment but still retains the ability to undergo cell division and further differentiation (Potten & Loeffler 2008).

Looking back at some of the early collections of reviews written as the proceedings of stem cell conferences, one regularly finds articles on the topic of cancer stem cells (McCulloch et al. 1988). However, these cells have only recently received widespread attention (Reya et al. 2001; Clarke et al. 2006; Dick 2008). The concept is very similar to the concept of normal tissue stem cells, namely that cells in tumours are heterogeneous, with only some, the cancer stem cells, or tumour initiating cells, being capable of tumour maintenance or regrowth following chemotherapy. The cancer stem cell concept is important because it suggests new approaches to anti-cancer therapies (figure2).

The cancer stem cell hypothesis. The upper tumour is shown as comprising a uniform population of cells, while the lower tumour contains both cancer stem cells and more differentiated cells. Successful or unsuccessful chemotherapy is interpreted according to the behaviour of cells within the tumour.

As in the case of tissue stem cells, it is important that cancer stem cell research is not sidetracked by arguments about definitions. It is quite likely that in some tumours all the cells are functionally equivalent, and there is no doubt that tumour cells, like normal stem cells, can behave differently under different assay conditions (Quintana et al. 2008). The oncogene dogma (Hahn & Weinberg 2002), that tumours arise through step-wise accumulation of oncogenic mutations, does not adequately account for cellular heterogeneity, and markers of stem cells in specific cancers have already been described (Singh et al. 2004; Barab et al. 2007; O'Brien et al. 2007). While the (rediscovered) cancer stem cell field is currently in its infancy, it is already evident that a cancer stem cell is not necessarily a normal stem cell that has acquired oncogenic mutations. Indeed, there is experimental evidence that cancer initiating cells can be genetically altered progenitor cells (Clarke et al. 2006).

In addition to adult tissue stem cells, stem cells can be isolated from pre-implantation mouse and human embryos and maintained in culture as undifferentiated cells (figure1). Such embryonic stem (ES) cells have the ability to generate all the differentiated cells of the adult and are thus described as being pluripotent (figure1). Mouse ES cells are derived from the inner cell mass of the blastocyst, and following their discovery in 1981 (Evans & Kaufman 1981; Martin 1981) have been used for gene targeting, revolutionizing the field of mouse genetics. In 1998, it was first reported that stem cells could be derived from human blastocysts (Thomson et al. 1998), opening up great opportunities for stem cell-based therapies, but also provoking controversy because the cells are derived from spare in vitro fertilization embryos that have the potential to produce a human being. It is interesting to note that, just as research on adult tissue stem cells is intimately linked to research on disease states, particularly cancer, the same is true for ES cells. Many years before the development of ES cells, the in vitro differentiation of cells derived from teratocarcinomas, known as embryonal carcinoma cells, provided an important model for studying lineage selection (Andrews et al. 2005).

Blastocysts are not the only source of pluripotent ES cells (figure1). Pluripotent epiblast stem cells, known as epiSC, can be derived from the post-implantation epiblast of mouse embryos (Brons et al. 2007; Tesar et al. 2007). Recent gene expression profiling studies suggest that human ES cells are more similar to epiSC than to mouse ES cells (Tesar et al. 2007). Pluripotent stem cells can also be derived from primordial germ cells (EG cells), progenitors of adult gametes, which diverge from the somatic lineage at late embryonic to early foetal development (Kerr et al. 2006).

Although in the past the tendency has been to describe ES cells as pluripotent and adult stem cells as having a more restricted range of differentiation options, adult cells can, in some circumstances, produce progeny that differentiate across the three primary germ layers (ectoderm, mesoderm and endoderm). Adult cells can be reprogrammed to a pluripotent state by transfer of the adult nucleus into the cytoplasm of an oocyte (Gurdon et al. 1958; Gurdon & Melton 2008) or by fusion with a pluripotent cell (Miller & Ruddle 1976). The most famous example of cloning by transfer of a somatic nucleus into an oocyte is the creation of Dolly the sheep (Wilmut et al. 1997). While the process remains inefficient, it has found some unexpected applications, such as cloning endangered species and domestic pets.

A flurry of reports almost 10 years ago suggested that adult cells from many tissues could differentiate into other cell types if placed in a new tissue environment. Such studies are now largely discredited, although there are still some bona fide examples of transdifferentiation of adult cells, such as occurs when blood cells fuse with hepatocytes during repair of damaged liver (Anderson et al. 2001; Jaenisch & Young 2008). In addition, it has been known for many years that adult urodele amphibians can regenerate limbs or the eye lens following injury; this involves dedifferentiation and subsequent transdifferentiation steps (Brockes & Kumar 2005).

The early studies involving somatic nuclear transfer indicated that adult cells can be reprogrammed to pluripotency. However, the mechanistic and practical applications of inducing pluripotency in adult cells have only become apparent in the last 2 or 3 years, with the emergence of a new research area: induced pluripotent stem cells (iPS cells). The original report demonstrated that retrovirus-mediated transduction of mouse fibroblasts with four transcription factors (Oct-3/4, Sox2, KLF4 and c-Myc; figure1) that are highly expressed in ES cells could induce the fibroblasts to become pluripotent (Takahashi & Yamanaka 2006). Since then, rapid progress has been made: iPS cells can be generated from adult human cells (Takahashi et al. 2007; Yu et al. 2007; Park et al. 2008a); cells from a range of tissues can be reprogrammed (Aasen et al. 2008; Aoi et al. 2008); and iPS cells can be generated from patients with specific diseases (Dimos et al. 2008; Park et al. 2008b). The number of transcription factors required to generate iPS cells has been reduced (Kim et al. 2008); the efficiency of iPS cell generation increased (Wernig et al. 2007); and techniques devised that obviate the need for retroviral vectors (Okita et al. 2008; Stadtfeld et al. 2008). These latter developments are very important for future clinical applications, since the early mice generated from iPS cells developed tumours at high frequency (Takahashi & Yamanaka 2006; Yamanaka 2007). Without a doubt, this is currently the most exciting and rapidly moving area of stem cell research.

In all the publicity that surrounds embryonic and iPS cells, people tend to forget that stem cell-based therapies are already in clinical use and have been for decades. It is instructive to think about these treatments, because they provide important caveats about the journey from proof-of-principle in the laboratory to real patient benefit in the clinic. These caveats include efficacy, patient safety, government legislation and the costs and potential profits involved in patient treatment.

Haemopoietic stem cell transplantation is the oldest stem cell therapy and is the treatment that is most widely available (Perry & Linch 1996; Austin et al. 2008). The stem cells come from bone marrow, peripheral blood or cord blood. For some applications, the patient's own cells are engrafted. However, allogeneic stem cell transplantation is now a common procedure for the treatment of bone marrow failure and haematological malignancies, such as leukaemia. Donor stem cells are used to reconstitute immune function in such patients following radiation and/or chemotherapy. In the UK, the regulatory framework put in place for bone marrow transplantation has now an extended remit, covering the use of other tissues and organs (Austin et al. 2008).

Advances in immunology research greatly increased the utility of bone marrow transplantation, allowing allograft donors to be screened for the best match in order to prevent rejection and graft-versus-host disease (Perry & Linch 1996). It is worth remembering that organ transplantation programmes have also depended on an understanding of immune rejection, and drugs are available to provide effective long-term immunosuppression for recipients of donor organs. Thus, while it is obviously desirable for new stem cell treatments to involve the patient's own cells, it is certainly not essential.

Two major advantages of haemopoietic stem cell therapy are that there is no need to expand the cells in culture or to reconstitute a multicellular tissue architecture prior to transplantation. These hurdles have been overcome to generate cultured epidermis to provide autologous grafts for patients with full-thickness wounds, such as third-degree burns. Proof-of-principle was established in the mid-1970s, with clinical and commercial applications following rapidly (Green 2008). Using a similar approach, limbal stem cells have been used successfully to restore vision in patients suffering from chemical destruction of the cornea (De Luca et al. 2006).

Ex vivo expansion of human epidermal and corneal stem cells frequently involves culture on a feeder layer of mouse fibroblastic cells in medium containing bovine serum. While it would obviously be preferable to avoid animal products, there has been no evidence over the past 30 years that exposure to them has had adverse effects on patients receiving the grafts. The ongoing challenges posed by epithelial stem cell treatments include improved functionality of the graft (e.g. through generation of epidermal hair follicles) and improved surfaces on which to culture the cells and apply them to the patients. The need to optimize stem cell delivery is leading to close interactions between the stem cell community and bioengineers. In a recent example, a patient's trachea was repaired by transplanting a new tissue constructed in culture from donor decellularized trachea seeded with the patient's own bone marrow cells that had been differentiated into cartilage cells (Macchiarini et al. 2008).

Whereas haemopoietic stem cell therapies are widely available, treatments involving cultured epidermis and cornea are not. In countries where cultured epithelial grafts are available, the number of potential patients is relatively small and the treatment costly. Commercial organizations that sell cultured epidermis for grafting have found that it is not particularly profitable, while in countries with publicly funded healthcare the need to set up a dedicated laboratory to generate the grafts tends to make the financial costbenefit ratio too high (Green 2008).

Clinical studies over the last 10 years suggest that stem cell transplantation also has potential as a therapy for neurodegenerative diseases. Clinical trials have involved grafting brain tissue from aborted foetuses into patients with Parkinson's disease and Huntington's disease (Dunnett et al. 2001; Wright & Barker 2007). While some successes have been noted, the outcomes have not been uniform and further clinical trials will involve more refined patient selection, in an attempt to predict who will benefit and who will not. Obviously, aside from the opposition in many quarters to using foetal material, there are practical challenges associated with availability and uniformity of the grafted cells and so therapies with pure populations of stem cells are an important, and achievable (Conti et al. 2005; Lowell et al. 2006), goal.

No consideration of currently available stem cell therapies is complete without reference to gene therapy. Here, there have been some major achievements, including the successful treatment of children with X-linked severe combined immunodeficiency. However, the entire gene therapy field stalled when several of the children developed leukaemia as a result of integration of the therapeutic retroviral vector close to the LMO2 oncogene locus (Gaspar & Thrasher 2005; Pike-Overzet et al. 2007). Clinical trials have since restarted, and in an interesting example of combined gene/stem cell therapy, a patient with an epidermal blistering disorder received an autologous graft of cultured epidermis in which the defective gene had been corrected ex vivo (Mavilio et al. 2006).

These are just some examples of treatments involving stem cells that are already in the clinic. They show how the field of stem cell transplantation is interlinked with the fields of gene therapy and bioengineering, and how it has benefited from progress in other fields, such as immunology. Stem cells undoubtedly offer tremendous potential to treat many human diseases and to repair tissue damage resulting from injury or ageing. The danger, of course, lies in the potentially lethal cocktail of desperate patients, enthusiastic scientists, ambitious clinicians and commercial pressures (Lau et al. 2008). Internationally agreed, and enforced, regulations are essential in order to protect patients from the dangers of stem cell tourism, whereby treatments that have not been approved in one country are freely available in another (Hyun et al. 2008).

Three questions in stem cell research are being hotly pursued at present. What are the core genetic and epigenetic regulators of stem cells? What are the extrinsic, environmental factors that influence stem cell renewal and differentiation? And how can the answers to the first two questions be harnessed for clinical benefit?

Considerable progress has already been made in defining the transcriptional circuitry and epigenetic modifications associated with pluripotency (Jaenisch & Young 2008). This research area is moving very rapidly as a result of tremendous advances in DNA sequencing technology, bioinformatics and computational biology. Chromatin immunoprecipitation combined with microarray hybridization or DNA sequencing (Mathur et al. 2008) is being used to identify transcription factor-binding sites, and bioinformatics techniques have been developed to allow integration of data obtained by the different approaches. It is clear that pluripotency is also subject to complex epigenetic regulation, and high throughput genome-scale DNA methylation profiling has been developed for epigenetic profiling of ES cells and other cell types (Meissner et al. 2008).

Oct4, Nanog and Sox2 are core transcription factors that maintain pluripotency of ES cells. These factors bind to their own promoters, forming an autoregulatory loop. They occupy overlapping sets of target genes, one set being actively expressed and the other, comprising genes that positively regulate lineage selection, being actively silenced (Jaenisch & Young 2008; Mathur et al. 2008; Silva & Smith 2008). Nanog stabilizes pluripotency by limiting the frequency with which cells commit to differentiation (Chambers et al. 2007; Torres & Watt 2008). The core pluripotency transcription factors also regulate, again positively and negatively, the microRNAs that are involved in controlling ES cell self-renewal and differentiation (Marson et al. 2008).

As the basic mechanisms that maintain the pluripotent state of ES cells are delineated, there is considerable interest in understanding how pluripotency is re-established in adult stem cells. It appears that some cell types are more readily reprogrammed to iPS cells than others (Aasen et al. 2008; Aoi et al. 2008), and it is interesting to speculate that this reflects differences in endogenous expression of the genes required for reprogramming or in responsiveness to overexpression of those genes (Hochedlinger et al. 2005; Markoulaki et al. 2009). Another emerging area of investigation is the relationship between the epigenome of pluripotent stem cells and cancer cells (Meissner et al. 2008).

Initial attempts at defining stemness by comparing the transcriptional profiles of ES cells, neural and haemopoietic stem cells (Ivanova et al. 2002; Ramalho-Santos et al. 2002) have paved the way for more refined comparisons. For example, by comparing the gene expression profiles of adult neural stem cells, ES-derived and iPS-derived neural stem cells and brain tumour stem cells, it should be possible both to validate the use of ES-derived stem cells for brain repair and to establish the cell of origin of brain tumour initiating cells. Furthermore, it is anticipated that new therapeutic targets will be identified from molecular profiling studies of different stem cell populations.

As gene expression profiling becomes more sophisticated, the question of what is a stem cell? can be addressed in new ways. Several studies have used single cell expression microarrays to identify new stem cell markers (Jensen & Watt 2006). Stem cells are well known to exhibit different proliferative and differentiation properties in culture, during tissue injury and in normal tissue homeostasis, raising the question of which elements of the stem cell phenotype are hard-wired versus a response to environmental conditions.

One of the growing trends in stem cell research is the contribution of mathematical modelling. This is illustrated in the concept of transcriptional noise: the hypothesis that intercellular variability is a manifestation of noise in gene expression levels, rather than stable phenotypic variation (Chang et al. 2008). Studies with clonal populations of haemopoietic progenitor cells have shown that slow fluctuations in protein levels can produce cellular heterogeneity that is sufficient to affect whether a given cell will differentiate along the myeloid or erythroid lineage (Chang et al. 2008). Mathematical approaches are also used increasingly to model observed differences in cell behaviour in vivo. In studies of adult mouse interfollicular epidermis, it is observed that cells can divide to produce two undifferentiated cells, two differentiated cells or one of each (figure3); it turns out that this can be explained in terms of the stochastic behaviour of a single population of cells rather than by invoking the existence of discrete types of stem and progenitor cell (Clayton et al. 2007).

The stem cell niche. Stem cells (S) are shown dividing symmetrically to produce two stem cells (1) or two differentiated cells (D) (2), or undergoing asymmetric division to produce one stem cell and one differentiated cell (3). Under some circumstances, a differentiated cell can re-enter the niche and become a stem cell (4). Different components of the stem cell niche are illustrated: extracellular matrix (ECM), cells in close proximity to stem cells (niche cells), secreted factors (such as growth factors) and physical factors (such as oxygen tension, stiffness and stretch).

There is strong evidence that the behaviour of stem cells is strongly affected by their local environment or niche (figure3). Some aspects of the stem cell environment that are known to influence self-renewal and stem cell fate are adhesion to extracellular matrix proteins, direct contact with neighbouring cells, exposure to secreted factors and physical factors, such as oxygen tension and sheer stress (Watt & Hogan 2000; Morrison & Spradling 2008). It is important to identify the environmental signals that control stem cell expansion and differentiation in order to harness those signals to optimize delivery of stem cell therapies.

Considerable progress has been made in directing ES cells to differentiate along specific lineages in vitro (Conti et al. 2005; Lowell et al. 2006; Izumi et al. 2007) and there are many in vitro and murine models of lineage selection by adult tissue stem cells (e.g. Watt & Collins 2008). It is clear that in many contexts the Erk and Akt pathways are key regulators of cell proliferation and survival, while pathways that were originally defined through their effects in embryonic development, such as Wnt, Notch and Shh, are reused in adult tissues to influence stem cell renewal and lineage selection. Furthermore, these core pathways are frequently deregulated in cancer (Reya et al. 2001; Watt & Collins 2008). In investigating how differentiation is controlled, it is not only the signalling pathways themselves that need to be considered, but also the timing, level and duration of a particular signal, as these variables profoundly influence cellular responses (Silva-Vargas et al. 2005). A further issue is the extent to which directed ES cell differentiation in vitro recapitulates the events that occur during normal embryogenesis and whether this affects the functionality of the differentiated cells (Izumi et al. 2007).

For a more complete definition of the stem cell niche, researchers are taking two opposite and complementary approaches: recreating the niche in vitro at the single cell level and observing stem cells in vivo. In vivo tracking of cells is possible because of advances in high-resolution confocal microscopy and two-photon imaging, which have greatly increased the sensitivity of detecting cells and the depth of the tissue at which they can be observed. Studies of green fluorescent protein-labelled haemopoietic stem cells have shown that their relationship with the bone marrow niche, comprising blood vessels, osteoblasts and the inner bone surface, differs in normal, irradiated and c-Kit-receptor-deficient mice (Lo Celso et al. 2009; Xie et al. 2009). In a different approach, in vivo bioluminescence imaging of luciferase-tagged muscle stem cells has been used to reveal their role in muscle repair in a way that is impossible when relying on retrospective analysis of fixed tissue (Sacco et al. 2008).

The advantage of recreating the stem cell niche in vitro is that it is possible to precisely control individual aspects of the niche and measure responses at the single cell level. Artificial niches are constructed by plating cells on micropatterned surfaces or capturing them in three-dimensional hydrogel matrices. In this way, parameters such as cell spreading and substrate mechanics can be precisely controlled (Watt et al. 1988; Thry et al. 2005; Chen 2008). Cells can be exposed to specific combinations of soluble factors or to tethered recombinant adhesive proteins. Cell behaviour can be monitored in real time by time-lapse microscopy, and activation of specific signalling pathways can be viewed using fluorescence resonance energy transfer probes and fluorescent reporters of transcriptional activity. It is also possible to recover cells from the in vitro environment, transplant them in vivo and monitor their subsequent behaviour. One of the exciting aspects of the reductionist approach to studying the niche is that it is highly interdisciplinary, bringing together stem cell researchers and bioengineers, and also offering opportunities for interactions with chemists, physicists and materials scientists.

Almost every day there are reports in the media of new stem cell therapies. There is no doubt that stem cells have the potential to treat many human afflictions, including ageing, cancer, diabetes, blindness and neurodegeneration. Nevertheless, it is essential to be realistic about the time and steps required to take new therapies into the clinic: it is exciting to be able to induce ES cells to differentiate into cardiomyocytes in a culture dish, but that is only one very small step towards effecting cardiac repair. The overriding concerns for any new treatment are the same: efficacy, safety and affordability.

In January 2009, the US Food and Drug Administration approved the first clinical trial involving human ES cells, just over 10 years after they were first isolated. In this trial, the safety of ES cell-derived oligodendrocytes in repair of spinal cord injury will be evaluated (http://www.geron.com). There are a large number of human ES cell lines now in existence and banking of clinical grade cells is underway, offering the opportunity for optimal immunological matching of donors and recipients. Nevertheless, one of the attractions of transplanting iPS cells is that the patient's own cells can be used, obviating the need for immunosuppression. Discovering how the pluripotent state can be efficiently and stably induced and maintained by treating cells with pharmacologically active compounds rather than by genetic manipulation is an important goal (Silva et al. 2008).

An alternative strategy to stem cell transplantation is to stimulate a patient's endogenous stem cells to divide or differentiate, as happens naturally during skin wound healing. It has recently been shown that pancreatic exocrine cells in adult mice can be reprogrammed to become functional, insulin-producing beta cells by expression of transcription factors that regulate pancreatic development (Zhou et al. 2008). The idea of repairing tissue through a process of cellular reprogramming in situ is an attractive paradigm to be explored further.

A range of biomaterials are already in clinical use for tissue repair, in particular to repair defects in cartilage and bone (Kamitakahara et al. 2008). These can be considered as practical applications of our knowledge of the stem cell microenvironment. Advances in tissue engineering and materials science offer new opportunities to manipulate the stem niche and either facilitate expansion/differentiation of endogenous stem cells or deliver exogenous cells. Resorbable scaffolds can be exploited for controlled delivery and release of small molecules, growth factors and peptides. Conversely, scaffolds can be designed that are able to capture unwanted tissue debris that might impede repair. Hydrogels that can undergo controlled solgel transitions could be used to release stem cells once they have integrated within the target tissue.

Although most of the new clinical applications of stem cells have a long lead time, applications of stem cells in drug discovery are available immediately. Adult tissue stem cells, ES cells and iPS cells can all be used to screen for compounds that stimulate self-renewal or promote specific differentiation programmes. Finding drugs that selectively target cancer stem cells offers the potential to develop cancer treatments that are not only more effective, but also cause less collateral damage to the patient's normal tissues than drugs currently in use. In addition, patient-specific iPS cells provide a new tool to identify underlying disease mechanisms. Thus stem cell-based assays are already enhancing drug discovery efforts.

Amid all the hype surrounding stem cells, there are strong grounds for believing that over the next 50 years our understanding of stem cells will revolutionize medicine. One of the most exciting aspects of working in the stem cell field is that it is truly multidisciplinary and translational. It brings together biologists, clinicians and researchers across the physical sciences and mathematics, and it fosters partnerships between academics and the biotech and pharmaceutical industries. In contrast to the golden era of developmental biology, one of stem cell research's defining characteristics is the motivation to benefit human health.

We thank all members of our lab, past and present, for their energy, fearlessness and intellectual curiosity in the pursuit of stem cells. We are grateful to Cancer Research UK, the Wellcome Trust, MRC and European Union for financial support and to members of the Cambridge Stem Cell Initiative for sharing their ideas.

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The therapeutic potential of stem cells - PMC

Worldwide, traditional, complementary, and integrative medicine (TCI) is central to health and well-being for millions of people. At the Permanent Mission of India in Geneva today the World Health Organization (WHO) signed a new multi million five-year agreement with the Ministry of AYUSH, Government of India to boost the development of new TCI technical documents by WHO.

This collaboration will play an important role not only in the globalization of evidence-informed traditional, complementary, and integrative medicine but also in mainstreaming proven TCI practices in national health systems towards achieving universal health coverage and well-being, said Dr. Bruce Aylward, Assistant Director-General of the Universal Health Coverage and Life Course Division of WHO.

His Excellency Shri Indra Mani Pandey, Permanent Representative of India to the United Nations and other International Organizations in Geneva, in signing the agreement stated Todays signing of this agreement will not only support the development of the TCI Global Strategy - but it will also support the integration of evidence-based Traditional and Complementary Medicine in national health systems, bio-diversity conservation and the sustainability of medicinal plants. India is committed to working with WHO to strengthen Traditional Medicine Systems globally and especially in supporting fellow developing countries in promoting their own traditional medicine systems.

The Government of India has been a long-time supporter of the WHO Traditional, Complementary, and Integrative medicine unit. The unit develops key benchmark documents, standardized terminologies, and other evidence-informed technical products to enhance the acceptability and credibility of the traditional medicine systems. The new agreement, the third in a series of such agreements, exemplifies Indias commitment to both technical and financial collaboration with WHO on this important area of work.

In August 2023 in Gujarat, India, where the new WHO Global Centre for Traditional Medicine was established, the first traditional medicine global summit launched the Gujarat Declaration. The declaration paves the way for collaborative efforts in making traditional medicine an important component of national health systems.

Through this new agreement, the Government of India will continue to support WHO to develop benchmarks for training and practice in Siddha, training modules on the quality and safety of herbal medicines, the international herbal pharmacopoeia among other activities over the next five years for the advancement of traditional medicine.

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Civil society organizations highlight the essential role of traditional, complementary and integrative health in addressing global health challenges, and opportunities for its integration into health systems

WHO Director-General, Dr Tedros Adhanom Ghebreyesus and a group of WHO senior officials met with representatives of civil society on 3 July, to discuss their priorities on traditional, complementary and integrative medicine, in the lead up to the WHO Traditional Medicine Global Summit on 17 and 18 August 2023 in Gandhinagar, Gujarat, India.

The virtual dialogue was coordinated by the Peoples Declaration for Traditional, Complementary and Integrative Healthcare, a worldwide civil society coalition of users and practitioners of traditional, complementary and integrative healthcare. Representatives of more than 300 civil society organisations (CSOs) and over 600 people were in attendance, bringing the full force of the knowledge and expertise on traditional, complementary and integrative health (TCIH) to the forefront.

In his opening remarks, Dr Tedros said: Traditional, complementary, and integrative health is rooted in the knowledge and resources of communities. For millions of people around the world, it is their first stop for health and well-being, and an integral part of their health system. It is for precisely these reasons that dialogues with civil society organizations are so important to WHO, as we shape our guidance and policy recommendations for countries.

WHO has started the development of the new traditional medicine strategy 2025-2034 as requested by its Member States at the World Health Assembly in May 2023, during which they have also extended WHO traditional medicine strategy: 2014-2023 for another two years, until 2025. Suggestions and proposals from the civil society will contribute to this important task, and will also inform the work of the WHO Global Centre for Traditional Medicine to harness the potential of traditional medicine from across the world to improve the health of people and the planet. Furthermore, the dialogue will contribute to WHO work on traditional, complementary and integrative medicine (TCIM), which seeks to respond to requests from countries for evidence and data to inform policies and practice, global standards and regulation to ensure safety, quality, equitable access and use, and support for scientific, innovation and technological advances in traditional medicine practices.

In the dialogue, CSOs stressed that traditional and complementary systems offer a holistic understanding of the human being and its interconnectedness with the world, and as such, can contribute to a positive vision of health that integrates the physical, the mental, the spiritual, and a social well-being. Recalling the Declaration of Astana and its specific references to the role of traditional knowledge in strengthening primary health care and improving health outcomes, CSOs stressed that the key questions is how to integrate and harness TCIH in a way that makes health services more health promoting and more in balance with the health of our planet.

Patients are demanding real choice in health care with the diversity of approaches that reflect and respect the individual, their culture and their beliefs and that are fully integrated into health care. When the health care services match the desire and choice of patients, this results in better health outcomes and greater satisfaction by patients. CSOs pointed to insufficient integration of TCIH into policy, especially in providing universal health coverage, citing the example of millions of TCIH practitioners and providers in the world, who often make health care accessible and affordable to many people.

Speaking about research, CSOs stressed that although there is an established evidence base for TCIH, integration into health systems has not yet happened and lack of evidence is often cited as a barrier. CSOs supported a dramatic increase in research activity, commensurate with TCIH use, and called for a more complex research agenda, to include products, practices and practitioners.

CSO representatives also spoke about the importance of training and continuous professional development of TCIH practitioners, and about specific registration, pathway and monitoring of TCIH products to ensure safely effectiveness and accessibility for all.

TCIH - which includes the diversity and complexity of Indigenous knowledge and traditional, complementary and integrative medicine systems - shows how the philosophical differences in practicing of medicine need to be respected, a speaker said. Indigenous knowledge must be protected, and Indigenous voices included, leading the process and upholding Indigenous world view where the collective is considered more important than the individual.

WHO respects the vast Indigenous knowledge systems and traditional complementary, integrative health approaches that have evolved over centuries in a diversity of contexts, in countries across the world, said Dr Shyama Kuruvilla, WHO lead for the Global Traditional Medicine Centre and Summit. WHOs role as the lead United Nations technical agency on health is to strengthen the evidence base and the data to support safe, scalable, effective, equitable, and optimal use, and to support equitable sharing of benefits.

Dr Kim Sungchol, head of WHO Traditional, Complementary and Integrative Medicine (TCIM) Unit said: Given the importance of the person-centred and integrative healthcare, WHO has already started working on developing policy guideline on integration of TCI into healthcare delivery system. The objective is to help and support Member States in formulating policies and programmes to maximize the potential contribution of TCI to achieving the highest possible level of health and wellbeing of the people, in line with their own contexts and realities.

Some of the civil societys asks - accelerating research agenda on TCIM, integration into health systems or regulation of TCIM products - are already embedded in WHO work, both in the TCIM strategy and in our operational and work planning as we go forward, noted Dr Bruce Aylward, Assistant Director-General, Universal Health Coverage and Life Course. After COVID-19, people value their health in a different way, and this represents an opportunity.

This virtual dialogue was part of a series of CSO Dialogues with the WHO Director-General to better understand civil society priorities and strengthen the important relationship between WHO and civil society organizations. Since October 2020, when the Dialogues first started, 16 have been organized on topics ranging from gender, youth, healthy ageing, social participation and accountability, climate and health, and more.

The Dialogues are CSO led civil society sets the agenda and presents their asks to WHO. Their objectives are to find concrete proposals and solutions to support the achievement of WHOs Triple Billion targets and to accelerate the attainment of Sustainable Development Goals.

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When it comes to the study of genes, there are two different approaches that scientists use to gain a deeper understanding of genetic makeup and its role in various biological processes. These approaches are genetics and molecular genetics. While both involve the analysis and research of genetic material, there are significant differences between the two.

Genetics, as a field of study, focuses on the inheritance and variation of genes in organisms. It involves the examination of traits, such as eye color or height, and the mapping of these traits to specific genes. This approach involves studying the DNA sequences and chromosomes to understand how genes are passed down from one generation to another.

On the other hand, molecular genetics takes a more detailed and intricate look at genes and their functions. It delves into the molecular mechanisms behind genetic processes, such as DNA replication, transcription, and translation. Molecular genetics employs advanced techniques and tools to analyze the structure and function of genes at the molecular level, including the study of specific gene mutations that may cause diseases.

Therefore, the main difference between genetics and molecular genetics lies in their scope and depth of analysis. While genetics looks at the broader picture of gene inheritance and variation, molecular genetics zooms in to elucidate the complex molecular processes and interactions that occur within genes. Both disciplines are valuable in their own right, complementing each other to provide a comprehensive understanding of how genes function and contribute to the diversity of life on Earth.

In the study of genetics, two main approaches are often compared: genetics and molecular genetics. Although these terms may sound similar, they represent different fields of analysis and study.

Genetics is the branch of biology that focuses on the study of genes, heredity, and variation in living organisms. It involves the analysis of inherited traits and the passing of genetic information from one generation to the next. Geneticists use various techniques, such as pedigree analysis and population genetics, to understand how genes are inherited and how they contribute to the diversity of organisms.

Molecular genetics, on the other hand, takes a more detailed and specific approach. It focuses on the study of the structure, function, and organization of genes at a molecular level. Molecular geneticists use advanced techniques, such as DNA sequencing and gene cloning, to analyze the DNA molecules and understand how specific genes function and interact. They study the mechanisms of gene expression, regulation, and mutations, and how they relate to genetic disorders and diseases.

While genetics provides a broad overview of inherited traits and genetic patterns in populations, molecular genetics delves deeper into the molecular mechanisms that underlie these patterns. It is a more specialized field that allows for a more detailed understanding of how genes function and how they contribute to the diversity of life forms.

In summary, genetics and molecular genetics are two distinct but related fields of study. Genetics provides a broader analysis of inherited traits and genetic patterns, while molecular genetics takes a more specific approach in understanding the molecular mechanisms of genes. Both fields are crucial for advancing our knowledge of genetics and its impact on living organisms.

In the field of genetics, the study of inheritance and variation in living organisms has been a subject of fascination since ancient times. However, it wasnt until the mid-20th century that the field took a major leap forward with the advent of molecular genetics.

Prior to the emergence of molecular genetics, the study of genetics primarily focused on observing and analyzing the hereditary traits of organisms through methods such as breeding experiments, statistical analysis, and observation of visible characteristics. This approach, known as classical genetics, provided valuable insights into the patterns of inheritance but had limitations in its ability to interrogate the underlying molecular mechanisms.

Molecular genetics, on the other hand, revolutionized the field by introducing a more detailed and comprehensive approach to the study of genetics. This approach involved the exploration of the structure and function of genes at the molecular level, analyzing the role of DNA and RNA in gene expression, and understanding the mechanisms of mutation and genetic variation.

The comparison between classical genetics and molecular genetics reveals significant differences in their research methodology and analysis. Classical genetics relied on observational data and statistical analysis to infer patterns of inheritance, while molecular genetics employs sophisticated laboratory techniques to manipulate and analyze DNA and RNA molecules.

Furthermore, the advent of molecular genetics has allowed researchers to delve deeper into the intricate mechanisms of genetic inheritance and variation. By studying molecular processes such as DNA replication, transcription, and translation, scientists have gained a more nuanced understanding of how genes interact and contribute to the development and functioning of organisms.

Overall, the emergence of molecular genetics as a distinct discipline has greatly expanded our knowledge of genetics and opened up new avenues of research. Its focus on the molecular level has provided invaluable insights into the complexities of genetic processes and has paved the way for advancements in fields such as biotechnology, genomics, and personalized medicine.

When it comes to the study of genetics and molecular genetics, there are several key concepts to understand. A comparison between genetics and molecular genetics provides insights into the differences in their approaches and analysis.

Genetics is the study of genes and heredity. It focuses on the inheritance of traits from one generation to another. Geneticists analyze the variations and similarities in genes to understand how certain traits are passed on and expressed through generations. They study the genes at a macroscopic level, observing the patterns of inheritance and the effects of genetic mutations on individuals.

Molecular genetics, on the other hand, takes a more microscopic approach. It zooms in on the molecular level to understand the structure and function of genes. By analyzing DNA and RNA sequences, molecular geneticists can unravel the intricacies of genetic information. They study the changes and interactions within genes and delve deeper into the mechanisms of gene expression and regulation.

The main difference between genetics and molecular genetics lies in their level of analysis. Genetics takes a broader approach, while molecular genetics focuses on a more detailed examination of genes and their molecular components. Both fields contribute to the understanding of how genetic information is inherited and expressed, but they employ different methodologies and perspectives.

In conclusion, genetics and molecular genetics are two related fields that study genes and heredity. Genetics focuses on the inheritance of traits at a macroscopic level, while molecular genetics delves into the molecular components and mechanisms of gene expression. By understanding the differences between these two approaches, scientists can gain a more comprehensive understanding of the complexities of genetic information.

In the research field of genetics, the methodology often involves the study of genetic variations and heredity patterns in living organisms. This can be achieved through various techniques such as pedigree analysis, gene mapping, and DNA sequencing. The primary focus of genetic research is to understand the differences and similarities between individuals and populations in terms of their genetic makeup.

On the other hand, molecular genetics is a more specific branch of genetics that focuses on the analysis of DNA and RNA molecules. It involves studying the structure, function, and regulation of genes at a molecular level. Molecular geneticists use techniques like PCR (polymerase chain reaction), gel electrophoresis, and DNA cloning to isolate and analyze specific genes or DNA sequences.

One key difference between genetic research and molecular genetics is the scale at which they operate. While traditional genetics looks at broader genetic traits and inheritance patterns, molecular genetics examines the specific molecular mechanisms behind these traits. This enables researchers to gain a more in-depth understanding of the biological processes involved.

Another difference is the level of detail in the analysis. Genetic research often involves observations at the organism level, such as comparing traits between individuals or populations. In contrast, molecular genetics focuses on the molecular level, analyzing DNA sequences and gene expression patterns.

In summary, the comparison between genetics and molecular genetics reveals the differences in the scope and approach of the two fields. While genetics provides a broader perspective on heredity and genetic variation, molecular genetics delves into the intricate molecular mechanisms underlying these phenomena. Both disciplines contribute valuable insights to our understanding of the genetic basis of life.

Both genetics and molecular genetics have important applications in the field of medicine. While there are some differences in their approaches and methodologies, they both contribute to our understanding of genetic diseases and provide valuable insights for diagnosis, treatment, and prevention.

Genetics is the study of heredity and the variation of inherited traits in organisms. It focuses on the examination of genes, their structures, functions, and how they are transmitted from one generation to another. Genetic analysis has long been used in the medical field to identify the genetic basis of diseases and to assess an individuals risk for developing certain conditions. It has played a crucial role in the identification of genetic disorders, such as Down syndrome and cystic fibrosis. Genetic counseling, which involves assessing an individuals risk for genetic conditions and providing information and support, is another important application of genetics in medicine.

Molecular genetics, on the other hand, takes a more focused approach by studying the structure and function of individual genes at a molecular level. It involves the analysis of DNA, RNA, and proteins to understand how genes are regulated and how they contribute to the development of diseases. The molecular analysis of genes has revolutionized the field of medicine by enabling researchers to identify specific molecular markers for diseases, develop targeted therapies, and predict therapeutic responses. This approach has led to personalized medicine, where treatments can be tailored to an individuals genetic profile.

Both genetics and molecular genetics have contributed significantly to the understanding and treatment of genetic diseases. While genetics provides a broader picture of genetic inheritance and variation, molecular genetics offers a more detailed analysis of genes and their functions. Together, they form a powerful combination for medical research and have paved the way for breakthroughs in the diagnosis, treatment, and prevention of genetic disorders.

When it comes to the field of agriculture, both genetics and molecular genetics play crucial roles in research and advancements. While they may have similarities in their approaches and analyses, there are also significant differences between the two.

Genetics, as a branch of biology, studies the inheritance and variation of traits in living organisms, including plants and animals. In agriculture, genetics is applied to breed selectively and improve the desirable traits of crops and livestock. Through traditional breeding methods, geneticists identify and cross-breed plants and animals with specific desirable traits to create new varieties with enhanced characteristics such as disease resistance, productivity, and quality.

The Role of Molecular Genetics in Agriculture

Molecular genetics takes a more advanced and precise approach compared to traditional genetics. It involves the study of genes and their functions at the molecular level, focusing on analyzing DNA and other genetic materials.

Advancements in molecular genetics have revolutionized the field of agriculture.

While traditional genetics relies on breeding experiments and observations, molecular genetics utilizes various laboratory techniques and technologies to directly manipulate and analyze genetic materials. This includes techniques such as polymerase chain reaction (PCR), gene cloning, and genome sequencing.

Analysis and Study of Genes

Molecular genetics allows for a more in-depth analysis and study of individual genes, their interactions, and their functions. This provides valuable insights into the underlying genetic mechanisms responsible for specific traits in plants and animals, leading to a better understanding of their biology and potential for improvement.

The study of molecular genetics also enables the identification and characterization of genes associated with valuable traits in crops, such as drought tolerance, nutrient efficiency, and pest resistance.

By identifying and manipulating these genes, scientists can develop genetically modified organisms (GMOs) with enhanced traits, such as genetically modified crops with increased yield or improved nutritional content.

In conclusion, both genetics and molecular genetics have significant applications in agriculture. While traditional genetics focuses on selective breeding, molecular genetics allows for a more advanced and precise understanding and manipulation of genes. Together, these fields contribute to the development of improved crops and livestock for a more sustainable and efficient agricultural industry.

In the field of forensics, the study of genetics and molecular genetics has been instrumental in solving crimes and identifying individuals involved in criminal activities. Through the analysis of genetic material found at crime scenes, forensic scientists are able to compare and identify differences in the genetic profiles of suspects. This approach allows for a more reliable and accurate comparison of DNA samples, which is crucial in criminal investigations.

Molecular genetics, with its focus on the study of molecular structure and function, offers a more detailed and precise approach to forensic analysis. By examining specific genes and their variations, forensic scientists can establish a genetic profile of an individual, providing important clues in identifying suspects or victims.

Genetics, on the other hand, takes a broader perspective in the study of inherited traits and the genetic makeup of individuals. It involves the comparison of DNA sequences, studying inherited variations, and determining the likelihood of individuals carrying certain traits or diseases. In forensics, genetic analysis is crucial in establishing familial relationships, such as paternity or kinship, which can be helpful in suspect identification or victim identification.

By comparing the genetic profiles of individuals found at crime scenes with those in a database, forensic scientists can identify potential suspects or rule out individuals who may be wrongly implicated. This process involves a comprehensive analysis of DNA samples, including the identification of specific genetic markers that are unique to each individual.

While both genetics and molecular genetics play vital roles in forensic analysis, there are differences in their approaches and focus. Genetics offers a broader perspective on inherited traits, while molecular genetics provides a more detailed and in-depth analysis of genetic material. The comparison of genetic profiles and the identification of unique markers are common goals in both fields.

Overall, the study of genetics and molecular genetics is essential in forensic research and analysis. By using these approaches, forensic scientists are able to provide valuable evidence and insights in solving crimes, identifying suspects, and bringing justice to victims and their families.

In the study of genetics and molecular genetics, researchers employ different approaches and techniques to gain insights into the complex world of genes and their functions. The analysis of genetic information requires a combination of both genetic and molecular research methods.

Genetics research typically focuses on the inheritance and variation of genes among different individuals or populations. It often involves studying the physical traits, hereditary patterns, and genetic disorders in order to understand the role of genes in biological processes. Geneticists use various techniques such as pedigree analysis, linkage analysis, and genetic mapping to uncover the inheritance patterns of specific traits.

Molecular genetics, on the other hand, delves deeper into the molecular mechanisms behind genetic processes. This field employs advanced techniques to analyze DNA, RNA, and protein molecules. Researchers in molecular genetics use tools like polymerase chain reaction (PCR), DNA sequencing, and gene expression analysis to study the structure, function, and regulation of genes at the molecular level.

While genetics research focuses on broader patterns of inheritance and genetic variation, molecular genetics provides a more detailed understanding of the molecular events governing gene expression and regulation. Both approaches are crucial for unraveling the complexities of genetic information and its implications for health and disease.

In summary, genetics research and molecular genetics employ distinct research techniques to study genes and their functions. Genetics research takes a broader approach, focusing on inheritance patterns and genetic variation, while molecular genetics provides a deeper analysis of the molecular mechanisms behind genetic processes. Combining these two approaches enables researchers to gain a comprehensive understanding of genetics and its role in various biological processes.

The study of genetics and molecular genetics has had a significant impact on the field of evolutionary biology. By comparing the differences between genetic information and its molecular analysis, scientists have been able to gain a deeper understanding of the processes and mechanisms that drive evolution.

In traditional genetic study, scientists focus on the study of genes and their inheritance patterns within populations. This approach allows researchers to track the transmission of specific traits and determine how they are passed down through generations. However, this method does not provide detailed information about the molecular mechanisms that underlie genetic changes.

In contrast, molecular analysis takes a more detailed and precise approach. It involves the study of DNA and other molecules that make up the genetic material. Molecular techniques such as DNA sequencing, PCR, and gene expression analysis allow scientists to analyze the structure and function of genes at the molecular level. By understanding the molecular differences between individuals and species, researchers can gain insights into the evolutionary processes that shape biodiversity.

By combining the study of genetics with molecular analysis, scientists can compare genetic information across different species and populations. This comparative approach provides valuable insights into the evolutionary relationships between organisms and helps researchers reconstruct the evolutionary history of species.

Molecular genetics also allows researchers to study the impact of genetic variation and genetic changes on evolution. By analyzing DNA sequences, scientists can identify mutations and other genetic changes that occur over time. These studies help researchers understand the mechanisms of adaptation, speciation, and genetic drift, all of which play crucial roles in shaping evolution.

In summary, the integration of genetics and molecular analysis has revolutionized the field of evolutionary biology. This interdisciplinary approach provides researchers with a powerful tool to decipher the genetic and molecular basis of evolutionary processes. By understanding the impact of genetic variation and molecular differences, scientists can gain a deeper understanding of how species evolve and adapt in response to their changing environments.

In the field of genetics, the role of molecular genetics plays a crucial part in the process of genetic counseling. Genetic counseling aims to provide individuals and families with accurate information about the risk of genetic conditions and to support them in making informed decisions about their health and reproduction.

Molecular genetics involves the analysis of DNA, genes, and chromosomes to understand the underlying causes of genetic disorders. This study uses a different approach compared to traditional genetics, which focuses on studying the observable differences and traits in individuals.

One of the key roles of molecular genetics in genetic counseling is to analyze genetic variations that may contribute to an individuals risk of developing a genetic condition. Through advanced research techniques and technologies, molecular geneticists can identify specific changes in an individuals DNA sequence that could be associated with inherited diseases.

This analysis helps genetic counselors provide accurate information about the chances of the condition being passed on to future generations, providing individuals with a better understanding of their genetic risks. With this information, individuals can make informed decisions about family planning, reproductive options, and potential medical interventions.

Another important aspect of molecular genetics in genetic counseling is the comparison of DNA sequences. By comparing DNA sequences of individuals with and without certain genetic conditions, researchers can identify genetic variations that may contribute to the development of these disorders.

Comparative analysis helps in the early detection, diagnosis, and treatment of various genetic conditions, allowing individuals and families to access appropriate medical care and support. This approach enables genetic counselors to provide personalized genetic counseling, tailoring their recommendations and support based on the specific genetic profile of each individual.

Conclusion

The role of molecular genetics in genetic counseling is essential for understanding the underlying genetic factors contributing to inherited diseases. By analyzing genetic variations and comparing DNA sequences, molecular genetics provides valuable information that can guide individuals and families in making informed decisions about their health and reproduction. Through this approach, genetic counseling becomes more accurate, personalized, and effective in supporting individuals and families.

The comparison between genetics and molecular genetics in the context of disease diagnosis is crucial for understanding the approach and differences in the analysis of genetic disorders.

Genetics, as a field of study, focuses on the inheritance and variation of genes in organisms. It examines the role of genes in transmitting traits, including disease susceptibility. Genetic analysis involves studying the genetic makeup of individuals to identify mutations or alterations in specific genes that may contribute to the development of diseases.

In disease diagnosis, the study of genetics plays a significant role. Genetic tests can determine the presence or absence of specific genes associated with certain diseases. These tests help identify individuals who may be at risk or carriers for genetic disorders. Genetic counseling based on these findings can aid in making informed decisions about treatment options or preventive measures.

Traditional genetics primarily focuses on the study of inherited diseases caused by mutations in specific genes. It involves analyzing the presence of these mutations within families or populations to understand the pattern of disease transmission. This information is valuable in identifying affected individuals, predicting disease outcomes, and providing appropriate interventions.

Molecular genetics takes a more detailed and advanced approach to disease diagnosis compared to traditional genetics. It involves analyzing the structure and function of genes at a molecular level, including DNA sequencing and gene expression studies. The study of molecular genetics allows for a deeper understanding of the underlying mechanisms behind the development and progression of genetic disorders.

Molecular genetic analysis can identify specific genetic mutations or alterations that may contribute to disease susceptibility. It provides insights into the molecular pathways involved in disease development, allowing for targeted therapies and personalized treatment approaches. This approach also enables the identification of potential therapeutic targets and the development of novel treatment strategies.

In conclusion, both genetics and molecular genetics play essential roles in disease diagnosis. While genetics focuses on the inheritance and variation of genes, molecular genetics provides a more detailed analysis at the molecular level. Understanding the differences between these approaches is crucial for advancing our knowledge of genetic disorders and improving disease diagnosis and treatment.

In drug development, both genetics and molecular genetics play an important role in understanding the mechanisms of action of drugs and their potential effects on individuals. However, there are some differences in their approach and focus on research and study.

Genetics: a field of study that focuses on the inheritance and variation of genes in individuals and populations. It explores how genes influence the development, functioning, and characteristics of organisms. In drug development, genetics research aims to identify genetic variations that may affect the response to drugs, such as the presence of specific genes associated with drug metabolism or drug targets.

Molecular Genetics: a subfield of genetics that involves the study of DNA, RNA, and other molecules involved in genetic information and gene expression. It focuses on understanding the molecular mechanisms underlying various genetic processes, such as DNA replication, gene transcription, and protein synthesis. In drug development, molecular genetics research aims to unravel the molecular pathways and targets that drugs interact with in order to develop more targeted and effective treatments.

While genetics provides a broader perspective on the influence of genes on drug response and potential side effects, molecular genetics delves deeper into the specific molecular mechanisms through which drugs exert their effects. By combining the knowledge gained from genetics and molecular genetics, researchers can better understand the genetic basis of diseases and tailor drug therapies to individual patients.

By comparing the genetic profiles of patients who respond well to a drug with those who do not, researchers can identify genetic markers that predict drug response. This information can then be used to develop diagnostic tests that help identify individuals who are more likely to benefit from a particular drug or who may experience adverse reactions. Additionally, the study of molecular genetics can uncover new drug targets and pathways that can be exploited for drug development.

When comparing genetics and molecular genetics, it is important to consider the ethical implications of both approaches. While genetics is the study of genes and heredity, molecular genetics takes a more focused and analytical approach by examining the structure and function of genes at a molecular level. This difference in approach can lead to different ethical considerations in research and analysis.

In genetics, ethical considerations often revolve around issues such as privacy and informed consent. Researchers may need to handle sensitive information related to an individuals genetic makeup, which raises concerns about data privacy and confidentiality. Additionally, when conducting genetic studies, informed consent from participants is crucial to ensure that they fully understand the risks and benefits of participating in the research.

There is also the issue of genetic discrimination, where individuals may face discrimination based on their genetic information. This can have significant social and psychological impacts, highlighting the need for policies and laws to protect individuals from such discrimination.

In molecular genetics, ethical considerations are more focused on the research and analysis techniques used. Molecular genetics often involves the manipulation and modification of genetic material in a laboratory setting. This raises concerns about the potential risks and consequences of such manipulations. It is important to ensure that these techniques are conducted safely and in accordance with ethical guidelines to minimize any harm to both humans and other organisms.

Furthermore, the use of genetically modified organisms (GMOs) in molecular genetics research also raises ethical concerns. GMOs can have ecological implications and may raise questions about the potential long-term effects on the environment and other organisms.

In conclusion, while both genetics and molecular genetics share similarities in their study of genes, they have different approaches that can lead to distinct ethical considerations. Understanding and addressing these ethical concerns is essential to ensure the responsible and ethical advancement of genetic and molecular genetics research.

The analysis of genetics and molecular genetics is a field of research and study that poses several challenges for scientists. Both approaches involve the study of genes and the hereditary material, but they differ in their methodologies and scope. Understanding these challenges is crucial for advancing our knowledge in genetics and molecular genetics.

One of the current challenges in the field of molecular genetics is understanding the complexity of the molecular mechanisms that govern gene expression and regulation. Molecular genetics focuses on studying the individual molecules, such as DNA, RNA, and proteins, that make up the genetic material. This approach requires advanced techniques and technologies to analyze and manipulate these molecules, as well as computational methods to interpret the vast amount of data generated. The complexity of these molecular interactions presents a challenge for researchers in understanding the underlying mechanisms that control gene expression.

Another challenge in genetics and molecular genetics is the comparative analysis of data obtained from different organisms. While genetics traditionally involves studying specific traits and genes within a particular species, molecular genetics allows for a broader comparison across species. However, comparing genetic information between organisms can be difficult due to variations in gene structure, gene function, and regulatory mechanisms. Researchers need to develop standardized approaches and tools to compare and analyze genetic data from diverse organisms, which can help identify common patterns and evolutionary relationships.

In conclusion, the study of genetics and molecular genetics faces various challenges. The molecular complexity of gene regulation and expression requires advanced techniques and computational methods. Additionally, the comparative analysis of genetic data across different organisms calls for standardized approaches and tools. Overcoming these challenges is essential for advancing our understanding of genetics and molecular genetics and their applications in various fields, including medicine and agriculture.

The study of genetics and molecular genetics has provided valuable insights into the complexities and mechanisms of inheritance and genetic variation. However, there is still much to be discovered and understood in these fields. As technology continues to advance, new approaches and techniques are being developed that will further enhance our understanding of genetics.

One future direction is the use of comparative analysis to deepen our understanding of genetics. By comparing the genomes of different organisms, scientists can identify similarities and differences in their genetic makeup. This comparative approach allows for a more comprehensive understanding of how genetics shape biological traits and functions.

Furthermore, comparative analysis can help us uncover the evolutionary relationships between species. By examining the similarities and differences in their genetic information, scientists can reconstruct the evolutionary history of different organisms, shedding light on how life has evolved over millions of years.

Another future direction is the integration of molecular and genetic approaches. While genetics focuses on the study of inheritance and variation at the organismal level, molecular genetics delves into the underlying molecular mechanisms that drive these processes.

By combining these two approaches, researchers can gain a more comprehensive understanding of the genetic basis of traits and diseases. Molecular genetics provides the tools and techniques to examine the specific genes and molecules involved, while genetics provides the broader context and understanding of how these genes and molecules interact within an organism.

This integration of molecular and genetic approaches will allow for a more nuanced and sophisticated analysis of genetic variation and inheritance, providing valuable insights into the differences and similarities between individuals and populations.

In conclusion, the future of genetics and molecular genetics lies in the continued exploration of comparative analysis and the integration of molecular and genetic approaches. These advancements will further enhance our understanding of the complexities of genetics and pave the way for breakthroughs in fields such as personalized medicine and genetic engineering.

In the field of genetics, data analysis plays a crucial role in understanding the complexities of genetic information. Both genetics and molecular genetics employ different approaches in the study of genes and genetic variations.

In traditional genetics, the analysis focuses on studying traits and heredity patterns within populations or families. It involves observing and quantifying physical characteristics, as well as tracking the inheritance of specific traits through generations. This approach relies on family trees, Punnett squares, and statistical methods to analyze the data.

On the other hand, molecular genetics takes a more detailed and precise approach in analyzing genetic data. It involves studying the structure and function of DNA, genes, and proteins at the molecular level. This field has revolutionized the study of genetics by introducing techniques such as DNA sequencing and polymerase chain reaction (PCR).

Molecular genetics uses advanced laboratory techniques to isolate, amplify, and analyze specific regions of DNA. This allows researchers to identify and study genetic variations, mutations, and gene expression patterns. Data analysis in molecular genetics often involves complex algorithms, bioinformatics tools, and statistical methods.

By comparing genetics and molecular genetics, it becomes evident that the main difference lies in the level of detail and precision in data analysis. Traditional genetics provides a broader perspective on genetic traits and inheritance patterns, while molecular genetics offers a deeper understanding of the molecular mechanisms underlying genetic variations and gene functions.

Overall, the comparison between genetics and molecular genetics highlights the evolving nature of genetic studies. While both approaches contribute to our understanding of genes and heredity, molecular genetics allows for a more in-depth analysis of genetic data, paving the way for new discoveries and advancements in the field.

Continued here:
Comparing Genetics and Molecular Genetics: What's the Difference?

Continuing Education Activity

Molecular diagnostics encompasses the analysis of human, viral, and microbial genomesand the products they encode. Molecular genetics utilizesmolecular biology's laboratory tools to relate genetic structureto protein function and, ultimately, health and disease.Variants identified during genetic testing are classified based on diverse evidence types, as the American College of Medical Genetics and Genomics recommends, emphasizing the need for board-certified geneticists to interpret the results.Integrating genetic testing methodologies with clinical expertise is crucial in translating molecular genetics advancements tobetter patient care.

The field of molecular genetic and genomic testing is undergoing rapid change due to improvements in our understanding of the molecular causes of uncommon and common illnesses and DNA analysis technologies.The advent of molecular genetics has revolutionized healthcare by offering unprecedented insights into the genetic basis of diseases, enabling personalized diagnostics, treatment strategies, and risk assessments. However, this progress brings with it the responsibility for healthcare providers to stay updated with the latest advancements and best practices in genetic testing.

This activity for healthcare professionals is designed to enhance learners' proficiency in identifying patients withindications for molecular genetics testing and interpreting genetic test results. Participants acquire a broader grasp of specimen collection, procedures, indications, potential diagnosis, normal and critical findings, interfering factors, and complications. Learners gain insights into thecomplexities of molecular genetics, preparing them to collaborate with an interprofessional team that aims to improve outcomes for patients who need molecular genetics testing.

Objectives:

Identify clinicalencounters appropriate for genetic molecular testing, distinguishing cases where such testing can contribute to diagnosis, prognosis, or treatment decisions.

Evaluate genetic test results accurately, discerning their clinical significance and relevance to patient management.

Differentiatebetween genetic testing methodologies, understanding their strengths, limitations, and optimal applications to diagnose patients.

Implement best interprofessional collaboration and communication practices to ensure that patientswho need molecular genetics testing receive comprehensive care that considers their medical, psychological, and social needs, thus improving outcomes.

Molecular genetics testing is fundamental in evaluating inherited disorders, somatic or acquired diseases with genetic associations, and pharmacogenetic responses. Genotyping can provide valuable disease diagnosis, prognosis, and progression indicators, guide treatment selection and response, and identify gene-specific therapeutic targets.[1]Human genetic material primarily consists of double-stranded, helical DNA. This molecule has a backbone composed of alternating sugar (deoxyribose) and phosphate groups, with hydrogen bonds linking nitrogenous base pairs. Specifically, adenine (purine) pairs with thymine (pyrimidine), while guanine (purine) pairs with cytosine (pyrimidine), forming the complementary base pairs within the DNA double helix.[2][3]

DNA in human cells is wrapped around histone proteins and packaged into nucleosome units, compacted further to form chromosomes.[4]Somatic cells normally have 23 chromosome pairs, with 1 pair comprised of the sex chromosomes X and Y. Each chromosome has DNA with a terminal stretch of short repeats called telomeres and additional repeats in the centromere region.[5]

Humans have 2 sets of 23 chromosomes, one derived from the mothers egg and the other from the father's sperm. Therefore, each egg and sperm is a single or haploid set of 23 chromosomes. Combining the 2 creates a diploid set of human DNA, allowing each individual to possess 2 different sequences, genes, and alleles on each chromosome.[6]Homologous recombination during meiosis generates unique allele combinations in gametes, leading to genetic diversity among offspring in the human population.[7]

The complete decoding of the human genome sequence and the development of powerful identification and cloning methods for genes linked to inherited diseases have transformed the practice of molecular genetics and molecular pathology. Advanced molecular analysis methods can now determine presymptomatic individuals' illness risk, detect asymptomatic recessive trait carriers, and prenatally diagnose conditions not yet evident in pregnancy.[8]Molecular genetics techniques are often the only approaches to these puzzles. Thus, genetic tests are powerful tools for diagnosis, genetic consultation, and prevention of heritable diseases.[9]

Many genetic testscan analyze gene, chromosome, and protein alterations. A clinician often considers several factors when selecting the appropriate test, including suspected conditions and their possible genetic variations. A broad genetic test is employed when a diagnosis is uncertain, while a targeted test is preferred for suspected specific conditions.[10]Molecular tests look for changes in 1 or more genes. These tests analyze the sequence of DNA building blocks (nucleotides) in an individual's genetic code, a process known as DNA sequencing, which can vary in scope.[11]

The targeted single variant test identifies a specific variant in a single gene known to cause a disorder, eg, the HBB gene variant causing-globin abnormalities that give rise to sickle cell disease. This test assesses the family members of an individual with the known variant to ascertain if they have the familial condition.[12]Single-gene tests examine genetic alterations in 1 gene to confirm or rule out a specific diagnosis, notably when many variants in the gene can cause the suspected condition. Gene panel tests look for variants in multiple genes to pinpoint a diagnosis when a person has symptoms that may fit various conditions or when many gene variants can cause the suspected condition.[13][14]

Whole-exome sequencing or whole-genome sequencing tests analyze the bulk of an individual's DNA to find genetic variations. This approach is useful when a single-gene or panel testing has not provided a diagnosis or when the suspected condition or genetic cause is unclear.[15]This sequencing method is often more cost- and time-effective than performing multiple single gene or panel tests.[16]

Chromosomal tests analyze whole chromosomes or long DNA lengths to identify significant alterations, including extra or missing chromosome copies (trisomy or monosomy), large chromosomal segment duplications or deletions, and segment rearrangements (translocations) (see Image. Trisomy 21on G-Banded Chromosomal Studies).[17]Chromosomal tests are employed when specific genetic conditions linked to chromosomal changes are suspected. For instance, Williams syndrome results from deleting a chromosome 7 segment.

Gene expression tests assess gene activation status in cells, indicating whether genes are active or inactive, with activated genes producing mRNA molecules that serve as templates for protein synthesis.[18]The mRNA produced helps determine which genes are highly active. Too much activity (overexpression) or too little activity (underexpression) of specific genes may suggest particular genetic disorders, including various cancer types.[19]Biochemical tests assess protein or enzyme levels and activity rather than directly analyzing DNA.[20]Abnormalities in these substances may indicate DNA changes underlying a genetic disorder.

Heritable mutations are detectable in all nucleated cells and are thus considered germline or constitutional genetic changes. Somatic genetic changes are characteristic of acquired or sporadic diseases like cancer.[21]Both scenarios are investigated using similar molecular biology methods to detect DNA and RNA variations, although the interpretation and utility of the laboratory results often differ significantly.[22]

Fluorescent in situ hybridization (FISH), chromosomal microarray analysis (CMA), and cytogenetic analysis (karyotyping) can be used to detect gross mutations like whole- and large-scale gene deletions, duplications, or rearrangements. Conventional karyotyping identifies rearrangements over 5 DNA megabases.[23]FISH has a resolution of 100 kilobases to 1 megabase. Minor alterations, such as single-base substitutions, insertions, and deletions, are detectable with single-strand conformation polymorphism (SSCP) and sequence analysis through next-generation sequencing (NGS). NGS uses genomic DNA (gDNA) or complementary DNA (cDNA) and has 3 modalities: whole genomic DNA, targeted, and exome sequencing.[24]

Denaturing high-performance liquid chromatography (DHPLC) can detect small deletions and duplications. Multiplex ligation-dependent probe amplification (MLPA) extends the range of deletions and duplications detected, bridging the gap between FISH or cytogenetic analysis and HPLC. MLPA is particularly useful in identifying complete or single and multiexon deletions or duplications.[25][26]

Peripheral blood is the specimen required for FISH, MLPA, DHPLC, and sequencing.Amniotic fluid cells and, more recently, cell-free fetal DNAmay be used for noninvasive prenatal testing.[27]Ethylenediaminetetraacetic acid is the most commonly used anticoagulant for molecular-based testing. However, acid citrate dextrose (ACD) is an acceptable alternative in cases where cellular form and function must be preserved.

ACD A and ACD B are the only ACD tube designations recognized, differing only by their additive concentrations.[28]Both enhancewhite blood cell viability and recovery for several days after specimen collection, making them suitable for molecular diagnostic and cytogenetic testing.

FISH utilizes fluorescent DNA probes to target specific gene sequences in interphase or metaphase cells, enabling their visualization and detection. Housekeeping gene probes always serve as positive internal controls. The probe must be large enough to hybridize specifically with the target without impeding the hybridization process. Conventional FISH involves pipetting the hybridization mix onto the cytological sample and incubating them together.

The technique can be applied to suspended cells, cultured cells, and frozen or formalin-fixed paraffin-embedded tissue sections, with subsequent cell sorting for fluorescence signal separation.[29]Preserving nucleic acid integrity and cell morphology is necessary during sample fixation. The experimental FISH procedure includes several preparatory steps, the hybridization reaction itself, and the removal of unbound probes.[30]The probe may be directly labeled with fluorophores or targeted for fluorescent detection using labeled antibodies or similar substrates. Different tags may be used, and different targets may be detected in the same sample simultaneously (multi-color FISH). Tagging is performed in various ways, including nick translation or polymerase chain reaction (PCR) using tagged nucleotides (see Image. Polymerase Chain Reaction). Probes can vary from 20 to 30 nucleotides to much longer sequences.

Locus-specific probes provide insight into gene amplification, deletion, or normal copy number status. Dual-fusion probes are adept at identifying frequently translocated gene regions associated with cancer development. These probes target regions spanning the breakpoints of translocation partners. Intact green and red signals are determined when they are closer than one signal's width. Conversely, a break in the gene sequence results in separate green and red signals.[31]

Break-apart probes target 2 areas of a specific gene sequence, using a green fluorescent label on one end and a red fluorescent label on the other. Intact gene sequences typically produce a yellow signal, known as a fusion signal. Whole-chromosome probes consist of smaller probes, each binding to different sequences along a chromosome.[32]Multiple probes, labeled with fluorescent dyes, enable unique color labeling of each chromosome, creating a spectral karyotypea full-color chromosome map identifyingall chromosome pairs.[33]Whole-chromosome probes are useful for examining chromosomal abnormalities, such as translocations.

Chromosomal microarray (CMA) consists of thousands of tiny probes, each representing small DNA fragments from known locations on the 46 chromosomes. CMA detects imbalances in chromosomal material between patient and control DNA samples, identifying copy number differenceswhether gains (duplications) or losses (deletions)in specific DNA segments.[34]These differences pinpoint the cause of the patient's health condition based on the location and type of change detected.[35]

Denaturing high-performance liquid chromatography (DHPLC) relies on differential chromatography retention of DNA heteroduplexes post-denaturation and renaturation. DNA heteroduplex migration is influenced by both molecule length and melting temperature, which is crucial for test sensitivity. DHPLC typically compares 2 PCR products amplified from 2 genes: 1 wild type and 1 mutated. These PCR products can originate from either RNA (cDNA) or genomic DNA. The PCR products are denatured at 95 C and gradually reannealed by cooling from 95 C to 65 C before chromatography. A major advantage of this technology is that multiple samples can be pooled together for variant detection.[36]Sequencing detects single-base substitutions and small deletions and insertions in DNA fragments ranging from 80 to 1500 base pairs, with close to 100% accuracy within minutes.

When a mismatch is present, both the original homoduplexes and 2 heteroduplexes are simultaneously produced. The original homoduplexes form from the reannealing of perfectly matching sense and antisense strands (25% each). The heteroduplexes form from the reannealing of the sense strand of one homoduplex with the antisense strand of the other (also 25% each). Heteroduplexes denature more extensively than homoduplexes, resulting in earlier elution from the chromatography column. The separation of all 4 species is based on their differences in stacking interactions with the chromatography column (solid phase). More detailed theoretical explanations of DHPLC are available in the literature.[37]

MLPA utilizes genomic DNA samples, with specific MLPA probes hybridizing with denatured genomic DNA. These probes are uniquely designed to hybridize adjacent to each other on the target DNA region and confer a distinct length to each amplified MLPA probe pair. Detection and quantification occur via capillary electrophoresis.[38]All MLPA probes are amplified using the same primer pair, with the abundance of each fragment proportional to its target's copy number in the sample.

NGS amplifies DNA with random priming, providing a genome-wide view of the patient's genetic background through millions of reads. Library generation begins with nucleic acid fragmentation, representing the individual's entire genome or transcriptome. Whole-exome sequencing uses cDNA fragments, whereas the whole-genome modality includes complete genomic DNA. Fragments join using enriched sequence adaptors. Only some genes (gene panel) are analyzed in targeted libraries. Fragments hybridize with cDNA fragments for the region or genes of interest and are specifically enriched.[39]During sequencing, nucleotide addition is detected by fluorescent dyes or pH changes from hydrogen ion release during DNA polymerization.[40]

Sanger sequencing begins with PCR-based target DNA amplification, followed by removing excess deoxynucleotide triphosphates (dNTPs) and PCR primers. The Sanger method has 99.99% base accuracy and is thus the "gold standard" for validating DNA sequences, including those from NGS. The test's stepsinclude denaturing the double-stranded DNA (dsDNA) into 2 single-stranded DNA (ssDNA), attaching a primer corresponding to one end of the sequence, and sequencing 4 polymerase solutions with 4 dNTPs. Only one type of ddNTP is incorporated, initiating DNA synthesis until termination. The resulting DNA fragments are denatured into ssDNA.

Denatured fragments undergo gel electrophoresis for sequence determination. DNA polymerase synthesizes DNA only in the 5 to 3 direction, initiating at a provided primer. Each terminal ddNTP corresponds to a specific nucleotide in the original sequence. For example, the shortest fragment must terminate at the first nucleotide from the 5 end, the second-shortest fragment must terminate at the second nucleotide from the 5 end, and so on. Reading gel bands from smallest to largest reveals the 5 to 3 sequence of the original DNA strand.[41]

In manual Sanger sequencing, the user reads all 4 gel lanes simultaneously, moving from bottom to top to identify the terminal ddNTP for each band. For instance, if the bottom band is found in the ddGTP column, then the smallest PCR fragment terminates with ddGTP, and the first nucleotide from the 5 end of the original sequence has a guanine (G) base.[42]Automated Sanger sequencing employs a computer to read each capillary gel band sequentially, using fluorescence to determine the terminal ddNTP identity. Laser activation of fluorescent tags emits light, detected by the computer, with each ddNTP tagged with a unique fluorescent label. The output is a chromatogram displaying fluorescent peaks corresponding to each nucleotide along the template DNA's length.[43]

Third-generation sequencing enables sequencing long DNA or RNA stretches without fragmentation. Single strands of DNA or RNA are directed through protein nanopores, with nucleotide bases distinguished by characteristic changes in electric current to determine the sequence.[44]Compared to 2nd-generation sequencing, 3rd-generation sequencing requires minimal sample preprocessing, enabling the design of smaller and more portable equipment.[45]

Molecular genetic testing has distinct indications, differing from traditional clinical and molecular biological testing used for diagnosing other diseases.[46]This modalitys applications encompass newborn screening, diagnostic testing for genetic or chromosomal conditions, carrier testing, prenatal testing, predictive and presymptomatic testing for adult-onset disorders, and forensic testing for legal identification purposes.[47]

FISH is employed for patients with a family history of known deletions and has been utilized to detect deletions in single blastomeres during preimplantation genetic diagnosis. FISH tests use gene-specific probe panels to investigate deletions, amplifications, and translocations in hematologic and solid tumors. FISH can also identify intracellular microorganisms and parasites.

CMA is recommended for individuals lacking specific clinical indicators to identify genetic or nongenetic causes of intellectual disability, developmental delay, autism spectrum disorder, or multiple congenital anomalies.[48]CMA can be helpful if prenatal structural anomalies are linked to particular microdeletions or microduplications. This modality can also evaluate copy number variants in cases of de novo balanced rearrangements or marker chromosomes.[49]

MLPA has diverse applications, such as mutation detection, single nucleotide polymorphisms (SNP) analysis, DNA methylation analysis, mRNA quantification, chromosomal characterization, gene copy number detection, and identification of duplications and deletions in cancer predisposition genes like BRCA1, BRCA2, hMLH1, and hMSH2. MLPA also holds promise for prenatal diagnosis, both invasive and noninvasive.[50]

DHPLC is well-suited for scanning genes for novel mutations and analyzing large sample sizes cost-effectively. This test is also useful for genotyping specific mutations or polymorphisms. DHPLC offers various applications beyond detecting genetic variants, including size-based double-strand DNA separation, single-strand DNA separation, and DNA purification analysis.[51]

NGS rapidly sequences whole genomes and target regions, employs RNA sequencing to identify novel RNA variants and splice sites, quantifies mRNAs for gene expression analysis, and analyzes epigenetic factors like DNA methylation and DNA-protein interactions. Sequence cancer samples study rare somatic variant tumor subclones and identify novel pathogens. Sanger sequencing, or the "chain termination method," determines DNA nucleotide sequences.

FISH swiftly diagnoses common fetal aneuploidies but with reduced sensitivity compared to cytogenetic analysis. FISH cannot identify cytogenetic abnormalities beyond the most common ones, such as translocations, inversions, and markers. DHPLC detects single nucleotide changes, small deletions, or insertions requiring subsequent confirmation by sequencing. This method identifies unknown mutations, making it advantageous for diseases with a high proportion of de novo mutations. Neurofibromatosis type 1 (NF1) is an example, as approximately 50% of cases arise from new mutations. CMAs are first-tier tests for developmental delays, intellectual disabilities, autism spectrum disorders, or multiple congenital disabilities, replacing karyotyping.

MLPA detects gene abnormalities, particularly small deletions in diseases like multiple endocrine neoplasia type 1 (MEN1partial or complete deletion). MLPA can also assess methylation alterations, such as in pseudohypoparathyroidism 1b (PHP1b), where deletion of 1 or 4 of four differentially methylated regions is common.

NGS generates millions of sequences, which are then processed, analyzed, and interpreted to identify variants. Bioinformatics analysis begins with raw data generated by nucleotide incorporation signal detection. Read quality is evaluated during primary data analysis. Sequences are aligned or mapped against a reference genome, with computational algorithms searching for the best match for each read while allowing for some mismatches to detect genetic variants.[52]

Sanger sequencing is reliable in detecting point mutations, small deletions, or duplications. This method has a long history of use across various settings, including tumor mutational spectrum analysis and diagnostic testing for constitutional variants. Primers can cover multiple regions (amplicons) or any desired region size.

The increasing demand for genetic testing has led to greater availability. Ensuring uniformity and standardization in communicating the complex results to referring clinicians is essential. Failure to include pertinent information is considered a deficiency in the molecular pathology laboratory accreditation inspection.[53]All molecular genetic laboratories offering clinical testing should be accredited according to the Clinical Laboratory Improvement Amendments and actively participate in proficiency testing.[54]

A comprehensive genetic report must include essential patient details such as name, medical record number or birth date, sex, and ethnicity. The report should also specify the type of specimen received, identification number, laboratory test requested, the performing laboratorys name and address, and referring healthcare professional or hospital. The date of the report, analytic result interpretation using standard nomenclature, detailed method description (including literature citations if applicable), and assay sensitivity and specificity should be provided. For example, sensitivity and specificity should be reported regarding the number of variants analyzed, the proportion of variants not detected, and the possibility of genetic heterogeneity and recombination.[55]Reports from clinical DNA laboratories should include a disclaimer due to the prevalence of laboratory-developed tests (LDTs) or procedures (LDPs) designed, developed, and validated internally by each laboratory but remain unapproved by the FDA.[56]

Fluorescent tags binding to chromosomes reveal chromosomal abnormalities in FISH. MLPA detects copy number variations by correlating peak intensity during capillary electrophoresis with sample copy numbers. An MLPA probe's amplification signals the presence of a mutation in the sample.

An MLPA test can yield two outcomes:

DHPLC detects mutations by identifying heteroduplexes compared to the reference genome in the same sample. NGS identifies various genetic variants, including single nucleotides, small insertions or deletions, and some structural variants, but their role in the disease is not implied. Clinical analysis and assessment of the pathological potential of detected variants require consideration in different contexts.[58]Sanger sequencing results interpretation depends on the target DNA strand and primer availability. If strand A is of interest but the primer suits strand B better, the output matches strand A. Conversely, if the primer suits strand A better, the output aligns with strand B, necessitating conversion back to strand A.

FISH probe specificity prevents unintended hybridization with nontarget genes. Some FISH preparations may exhibit autofluorescence, necessitating thorough cell washing to remove fluorescent residues and reduce background fluorescence.

MPLA has limitations, including its ability to detect only known mutations designed into probes, making gene rearrangements like inversions and translocations undetectable. Sample purity is essential as contaminants such as phenol can interfere with the ligation step. MLPA may yield false positive or negative results due to rare sequence variants in target regions detected by probes. Reduced probe binding efficiency from point mutations or polymorphisms candiminishthe relative peak areas height. Confirmation of single exon deletions detected by MLPA is thus recommended using other methods like multiplex PCR or sequencing.[59]

DHPLC sensitivity relies on melting temperature. Computational algorithms can predict the melting temperature, and the procedure typically involves at least 2 melting temperatures for increased sensitivity. CMA does not detect point mutations, small DNA segment changes (eg, in Fragile X syndrome), or balanced chromosomal rearrangements (eg, balanced translocations, inversions).

NGS technologies continue to evolve to address various challenges. Some large sequencers can detect large insertions, duplications, and deletions, while sequencing long homopolymer regions remains problematic. However, establishing the infrastructure and expertise for data analysis remains a significant challenge in clinical settings. The primary limitation of implementing NGS in clinical settings is the requirement for adequate infrastructure, including computational resources, storage capacity, and skilled personnel for comprehensive data analysis and interpretation.

Despite automation, Sanger sequencing remains labor-intensive, time-consuming, and expensive, relying on specialized equipment. Sanger sequencing exhibits reduced sensitivity in detecting point mutations when 20% of mutant DNA is of a wild-type background. Additionally, it lacks quantifiability, making it impossible to differentiate mutation prevalence accurately based solely on peak sizes, necessitating supplementary testing approaches.

Peripheral blood collection via venipuncture infrequently leads to serious complications. Some patients, especially children, may experience hematomas, pain, and fear, which are expected. In contrast, procedures like amniocentesis are more invasive, thus posing more serious risks such as infection, preterm delivery, respiratory distress, trauma, and alloimmunization, though these complications are also infrequent.[60]Genetic tests using NGS of free-cell DNA from maternal peripheral blood offer an alternative to diagnosis using amniocentesis fluid.[61]

Molecular testing may give rise to legal, medical, psychological, and ethical issues besides the sampling procedures potential complications.[62]While molecular testing primarily aims to demonstrate a genetic trait associated with a disease, the current recommendation is to integrate the results into genetic counseling.[63]

Genetic counseling, led by a team including genetic counselors and other professionals, begins with clinically identifying suspected diseases to guide molecular testing. Patients are informed about the testing procedure, potential results, and legal considerations like informed consent, particularly for children.[64]Patient education is integral to this process.

NGS technologies applied to genetic counseling yield complex results surpassing traditional tests, necessitating informed patient discussions due to the considerable information and ethical implications involved.[65]Laboratories conducting molecular genetic tests should address preexamination, examination, and postexamination considerations, tailoring methodology and interpretation to each test's indication, application, and ethical implications.

Any permanent alteration in a gene's nucleotide sequence compared to a reference genome is deemed a genetic change or mutation. Variants identified through a tiered protocol must undergo sequencing confirmation, and their role in disease pathology must be assessed. Genetic testing may reveal variants classified as benign, likely benign, pathogenic, likely pathogenic, or of uncertain significance.[66]Variants must be rigorously classified based on various types of evidencepopulation, computational, functional, or segregation datato determine clinical significance.[67]

The American College of Medical Genetics and Genomics recommends this nomenclature and classification for genetic test findings, covering genotyping, single genes, panels, exomes, and genomes. NGS applications have deepened our understanding of genetic diseases and led to the discovery of variants requiring further study of their disease implications.[68]Interprofessional collaboration is essential for leveraging genetic tests for patient benefit, withan expertpanel advocating for results interpretation by a board-certified geneticist.[69]

Molecular genetic testing advanced significantly with PCR and NGS, providing genome-wide data.[70]Multidisciplinary teams collaborate to integrate various testing methods with clinical, pathological, functional, computational, ethical, and social aspects of diseases for patient benefit.[71]

Polymerase Chain Reaction. This diagram shows the polymerase chain reaction steps. Enzoklop,Public Domain via Wikimedia Commons

Trisomy 21 on G-Banded Chromosome Studies. This karyogram depicts trisomy 21 resulting from an inherited Robertsonian translocation between chromosomes 14 and 21. The infant's father was a carrier of the translocation in a balanced form. Crotwell PL, (more...)

Disclosure: Cecilia Ishida declares no relevant financial relationships with ineligible companies.

Disclosure: Muhammad Zubair declares no relevant financial relationships with ineligible companies.

Disclosure: Vikas Gupta declares no relevant financial relationships with ineligible companies.

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Molecular Genetics Testing - StatPearls - NCBI Bookshelf

The complex molecular underpinnings of genetic and rare diseases offer a promising avenue for scientific exploration and innovation. This research topic explores the intricate molecular mechanisms driving these conditions, underlining the latest developments in diagnostic methodologies and therapeutic approaches. Due to the rapid advancement in molecular diagnostics, including Next-Generation Sequencing, our insights into rare and genetic diseases have expanded significantly.

Through this research topic, our aim is to uncover genetic and molecular mechanisms driving the onset and progression of genetic and rare diseases, with a particular focus on unraveling actionable therapeutic targets. By integrating advanced molecular biology methods, including Next-Generation Sequencing, CRISPR, and other cutting-edge technologies, this topic emphasizes the development of novel therapeutic approaches. The goal is to translate these molecular insights into innovative, personalized therapies that address the specific challenges of genetic and rare diseases, ultimately improving patient outcomes and advancing the field of precision medicine.

This Research Topic addresses significant challenges in accurately diagnosing and effectively treating genetic and rare diseases. Despite advances in molecular diagnostics, many of these conditions remain underdiagnosed or misdiagnosed, delaying crucial interventions. Recent developments in technologies like Next-Generation Sequencing have revolutionized our ability to detect genetic anomalies, but there is still a need to bridge the gap between these diagnostics and targeted therapeutic strategies. This topic seeks to explore how integrating advanced molecular tools with therapeutic innovations can lead to more precise and personalized treatments, ultimately improving outcomes for patients with genetic and rare diseases.

We welcome submissions of original research articles, in-depth reviews, case studies, and perspective pieces that advance the understanding of the genetic and molecular foundations of genetic and rare diseases. Contributions that explore novel diagnostic tools, therapeutic strategies, and translational research are particularly encouraged.

This Research Topic will cover a wide range of themes related to genetic and rare diseases, including but not limited to:

Identification and characterization of novel genetic mutations and their clinical implications;

Advances in molecular diagnostic technologies, including Next-Generation Sequencing and multi-omics approaches;

Development of targeted therapies and personalized treatment strategies for rare and genetic disorders;

Translational research bridging molecular diagnostics and therapeutic applications;

Ethical and clinical considerations in the treatment of genetic and rare diseases.

Keywords:Rare Diseases, Genetic Diseases, Next-Generation Sequencing, Molecular Diagnostics, Clinical Genomics

Important Note: All contributions to this Research Topic must be within the scope of the section and journal to which they are submitted, as defined in their mission statements. Frontiers reserves the right to guide an out-of-scope manuscript to a more suitable section or journal at any stage of peer review.

Through this research topic, our aim is to uncover genetic and molecular mechanisms driving the onset and progression of genetic and rare diseases, with a particular focus on unraveling actionable therapeutic targets. By integrating advanced molecular biology methods, including Next-Generation Sequencing, CRISPR, and other cutting-edge technologies, this topic emphasizes the development of novel therapeutic approaches. The goal is to translate these molecular insights into innovative, personalized therapies that address the specific challenges of genetic and rare diseases, ultimately improving patient outcomes and advancing the field of precision medicine.

This Research Topic addresses significant challenges in accurately diagnosing and effectively treating genetic and rare diseases. Despite advances in molecular diagnostics, many of these conditions remain underdiagnosed or misdiagnosed, delaying crucial interventions. Recent developments in technologies like Next-Generation Sequencing have revolutionized our ability to detect genetic anomalies, but there is still a need to bridge the gap between these diagnostics and targeted therapeutic strategies. This topic seeks to explore how integrating advanced molecular tools with therapeutic innovations can lead to more precise and personalized treatments, ultimately improving outcomes for patients with genetic and rare diseases.

We welcome submissions of original research articles, in-depth reviews, case studies, and perspective pieces that advance the understanding of the genetic and molecular foundations of genetic and rare diseases. Contributions that explore novel diagnostic tools, therapeutic strategies, and translational research are particularly encouraged.

This Research Topic will cover a wide range of themes related to genetic and rare diseases, including but not limited to:

Identification and characterization of novel genetic mutations and their clinical implications;

Advances in molecular diagnostic technologies, including Next-Generation Sequencing and multi-omics approaches;

Development of targeted therapies and personalized treatment strategies for rare and genetic disorders;

Translational research bridging molecular diagnostics and therapeutic applications;

Ethical and clinical considerations in the treatment of genetic and rare diseases.

Keywords:Rare Diseases, Genetic Diseases, Next-Generation Sequencing, Molecular Diagnostics, Clinical Genomics

Important Note: All contributions to this Research Topic must be within the scope of the section and journal to which they are submitted, as defined in their mission statements. Frontiers reserves the right to guide an out-of-scope manuscript to a more suitable section or journal at any stage of peer review.

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Molecular Underpinnings of Genetic and Rare Diseases: From ... - Frontiers