Induced pluripotent stem (iPS) cells are laboratory-produced cells that combine the biological advantages of somatic adult and stem cells for cell-based therapy. The reprogramming of cells, such as fibroblasts, to an embryonic stem cell-like state is done by the ectopic expression of transcription factors responsible for generating embryonic stem cell properties. These primary factors are octamer-binding transcription factor 4 (Oct3/4), sex-determining region Y-box 2 (Sox2), Krppel-like factor 4 (Klf4), and the proto-oncogene protein homolog of avian myelocytomatosis (c-Myc). The somatic cells can be easily obtained from the patient who will be subjected to cellular therapy and be reprogrammed to acquire the necessary high plasticity of embryonic stem cells. These cells have no ethical limitations involved, as in the case of embryonic stem cells, and display minimal immunological rejection risks after transplant. Currently, several clinical trials are in progress, most of them in phase I or II. Still, some inherent risks, such as chromosomal instability, insertional tumors, and teratoma formation, must be overcome to reach full clinical translation. However, with the clinical trials and extensive basic research studying the biology of these cells, a promising future for human cell-based therapies using iPS cells seems to be increasingly clear and close.

Keywords: induced pluripotent stem cells, regeneration, cellular therapy, stem cells, muscular dystrophy

Stem cells can be classified as totipotent, pluripotent, or multipotent cells according to their biological source and the capacity to differentiate into other cell types. Totipotent stem cells are found very early in embryonal development and can differentiate into all cell types in the organism, as well as into extraembryonic tissues. Pluripotent cells can be isolated from blastocysts or the umbilical cord immediately after birth, and are also able to differentiate into all tissue cells, except extraembryonic structures. However, certain disadvantages must be observed when considering these stem cells in regenerative medicine. These include the high risk of rejection and ethical issues when the isolation is performed from embryos. On the other hand, due to their high plasticity, pluripotent stem cells are considered ideal to obtaining the multiple cell types required after stem cell-based therapies.

Multipotent stem cells are isolated from adult tissues and have no ethical issues involved. These include hematopoietic, mesenchymal, and neural stem cells. Multipotent stem cells can be isolated from the patients subjected to treatment, with no risk of rejection, and be expanded in vitro for transplant. However, these cells display reduced plasticity, as they can only differentiate into specialized cell types present in specific tissues or organs, their main disadvantage. The ideal cellular population best suited for stem cell-based therapies should combine the high plasticity of embryonic stem cells and the convenient isolation from patients under treatment. To this end, induced pluripotent stem (iPS) cells were generated using embryonic or adult somatic cells. The somatic cells are subjected to the ectopic expression of transcription factors that induce the stem cell-like properties and the high plasticity required for cell therapy. Therefore, iPS cells can potentially revolutionize the field of regenerative medicine and provide new tools for stem cell research.

In the nineties, it was demonstrated that somatic cells could be reprogrammed to an undifferentiated state by transferring their nuclear content into unfertilized oocytes [1]. These results showed that cellular differentiation is reversible. Later, the resetting of a somatic epigenotype to a totipotent state was successfully achieved when adult thymocytes were fused with embryonic stem cells [2]. These and other pioneering studies [3] paved the way for the Nobel prize-awarded paper published by Takahashi and Yamanaka [4], who hypothesized that factors that play important roles in the maintenance of embryonic stem cell identity also play pivotal roles in the induction of pluripotency in somatic cells. In this study, mouse embryonic and adult fibroblasts were genetically reprogrammed to a pluripotent state, and the authors coined the term iPS cells. These cells were generated by using a retrovirus-based gene transfer system carrying the octamer-binding transcription factor 4 (Oct3/4), sex determining region Y-box 2 (Sox2), Krppel-like factor 4 (Klf4), and c-Myc transcription factors, all involved in pluripotency maintenance in embryonic stem cells [4].

IPS cell technology brings great promise to medicine, such as personalized cell therapy, disease modeling, and new drug development and screening. However, some challenges must be circumvented, such as reprogramming efficiency and the risks associated with chromosomal instability, insertional tumor development, and teratoma formation. In this context, here, we review the literature, present the main methods of cell reprogramming, and show some initial results of clinical tests. Besides, we discuss the possibility of applying iPS cells in the treatment of muscular dystrophies.

Various delivery methods have been used to insert reprogramming factors into somatic cells. These approaches can be divided into integrative, which involves the insertion of exogenous genetic material into the host genome, and non-integrative methods. The integrative systems include the use of viral vectors (lentivirus, retrovirus, and inducible or excisable retro or lentivirus) and non-viral vectors (linear or plasmid DNA fragments and transposons). Likewise, non-integrative systems include viral (Sendai virus and adenovirus) and non-viral vectors (episomal DNA vectors, RNAs, human artificial chromosomes (HAC), proteins, and small molecule compounds) [5,6] (Figure 1 and Table 1).

Somatic Cells Reprogramming Methods. The methods used to produce iPS cells can be classified into integrative viral, such as retrovirus (a), lentivirus (b), or inducible retro or lentivirus (c); and integrative non-viral, such as linear or circular DNA fragments (d) or transposons (e). In regards to non-integrative methods, they can also be separated as viral, such as adenovirus (f) or Sendai virus (g). Non-integrating non-viral methods are episomal DNA (h), RNAs (i), human artificial chromosome (HAC) (j), proteins (k), or small molecules (compounds) (l). The red DNA represents epigenetic inserted sequences for cellular reprogramming.

Comparison of multiple reprogramming techniques.

The expression of primarily just four transcription factors (c-Myc, Klf4, Oct4, and Sox2) is sufficient to reprogram somatic cells into a pluripotent state. The discovery of those factors related to the embryonic stem cell phenotype allowed the production of embryonic stem-like cells first from mouse embryonic and adult murine fibroblasts [4] and then from adult human dermal fibroblasts [7,8]. The Oct4 seems to be the most important reprogramming factor, whereas Klf4 and c-Myc can be replaced by Nanog and Lin28, for example [9].

The first experiments achieved the conversion of somatic cells into iPS cells using retroviral or lentiviral transduction of the transcription factors. However, these vectors become integrated into the cell genome and represent a risk of insertional mutagenesis [10]. Moreover, they may leave residual transgene sequences as part of the host genome, leading to unpredictable alterations in the phenotype of downstream applications. To reduce multiple proviral integrations of the transcription factors and to increase the efficiency of the retrovirus- or lentivirus-based reprogramming process, polycistronic RNA viral vectors were created. These constructs allowed the expression of all reprogramming factors driven by a single promoter, reducing the number of genomic insertions [11]. Once the integration of the reprogramming factors is achieved, it is also essential to control the extent of expression. To this end, the use of excisable Cre-loxP technology for site-specific recombination and inducible tetracycline- or doxycycline-based vector systems allowed greater control of inserted genes expression, reducing inefficient silencing and uncontrolled reactivation [5].

It is important to highlight that other factors have been described as being able to induce cellular pluripotency and self-renewal. Besides, several types of somatic cells have also been subjected to in vitro reprogramming, such as pancreatic cells, neural stem cells, stomach and liver cells, mature B lymphocytes, melanocytes, adipose stem cells, and keratinocytes. These results are summarized in the review published by Oldole and Fakoya [5].

The integrative non-viral technologies used to obtain iPS cells are based on the transference of DNA sequences using liposomes or electroporation [5], for example. It was possible to reprogram both mouse and human fibroblasts using a single multiprotein expression vector comprising the coding sequences of c-Myc, Klf4, Oct4, and Sox2 linked with 2A peptide [24]. When this single vector-based reprogramming system was combined with a piggyBac transposon, the authors successfully established reprogrammed human cell lines from embryonic fibroblasts with sustained pluripotency markers expression. PiggyBac is a mobile genetic element that includes a transposase enzyme that mediates gene transfer by targeted insertion and excision in the DNA. Moreover, Woltjen and collaborators showed the efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PiggyBac transposition. The authors also showed that the individual PiggyBac insertions could be removed from the iPS cell lines [15], being completely excised from its integration site in the original DNA sequence [25], which is a significant advantage.

The integrative methods for random or site-specific DNA insertion can affect normal cell function and physiology, including the transformation for tumorigenic cells, proliferation, and apoptosis control. Therefore, non-integrating viral vectors were constructed to generate iPS cells, the most promising of which is the Sendai virus, a negative-strand RNA virus [26]. The Sendai virus has the advantage of being an RNA virus that does not enter the nucleus and can produce large amounts of proteins [27]. Adenoviruses are also non-integrating viruses that appear to be excellent expression vehicles to generate iPS cells. They show DNA demethylation (a characteristic of reprogrammed cells), express endogenous pluripotency genes, and can generate multiple cells and tissues. However, the reprogramming efficiency of adenoviral vectors is only 0.001%0.0001% in mouse [28] and 0.0002% in human cells [29], several orders of magnitude lower, when compared to lentiviruses or retroviruses [5]. The use of viruses, even in non-integrating systems, requires refined steps to exclude reprogrammed cells with active replicating viruses. Moreover, viral vectors may elicit an innate and adaptive immune response against viral antigens after the transplant to patients. In this case, the transplanted cells would become the target of molecular and cellular cytotoxic pathways, directly compromising the engraftment and therapy success.

Non-integrating non-viral systems include the transient expression of reprogramming factors inserted as combined episomal minicircles or plasmids. These contain the complementary DNA (cDNA) of Oct3/4, Sox2, and Klf4 and another plasmid containing the c-Myc cDNA, for example. This technique resulted in iPS cells with no evidence of plasmid integration [16], suggesting that episomal plasmids may be the best option for clinical translation. This technique has already been used in the autologous induced stem cell-derived retinal treatment for macular degeneration [30]. Moreover, minicircle vectors are also used as a method for cellular reprogramming and consist of minimal vectors containing only the eukaryotic promoter and the cDNA(s) that will be expressed. This technique was able to reprogram human adipose stromal cells, but the reprogramming efficiency is substantially lower (~0.005%) when compared to lentiviral-based techniques, for example [31].

HACs are also non-integrative systems for gene delivery with the main advantage of being able to transfer multiple genes and large sequences, which can be combined with sequences that increase therapy security and expression control. The authors constructed two different HACs, and the reprogramming of mouse embryonic fibroblasts into iPS cells was better achieved when the artificial chromosome also encoded a p53-knockdown cassette. The iPS cells were uniformly generated, and a built-in safeguard system was included, consisting of a reintroduced HAC encoding the Herpes Simplex virus thymidine kinase, which allowed the targeted elimination of reprogrammed cells by ganciclovir treatment [19].

Another promising strategy focusing on non-integrative non-viral reprogramming methods for iPS cell generation is through RNA molecules, such as micro-RNAs. These sequences are small endogenous non-coding RNAs that play important post-transcriptional regulatory roles [32]. They also repress gene expression through translational inhibition or by promoting the degradation of mRNAs [33]. One study showed that normal human hair follicles could be reprogramed into human iPS cells via doxycycline-inducible pTet-On-tTS vectors inserted by electroporation. These constructs contained pre-microRNA members of the miR-302 cluster, including pre-miR-302a, 302b, 302c, and 302d [34]. Although the reprogramming efficiency was not reported in this study, it is known that iPS cells induced by micro-RNAs have a reprogramming efficiency above 10% and also have the lowest tumorigenicity rate. Although this approach has not yet been used in any clinical test, it may help in future developments in regenerative medicine [33]. More recently, micro-RNAs were used in combination with other reprogramming methods to increase reprogramming efficiency [5].

Another promising transgene-free approach is the direct mRNA transfection of synthetic modified coding sequences of the Yamanaka factors (c-Myc, Klf4, Oct4, and Sox2). This is a non-integrating method that can reprogram multiple human cell types to pluripotency very efficiently, avoiding the antiviral immune response. The authors further showed that the same technology efficiently directed the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells [35]. The method of the direct delivery of synthetically transcribed mRNAs triggered somatic cell reprogramming with higher efficiency when compared to retroviruses [35]. These mRNAs are commercially available, and the authors used cationic lipid delivery vehicles for transfection in cell culture for seven days [27]. Similar alternatives are emerging as the cellular introduction of all reprogramming factors via a single synthetic polycistronic RNA replicon that requires single transfection [36]. In this case, the transfection of adult fibroblasts resulted in an efficient generation of iPS cells with the expression of all stem cell markers tested, consistent global gene expression profile, and in vivo pluripotency for all three germ layers.

Transgene-free cellular reprogramming has also been achieved using recombinant proteins. In this case, the generation of stable iPS cells was possible by directly delivering the four reprogramming proteins fused with a cell-penetrating peptide [22]. However, it has been technically challenging to synthesize large amounts of bioactive proteins that can cross the plasma membrane. This problem associated with low efficiency shows that much remains to be done for the use of recombinant proteins as a viable method. Two research groups were able to make enough bioactive proteins in an E. coli expression system and to reprogram mouse [37] and human fibroblasts [22]. More recently, Weltner and collaborators also used Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 nuclease (CRISPR-Cas9)-based gene activation (CRISPRa) for reprogramming human skin fibroblasts into iPS cells [38]. CRISPR/Cas9 is a genome-editing tool powered by the design principle of the guide RNA that targets Cas9 to the desired DNA locus and by the high specificity and efficiency of CRISPR/Cas9-generated DNA breaks [39].

Another system for cellular reprogramming to generate iPS cells was the use of small-molecule compounds, which was developed by Hou and collaborators [23]. These authors used a combination of seven small molecules, but the efficiency achieved was only 0.2%. Small molecules have some advantages such as structural versatility, reasonable cost, easy handling, and no immune response. They can boost the application of iPS cells in disease therapy and drug screening. Some of these chemical compounds are valproic acid, trichostatin A (TSA), and 5-azacytidine, all capable of enhancing iPS cell generation [40]. One of the main advantages is that small (chemical) molecules can stimulate endogenous human cells to make tissue repair and regeneration in vivo, with no ectopic expression of factors. On the other hand, the method is time-consuming, and there is still a risk of genetic instability [6] to be overcome in future studies.

Despite all developments in the field of iPS cells, viral vector-based methods remain most popular among researchers [41]. Still, non-integrating non-viral self-excising vectors are more likely to be clinically applicable. To select an iPS cell reprogramming method, it is essential to maximize the capacity of cellular expansion in vitro, validate the detection and removal of incompletely differentiated cells, and search for genomic and epigenetic alterations. Probably, different somatic cell types will require different reprogramming methods to differentiate into the required terminal cell type in vivo.

Regardless of the reprogramming method, the risk of teratoma formation is inherent to iPS cells, as residual undifferentiated pluripotent cells retain very high plasticity. Although this risk has been reduced by highly sensitive methods for detecting remaining undifferentiated cells, teratoma formation cannot be ruled out [42]. Besides, c-Myc, one of the factors used for cellular reprogramming, is a well-known proto-oncogene, and its reactivation can give rise to transgene-driven tumor formation [43].

IPS cells can differentiate into cells from any of the three primary germ layers [44], with great potential for clinical applications. Neurodegenerative disorders, for example, and diseases in which in vitro differentiation and transplant protocols have been established using conventional embryonic stem cells, are areas of immediate interest for iPS-based cell therapy. IPS cell lines can be generated in virtually unlimited numbers from patients affected by diseases of known or unknown causes. These cells can differentiate in vitro into the disease-affected cell type and offer an opportunity to gain insight into the disease mechanism to identify novel disease-specific drugs. In Table 2, we show examples of iPS cells generated from patients with sporadic or genetic diseases.

Examples of terminally differentiated cells generated from induced pluripotent stem (iPS) cells.

Some drugs that are in clinical trials were derived from iPS cell studies. For example, cardiomyocyte-derived iPS cells obtained from patients with type-2 long QT syndrome were used to test the efficacy and potency of new and existing drugs [51]. In regenerative medicine, iPS cells can be used for tissue repair or replacement of injured tissues after cell transplantation. Early trials using iPS cell transplantation focused on age-related macular degeneration, and this is a refractory ocular disease that causes severe deterioration in the central vision due to senescence in the retinal pigment epithelium (RPE). Preclinical studies showed good results in various animal models and corroborated the first clinical trial that began in 2014 [54]. Kamao and collaborators generated human iPS cells derived from RPE (hiPSC-RPE) cells that met clinical use requirements, including cellular quality and quantity, reproducibility, and safety. After the transplant, autologous non-human primate iPSC-RPE cell sheets showed no immune rejection or tumor formation [55]. Then, in the clinical trial using iPS cells, the cells were generated from skin fibroblasts obtained from patients with advanced neovascular age-related macular degeneration and were differentiated into RPE cells. In this test, autologous iPS cell-based therapy did not cause any significant adverse event [30]. However, the test with the second patient was discontinued due to genetic aberrations detected in the autologous iPS cells. With the rapid progress of genomic technologies, genetic aberrations in iPS cells will probably be reduced to a minimal level, with technological advances also focusing on automated closed culture systems [56].

Recent advances in genome editing technology have made it possible to repair genetic mutations in iPS cell lines derived from patients. Special attention has recently been focused on organoids derived from iPS cells, which are three-dimensional cellular structures mimicking part of the organization and functions of organs or tissues. Organoids were generated for various organs from both mouse and human stem cells, generating intestinal, renal, brain, and retinal structures, as well as liver organoid-like tissues, named liver buds [57]. Therefore, iPS cells-derived organoids can also be useful for drug testing and in vitro studies based on more complex cell models.

Moreover, iPS cells derived from cancer cells (cancer-iPS cells) can be a novel strategy for studying cancer. Primary cancer cells have been reprogrammed into iPS cells or at least to a pluripotent state, allowing the study and elucidation of some of the molecular mechanisms associated with cancer progression [58].

The possibility of using iPS cells in the treatment of various diseases has brought hope regarding their potential to treat an increasing number of conditions. As iPS cells can be differentiated into all different cell types, new prospects for studying diseases and developing treatments by regenerative medicine and drug screening have emerged. Therefore, a large number of clinical and preclinical trials are being carried out [59] to treat human diseases using iPS cells.

The reprogramming of somatic cells was demonstrated using different animal species, including mouse, rat [60], dog [61], a variety of non-human primate species [62], pig [63], horse [64], cow [65], goat [66], and sheep [67]. However, once the goal of pre-clinical trials is the clinical use of iPS cells, a number of these trials are being conducted using human iPS cells. For specific applications, however, human cells are expected to be rejected by the animal hosts, and immunosuppressive protocols are required for long-term observation. On the other hand, immunomodulating drugs may affect the disease phenotype, and careful planning of every step is necessary. Any stem-cell-based clinical trial must follow all precedents already established for the evaluation of small biological molecules or human tissue remodeling and must be safe and effective. The production of cells must be carried out in facilities that follow the current Good Manufacturing Practices (GMP) and have stringent quality control for reagents with well-defined product release and potency assays. GMP is a set of conditions that define the principles and details of the manufacturing process, quality control, evaluations, and documentation for a particular product. Moreover, the best delivery system of iPS cells must be evaluated for each disease, which can be the use of intravascular catheters or surgical injection, for example.

Human-derived iPS cell lines successfully repopulated the murine cirrhotic liver tissue with hepatic cells at various differentiation stages. They also secreted human-specific liver proteins into mouse blood at concentrations comparable to those of proteins secreted by human primary hepatocytes [68]. In other preclinical studies, iPS cells were generated using adult dsRed mouse dermal fibroblasts via retroviral induction, following transplantation into the eye of immune-compromised retinal degenerative mice. After thirty-three days of differentiation, a large proportion of the cells expressed the retinal progenitor cell marker Pax6 and photoreceptor markers. Therefore, adult fibroblast-derived iPS cells are a viable source for the production of retinal precursors to be used for transplantation and treatment of retinal degenerative disease [69]. IPS cells were also generated from nonobese diabetic mouse embryonic fibroblasts or nonobese diabetic mouse pancreas-derived epithelial cells and differentiated into functional pancreatic beta cells. The differentiated cells expressed diverse pancreatic beta-cell markers and released insulin in response to glucose and KCl stimulation. Moreover, the engrafted cells responded to glucose levels by secreting insulin, thereby normalizing blood glucose levels, showing that these cells may be an important tool to help in the treatment of diabetic patients [70]. Human cardiomyocytes derived from iPS cells are another source of cells capable of inducing myocardial regeneration for the recovery of cardiac function. These cells were established using human dermal fibroblasts transfected with a retrovirus carrying the conventional factors Oct3/4, Sox2, Klf4, and c-Myc. When the iPS cells were transplanted into the myocardial infarcted area in a porcine model of ischemic cardiomyopathy, the activation of WNT signaling pathways induced cardiomyogenic differentiation. It was also observed that the transplanted cells significantly improved cardiac function and attenuated left ventricular remodeling [71]. In another study, dopaminergic neurons derived from protein-induced human iPS cells exhibited gene expression, physiology, and electrophysiological properties similar to the dopaminergic neurons found in the midbrain. The transplantation of these cells significantly rescued the motor deficits of rats with striatal lesions, an experimental model of Parkinsons disease [72]. Moreover, after stroke-induced brain damage, adult human fibroblast-derived iPS cells were transplanted into the cortical lesion and, one week after the transplantation, there was the initial recovery of the forepaw movements. Moreover, engrafted cells exhibited electrophysiological properties of mature neurons and received synaptic input from host neurons [73].

In October 2018, 2.4 million iPS cells reprogrammed into dopaminergic precursor cell neurons were implanted into the brain of a patient in his 50s. In the three-hour procedure, the team deposited the cells into twelve sites, known to be centers of dopamine activity. The patient showed no significant adverse effects [74]. The first allogeneic clinical trial using iPS cells derived from mesenchymal stem cells for the treatment of graft-versus-host disease has also been reported, and no treatment-related serious adverse effects were observed [75]. Other clinical studies using iPS cells are being conducted in patients with heart failure [76,77]. Moreover, other tests have been approved for neural precursor cells for spinal cord injuries [78] and corneal epithelial cell sheets for corneal epithelial stem cell deficiency [79]. Thus, ongoing clinical tests provide a better understanding of clinical aspects involving immunosuppressants and fundamental elements such as genomic data that will pave the way for therapies using iPS cells.

The iPS cells have the potential to revolutionize the field of neurodegenerative diseases, which are characterized by the progressive deterioration of neuronal function. Therefore, multiple capacities are affected, leading to cognitive impairment, memory deficits, deficiency in motor function, loss of sensitivity, dysfunction of the autonomous brain system, changes in perception, and mood [80]. Among neurodegenerative diseases, Alzheimers disease is the most prevalent form of dementia, characterized by the accumulation of amyloid-beta (A) plaques and Tau-laden neurofibrillary tangles. Tau is a microtubule-associated protein found in the axons of the nerve cells, and these aggregates and tangles are the histopathological hallmarks of the disease [81]. The dysfunction and degeneration of neurons indeed underlie much of the observed decline in cognitive function, but various other types of non-neuronal cells are increasingly being implicated in the disease progression [82]. Therefore, iPS cells are emerging as an invaluable tool to better modeling the complex interactions that occur between multiple cell types in vivo. 3D and co-culture systems of iPS-derived cells in vitro hold promise to better understand the relevance of multiple cell types and the pathomechanisms that underlie the disease progression. Therefore, iPS cells have been generated from patients and healthy donors to study multiple genetic mutations in neurons, astrocytes, oligodendrocytes, microglia, pericytes, and vascular endothelial cells [83]. Moreover, a mutant Tau model derived from iPS cells was generated and showed several phenotypes associated with this neurodegenerative disease, including the pathogenic accumulation of Tau for drug screening [84]. Choi et al., 2014 showed a 3D culture model based on iPS cells that histopathologically reproduces the hallmarks of Alzheimers disease, including a robust extracellular deposition of A. This model was sensitive to drugs, which reversed the pathological phenotype [85]. The use of neural models derived from iPS cells can validate molecular mechanisms identified in the disease models in rodents, for example, and play an important role in the discovery and screening of new drugs [86].

Parkinsons disease is another important disease; being the second most common neurodegenerative disorder, it affects 2% to 3% of the population over 65 years of age. Characteristic features of Parkinsons disease include neuronal loss in specific areas of the substantia nigra and widespread intracellular protein (-synuclein) accumulation [87]. Due to the loss of dopaminergic neurons in localized regions of the brain, the use of human cells for therapeutic purposes has been studied with special attention. These assessments include iPS cells, whose good results supported the deployment of some studies that are already in the clinical phase. Pre-clinical studies have shown the efficient generation of iPS cells-derived dopaminergic motor neurons from non-human primates. Then, these cells were efficiently transplanted into a model of Parkinsons disease in rats [88]. Several new protocols have improved the efficiency of obtaining dopaminergic neurons from iPS cells for the study and modeling of Parkinsons disease [89]. The iPS cells used in some studies were mainly from patients carrying mutations in synuclein alpha, leucine-rich repeat kinase 2, PTEN-induced putative kinase 1, parkin RBR E3 ubiquitin-protein ligase, cytoplasmic protein sorting 35, and variants in glucosidase beta acid [90]. Although improvements are still needed, iPS cells make it possible to develop patient-specific disease models using disease-relevant cell types. Interestingly, using a human iPS cells-derived model of Parkinsons disease, it was found that the myocyte enhancer factor 2C-peroxisome proliferator-activated receptor- coactivator-1 (MEF2C-PGC1) pathway may be a new therapeutic target for Parkinsons disease. The data from this study provided mechanistic insight into geneenvironmental interaction in the pathogenesis of the disease [91]. Thus, it is important to develop models of neurodegenerative diseases using iPS cells because they involve a complex interplay of genetic alterations, transcriptional feedback, and endogenous control by transcription factors. Probably, the combination of different experimental approaches, using cellular systems and animal models, will increase the successful translation to the clinical practice [92].

In a successful pre-clinical study, the authors demonstrated that human dopaminergic neurons generated from iPS cells, and transplanted into a primate model of Parkinsons disease, established connections with the host monkey brain cells with no tumor formation after two years [93]. Immediately after the successful animal experiments, the Japanese research group implanted reprogrammed stem cells into the brain of a patient with Parkinsons disease for the first time in 2018 (as NEWS Reported by Nature https://www.nature.com/articles/d41586-018-07407-9).

Recently, extracellular vesicles/exosomes derived from iPS cells of different lineages were involved in neurological diseases. Extracellular vesicles are lipid-enclosed structures with a diameter of 301000 nm, carrying transmembrane and cytosolic proteins. Exosomes are a subset of extracellular vesicles, with a diameter ranging between 30 and 200 nm. Functionally, they play an important role in intercellular communications, immune modulation, senescence, proliferation, and differentiation in various biological processes, and are vital in maintaining tissue homeostasis [94]. On the other hand, and as cited before, abnormal protein aggregation has been implicated in many neurodegenerative processes that lead to human neurological disorders. Recent reports suggested that exosomes combine these two important characteristics, as they are involved in the intercellular transfer of macromolecules, such as proteins and RNAs, and seem to play an important role in the aggregate transmission among neurons [95]. The authors showed that extracellular vesicles from iPS cells carry proteins and mRNA that can induce or maintain pluripotency, which can be used in regenerative strategies for neural tissue [96]. If this is true, extracellular vesicles/exosomes derived from corrected iPS cells, which do not accumulate protein aggregates, may be safer for human treatment than iPS cells themselves [94]. The infusion of neuronal exosomes into the brains of a murine model of Alzheimers disease decreased the A peptide and amyloid depositions [97]. Moreover, exosomes obtained from stem cells were able to rescue dopaminergic neurons from apoptosis [98]. The authors showed that extracellular vesicles from mesenchymal stem cells, when injected into a mouse model of Alzheimers disease, reduced the A plaque burden and the number of dystrophic neurites in the cortex and hippocampus [99]. Extracellular vesicles were also derived from human iPS neural stem cells and used for stroke treatment [100]. The results using extracellular vesicles/exosomes obtained from iPS cells point to a promising future in the treatment of neurodegenerative diseases.

Muscular dystrophies (MD) are a group of genetic diseases that lead to skeletal muscle wasting and may affect many organs (multisystem) [101]. The terminal pathology often shows muscle fibers necrosis and muscle tissue replacement by fibrotic or adipose tissues. Currently, there is no cure for MD, and the available treatments are palliative or of limited effectiveness [102]. The most frequent and one of the most severe forms of all MD is the Duchenne muscular dystrophy (DMD), a muscle pathology caused by the lack of the protein dystrophin. In this case, previous cell-based therapies did not show satisfactory results after myoblast transplantation [103]. Myoblasts are the progeny of muscle satellite cells (SC), the main stem cell population found in adult skeletal muscles. Quiescent SCs are triggered to reenter into the cell cycle mainly by muscle damage, and the SC-derived myoblasts proliferate and fuse to form new multinucleated myofibers [101]. In most myoblast-based therapies, allogeneic cells were obtained from muscle biopsies from healthy donors, resulting in transplanted cell rejection by the immune system, with low surviving rates, poor dispersion, and differentiation [103,104,105]. With the advances of iPS cell technology, some of these issues are being addressed (Figure 2).

iPS cells in Duchene muscular dystrophy cell therapy. The somatic cells derived from specific patients with Duchenne muscular dystrophy (DMD) can be reprogrammed into iPS cells with reprogramming factors. These cells are then genetically corrected to express the protein dystrophin for the autologous muscular injection of muscle-committed cells.

One of the main problems in the application of stem cell therapy in muscle diseases is to obtain large numbers of cells for sufficient engraftment, and the use of iPS cells may overcome this obstacle. For this purpose, Darabi and colleagues [106] applied the conditional expression of Pax7 to iPS cells, a transcription factor that plays a role in SC proliferation. Then, Pax7+ iPS cells were obtained on a larger scale for transplant into a mouse dystrophic muscle, which showed dystrophin+ fibers with superior strength [106]. Moreover, the authors genetically restored the dystrophin expression in autologous iPS cells derived from DMD patients. For this, three corrective methods were used, which were exon knock-in, exon skipping, and frameshifting, and the exon knock-in was the most effective approach [107]. The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is a technology that has also emerged as an approach capable of targeting the mutated dystrophin gene, aiming to rescue its expression in vitro in iPS cells derived from selected patients [108].

Moreover, using CRISPR-Cas9 technology with single guide RNA, dystrophin expression was restored by exon skipping through myoediting in iPS cells. The genetic alterations observed in the multiple patients included large deletions, point mutations, or duplications within the DMD gene. The corrected iPS cells efficiently restored the expression of dystrophin and the corresponding mechanical contraction force in derived cardiomyocytes [109]. In summary, several methods of gene editing have been applied for the correction of the DMD gene to allow the transplantation of genetically corrected autologous iPS cells. Of these, the CRISPR-Cas9 system, in particular, has passed multiple proof-of-principle tests and is now being used in pre-clinical trials (Figure 2).

Reprogrammed fibroblast- and myoblast-derived iPS cells were also obtained from patients with limb-girdle muscular dystrophy type 2D (LGMD2D). This disease is a sarcoglycanopathy caused by mutations in the SCGA gene, which provides instructions for making the alpha component of the sarcoglycan protein complex. This multiprotein complex plays a role in the anchoring of the dystrophin-glycoprotein complex (DGC) to the extracellular matrix and helps to maintain muscle fiber membrane integrity. The iPS cells were expanded and genetically corrected in vitro with a lentiviral vector carrying the human gene encoding the -sarcoglycan. Finally, the transplantation of mouse iPS cells into -sarcoglycan-null immunodeficient mice, an experimental model of the disease, resulted in the amelioration of the dystrophic phenotype [110]. This transplant also showed that iPS cells restored the compartment of SC, an essential checkpoint for sustained muscle regeneration.

Recently, Perepelina and collaborators generated iPS cells from EmeryDreifuss muscle dystrophy associated with the genetic variant LMNAp.Arg527Pro. Patient-specific peripheral blood mononuclear cells were reprogrammed using the Sendai virus system, and the authors comment that this is a useful tool to study laminopathies in vitro [111]. Moreover, using three-dimensional (3D) tissue engineering techniques, artificial skeletal muscle tissue was generated using iPS cells from patients with Duchenne, limb-girdle, or congenital muscular dystrophies [112]. In this way, artificial muscles recapitulated characteristics of human skeletal muscle tissue, providing an invaluable tool to study pathological mechanisms, drug testing, cell therapy, and the development of tissue replacement protocols.

The use of iPS cells still has many challenges ahead before they can be clinically used in the supportive treatment of patients with MD. Among these, we can cite the injection of iPS cells (or muscle-committed iPS-derived cells) into large muscles, the immunological recognition of proteins expressed only after the genetic correction, the capacity of cellular dispersion through the muscle, the number of therapeutic interventions needed to replenish cellular muscle populations, the ability to produce corrected SC for sustained muscle recovery, and the control of transplanted cells death.

To address these and other limitations, we propose that autologous iPS cells be submitted to multiple treatments aiming to improve cellular engraftment and clinical use. Besides the genetic correction of underlying pathological mutations, these cells can be further treated in culture to boost cell proliferation, long-term survival, dispersion in the muscle, differentiation into muscle fibers, and others. We proposed before the use of multiple combined in vitro treatments for adoptively transferred myoblasts for cell-based therapy, and these are summarized in [101]. These treatments include vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (bFGF), Wnt7a, Ursolic acid, and extracellular matrix components. Moreover, the recipient muscle to be injected with the corrected and boosted iPS cells can also be treated to favor the engraftment. These treatments include actinin receptor type 2B inhibitor, IL-6, JAK/STAT 3 inhibitor, growth factors, the coinjection of other supportive cell types, such as macrophages and fibroblasts, and others.

We believe that the correct choice for the ideal combination of the cell type to be reprogrammed into iPS cells, the technical procedure for genetic correction, the in vitro treatments to boost iPS cells, and the in vivo preparation of recipients muscles, hold the key for a more successful application of iPS cells in clinical translation. However, we believe that systemic treatments consisting of the injection of cells will not lead to individual muscle damage and strength improvement. The transplanted cells do not express the required repertoire of molecules necessary for endothelial transmigration. Probably, selected individual and more affected muscles are more likely to benefit from cellular-based therapies, followed by treatments that can increase injected cell dispersion within the muscle.

Currently, publicprivate partnership consortia are providing resources to form iPS cell banks for clinical and research purposes. These banks have coordinated standards to meet international criteria for quality-controlled repositories of iPS cells. Although the use of iPS cells for autologous therapy seems more appropriate, having allogeneic banks of iPS cells already generated and tested would reduce the time needed to start treatment, decrease costs, and increase the chances of recovery of treated individuals [113]. Thus, although many technical challenges must still be overcome, the technology of iPS cells has already taken a marked leap in clinical management and in vitro models to study and treat diseases.

D.G.B.: manuscript preparation and review; S.I.H.: manuscript review and preparation of figure; C.M.C.: manuscript preparation; L.A.A.: manuscript review and figure preparation; A.H.-P.: manuscript and figure preparation and review. All authors have read and agreed to the published version of the manuscript.

This work was funded by CNPq (Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico) grant numbers 407711/2012-0 and 421803/2017-7 and Fundao Oswaldo Cruz.

The authors declare no conflict of interest.

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March 11th, 2025

Stem cell therapy side effects & risks at clinics – The Niche

By daniellenierenberg

What are possible side effects of stem cell therapy ? Patients often reach out to ask about such risks They usually refer to unproven stem cell clinics.

Todays post addresses the scope of stem cell therapy side effects and risks based on available hard data. Its also important to discuss possible unknown risks.

In this post I am focusing on the risks primarily associated with unproven stem cell clinics. Not for established methods like bone marrow transplantation, which have their own risks including the shared one of infection.

Recent publications in journals have helped clarify risks. This literature includes a study by my UC Davis colleague Gerhard Bauer and a special report by The Pew Charitable Trust. Gerhards paper presents the types of side effects that appear more common after people go to stem cell clinics. After closely following this area for a decade I was familiar with many of the examples of problems.

One of the highest profile examples of bad outcomes was the case where three people lost their vision due to stem cells injected by a clinic.

I have included a YouTube video below on stem cell therapy side effects as well.

Why do stem cells pose risks?

One major reason is that stem cells are uniquely powerful cells.

By definition they can both make more of themselves and turn into at least one other kind of specialized cells. This latter attribute is called potency and the process of becoming other cells is called differentiation. The ability to make more of themselves is called self-renewal.

The most powerful stem cells are totipotent stem cells that can literally make any kind of differentiated cell. The fertilized human egg is the best example of a cell having totipotency. The first few cell divisions after that retain the totipotency. Next in the power lineup are pluripotent stem cells including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). These cells are not directly used in therapies.

Adult stem cells are multipotent, which means they can make just a few types of specialized cells.

What is the best type of stem cell? The best type of stem cell depends on the condition that is trying to be treated and may not be the most powerful.

In any case, the power of stem cells is one reason they also pose risks along with mishandling that can cause infections. Stem cells are not always easy to control and misdirected power can do harm.

Let me explain and start with the side effect that seems most concerning to most people but is probably the rarest. Tumor formation.

If someone injects a patient with stem cells, its possible that the self-renewal power of stem cells just wont get shut off. In that scenario, the stem cells could drive the formation of a tumor.

Why wouldnt a transplanted stem cell always eventually hit the brakes on self-renewal? It could be that the stem cell has one or more mutations. For any stem cells grown in a lab, within the population of millions of cells in a dish, there are going to be at least a few with mutations that crop up. Thats just the way it goes with growing cells in a lab. The longer you grow them the more mutations they will have on average.

Even stem cells not grown in the lab have the same spectrum of mutations as the person they were isolated from. It may seem odd to think about, but we all have some mutations.

Research suggests it takes more than one cell with cancer-causing potential to make a tumor in experiments in the lab, but in actual people, we just dont know. Many cancers may arise from one stem cell gone awry. If a clinic injects 100 or 500 million cells, a one-in-a-million rate of potentially dangerous cells means that 100-500 such risky cells end up in the patient. The risk of getting an actual tumor may still be low but I wouldnt take those odds.

The encouraging news here is that the incidence of tumors in stem cell clinic customers, particularly in the U.S., appears extremely low.

The odds of getting a tumor are far lower for cells never grown in a lab but its still possible. Oddly, receiving someone elses stem cells (we call this allogeneic) might pose a lower cancer risk because your immune system is going to see those cells as foreign from the start. Itll reject them. Still, an immunocompromised state could play a role.

Some stem cells, especially those with mutations, might be able to somewhat fly under the radar of the immune system to some extent. This could allow them to grow into a tumor.

The Pew report does a nice job of summarizing risks and there are several reports of tumors.

The possibility of infections after stem cell injections is another risk that is often discussed. Infections from injections of stem cells or other biologics are probably the most common type of side effect. Bacteria can sometimes already be in the product that is injected. Or germs can be introduced by poor injection or preparation methods by the one doing the procedure.

The distributor Liveyon had a product contaminated with bacteria that sickened at least a dozen people who were hospitalized. Some of them ended up in the ICU. A few may even have permanent issues.

Infection risk usually does not arise from the cells themselves.

Another risk is the potential for blood clots.

In the case of adipose biologics life SVF, they mostly consist of a mixture of a dozen or so other kinds of cells found in fat. Fat cells just live in fat so they arent supposed to be floating around in your blood. As a result, after IV injection, many fat cells are thought to get killed right away by the immune system or the microenvironment. While in the blood, fat and other stromal-type cells, whether dead or alive, may catalyze clot formation, which is dangerous.

Some of these cells end up landing in the lungs. There many cells are probably being killed and theres also risk of blood clot formation.

Unproven clinics mainly sell MSCs.

MSCs could have some powerful uses in medicine. I can already see a few rigorous clinical trials that look exciting.

However, the way some unproven clinics use MSCs can be highly risky.

Such cells just shouldnt be injected willy-nilly into dozens of places in patients including into peoples eyes. Further, what are called MSCs by some unproven clinics may also not meet even basic lab standards and may not have the potential of other MSC preps. Some such clinic preps are likely just fibroblasts or mostly dead cells.

MSCs produced in a rigorous manner in clean labs by experienced teams are likely to be a far superior product than that typically made by just any strip mall clinic. I dont endorse any cell therapy clinic selling MSCs at this time, but some are doing far better than others. They do research and publish papers.

Properly conducted injections of unmodified, high quality MSC-type cells or marrow cells into joints or for other orthopedic conditions by qualified providers in theory should pose almost zero risk of pulmonary emboli or cancer. Clinics using excellent procedures and cell products also should pose a very low risk of infection, a risk more similar to getting medical procedures in general even unrelated to stem cells.

Overall, Im not sure I believe such MSCs even from the best clinics can provide lasting benefit for diverse orthopedic conditions, but the overall risks associated with them should be quite low there relatively speaking.

Patients have also asked me about other potential risks of cell injections.

I wrote a post about possible graft versus host disease in stem cell recipients. This would only happen in people receiving someone elses stem cells and probably only with IV administration. Its not clear if GvHD is something that happens to patients after going to clinics selling allogeneic cells. With no immunosuppression, it should be highly unlikely.

Beyond outright tumor formation, it is also possible that stem cells may become an undesired or even dangerous tissue type that isnt technically an actual tumor. The example that comes to mind is the practice mentioned earlier of some clinics injecting fat cells into peoples eyes. What seems to have happened in some cases is that the mesenchymal cells (MSCs) or other cells like fibroblasts that were injected turned into scar tissue, which caused retinal detachment.

In addition, we have seen indications that patients getting IV infusions of stem cells might be at some risk for heart attacks. Perhaps via clot formation. For example, read this piece: Cellular Performance Institute death.

One of the challenges is that it can be difficult to figure out if heart attacks or other outcomes were linked to the actual stem cell procedures or just incidental. Many patients getting stem cells may already be at higher risks for these issues. In any particular case, one can ask: was the cell infusion linked to the death? Im not sure we could ever know. Such outcomes should be carefully tracked and analyzed. One challenge is that adverse events at hundreds of unproven clinics may never be reported or otherwise come to light.

Specific routes of administration may pose unique risks as well. For instance, intranasal stem cells are getting popular with some unproven clinics and could lead to cells or other material ending up in the brain. Intranasal delivery of stem cells could have real promise such as for treating brain conditions, but you need rigorous clinical data to back that up. You need to work with the FDA and send them data. Clinics without such data are already selling the procedure.

Other products in the regenerative sphere that are not stem cells may be risky as well for various reasons. For instance, an exosome product harmed quite a few people in Nebraska. Some problems may relate to product contamination. Here again, exosomes may have promise for some conditions but should not be sold already as therapies at this time.

Finally, stem cells and other cell therapies also pose unknown risks because of their newness and power.

We also just dont have long-term follow-up data to have a clear sense of all major risks after people go to clinics.

In general, so much depends on collecting good data before trying to make money form vulnerable patients.

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Stem cell therapy side effects & risks at clinics - The Niche

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March 11th, 2025

Stem Cell Use to Treat Dermatological Disorders – IntechOpen

By daniellenierenberg

1. Introduction

Stem cells are unspecialized cells and are the essential building blocks of all organisms. They can differentiate into any specialized cell within an organism [1]. In this capacity, stem cells possess the ability to self-renewal, in addition to differentiating into all cells within tissues and ultimately organ systems [2, 3, 4]. Stem cells exist from conception and remain functional through adulthood, with many regulatory factors responsible for their specialization. As stem cells mature, differentiation becomes more limited which is referred to as commitment to a specific lineage. This means a unipotent stem cell is restricted in differentiation compared to a pluripotential stem cell (PSC) that can produce a variety of lineage specific cells. Thus, PSCs are more restricted when compared to a totipotent stem cell (TSC) [5, 6].

TSCs are capable of cell division with the ability to differentiate into mature cells comprising all the physiological systems associated with an intact and complete organism [6]. TSCs have unlimited potential to fully differentiate. This property allows TSCs to form both embryonic and extra-embryonic structures such as the placenta and the tissues associated with pregnancy [7, 8]. An example of a TSC is the zygote that forms after a sperm fertilizes an egg. TSCs will form a blastocyst which develops the inner cell mass (ICM). The ICM contains a unique population of stem cells known as embryonic stem cells (ESCs). ESCs are capable of remaining pluripotent in vitro [9, 10]. ESCs form the three germ layers associated with developmental biology, i.e., ectoderm, mesoderm, and endoderm [10], thus providing the core foundation of an organism through each germ layer by providing all the anatomical and physiological systems of the organism [11].

Pluripotential stem cells (PSCs) form structures associated with only the germ layers [11]. Another example of stem cells possessing pluripotency was achieved following the reprogramming capability to produce induced pluripotent stem cells (iPSCs) [12]. iPSC pluripotency is a continuum, starting from totipotent cells to cells possessing less potency as in multi-, oligo- or unipotent cells. The independence of iPSCs allows for using improved methods that are more promising for therapeutic stem cell use now and for future applications as defined in regenerative medicine [13].

Within their respective lineages, multipotent stem cells can generate more specialized cells. It differentiates blood cell development to form a variety of diverse cells such as erythrocytes, leukocytes, and thrombocytes [14]. A myeloid stem cell is an example where a stem cell may differentiate into different types of leukocytes, e.g., white blood cells such as granulocytes or monocytes, but never erythrocytes or platelets [15].

As mentioned above, during embryogenesis, stem cells form aggregates referred to as germ layers [16]. Once hESCs differentiate into a specific germ layer, they become multipotent stem cells and can only differentiate according to their respective layer. Pluripotent stem cells are present throughout the life of any organism existing as undifferentiated cells [17]. Regulatory signals influence stem cell specialization to create specific tissues that are produced via physical contact between cells through the microenvironment/stroma or as stimulators in the form of cytokines, interleukins, and/or tissue factors secreted by surrounding tissues. These factors from internal sources are controlled via the presence of the genome, i.e., genes, thus DNA acting through transcription translation reactions [11]. Stem cells provide a mechanism designed to function as the bodys internal repair system. For as long as an organism remains functional, its stem cells will continue to provide differentiation pathways to replace more mature cell lineages. This is the repair and replenishment aspect of stem cell vitality [11, 18].

The growth and development of an organism depends on the presence of stem cells. Overall, somatic stem cells such as ESCs can be distinguished based upon their characteristic lineage line of development. ESCs can be derived without isolating them from the inner cell mass; however, their growth potential is limited [11]. ESCs can be propagated in vitro using tissue culture conditions indefinitely without restriction if their growth requirements are maintained [19, 20]. ESCs can be propagated in culture with appropriate culture medium containing essential nutrients [19]. Passage of ESCs is an adequate method of sub-culturing stem cells to propagate their numbers over time. Because ESCs are totipotent, they can differentiate into every cell type required in any organ cell system [21]. However, because totipotent stem cells demonstrate immortality, ethical restrictions restrict the procurement of these cells. The origin of these totipotent stem cells is from the ICM of the blastocyst present in embryos. Thus, the procedure to obtain them destroys the viability of that embryo from further development. However, most ESCs are derived from fertilized eggs in an in vitro clinic rather than from eggs harvested from pregnant women [22].

Among the many stem cell types that exist are as follows:

Hematopoietic stem cells have the potential to differentiate into many types of blood cells, e.g., erythrocytes, leukocytes, and thrombocytes.

Mesenchymal stem cells are found in multiple types of tissues. They can differentiate into multiple lineages such as bone, adipose, vascular, and cartilage tissue. They can be harvested from sources including but not limited to the umbilical cord, bone marrow, and endometrial polyps [23].

Neural stem cells develop into glial or neuronal cells such as nerve cells, oligodendrocytes, and astrocytes. These cells have been used in treatments regarding Parkinsons disease through transplants [24].

Skin stem cells (SSCs) consist of several types that are separated into their own niches including hair follicle stem cells, melanocyte stem cells, and dermal stem cells. SSCs have greater potential to be used for stem cell therapies and treatments since these cells can differentiate into more cell lineages [25].

Human ESCs are involved in whole-body development and can eventually become pluripotent, multipotent, and unipotent stem cells. Compared to adult somatic stem cells, they also have a quicker proliferation time and greater range of differentiation causing them to be more ideal and preferred in therapies [26].

Stem cells can also be taken from the placenta. Placental fetal mesenchymal stem cells can differentiate into a wide variety of cells and are abundant, not requiring invasive procedures to procure. They are not surrounded with ethical issues that ESCs have since the placenta is usually considered medical waste after birth, making it favorable for use as treatment. They can produce ectodermal, endodermal, and mesodermal lineages in vitro and contain the same cell markers as ESCs, making them very similar. Placental stem cells are pluripotent and have low immunogenicity which allows them to be ideal for therapies and treatments [27].

Differentiation was thought to be restricted and non-reversible. However, after several major experiments through cloning, even differentiated cells can be reprogrammed or induced to be pluripotent. Two major cloning-related discoveries were made in 1962 and 1987. The first was done by John Gurdon who cloned frogs through the process of somatic nuclear cell transfer (SNCT) into an enucleated frog egg [28]. This showed that the nucleus of a specialized somatic cell could be reverted and develop cells that could eventually produce an entirely new organism [29]. The specialized somatic cell became pluripotent which, before this experiment, was thought to be impossible [30, 31]. This technique was famously used successfully in the cloning of Dolly, the sheep [28]. The 1987 experiment focused on gene expression. The forced expression of one gene, known as myogenic differentiation 1 (Myod1), could cause fibroblasts to turn into myoblasts [32]. This was another example of transforming cells, but this was done through programming the cell in the DNA.

These discoveries provided the turning point in stem cell research by advancing the therapeutic application of stem cells when a Japanese team of scientists showed adult multipotent stem cells could be reverted into a pluripotent state. These cells functioned like ESCs but did not need to be acquired from embryos. This discovery created a process to avoid endangering the life of a fetus to obtain ESCs. The determining factor in the process using murine fibroblasts was incorporating a retrovirus-mediated transduction system containing four transcription factors found in ESCs known as Oct-3/4, Sox2, KLF4, and c-Myc [17]. These factors induced the fibroblasts to become pluripotent. The newly formed reprogrammed stem cells were named induced pluripotent stem cells (iPSCs). A later study succeeded using human cells [33]. This technological breakthrough created a new line of research in stem cell biology that coincided with the generation of iPSC cell lines. Importantly, as mentioned, iPSCs can be made biocompatible with any patient, thus dramatically improving the therapeutic potential of this newly created cellular therapy [13]. ESCs are still the only naturally occurring pluripotent cells, but from these experiments, terminally differentiated cells can be induced into pluripotency to become iPSCs. Still, reprogramming cells comes with risks to cellular development due to histone alteration. However, an experiment was done by sequencing DNA from murine iPSCs and confirmed that although mutations were introduced, reprogramming cells could create iPSCs that were not seriously genetically affected or produce ill-functioning cells [11, 34].

As these cells are manufactured, controlling the quality of iPSC lines is necessary for use as treatments. Ways that they are controlled for their quality are as follows (Table 1) [35]:

Different ways that stem cells can be verified and tested during growth to ensure their quality and viability.

A common source for iPSCs includes fibroblasts. Especially in treatments, taking the patients own fibroblasts for the treatment has shown to be beneficial as the autologous cells do not risk being rejected. However, at first, they were the only source that could be used, and obtaining these cells required a biopsy. Thus, further research was conducted to enhance the techniques efficiency. Other cell types have also been reprogrammed, but fibroblasts are still preferred since their stimulation can be fast and controlled [36, 37].

Stem cells are only potentially useful if they can be differentiated into specific lineages. If not, they can form a teratoma in vivo. However, this condition can be regulated; therefore, if the process can be controlled, it allows clinicians and researchers to improve their therapeutic use when using specific signaling pathways for differentiation. In regenerative medicine, it is important to ensure that these cells will then differentiate in a timely and efficient manner. Directed differentiation exists to push the ESCs to differentiate. As cells develop, they send out signals within their surroundings [38]. Messages from the extracellular environment can also control the differentiation of stem cells which has been shown in in vitro cultures [39]. This can be done easily in in vitro cultures by controlling the conditions in culture. However, replicating such environments in vivo, has been challenging, requiring strict culture conditions [11].

For hESC treatments to be used on patients, the therapies must be culture-free, meaning the stem cells are not contaminated with any feeder or animal cell components [40]. The FDA requires this pertaining to procurement and storage of any type of stem cells contemplated for human use [41]. A difficulty in procuring these treatments is that great amounts of these cells used for treatment must be cultivated in the absence of feeder cells.

Directed differentiation protocols replicate the development of the ICM during embryogenesis. Pluripotent stem cells differentiate into derived progenitors from each of the three germ layers, just as is observed in vivo. Specific molecules act as growth factors to induce stem cells to become specific progenitor cells eventually to develop into a specific cell type. Growth factors function as important regulatory molecules that affect germ layer development in vivo; examples include bone morphogenic proteins (BMP) [42, 43], fibroblast growth factors (FGFs) [44], transcription factors of the Wnt family [45], or transforming growth factors-beta (TGF). How each factor influences germ cell differentiation is unclear and research is ongoing.

The concentration levels and duration of action of a targeted signaling molecule such as a growth factor produces a variety of outcomes. However, the high cost of recombinant molecules currently restricts their routine use in therapy limiting their clinical application. A more promising approach is to focus on using small molecules, thereby activating or deactivating specific signaling pathways [46]. These methods are effective in improving reprogramming efficiency by helping to generate cells that are compatible with the target tissue type. Also, they offer a more cost-effective and non-immunogenic therapy method [47]. Endogenously generated small molecules, e.g., retinoic acid is effective for patterning nervous system development in vivo. It functions effectively in embryonic development where it is used in vitro in culture systems to induce the differentiation of somatic cells [48, 49]. These cells can also induce retinal cell formation when hESCs are used [50]. Through the control of biochemical signals and the environment as important factors can be essential to achieve optimal hESC differentiation when culturing stem cells.

Culture systems have been regulated by multiple agencies around the world including the Food and Drug Administration (FDA) and the European Medicine Agency (EMA). Initially, animal-derived products were utilized, however, that introduced possible animal pathogens. Some stem cell lines derived from embryos and human feeder cell lines have been established which include stem cell-derived cardiac progenitors and mesenchymal stem cells. Xeno-free culture systems also include the development of human foreskin fibroblasts (HFFs) [11, 51, 52, 53].

Stem cells hold immense promise as an important therapeutic option for the future of medicine. Beyond their crucial role in regenerative medicine, stem cell research has demonstrated their intricate processes when involved in growth development. In stem cells, DNA is loosely organized, allowing genes to remain active. Differentiated cells differ in that these cells deactivate certain genes and activate others that are essential to the signals that the cell receives. This process is reversible, demonstrating that pluripotency can be induced through specific gene modifications. Several core transcription factors including Oct3/4, (SRY)-box 2, and Nanog genes have been found to keep these cells pluripotent [17, 54]. Nuclear transcription factors Oct3/4 and Sox2 are crucial for producing iPSCs [54].

Presently, various therapies using stem cells are offered as treatments for conditions like spinal cord injuries, heart failure, retinal and macular degeneration, tendon ruptures, and type 1 diabetes [52, 55, 56, 57, 58]. Stem cell research improves our understanding of stem cell physiology, potentially leading to new treatments for presently untreatable diseases. Many of which are dermatological disorders which were previously thought to have no good solution. This chapter focuses on the application of stem cells treating various dermatological disorders and compliments recent reviews on the same topic [11, 59].

Stem cell therapy has not been actively used as a solution for restoring hair growth, but current results are promising. One study used harvested autologous adipose-derived stromal vascular cells through injected into the scalp of 20 patients with alopecia areata (AA) [60]. At three and six months of follow-up, all patients produced statistically significant hair growth. Adipose-derived stem cell conditioned medium (ADS-CM) contains growth factors essential for hair follicle regrowth such as basic fibroblast growth factor, hepatocyte growth factor, platelet-derived growth factor, vascular endothelial growth factor, and transforming growth factor-beta (TGF-) [61]. Another study isolated human adult stem cells by centrifuging human hair follicles obtained through punch biopsy and injected them into the scalps of 11 androgenetic alopecia (AGA) patients resulting in an increase in hair density and count compared to baseline and placebo [62]. In a larger study with 140 AGA patients, autologous cellular micrografts containing HFSCs were used as a treatment. Within one session, over two-thirds of the patients showed positive results while there was significant increase in their regrowth and thickness [63, 64].

A study randomly assigned 40 patients (20 with AGA and 20 with AA) to receive either autologous bone marrow-derived mononuclear cells or autologous follicular stem cell injections into the scalp, found significant improvement in hair loss with no significant difference between the two preparations [65]. An investigation introduced a novel stem cell method, termed stem cell educator therapy in which patients mononuclear cells are separated from whole blood and allowed to interact with human cord bloodderived multipotent stem cells, thus educating these stem cells after returning them to patients [61]. In nine patients with severe AA, all but one experienced improved hair regrowth of varying degrees. Two patients (one with alopecia totalis and one with patchy AA) experienced complete hair regrowth at 12weeks without relapse after two years. A combination of platelet-rich plasma and stem cell technology also showed promising results [61].

Numerous murine studies have demonstrated the progression of allergies in atopic dermatitis (AD) can be inhibited by using umbilical cord blood mesenchymal stem cells (UCB-MSCs), bone marrow mesenchymal stem cells (BM-MSCs), or adipose-derived mesenchymal stem cells (AD-MSCs) [66, 67, 68, 69]. It is important to consider the type of stem cell used, the number of cells transplanted, the preconditioning of the cell preparation, the therapys relevant targets, and the route and frequency of administration. One example highlighting the complexity of stem cell-based therapy was shown in a study where human UCB-MSCs were pre-treated with mast cell granules [68]. This pre-treatment method enhanced their therapeutic effectiveness, as evidenced by the reduced signs of AD in a NC/Nga mouse model. It was found that hUCB-MSCs primed with mast cell granules were more effective in suppressing the activation of mast cells and B lymphocytes compared to nave MSCs, both in vitro and in vivo [70].

Despite promising results from murine studies in AD, only a few clinical trials have been conducted. In one study, a single subcutaneous administration of hUCB-MSCs was given to 34 adult participants with moderate-to-severe AD [66]. The improvement in AD symptoms was measured using the eczema area and severity index (EASI) score. Treatments for both low and high doses of hUCB-MSCs showed symptom improvement. In the higher dose group, six out of 11 subjects experienced a 50% reduction in EASI score, with no reported side effects. Additionally, typical biomarkers of AD, such as serum IgE levels and the number of eosinophils, decreased after treatment.

A later clinical trial had the injection of clonal mesenchymal stem cells (MSCs) into five patients with atopic dermatitis (AD) who had not responded to conventional treatments [71]. Patients received either one or two cycles of MSC treatment. Effective treatment was evaluated using cytokine biomarkers (CCL-17, CCL-22, IL-13, IL-18, IL-22, and IgE) and EASI scores. Results showed four out of five patients achieved more than a 50% reduction in EASI scores after one treatment cycle. Additionally, significant decreases in IL-13 and IL-22 levels were observed with other biomarkers showing decreasing trends during the studies.

In a more recent phase 1 clinical trial published in 2024, 20 subjects were treated intravenously with human clonal MSCs, given a low dose of cells in Arm 1 and a higher dose in Arm 2. There was an overall improvement for both arms, and the difference in dosage did not make a statistically significant effect. A phase 2 trial proceeded and was randomized, double-blind, and placebo controlled. In this, 72 subjects were tested. The half given the treatment were given the high dosage of hcMSCs originally tested in phase 1. Compared to the placebo group, the treated group had a statistically significant improvement response [72]. These findings suggest MSC administration might help normalize the immune system in AD patients. However, further studies are needed to understand the long-term mechanisms and effects of MSC treatment in this context.

Dermatomyositis remains a mystery with its exact etiology still unknown. Research using stem cells to treat the disease is limited with few studies and case reports available. One report detailed successful autologous stem cell transplants for two patients with juvenile dermatomyositis who had not responded to initial treatments [73]. In the first patient, the procedure involved transferring CD3/CD19-depleted mobilized peripheral blood mononuclear cells (PBMCs), which included 7.5106/kg CD34+ stem cells and 2.9104/kgT cells. Following a 26-month follow-up period, significant improvements were observed. The Childhood Myositis Assessment Scale (CMAS) score increased from 6 to 51and the manual muscle testing (MMT) score rose from 61 to 150. These results demonstrated a substantial improvement in symptoms with the patient regaining the ability to walk and showing significant reductions in inflammatory reactions after the autologous stem cell transplant.

In the second patient, a similar response was observed. The patient was treated with CD3/CD19-depleted autologous PBMC graft (7.51106/kg CD34+; 1.6104/kg CD3+). After three months of treatment, the patient had less muscle pain and contractures, and she began also regained the ability to walk [73].

An uncontrolled study in which 10 patients received allogenic mesenchymal stem cell therapy was reported where one or two MSC infusions were given to patients depending on whether they had disease recurrence within a short time after initial treatment. Out of the 10 patients, eight showed significant clinical improvement, with their symptoms improving after MSC therapy [74]. However, further research is required to evaluate the long-term effects of MSC treatment in patients with dermatomyositis.

Epidermolysis bullosa (EB) is a genetic condition that currently has no treatment, but stem cell therapy is one cell-based therapy under investigation that may be able to correct the skin and its underlying genetic component. Autologous or allogenic stem cells are options that can be used, with mesenchymal stem cell therapy showing potential; therefore, they may be more useful in alleviating some symptoms when tested in additional studies.

One study followed two patients with severe generalized recessive dystrophic epidermolysis bullosa (EB) treated with intradermal administration of allogenic mesenchymal stem cells from bone marrow showed complete healing of ulcers around the treated site by 12weeks [75]. Type VII collagen was detected along the basement membrane zone and the dermal-epidermal junction was continuous in the treated site 1week after treatment. Unfortunately, the clinical effect lasted for only 4months in both patients.

In the case of junctional EB treated with primary cultured keratinocytes, it showed normal morphology and the absence of spontaneous and induced blisters or erosions at 21months of follow-up [76]. Studies using BMSCs to treat recessive dystrophic EB have also shown promise [77, 78]. One study investigated 10 recessive dystrophic EB children treated with intravenous allogeneic bone marrow-derived mesenchymal stem cells and found that the procedure was well tolerated with minimal side effects over the nine-month period [79]. However, skin biopsies performed at the two-month time point showed no increase in type VII collagen and no new anchoring fibrils. While the initial clinical improvement was favorable, it was not maintained over time due to insufficient production of durable proteins like collagen and laminins. The current evidence for stem cell therapy in treating EB is limited because few patients have been treated. This underscores the need for additional research to assess the therapys effectiveness and the balance of its risks and benefits [80].

Despite significant progress in understanding psoriasis pathogenesis in recent years, it remains unclear what is the exact etiology. Current research suggests that dysfunction in certain types of stem cells might be a primary cause of the inflammatory response dysregulation in psoriasis [81]. This hypothesis came after observing long-term remission in psoriasis patients who underwent hematopoietic stem cell therapy [82, 83]. Conversely, there have been reports of acquired psoriasis in patients who received bone marrow transplants from donors with psoriasis, indicating a significant role of hematopoietic stem cells in disease pathogenesis [84, 85]. MSCs have also shown success in treatment likely due to their engraftment, paracrine, or immunomodulatory effects [86]. However, the availability of cost-effective and safe alternatives limits the use of stem cell transplantation as a practical option for treating psoriasis.

Scleromyxedema is a chronic fibro-mucinous disorder that can result in respiratory complications. A study conducted on five patients who underwent high-dose chemotherapy followed by stem cell rescue led to durable remission in most cases, although it did not cure the disease [87]. Another study showed scleromyxedema was successfully treated with chemotherapy and autologous stem cell transplantation [88]. The patient achieved complete recovery within six months and remained in remission for 3years post-transplantation. In a 2022 report, a male patient underwent an autologous hematopoietic stem cell (HSC) transplant after previous therapies failed to improve his symptoms. Improvements were seen in the patients skin, but the renal and pulmonary complications required the use of steroids and plasmapheresis. Unfortunately, the patient contracted SARS-CoV-2 virus and died [89]. More studies still need to be done to determine if stem cell therapy might be useful alone or combined with other therapies to treat scleromyxedema.

Systemic sclerosis (SSc) is an autoimmune disease characterized by excess collagen in the internal organs and skin, causing ulcers and organ damage. HSC therapy and MSC therapy have been tested and found to improve pain, blood flow, lung function, among other symptoms of the disease [90]. Autologous hematopoietic stem cell therapy is preferred over allogeneic therapy due to its lower treatment-related mortality and absence of graft-vs.-host disease [91].

Stem cell therapy has been extensively studied in three randomized controlled trials: the American Scleroderma Stem Cell versus Immune Suppression Trial (ASSIST, phase 2, 19 patients), the Autologous Stem Cell Transplantation International Scleroderma Trial (ASTIS, phase 3, 156 patients), and the Scleroderma Cyclophosphamide or Transplantation study (SCOT, phase 3, 75 patients), with several pilot and case studies [92, 93, 94]. These studies have demonstrated autologous hematopoietic stem cell therapy is an effective and safe treatment for systemic sclerosis. However, patients with severe major organ involvement (pulmonary, cardiac, or renal) or serious comorbidities were excluded from all three trials due to contraindications [59].

MSC therapy has the ability to suppress innate and adaptive immunity and can differentiate into a wide variety of tissues, making it seem like an ideal choice for SSc [95]. However, if donors are not carefully chosen, there is the chance that collagen production can be increased, thus this therapy can worsen symptoms [96]. This research suggests that autologous MSCs from patients that have advanced stage SSc should not be used for treatment. On the other hand, allogenic MSC therapy has lived up closer to the promises of stem cell therapy. Allogenic MSCs were administered intravenously in a female patient, where her skin condition improved, reducing the appearance of ulcers and her pain score [95]. In a clinical trial, combining MSC therapy with plasmapheresis was shown to improve lung function and skin thickness shown in improved modified Rodnan Skin Scores. The current research suggests that MSC therapy may be most effective when paired with another therapeutic option, but research still needs to be done to explore this.

Stem cell therapy has been found to be more effective than conventional immunosuppressive drugs and is currently the only disease-modifying strategy that improves long-term survival, prevents organ deterioration, enhances skin and pulmonary function, and improves overall quality of life.

The European Society for Blood and Marrow Transplantation (ESBMT) and the British Society of Blood and Marrow Transplantation (BSBMT) classify autologous hematopoietic stem cell therapy in severe resistant cases as a clinical option, requiring a risk-benefit assessment [97, 98]. Guidelines from the American Society for Blood and Marrow Transplantation (ASBMT) categorize this therapy as standard of care, rare indication for children (indicating it is an option for individual patients after careful risk-benefit evaluation) and developmental for adults [98]. Patients with acute onset rapidly progressive disease refractory to conventional therapy and mild initial organ damage carry a better prognosis after HSC therapy. Patients with long standing conditions, indolent course and/or irreversible organ damage are contraindications to this therapy [99]. Thus, the challenge is to identify patients who are likely to be benefitted with HSC therapy.

HSC therapy has been tested in patients with refractory systemic lupus erythematosus (SLE). Many observational studies and clinical trials have been aimed at assessing the effectiveness and safety of this transplant approach [100, 101, 102]. In a long-term follow-up of a female patient who underwent allogenic BM-HSC treatment, her systemic lupus erythematosus disease activity index (SLEDAI) score was found to improve, pain improved, and engraftment remained functional [103]. Collectively, these reports show HSCs to be beneficial for patients with a shorter duration of refractory disease suggesting that earlier intervention might lead to better outcomes [104].

The therapeutic potential of MSCs has been investigated for various autoimmune diseases including SLE [105]. In a recent study, six refractory SLE patients were treated with an intravenous infusion of MSCs. Five of the patients reached the threshold for improvement, achieving an SLE Responder Index (SRI) of 4 [106]. In a separate long-term follow-up study done in 2021, 81 patients were treated with allogenic BM-MSC and/or UC-MSCs. After 5years, 37 patients had achieved clinical remission. MSC therapy has been shown to improve patient survival and reduce the severity of the disease as it has been shown to be safe and effective in treatments [107]. MSCs have been shown to alleviate SLE severity, improve renal function, decrease autoantibody production, upregulate peripheral T-cells, and restore balance between Th1- and Th2-related cytokines [108]. These collective immunomodulatory and regenerative properties position MSCs as a promising treatment for SLE.

Steroid topical treatment is the first line of therapy for vitiligo, but when it proves ineffective, surgical options may be viewed next [109, 110]. Cellular grafts using autologous non-cultured outer root sheath hair follicle cell suspension (NCORSHFS) have been tested as a method to treat vitiligo [111]. This method utilizes the regenerative capacity of hair follicle melanocytes, as they can repigment areas where vitiligo has caused depigmentation by allowing melanocyte precursors to proliferate into the areas that lack melanocytes, making them preferable over epidermal melanocytes for cell-based vitiligo treatments. One study reported NCORSHFS achieved an average repigmentation rate of 65.7%, with more than 75% repigmentation observed in nine out of 14 patients [112]. Another study investigated factors affecting therapeutic outcomes in 30 patients with 60 target lesions treated with NCORSHFS [111]. They found that 35% of the lesions achieved repigmentation greater than 75%. The study showed patients who achieved optimal repigmentation had significantly higher numbers of transplanted melanocytes and hair follicle stem cells. Also, the absence of dermal inflammation was a significant predictor of successful repigmentation. These results emphasize the importance of specific cellular components, and a favorable dermal environment is necessary for the effective treatment of vitiligo with NCORSHFS.

Another promising stem cell treatment for vitiligo is multilineage-differentiating stress-enduring (MUSE) cells [113]. In three-dimensional skin culture models, ex vivo studies have identified factors that encourage MUSE cells to differentiate into melanocytes. The melanocytes are integrated into the epidermis, promoting melanogenesis. However, the impact of MUSE cells in vivo remains to be determined [114].

Chronic or non-healing skin wounds present an ongoing challenge in advanced wound care. Current wound healing treatments remain insufficient. Stem cell therapy has emerged as a promising new approach for wound healing using MSCs [115]. MSCs are an attractive cell type for cell-based therapy due to their ease of isolation, vast differentiation potential, and immunomodulatory effects during transplantation. MSCs are known to play a key role in the wound healing process making them an obvious candidate for clinical use. When introduced into the wound bed, MSCs have been shown to promote fibroblast migration, stimulate extracellular matrix (ECM) deposition, facilitate wound closure, initiate re-epithelialization, enhance angiogenesis, and mitigate inflammation in preclinical animal models. MSC efficacy and safety use for the treatment of chronic wounds was further confirmed by several clinical studies involving human subjects which yielded similar positive results with no adverse side effects [116]. However, while MSCs appear to be a promising resource for chronic wound care, additional studies are needed to determine optimal cell source and route of delivery before this treatment can be recommended for clinical use.

MSCs for the treatment of chronic wounds has proven to be feasible, effective, and safe, reported through preclinical and clinical trials [117]. MSCs stimulate the healing process in chronic wounds through several biological and molecular mechanisms. One of the primary roles of MSCs is to promote the directional migration of fibroblast cells to the injury site where they can localize in the wound bed [115, 118]. Once localized fibroblasts facilitate wound closure and synthesize the necessary components of the ECM such as collagen. MSCs can also downregulate MMP-1, a type of collagenase primarily responsible for ECM degradation. MSCs function to preserve ECM and maintain dermal structure. MSC-treated wounds have increased elastin levels which provides recovering tissue with resiliency that is not typically seen in normal wound healing [116]. MSCs play a role in the re-epithelialization process by activating the proliferation, differentiation, and migration of keratinocytes that support the formation of a multi-layered and well-differentiated epidermis [117, 119].

MSCs are believed to stimulate the development of new hair follicles and sweat glands, which suggests these stem cells are capable of not only accelerating wound healing but also improving the quality of wound healing. MSCs use for chronic wounds supports angiogenesis by upregulating VEGF and Ang-1 increasing microvessels throughout the wound bed [120]. This allows the nutrient and oxygen transport to developing cells enhancing their longevity. Also, MSCs help to modulate the wound environment and in turn support proper healing by mitigating inflammation at the site of injury. Importantly, MSCs decrease infiltration of inflammatory cells and pro-inflammatory cytokines and initiate the polarization of M1 macrophages to anti-inflammatory M2 macrophages. MSCs also downregulate ICAM1, a protein involved in inflammation, and upregulate superoxide dismutase, an enzyme which breaks down harmful superoxide radicals [118, 121]. By supporting wound healing MSCs by optimizing the healing environment can produce efficient wound closure.

Several clinical trials in human subjects have generated positive results when MSCs were applied to chronic or non-healing wounds [122]. No adverse side effects have been observed which confirms the safety and feasibility of this cellular therapy for human application. However, further research is needed to determine the best cell source and route of delivery before this procedure can be recommended for human use clinically.

MSCs can be isolated from various tissue types including bone marrow, adipose tissue, cord blood, and placenta. MSCs demonstrate unique properties. Several comparative studies have reported MSCs as the most promise for cell therapy due to their abundance and ease of isolation as well as their regenerative and immunomodulatory properties [123]. How these MSCs are delivered into the wound is the critical question. MSCs can be delivered locally to the wound bed via injection, topical application, or incorporation into a 3D scaffold to avoid issues related to low engraftment efficiency observed following IV injection [124, 125]. Investigating local delivery methods, MSCs seeded into a biomaterial scaffold appears to hold promise as it allows for the localization of the cells into the wound bed and provides donor cells with protection and structure [126, 127]. Following additional research, the application of MSCs for chronic or non-healing wounds could provide a major development in advanced wound care.

Epidermal stem cells have potential to regenerate the epidermis and differentiate under appropriate stimuli into various skin cell types and tissues [128]. This property can be used to initiate and accelerate healing of chronic non-healing wounds. MSCs promote wound healing by decreasing inflammation, promoting angiogenesis, and decreasing scarring [129]. One study successfully applied human MSCs to non-healing and acute wounds using a specialized fibrin spray system [130]. Another study demonstrated the efficacy of stem cell therapy in diabetic foot ulcers [131].

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March 1st, 2025

Skin Regeneration and Rejuvenation | Harvard Stem Cell Institute (HSCI)

By daniellenierenberg

Whether through injury or simple wear and tear, the skins integrity and function can be easily compromised. Although this impacts billions of people worldwide, little is known about how to prevent skin degeneration.

The Harvard Stem Cell Institute (HSCI) Skin Program is committed to understanding why skin sometimes fails to heal or forms scars, as well as why skin inevitably becomes thin, fragile, and wrinkled with age. The Skin Programs ultimate goal is to identify new therapies for skin regeneration and rejuvenation.

Wound healing is a major problem for many older individuals. Furthermore, chronic, non-healing skin ulcers are a major source of health care costs and patient morbidity and mortality.

Human skin repairs itself slowly, via the formation of contractile scars which may cause dysfunction. In contrast, the axolotl salamander can readily regrow a severed limb, the spiny mouse has densely haired skin that heals with remarkable speed, and the skin of the growing human embryo can regenerate after trauma without the need for any scar formation. By studying these examples, scientists are finding clues for how to enhance skin healing through a more regenerative response.

During normal wound healing, scars form from dermal cells that align in parallel. But when this alignment is disrupted by a biodegradable scaffold that directs cells to grow in a random orientation, the cells follow the diverse differentiation program necessary for true regeneration.

HSCI scientists have also identified biomarkers for the key cells involved in skin regeneration, and are developing therapeutic strategies for their enrichment and activation. Ongoing clinical trials are using skin stem cells to treat chronic, non-healing ulcers, and early results are promising.

Additional approaches include 3D bioprinting, where skin stem cells are layered into a complex structure that mimics skin and could be potentially used for transplantation.

Skin aging can be thought of as a form of wounding, in which stem cells no longer maintain normal skin thickness, strength, function, and hair density. Understanding how to harness stem cells for scarless wound healing will also provide key insights into regenerating aged skin, a process termed rejuvenation. Multidisciplinary collaborators in the HSCI Skin Program are investigating the biological basis for how the skin ages over time and when exposed to ultraviolet radiation.

In addition to aging, skin stem cells also may mistake normal regions of the skin as wounds, then erroneously attempt to fill them. HSCI investigators are exploring whether this may be one of the underpinnings of psoriasis, a common and devastating disorder.

These areas of investigation are just the beginning. Skin stem cell biology has the potential to provide key insights into the mechanisms of regeneration for other organs in the body.

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Skin Regeneration and Rejuvenation | Harvard Stem Cell Institute (HSCI)

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March 1st, 2025

A Beginners Introduction to Skin Stem Cells and Wound Healing – MDPI

By daniellenierenberg

Covering an average surface area of 1.85 m2, and accounting for ~15% of total body weight, the skin is considered the largest organ in the human body. Its primary function is that of a physical barrier against microbial pathogens, toxic agents, UV light, and mechanical injury [1]. However, this function can also extend into other vital functions, such as thermoregulation, protection against dehydration, and the excretion of waste metabolites [2]. Moreover, the skin also represents a major metabolic site, yielding a broad range of biomolecules, e.g., vitamin D [3].The skin is composed of two main layers, i.e., the epidermis and the dermis. Previously, another layer had been described within the skin, i.e., hypodermis [4]; however, there is an ongoing controversy in this regard and the hypodermis is now considered as part of the dermis. The skin contains accessories, such as hair, nails, and sweat, and sebaceous glands [5]. In addition, the skin is also populated by nerve receptors that can be triggered by external stimuli (e.g., touch, heat, pain, and pressure) [6]. The skin layers have different thickness according to their anatomical location; for example, the epidermis can be very thin in the eyelids (0.1 mm) whereas it can be thicker in the palms and soles of the feet (1.5 mm). In contrast, the dermis can be ~3040 times thicker in the dorsal area than the corresponding epidermal layer [2].The epidermis can be further sub-divided into strata with a unique cell composition, i.e., keratinocytes, dendritic cells, melanocytes, Merkels cells, and Langerhans cells. These epidermal layers are known as stratum germinativum, stratum spinosum, stratum granulosum, stratum lucidum, and stratum corneum. The first of these strata, also known as the basal cell layer, conforms the inner-most part of the epidermis [2,7]. It is in this layer that different populations of stem cells (SCs) are located, and which, through extensive proliferation and differentiation, provide the great regeneration capacity of the skin and enable the generation of auxiliary structures, e.g., nails and sweat glands [8]. It must be mentioned that the basal cell layer is not the only stem cell niche within the skin as these cells can also be found within the hair follicle (HF), interfollicular epidermis (IFE), and sebaceous glands [8], all of which are contained within the basal layer itself. The stem cells within the skin are usually named after the niche in which they reside in, i.e., hair follicle stem cells (HFSCs), melanocyte stem cells (MeSCs), interfollicular epidermis stem cells (IFESCs), and dermal stem cells (DSCs). Regardless of their niche, these cells are collectively known as skin stem cells (SSCs) (Figure 1).The main task of these SSCs is to replace, restore, and regenerate the epidermal cells that may have been lost, damaged, or have become pathologically dysfunctional [9,10]. For such end, a carefully orchestrated cell division, both symmetrical and asymmetrical, is required to both maintain the stem cell pool and produce lineage-committed cell precursors [11]. Initially, SSCs were thought to be age-resistant, mostly because their number does not seem to dwindle through time [12,13]. However, despite their longevity, SSCs eventually become unstable or dysfunctional and display a lower differentiation and self-renewal capacity [14].As previously mentioned, SSCs are found in diverse niches within the skin, of which the hair follicle has been the most studied. The distinct anatomical zones of the HF can house different stem cell types, such as HFSCs and MSCs [15,16]. The bulge region of the HF contains different stem cell populations; however, the exact identity of these cells is still unclear. Regardless, the presence of both proliferative (CD34+/LGR5+) and quiescent (CD34+/LGR5) stem cells has been described in previous research [16,17].Overall, the diverse subpopulations of SSCs have specific characteristics that set them apart from one another. For instance, HFSCs are mostly quiescent until triggered by several factors secreted by their progeny and by adjacent dermal cells [18]. Regarding the former, their isolation has been so far complicated by the lack of specific markers to identify them [19]. In addition to the hair follicle bulge, SSCs can also populate the sebaceous glands; however, these stem cells are thought to be unipotent and dedicated exclusively to the renewal of the sebocytes pool [16,20]. Other proposed niches are found within the compartments of the dermal papilla (DP) and the dermal sheath (DS) [9,16] and, unlike the stem cells located in the sebaceous gland, those located in both the DP and DS display a greater differentiation capacity, even being able to differentiate into cells of hematopoietic lineages [9], and have also been involved in the maintenance and repair of the dermal tissue. Melanocyte stem cells (MeSCs) are also located in the bulge and hair germ of the HF. Interestingly, their proliferation and differentiation seem to be closely tied to that of HFSCs [21]. Therefore, the concurrent activation of both MeSCs and HFSCs by the signals originating from the latter is hardly surprising. Due to their embryonic origin (i.e., neural crest), MeSCs possess high proliferative and multipotent capacity, which makes them interesting for regenerative medicine [22] and stem cell-based therapies [15,23]. In this regard, dermal stem cells (DSCs) are also considered as an accessible and abundant source for stem cell-based therapies [24] as they display great plasticity and the potential to differentiate into cells of ectodermal, mesenchymal, and endodermal lineages [24,25]. Consistently, the niche of these cells has been localized to the DP and DS [26]. IFESCs, on the other hand, are difficult to isolate and identify due to their unclear location within the basal layer. Therefore, their study has been mostly conducted through indirect means, such as screening with cell surface markers [27,28] or lineage analysis and tissue regeneration assays [29].Before delving further and in trying to bring greater clarity to the previous paragraph, let us recapitulate the existing models for skin stem cells that are currently being considered. The earliest model describing the hierarchy of stem cells in the interfollicular epidermis suggests the columnar arrangement of keratinocytes stacked in what is known as epidermal proliferative units (EPU) [30]. According to this model, stem cell clones are similar in size and their number remains rather constant during homeostasis. Relatively few basal cells have stem cell properties and can create transit amplifying (TA) cells, which constitute the majority of basal cells. This model suggests that TA cells go through several proliferation cycles before leaving the basal cell layer and follow their terminal differentiation program [31].Despite the seeming adequacy of this model, a relatively recent study showed that the size of epidermal clones increases over time, which contradicts the previous EPU model. Therefore, a stochastic model was proposed where the basal cells have inherent progenitor characteristics and their differentiation occurs at random. This apparent asymmetry in the cell population results in a scaling behavior in clone size and distribution. Thus, according to this model cell clones become fewer in number and have variable size [32]. Further, this model proposes the existence of a quiescent stem cell population with as few as four to six divisions per year and where progenitor cells present a balanced, although still random, differentiation pattern. However, one in five mitotic cycles would result in progenitor loss, thus suggesting that the population of both stem cells and progenitors might be heterogeneous and with different degree of competence [33].The validity of these theories was later tested in a mathematical simulation in which both the classical hierarchical model and the stochastic model described above would result in stem cell depletion [34]. Therefore, a third model proposed the existence of both a quiescent stem cell population and a committed progenitor population with stochastic differentiation fate [35]. Interestingly, this model could also explain the diminished healing capacity observed in the later stages of life, as the number of stem cells would decline with age. However, it must be kept in mind that all of these models are based in murine models and are not fully applicable in humans. Thereby, further research in this regard is still needed. Due to the extensive nature of this subject in particular, we suggest an excellent review by Dr. Helena Zomer et al. providing greater detail and context [36].

Due to the extensive and complex nature of the subject, the present review conveys a broad overview on SSCs, wound healing and the signaling pathways involved therein, as well as some of the current strategies in stem-cell based treatment strategies for wound healing.

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A Beginners Introduction to Skin Stem Cells and Wound Healing - MDPI

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March 1st, 2025

Researchers Turn Skin Cells Into Stem Cells | Science | AAAS

By daniellenierenberg

Scientists have managed to reprogram human skin cells directly into cells that look and act like embryonic stem (ES) cells. The technique makes it possible to generate patient-specific stem cells to study or treat disease without using embryos or oocytes--and therefore could bypass the ethical debates that have plagued the field.

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Researchers Turn Skin Cells Into Stem Cells | Science | AAAS

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Bone Marrow Transplantation Market Poised to Achieve USD 14,336 Billion by 2031 – Persistence Market Research – openPR

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Blood and Marrow Transplantation and Cellular Therapy Center – NYU Langone Health

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BioRestorative Therapies to Report 2024 Financial Results and Host Conference Call on March 27, 2025 – The Manila Times

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Boost in cancer treatment: PGI working on lab for stem cell, gene therapies – The Times of India

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Man Cured Of Sickle Cell Disease In New York Thanks To New Gene Therapy – Forbes

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Dame Sandra Biskind And Stem Cell Rejuvenation – Anti Aging News

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Skin cells can now be directly converted into neurons – Earth.com

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Japan team says stem cell treatment helped improve spinal cord injuries | NHK WORLD-JAPAN News – NHK WORLD

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Keio University team says stem cell treatment helped improve spine injuries – The Japan Times

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Clonal dynamics and somatic evolution of haematopoiesis in mouse – Nature.com

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Garuda Therapeutics Closes $50 Million Series A-1 Financing to Advance Off-the-Shelf Blood Stem Cell Therapies – GlobeNewswire

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TET2 deficiency increases the competitive advantage of hematopoietic stem and progenitor cells through upregulation of thrombopoietin receptor…

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Mitochondria-enriched hematopoietic stem cells exhibit elevated self-renewal capabilities, thriving within the context of aged bone marrow -…

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Abu Dhabi Stem Cells Center and Yas Clinic first in UAE to receive AABB accreditation for haematopoietic progenitor cell collection – Abu Dhabi Media…

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Induced Pluripotent Stem Cells: Hope in the Treatment of Diseases …

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Stem cell therapy side effects & risks at clinics – The Niche

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Stem Cell Use to Treat Dermatological Disorders – IntechOpen

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Skin Regeneration and Rejuvenation | Harvard Stem Cell Institute (HSCI)

By daniellenierenberg

A Beginners Introduction to Skin Stem Cells and Wound Healing – MDPI

By daniellenierenberg

Researchers Turn Skin Cells Into Stem Cells | Science | AAAS

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