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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March 11th, 2025

Induced Pluripotent Stem Cells and Their Potential for Basic and …

By daniellenierenberg

Abstract

Induced pluripotent stem (iPS) cells, are a type of pluripotent stem cell derived from adult somatic cells. They have been reprogrammed through inducing genes and factors to be pluripotent. iPS cells are similar to embryonic stem (ES) cells in many aspects. This review summarizes the recent progresses in iPS cell reprogramming and iPS cell based therapy, and describe patient specific iPS cells as a disease model at length in the light of the literature. This review also analyzes and discusses the problems and considerations of iPS cell therapy in the clinical perspective for the treatment of disease.

Keywords: Cellular therapy, disease model, embryonic stem cells, induced pluripotent stem cells, reprogramm.

Induced pluripotent stem (iPS) cells, are a type of pluripotent stem cell derived from adult somatic cells that have been genetically reprogrammed to an embryonic stem (ES) cell-like state through the forced expression of genes and factors important for maintaining the defining properties of ES cells.

Mouse iPS cells from mouse fibroblasts were first reported in 2006 by the Yamanaka lab at Kyoto University [1]. Human iPS cells were first independently produced by Yamanakas and Thomsons groups from human fibroblasts in late 2007 [2, 3]. iPS cells are similar to ES cells in many aspects, including the expression of ES cell markers, chromatin methylation patterns, embryoid body formation, teratoma formation, viable chimera formation, pluripotency and the ability to contribute to many different tissues in vitro.

The breakthrough discovery of iPS cells allow researchers to obtain pluripotent stem cells without the controversial use of embryos, providing a novel and powerful method to "de-differentiate" cells whose developmental fates had been traditionally assumed to be determined. Furthermore, tissues derived from iPS cells will be a nearly identical match to the cell donor, which is an important factor in research of disease modeling and drug screening. It is expected that iPS cells will help researchers learn how to reprogram cells to repair damaged tissues in the human body.

The purpose of this paper is to summarize the recent progresses in iPS cell development and iPS cell-based therapy, and describe patient specific iPS cells as a disease model, analyze the problems and considerations of iPS therapy in the clinical treatment of disease.

The methods of reprogramming somatic cells into iPS cells are summarized in Table 1. It was first demonstrated that genomic integration and high expression of four factors, Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28 by virus, can reprogram fibroblast cells into iPS cells [1-3]. Later, it was shown that iPS cells can be generated from fibroblasts by viral integration of Oct4/Sox2/Klf4 without c-Myc [4]. Although these iPS cells showed reduced tumorigenicity in chimeras and progeny mice, the reprogramming process is much slower, and efficiency is substantially reduced. These studies suggest that the ectopic expression of these three transcription factors (Oct4/Klf4/Sox2) is required for reprogramming of somatic cells in iPS cells.

Various growth factors and chemical compounds have recently been found to improve the induction efficiency of iPS cells. Shi et al., [5] demonstrated that small molecules, able to compensate for Sox2, could successfully reprogram mouse embryonic fibroblasts (MEF) into iPS cells. They combined Oct4/Klf4 transduction with BIX-01294 and BayK8644s and derived MEF into iPS cells. Huangfu et al., [6, 7] reported that 5-azacytidine, DNA methyltransferase inhibitor, and valproic acid, a histone deacetylase inhibitor, improved reprogramming of MEF by more than 100 folds. Valproic acid enables efficient reprogramming of primary human fibroblasts with only Oct4 and Sox2.

Kim et al. showed that mouse neural stem cells, expressing high endogenous levels of Sox2, can be reprogrammed into iPS cells by transduction Oct4 together with either Klf4 or c-Myc [19]. This suggests that endogenous expression of transcription factors, that maintaining stemness, have a role in the reprogramming process of pluripotency. More recently, Tsai et al., [20] demonstrated that mouse iPS cells could be generated from the skin hair follicle papilla (DP) cell with Oct4 alone since the skin hair follicle papilla cells expressed endogenously three of the four reprogramming factors: Sox2, c-Myc, and Klf4. They showed that reprogramming could be achieved after 3 weeks with efficiency similar to other cell types reprogrammed with four factors, comparable to ES cells.

Retroviruses are being extensively used to reprogram somatic cells into iPS cells. They are effective for integrating exogenous genes into the genome of somatic cells to produce both mouse and human iPS cells. However, retroviral vectors may have significant risks that could limit their use in patients. Permanent genetic alterations, due to multiple retroviral insertions, may cause retrovirus-mediated gene therapy as seen in treatment of severe combined immunodeficiency [25]. Second, although retroviral vectors are silenced during reprogramming [26], this silencing may not be permanent, and reactivation of transgenes may occur upon the differentiation of iPS cells. Third, expression of exogenous reprogramming factors could occur. This may trigger the expression of oncogenes that stimulate cancer growth and alter the properties of the cells. Fourth, the c-Myc over-expression may cause tumor development after transplantation of iPS derived cells. Okita et al. [10] reported that the chimeras and progeny derived from iPS cells frequently showed tumor formation. They found that the retroviral expression of c-Myc was reactivated in these tumors. Therefore, it would be desirable to produce iPS cells with minimal, or free of, genomic integration. Several new strategies have been recently developed to address this issue (Table 1).

Stadtfeld et al. [16] used an adenoviral vector to transduce mouse fibroblasts and hepatocytes, and generated mouse iPS cells at an efficiency of about 0.0005%. Fusaki et al. [22] used Sendai virus to efficiently generate iPS cells from human skin fibroblasts without genome integration. Okita et al. [27] repeatedly transfected MEF with two plasmids, one carrying the complementary DNAs (cDNAs) of Oct3/4, Sox2, and Klf4 and the other carrying the c-Myc cDNA. This generated iPS cells without evidence of plasmid integration. Using a polycistronic plasmid co-expressing Oct4, Sox2, Klf4, and c-Myc, Gonzalez et al., [28] reprogrammed MEF into iPS cells without genomic integration. Yu et al. [29] demonstrated that oriP/EBNA1 (EpsteinBarr nuclear antigen-1)-based episomal vectors could be used to generate human iPS cells free of exogenous gene integration. The reprogramming efficiency was about 36 colonies/1 million somatic cells. Narsinh et al., [21] derived human iPS cells via transfection of human adipocyte stromal cells with a nonviral minicircle DNA by repeated transfection. This produced hiPS cells colonies from an adipose tissue sample in about 4 weeks.

When iPS cells generated from either plasmid transfection or episomes were carefully analyzed to identify random vector integration, it was possible to have vector fragments integrated somewhere. Thus, reprogramming strategies entirely free of DNA-based vectors are being sought. In April 2009, it was shown that iPS cells could be generated using recombinant cell-penetrating reprogramming proteins [30]. Zhou et al. [30] purified Oct4, Sox2, Klf4 and c-Myc proteins, and incorporated poly-arginine peptide tags. It allows the penetration of the recombinant reprogramming proteins through the plasma membrane of MEF. Three iPS cell clones were successfully generated from 5x 104 MEFs after four rounds of protein supplementation and subsequent culture of 2328 days in the presence of valproic acid.

A similar approach has also been demonstrated to be able to generate human iPS cells from neonatal fibroblasts [31]. Kim et al. over-expressed reprogramming factor proteins in HEK293 cells. Whole cell proteins of the transduced HEK293 were extracted and used to culture fibroblast six times within the first week. After eight weeks, five cell lines had been established at a yield of 0.001%, which is one-tenth of viral reprogramming efficiency. Strikingly, Warren et al., [24] demonstrated that human iPS cells can be derived using synthetic mRNA expressing Oct3/4, Klf4, Sox2 and c-Myc. This method efficiently reprogrammed fibroblast into iPS cells without genome integration.

Strenuous efforts are being made to improve the reprogramming efficiency and to establish iPS cells with either substantially fewer or no genetic alterations. Besides reprogramming vectors and factors, the reprogramming efficiency is also affected by the origin of iPS cells.

A number of somatic cells have been successfully reprogrammed into iPS cells (Table 2). Besides mouse and human somatic cells, iPS cells from other species have been successfully generated (Table 3).

The origin of iPS cells has an impact on choice of reprogramming factors, reprogramming and differentiation efficiencies. The endogenous expression of transcription factors may facilitate the reprogramming procedure [19]. Mouse neural stem cells express higher endogenous levels of Sox2 and c-Myc than ES cells. Thus, two transcription factors, exogenous Oct4 together with either Klf4 or c-Myc, are sufficient to generate iPS cells from neural stem cells [19]. Ahmed et al. [14] demonstrated that mouse skeletal myoblasts endogenously expressed Sox2, Klf4, and c-Myc and can be easily reprogrammed to iPS cells.

It is possible that iPS cells may demonstrate memory of parental source and therefore have low differentiation efficiency into other tissue cells. Kim et al. [32] showed that iPS cells reprogrammed from peripheral blood cells could efficiently differentiate into the hematopoietic lineage cells. It was found, however, that these cells showed very low differentiation efficiency into neural cells. Similarly, Bar-Nur et al. found that human cell-derived iPS cells have the epigenetic memory and may differentiate more readily into insulin producing cells [33]. iPS cells from different origins show similar gene expression patterns in the undifferentiated state. Therefore, the memory could be epigenetic and are not directly related to the pluripotent status.

The cell source of iPS cells can also affect the safety of the established iPS cells. Miura et al. [54] compared the safety of neural differentiation of mouse iPS cells derived from various tissues including MEFs, tail-tip fibroblasts, hepatocyte and stomach. Tumorigenicity was examined. iPS cells that reprogrammed from tail-tip fibroblasts showed many undifferentiated pluripotent cells after three weeks of in vitro differentiation into the neural sphere. These cells developed teratoma after transplantation into an immune-deficient mouse brain. The possible mechanism of this phenomenon may be attributable to epigenetic memory and/or genomic stability. Pre-evaluated, non-tumorigenic and safe mouse iPS cells have been reported by Tsuji et al. [55]. Safe iPS cells were transplanted into non-obese diabetic/severe combined immunodeficiency mouse brain, and found to produce electrophysiologically functional neurons, astrocytes, and oligodendrocytes in vitro.

The cell source of iPS cells is important for patients as well. It is important to carefully evaluate clinically available sources. Human iPS cells have been successfully generated from adipocyte derived stem cells [35], amniocytes [36], peripheral blood [38], cord blood [39], dental pulp cells [40], oral mucosa [41], and skin fibroblasts (Table 2). The properties and safety of these iPS cells should be carefully examined before they can be used for treatment.

Shimada et al. [17] demonstrated that combination of chemical inhibitors including A83-01, CHIR99021, PD0325901, sodium butyrate, and Y-27632 under conditions of physiological hypoxia human iPS cells can be rapidly generated from adipocyte stem cells via retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Miyoshi et al., [42] generated human iPS cells from cells isolated from oral mucosa via the retroviral gene transfer of Oct4, Sox2, c-Myc, and Klf4. Reprogrammed cells showed ES-like morphology and expressed undifferentiated markers. Yan et al., [40] demonstrated that dental tissue-derived mesenchymal-like stem cells can easily be reprogrammed into iPS cells at relatively higher rates as compared to human fibroblasts. Human peripheral blood cells have also been successfully reprogrammed into iPS cells [38]. Anchan et al. [36] described a system that can efficiently derive iPS cells from human amniocytes, while maintaining the pluripotency of these iPS cells on mitotically inactivated feeder layers prepared from the same amniocytes. Both cellular components of this system are autologous to a single donor. Takenaka et al. [39] derived human iPS cells from cord blood. They demonstrated that repression of p53 expression increased the reprogramming efficiency by 100-fold.

All of the human iPS cells described here are indistinguishable from human ES cells with respect to morphology, expression of cell surface antigens and pluripotency-associated transcription factors, DNA methylation status at pluripotent cell-specific genes and the capacity to differentiate in vitro and in teratomas. The ability to reprogram cells from human somatic cells or blood will allow investigating the mechanisms of the specific human diseases.

The iPS cell technology provides an opportunity to generate cells with characteristics of ES cells, including pluripotency and potentially unlimited self-renewal. Studies have reported a directed differentiation of iPS cells into a variety of functional cell types in vitro, and cell therapy effects of implanted iPS cells have been demonstrated in several animal models of disease.

A few studies have demonstrated the regenerative potential of iPS cells for three cardiac cells: cardiomyocytes, endothelial cells, and smooth muscle cells in vitro and in vivo. Mauritz [56] and Zhang [57] independently demonstrated the ability of mouse and human iPS cells to differentiate into functional cardiomyocytes in vitro through embryonic body formation. Rufaihah [58], et al. derived endothelial cells from human iPS cells, and showed that transplantation of these endothelial cells resulted in increased capillary density in a mouse model of peripheral arterial disease. Nelson et al. [59] demonstrated for the first time the efficacy of iPS cells to treat acute myocardial infarction. They showed that iPS cells derived from MEF could restore post-ischemic contractile performance, ventricular wall thickness, and electrical stability while achieving in situ regeneration of cardiac, smooth muscle, and endothelial tissue. Ahmed et al. [14] demonstrated that beating cardiomyocyte-like cells can be differentiated from iPS cells in vitro. The beating cells expressed early and late cardiac-specific markers. In vivo studies showed extensive survival of iPS and iPS-derived cardiomyocytes in mouse hearts after transplantation in a mouse experimental model of acute myocardial infarction. The iPs derived cardiomyocyte transplantation attenuated infarct size and improved cardiac function without tumorgenesis, while tumors were observed in the direct iPS cell transplantation animals.

Strategies to enhance the purity of iPS derived cardiomyocytes and to exclude the presence of undifferentiated iPS are required. Implantation of pre-differentiation or guided differentiation of iPS would be a safer and more effective approach for transplantation. Selection of cardiomyocytes from iPS cells, based on signal-regulatory protein alpha (SIRPA) or combined with vascular cell adhesion protein-1 (VCAM-1), has been reported. Dubois et al. [60] first demonstrated that SIRPA was a marker specifically expressed on cardiomyocytes derived from human ES cells and human iPS cells. Cell sorting with an antibody against SIRPA could enrich cardiac precursors and cardiomyocytes up to 98% troponin T+ cells from human ESC or iPS cell differentiation cultures. Elliott et al. [61] adopted a cardiac-specific reporter gene system (NKX2-5eGFP/w) and identified that VCAM-1 and SIRPA were cell-surface markers of cardiac lineage during differentiation of human ES cells.

Regeneration of functional cells from human stem cells represents the most promising approach for treatment of type 1 diabetes mellitus (T1DM). This may also benefit the patients with type 2 diabetes mellitus (T2DM) who need exogenous insulin. At present, technology for reprogramming human somatic cell into iPS cells brings a remarkable breakthrough in the generation of insulin-producing cells.

Human ES cells can be directed to become fully developed cells and it is expected that iPS cells could also be similarly differentiated. Stem cell based approaches could also be used for modulation of the immune system in T1DM, or to address the problems of obesity and insulin resistance in T2DM.

Tateishi et al., [62] demonstrated that insulin-producing islet-like clusters (ILCs) can be generated from the human iPS cells under feeder-free conditions. The iPS cell derived ILCs not only contain C-peptide positive and glucagon-positive cells but also release C-peptide upon glucose stimulation. Similarly, Zhang et al., [63] reported a highly efficient approach to induce human ES and iPS cells to differentiate into mature insulin-producing cells in a chemical-defined culture system. These cells produce insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets. Most of these cells co-expressed mature cell-specific markers such as NKX6-1 and PDX1, indicating a similar gene expression pattern to adult islet beta cells in vivo.

Alipo et al. [64] used mouse skin derived iPS cells for differentiation into -like cells that were similar to the endogenous insulin-secreting cells in mice. These -like cells were able to secrete insulin in response to glucose and to correct a hyperglycemic phenotype in mouse models of both T1DM and T2DM after iPS cell transplant. A long-term correction of hyperglycemia could be achieved as determined by hemoglobin A1c levels. These results are encouraging and suggest that induced pluripotency is a viable alternative to directing iPS cell differentiation into insulin secreting cells, which has great potential clinical applications in the treatment of T1DM and T2 DM.

Although significant progress has been made in differentiating pluripotent stem cells to -cells, several hurdles remain to be overcome. It is noted in several studies that the general efficiency of in vitro iPS cell differentiation into functional insulin-producing -like cells is low. Thus, it is highly essential to develop a safe, efficient, and easily scalable differentiation protocol before its clinical application. In addition, it is also important that insulin-producing b-like cells generated from the differentiation of iPS cells have an identical phenotype resembling that of adult human pancreatic cells in vivo.

Currently, the methodology of neural differentiation has been well established in human ES cells and shown that these methods can also be applied to iPS cells. Chambers et al. [65] demonstrated that the synergistic action of Noggin and SB431542 is sufficient to induce rapid and complete neural conversion of human ES and iPS cells under adherent culture conditions. Swistowsk et al. [66] used a completely defined (xenofree) system, that has efficiently differentiated human ES cells into dopaminergic neurons, to differentiate iPS cells. They showed that the process of differentiation into committed neural stem cells (NSCs) and subsequently into dopaminergic neurons was similar to human ES cells. Importantly, iPS cell derived dopaminergic neurons were functional as they survived and improved behavioral deficits in 6-hydroxydopamine-leasioned rats after transplantation. Lee et al. [67] provided detailed protocols for the step-wise differentiation of human iPS and human ES into neuroectodermal and neural crest cells using either the MS5 co-culture system or a defined culture system (Noggin with a small-molecule SB431542), NSB system. The average time required for generating purified human NSC precursors will be 25 weeks. The success of deriving neurons from human iPS cells provides a study model of normal development and impact of genetic disease during neural crest development.

Wernig et al., [68] showed that iPS cells can give rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinson's disease (PD) upon transplantation into the adult brain. This study highlights the therapeutic potential of directly reprogrammed fibroblasts for neural cell replacement in the animal model of Parkinsons disease.

Tsuji et al., [55] used pre-evaluated iPS cells derived for treatment of spinal cord injury. These cells differentiated into all three neural lineages, participated in remyelination and induced the axonal regrowth of host 5HT+ serotonergic fibers, promoting locomotor function recovery without forming teratomas or other tumors. This study suggests that iPS derived neural stem/progenitor cells may be a promising cell source for treatment of spinal cord injury.

Hargus et al., [69] demonstrated proof of principle of survival and functional effects of neurons derived from iPS cells reprogrammed from patients with PD. iPS cells from patients with Parkinsons disease were differentiated into dopaminergic neurons that could be transplanted without signs of neuro-degeneration into the adult rodent striatum. These cells survived and showed arborization, and mediated functional effects in an animal model of Parkinsons disease. This study suggests that disease specific iPS cells can be generated from patients with PD, which be used to study the PD development and in vitro drug screen for treatment of PD.

Reprogramming technology is being applied to derive patient specific iPS cell lines, which carry the identical genetic information as their patient donor cells. This is particularly interesting to understand the underlying disease mechanism and provide a cellular and molecular platform for developing novel treatment strategy.

Human iPS cells derived from somatic cells, containing the genotype responsible for the human disease, hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. The differentiated cells from reprogrammed patient specific human iPS cells retain disease-related phenotypes to be an in vitro model of pathogenesis (Table 4). This provides an innovative way to explore the molecular mechanisms of diseases.

Disease Modeling Using Human iPS Cells

Recent studies have reported the derivation and differentiation of disease-specific human iPS cells, including autosomal recessive disease (spinal muscular atrophy) [70], cardiac disease [71-75], blood disorders [13, 76], diabetes [77], neurodegenerative diseases (amyotrophic lateral sclerosis [78], Huntingtons disease [79]), and autonomic nervous system disorder (Familial Dysautonomia) [80]. Patient-specific cells make patient-specific disease modeling possible wherein the initiation and progression of this poorly understood disease can be studied.

Human iPS cells have been reprogrammed from spinal muscular atrophy, an autosomal recessive disease. Ebert et al., [70] generated iPS cells from skin fibroblast taken from a patient with spinal muscular atrophy. These cells expanded robustly in culture, maintained the disease genotype and generated motor neurons that showed selective deficits compared to those derived from the patients' unaffected relative. This is the first study to show that human iPS cells can be used to model the specific pathology seen in a genetically inherited disease. Thus, it represents a promising resource to study disease mechanisms, screen new drug compounds and develop new therapies.

Similarly, three other groups reported their findings on the use of iPS cells derived cardiomyocytes (iPSCMs) as disease models for LQTS type-2 (LQTS2). Itzhaki et al., [72] obtained dermal fibroblasts from a patient with LQTS2 harboring the KCNH2 gene mutation and showed that action potential duration was prolonged and repolarization velocity reduced in LQTS2 iPS-CMs compared with normal cardiomyocytes. They showed that Ikr was significantly reduced in iPS-CMs derived from LQTS2. They also tested the potential therapeutic effects of nifedipine and the KATP channel opener pinacidil (which augments the outward potassium current) and demonstrated that they shortened the action potential duration and abolished early after depolarization. Similarly, Lahti et al., [73] demonstrated a more pronounced inverse correlation between the beating rate and repolarization time of LQTS2 disease derived iPS-CMs compared with normal control cells. Prolonged action potential is present in LQT2-specific cardiomyocytes derived from a mutation. Matsa et al., [74] also successfully generated iPS-CMs from a patient with LQTS2 with a known KCNH2 mutation. iPS-CMs with LQTS2 displayed prolonged action potential durations on patch clamp analysis and prolonged corrected field potential durations on microelectrode array mapping. Furthermore, they demonstrated that the KATP channel opener nicorandil and PD-118057, a type 2 IKr channel enhancer attenuate channel closing.

LQTS3 has been recapitulated in mouse iPS cells [75]. Malan et al. [75] generated disease-specific iPS cells from a mouse model of a human LQTS3. Patch-clamp measurements of LQTS 3-specific cardiomyocytes showed the biophysical effects of the mutation on the Na+ current, withfaster recovery from inactivation and larger late currents than observed in normal control cells. Moreover, LQTS3-specific cardiomyocytes had prolonged action potential durations and early after depolarizations at low pacing rates, both of which are classic features of the LQTS3 mutation.

Human iPS cells have been used to recapitulate diseases of blood disorder. Ye et al. [13] demonstrated that human iPS cells derived from periphery blood CD34+ cells of patients with myeloproliferative disorders, have the JAK2-V617F mutation in blood cells. Though the derived iPS cells contained the mutation, they appeared normal in phenotypes, karyotype, and pluripotency. After hematopoietic differentiation, the iPS cell-derived hematopoietic progenitor (CD34+/CD45+) cells showed the increased erythropoiesis and expression of specific genes, recapitulating features of the primary CD34+ cells of the corresponding patient from whom the iPS cells were derived. This study highlights that iPS cells reprogrammed from somatic cells from patients with blood disease provide a prospective hematopoiesis model for investigating myeloproliferative disorders.

Raya et al., [76] reported that somatic cells from Fanconi anaemia patients can be reprogrammed to pluripotency after correction of the genetic defect. They demonstrated that corrected Fanconi-anaemia specific iPS cells can give rise to haematopoietic progenitors of the myeloid and erythroid lineages that are phenotypically normal. This study offers proof-of-concept that iPS cell technology can be used for the generation of disease-corrected, patient-specific cells with potential value for cell therapy applications.

Maehr et al., [77] demonstrated that human iPS cells can be generated from patients with T1DM by reprogramming their adult fibroblasts. These cells are pluripotent and differentiate into three lineage cells, including insulin-producing cells. These cells provide a platform to assess the interaction between cells and immunocytes in vitro, which mimic the pathological phenotype of T1DM. This will lead to better understanding of the mechanism of T1DM and developing effective cell replacement therapeutic strategy.

Lee et al., [80] reported the derivation of human iPS cells from patient with Familial Dysautonomia, an inherited disorder that affects the development and function of nerves throughout the body. They demonstrated that these iPS cells can differentiate into all three germ layers cells. However gene expression analysis demonstrated tissue-specific mis-splicing of IKBKAP in vitro, while neural crest precursors showed low levels of normal IKBKAP transcript. Transcriptome analysis and cell-based assays revealed marked defects in neurogenic differentiation and migration behavior. All these recaptured familial Dysautonomia pathogenesis, suggesting disease specificity of the with familial Dysautonomia human iPS cells. Furthermore, they validated candidate drugs in reversing and ameliorating neuronal differentiation and migration. This study illustrates the promise of disease specific iPS cells for gaining new insights into human disease pathogenesis and treatment.

Human iPS cells derived reprogrammed from patients with inherited neurodegenerative diseases, amyotrophic lateral sclerosis [78] and Huntingtons disease 79, have also been reported. Dimos et al., [78] showed that they generated iPS cells from a patient with a familial form of amyotrophic lateral sclerosis. These patient-specific iPS cells possess the properties of ES cells and were reprogrammed successfully to differentiate into motor neurons. Zhang et al., [79] derived iPS cells from fibroblasts of patient with Huntingtons disease. They demonstrated that striatal neurons and neuronal precursors derived from these iPS cells contained the same CAG repeat expansion as the mutation in the patient from whom the iPS cell line was established. This suggests that neuronal progenitor cells derived from Huntingtons disease cell model have endogenous CAG repeat expansion that is suitable for mechanistic studies and drug screenings.

Disease specific somatic cells derived from patient-specific human iPS cells will generate a wealth of information and data that can be used for genetically analyzing the disease. The genetic information from disease specific-iPS cells will allow early and more accurate prediction and diagnosis of disease and disease progression. Further, disease specific iPS cells can be used for drug screening, which in turn correct the genetic defects of disease specific iPS cells.

iPS cells appear to have the greatest promise without ethical and immunologic concerns incurred by the use of human ES cells. They are pluripotent and have high replicative capability. Furthermore, human iPS cells have the potential to generate all tissues of the human body and provide researchers with patient and disease specific cells, which can recapitulate the disease in vitro. However, much remains to be done to use these cells for clinical therapy. A better understanding of epigenetic alterations and transcriptional activity associated with the induction of pluripotency and following differentiation is required for efficient generation of therapeutic cells. Long-term safety data must be obtained to use human iPS cell based cell therapy for treatment of disease.

These works were supported by NIH grants HL95077, HL67828, and UO1-100407.

The authors confirm that this article content has no conflicts of interest.

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February 20th, 2025

iPS cell therapy 2.0: Preparing for next-generation regenerative …

By daniellenierenberg

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This year marks the tenth anniversary of the world's first transplantation of tissue generated from induced pluripotent stem cells (iPSCs). There is now a growing number of clinical trials worldwide examining the efficacy and safety of autologous and allogeneic iPSC-derived products for treating various pathologic conditions. As we patiently wait for the results from these and future clinical trials, it is imperative to strategize for the next generation of iPSC-based therapies. This review examines the lessons learned from the development of another advanced cell therapy, chimeric antigen receptor (CAR) T cells, and the possibility of incorporating various new bioengineering technologies in development, from RNA engineering to tissue fabrication, to apply iPSCs not only as a means to achieve personalized medicine but also as designer medical applications.

Keywords: bioengineering; cell therapy; clinical trials; iPS cells; regenerative medicine; transplantation.

2024 Wiley Periodicals LLC.

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Ryan R, Hill S. Ryan R, et al. Cochrane Database Syst Rev. 2019 Oct 23;10(10):ED000141. doi: 10.1002/14651858.ED000141. Cochrane Database Syst Rev. 2019. PMID: 31643081 Free PMC article.

Showell MG, Mackenzie-Proctor R, Jordan V, Hart RJ. Showell MG, et al. Cochrane Database Syst Rev. 2020 Aug 27;8(8):CD007807. doi: 10.1002/14651858.CD007807.pub4. Cochrane Database Syst Rev. 2020. PMID: 32851663 Free PMC article.

Triana L, Palacios Huatuco RM, Campilgio G, Liscano E. Triana L, et al. Aesthetic Plast Surg. 2024 Oct;48(20):4217-4227. doi: 10.1007/s00266-024-04260-2. Epub 2024 Aug 5. Aesthetic Plast Surg. 2024. PMID: 39103642 Review.

Petty S, Allen S, Pickup H, Woodier B. Petty S, et al. Autism Adulthood. 2023 Dec 1;5(4):437-449. doi: 10.1089/aut.2022.0073. Epub 2023 Dec 12. Autism Adulthood. 2023. PMID: 38116056 Free PMC article.

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February 20th, 2025

iPSC Therapy: Advances and Clinical Potential – BiologyInsights

By daniellenierenberg

Induced pluripotent stem cell (iPSC) therapy is a promising frontier in regenerative medicine, offering potential treatments for a variety of diseases. By reprogramming adult cells to an embryonic-like state, iPSCs can differentiate into any cell type, paving the way for patient-specific therapies and reducing immune rejection risks.

As research progresses, understanding how these cells are generated, their molecular dynamics, and differentiation mechanisms is crucial. This article explores recent advances in iPSC technology and its clinical applications, highlighting key developments that could transform therapeutic approaches soon.

The creation of iPSCs involves sophisticated techniques that revert adult somatic cells to a pluripotent state. Understanding these methods is essential to developing efficient and safe therapies.

The initial method for generating iPSCs involves introducing specific transcription factors into adult cells. The groundbreaking work by Takahashi and Yamanaka in 2006 demonstrated that four transcription factorsOct3/4, Sox2, Klf4, and c-Myccould reprogram fibroblasts into pluripotent stem cells. This method typically employs viral vectors, such as retroviruses or lentiviruses, to deliver these factors into the host genome, initiating the reprogramming process. While effective, this approach poses risks, including insertional mutagenesis, which can disrupt host genes and potentially lead to tumorigenesis. Recent advancements have focused on optimizing vector systems, such as using polycistronic vectors, to enhance reprogramming efficiency and reduce genomic integration. Ongoing research aims to refine these methods further to ensure the safety and reliability of iPSC generation for clinical applications.

Chemical reprogramming offers a promising alternative, utilizing small molecules to induce pluripotency. These molecules can modulate signaling pathways and epigenetic states, effectively replacing transcription factors. Compounds like valproic acid, a histone deacetylase inhibitor, and CHIR99021, a GSK3 inhibitor, enhance reprogramming efficiency when used with reduced sets of transcription factors. Studies have shown that certain chemical cocktails can even reprogram somatic cells without genetic modification, minimizing genomic instability risks. A notable example includes a seven-compound cocktail to generate iPSCs from mouse somatic cells, reported in a 2013 study in Science. This method holds significant potential for generating safer iPSCs, though it requires further refinement and validation in human cells.

To address safety concerns associated with integrating vectors, nonintegrating vectors have emerged as a viable alternative for iPSC generation. These vectors, including Sendai virus, episomal vectors, and synthetic mRNA, enable transient expression of reprogramming factors, eliminating the risk of insertional mutagenesis. Sendai virus vectors, for example, are advantageous due to their cytoplasmic replication, preventing integration into the host genome. This method has been successfully used to generate clinical-grade iPSCs, as evidenced by a 2015 study in Cell Stem Cell, which highlighted the robust pluripotency and differentiation potential of these cells. Another promising approach involves synthetic mRNA, allowing precise temporal control of factor expression and producing iPSCs with high efficiency and minimal off-target effects. These nonintegrating methods are increasingly preferred in clinical settings, offering a safer pathway for therapeutic applications.

Reprogramming adult somatic cells into iPSCs initiates intricate molecular events that reshape cellular identity. Epigenetic modifications, such as DNA methylation and histone modifications, lead to the activation of pluripotency-associated genes and the silencing of lineage-specific genes. Studies in Nature Communications have shown that these alterations are actively orchestrated by reprogramming factors, which recruit chromatin remodelers and modify histone marks to establish a pluripotent state.

The regulatory network of genes and signaling pathways undergoes a transformation during reprogramming. The Wnt/-catenin and TGF- pathways play significant roles in maintaining pluripotency and facilitating the transition from a somatic to a pluripotent state. Research in Cell Reports has shown that modulation of these pathways can enhance reprogramming efficiency and stability. The cellular stress response, often triggered by reprogramming factors, influences reprogramming dynamics, affecting cell survival and genomic integrity.

As iPSCs transition from a somatic to a pluripotent state, metabolic reprogramming occurs, crucial for sustaining the high proliferative capacity of these cells. The shift from oxidative phosphorylation to glycolysis mirrors the metabolic profile of embryonic stem cells and supports the energy demands of rapid cell division. Detailed analysis in Journal of Cell Biology has elucidated how this metabolic switch is regulated by key transcription factors and enzymes, ensuring the maintenance of pluripotency. Understanding these metabolic changes provides potential targets for enhancing reprogramming efficiency and iPSC quality.

Once iPSCs are generated, their ability to differentiate into specific cell types is a cornerstone of their therapeutic potential. Understanding the pathways and conditions that guide iPSCs into becoming specialized cells is essential for developing effective treatments for various diseases.

Differentiating iPSCs into neural cells involves steps that mimic embryonic neural development. Key signaling pathways, such as Notch, Wnt, and Sonic Hedgehog, guide iPSCs towards a neural lineage. Protocols often begin with the formation of neural progenitor cells, which can further differentiate into neurons, astrocytes, and oligodendrocytes. A study published in Nature Neuroscience in 2022 demonstrated the use of dual-SMAD inhibition to efficiently generate neural progenitors from iPSCs, providing a robust platform for modeling neurological diseases and testing potential therapies. Additionally, small molecules and growth factors like retinoic acid and brain-derived neurotrophic factor (BDNF) enhance the maturation and functionality of iPSC-derived neurons.

iPSC-derived cardiac cells hold promise for treating heart diseases, as they can potentially regenerate damaged heart tissue. The differentiation process involves activating mesodermal and cardiac-specific pathways, including BMP, Activin/Nodal, and Wnt. Recent advancements have focused on optimizing the timing and concentration of these signaling molecules to improve the yield and purity of cardiomyocytes. A 2023 study in Circulation Research highlighted a chemically defined protocol that enhances cardiac differentiation efficiency by modulating the Wnt pathway at specific stages. This approach improves the production of functional cardiomyocytes and reduces variability in differentiation outcomes. The resulting iPSC-derived cardiomyocytes exhibit electrophysiological properties and contractile functions similar to native heart cells.

Differentiating iPSCs into pancreatic cells, particularly insulin-producing beta cells, offers a promising strategy for diabetes treatment. This process involves recapitulating the stages of pancreatic development, guided by signaling pathways such as Activin/Nodal, FGF, and Notch. Protocols typically start with the induction of definitive endoderm, followed by pancreatic progenitors and their maturation into functional beta cells. A 2021 study in Cell Stem Cell demonstrated a stepwise differentiation protocol incorporating specific growth factors and small molecules to enhance iPSC-derived beta cell efficiency and functionality. These cells have shown the ability to secrete insulin in response to glucose, providing a potential source for cell replacement therapies in diabetes.

Culturing iPSCs requires a meticulous approach to ensure their viability and functionality. Selecting an appropriate culture medium is crucial, providing the necessary nutrients and growth factors to maintain pluripotency. Commercially available media, like mTeSR1 and Essential 8, support robust growth and reduce the need for frequent media changes. These formulations are often supplemented with factors like bFGF to sustain the pluripotent state and prevent spontaneous differentiation.

The substrate on which iPSCs are cultured also plays a significant role in their growth and differentiation potential. Traditionally, iPSCs were cultured on feeder layers of mouse embryonic fibroblasts, but this can introduce variability and potential contamination. To address this, synthetic or recombinant extracellular matrix proteins, such as vitronectin and laminin, are now widely used. These matrices provide a more defined environment, enhancing reproducibility and scalability, particularly beneficial for clinical-grade production of iPSCs.

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February 11th, 2025

I Peace establishes and offers low immunoreaction risk GMP iPS Cells – PR Newswire

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I Peace establishes and offers low immunoreaction risk GMP iPS Cells PR Newswire

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February 11th, 2025

Induced Pluripotent Stem Cells (iPSCs)Roles in Regenerative Therapies …

By daniellenierenberg

The science around terminal inactivation and deletion of genetic codes of heredity in somatic cells was postulated by the Weismann barrier theory [1]. The somatic cell nuclear transfer (SCNT) demonstration asserted the fact that the genetic code in somatic cells is not discarded, and that reactivation of the same is a possibility through careful manipulations [2]. Developmental biology entered a new dimension of achievement when the discovery of embryonic stem cells (ESCs) and their pluripotency was exhibited, and further research identified that on fusion of somatic cells like fibroblasts, and T-lymphocytes with ESCs, reprogramming of the former through expression of genes associated with pluripotency becomes a possibility [3,4]. The findings around SCNT and ESC fusion identified the possibility of reversion in somatic cells indicating the presence of reprogramming factors that bear the potential to act as epigenetic memory erasing factors [5]. The earliest study around generation of pluripotent stem cells from fibroblasts was linked to introduction of four crucial transcription factors including octamer binding transcription factor 3/4 (Oct3/4), sex determining region Ybox 2 (SRY-Sox2), Krppel-like factor 4 (Klf4), and cellular-Myelocytomatosis (c-Myc) (OSKM) [6]. The allogenic trait of ESCs, risk of immune rejection in the recipient along with need for lifetime immunosuppression, and the ethicality around using the same, makes human induced pluripotent stem cells (iPSCs) an established candidate for regenerative therapies as they were found to not impact the host immune system [7]. The introduction of the iPSCs technology happened in the year 2006, and since then multiple observational studies have recounted its impact on cardiac diseases, ophthalmic conditions, as well as neurological disorders [8,9,10]. Figure 1 highlights the process of generating iPS cells.

Showing the process of progression and generating iPSC cells. Detailed description of creating iPSCs with reprogramming factors and differentiating them into a variety of cell types.

The nuclear reprogramming strategies, without compromising on safety and quality for therapeutic applications, include the integrative or nonintegrative transfer systems using viral or nonviral vectors. The first iPSCs were generated by integrating viral vectors, more popularly the retrovirus wherein the resultant iPSCs exhibited failure in complete expression of endogenous genes of pluripotency [11]. The more efficient viral vector has been documented to be the lentiviral vector (LV), which has recorded a reprogramming efficiency of between 0.11% [12,13,14]. To ensure increased safety for therapeutics, nonviral integrative systems have also been worked upon involving use of two plasmids; once encoding for c-Myc, and the other for the four reprogramming factors [15]. However, this system was also shown to have risk of integration, and low reprogramming efficiency. In case of nonintegrative nonviral systems for reprogramming, delivery of pluripotency marker genes has been done using self-replicating vectors, and cytoplasmic RNA. Though easy to work with, the reprogramming efficiency has been found to be lower than LV [16]. Today, research has identified possibility of successful reprogramming using microRNAs (miRNAs) which exhibit improved efficiency, wherein use of c-Myc has been replaced with miR-291-3p, miR-294, and miR-295 to generate homogenous colonies of human iPSCs [17]. The reprogramming methods have been highlighted in Table 1.

Reprogramming strategies for iPSCs in human species. Various programming strategies with ensuring safety and quality for therapeutic applications include the integrative or nonintegrative transfer systems using viral or nonviral.

There are many assays, including molecular and functional, to evaluate the developmental efficiency of iPSCs. These include alkaline phosphatase staining of pluripotency markers, DNA demethylation, retroviral silencing, and factor independence involving assessment of self-renewal in the absence of dox-inducible trans genes. The functional assays include teratoma formation, chimera development, tetraploid complementation, germline transmission, and in vitro differentiation [14]. Considering the low reprogramming efficiency in iPSCs, many studies have identified blocks in lineage conversion. Reprogramming pathway studies in fibroblasts have identified the repel factor to be involved in mesenchymal-to-epithelial transition (MET) and BMP receptor signaling [27,28]. Further studies on the refractory fibroblasts indicate negative iPSC generation in spite of prolonged culturing and presence of homogeneous factor expression indicating loss of somatic program, and activation of endogenous pluripotency genes to be the main roadblocks in formation of iPSCs [14]. The other limiting factor has been linked to expression levels of Nanog locus which are activated late in the reprogramming process and thus limit efficiency of conversion [29]. Gene silencing by DNA methylation, involving the pluripotency genes nanog and Oct4 which causes blockage in binding of transcription factors, has also been linked to causing interference in reprogramming [30]. Though the four most popular reprogramming factors have been Oct4, Sox2, Klf4, and c-Myc, human iPSCs have also been derived using expression of Oct4, Sox2, Nanog, and Lin28, indicating that pluripotent ground state becomes achievable through activation of different transcription factors [21]. The detailed derivation of iPSC along with the assay has been highlighted in Figure 2.

Schematic representation on derivation and assay for human iPSCs. Detailed schematic representation of derivation of iPSC with the various assays to evaluate the developmental efficiency.

The therapeutic potential of iPSC towards personalized cell therapy and disease modelling, has extended the functionality beyond laboratory tables as a research tool in murine and human models. Animal studies have identified promising potential of iPSC around treatment of genetic disorders, including sickle cell anemia; disease modelling of complex degenerative conditions like diabetes, Alzheimers disease, and the feasibility to be used in organ transplantation without risk of rejection and need of immunosuppression [14,31]. Few highlights on the therapeutic potential of iPSCs have been summarized in Table 2. The focus of the current review is to highlight and discuss the therapeutic roles of human iPSCs in different conditions and the future.

Few highlights of iPSC-disease models and the investigated therapy. The example of therapeutic potential of iPSC towards personalized cell therapy and disease modelling, has extended the functionality of the pluripotency beyond laboratory tables as a research tool in murine and human models.

Pluripotency and self-renewal are unique characteristics of iPSC that make them ideal for disease modelling and regenerative medicine. Their ability to indefinitely differentiate into cells of all the three germ layers makes them an important source for treating injuries as well as diseases. The availability of generating patient-specific iPSC with high efficiency and safety through protocols involving biochemical and epigenetic aspects expands the therapeutic potential of this tool. This can be assessed from the fact that a clinical trial involving iPSC-derived dopaminergic neurons have been initiated for Parkinsons disease after successful in vivo studies involving immunodeficient mice highlighted no risk of tumorigenicity [43]. Further, tissue resident macrophages, which are critical for immunity and derived from human-iPSCs, have been found to be immunologically different and better than the traditional monocyte-derived macrophages. Studies have shown human iPSC macrophages to restrict Mycobacterium tuberculosis growth in vitro by >75%, and were found to be capable of mounting antibacterial response when challenged with pathogens [44]. The greatest niche for iPSCs is the ability to generate the same from different donor categories including the diseased, and healthy making its application in the clinical setting at any stage a feasibility without the ethical issues around the ESCs.

The fundamental use of iPSC in regenerative medicine remains undisputed, but the tumorigenic potential of residual undifferentiated stem cells necessitates the need to devise strategies to remove the same from differentiated cells. Different study reports multiple treatment methodologies for eliminating undifferentiated iPSCs and one such recent publication identified undifferentiated hiPSCs to be sensitive to treatment involving medium supplemented with high concentration of L-alanine [45]. Another study assessed the efficacy of plasma-activated medium (PAM) in eliminating undifferentiated hiSPCs through inducing oxidative stress. This study found PAM to selectively eliminate undifferentiated hiPSCs cocultured with normal human dermal fibroblasts, which were the differentiated cells. Lower expression of oxidative-stress related genes in the undifferentiated hiPSCs were found to be the underlying cause for PAM-selective cell death [46]. A recent study report describes the use of salicylic diamines to remove residual undifferentiated cells from iPSC-derived cardiomyocytes. Salicylic diamines were found to exert their specific cytotoxic activity in the pluripotent stem cells by inhibiting the oxygen consumption rate. Teratoma formation was also found to be abolished in comparison to untreated cells [47].

Non-communicable diseases, including cardiovascular conditions, have emerged to be one of the leading causes for mortality in developed as well as developing nations. The trigger for myriad heart conditions exists both in genetics and the environment, which makes studying disease etiology in animal models complicated and inefficient. Animal model studies indicate up to 90% failure in new drug clinical trials, highlighting the limitation around prediction of safety and efficacy among humans. The iPSCs-based disease models have been studied for cardiac channelopathies including hereditary long QT syndrome (LQTS), dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC); the endothelial cell disease including familial pulmonary arterial hypertension (FPAH); the smooth muscle cell condition including Williams-Beuren syndrome (WBS), and Marfan syndrome (MFS) [8].

LQTS is an inherited fatal arrhythmia syndrome and around 17 genes have been associated with congenital LQTS, including the three main genes; KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3), together which account for ~75% of clinically definite cases. The current therapeutic intervention includes -blockers and a surgical procedure named left cardiac sympathetic denervation. Though genetic markers have been defined, the occurrence of variance of unknown significance (VUS) in 1 of 3 patients adds to the dilemma of inconclusive diagnosis. The need for better diagnostic platforms to assess outcome of genetic variants as well as different therapeutics led to the introduction of iPSCs. Many studies have worked to improve the differentiation efficiency, cellular maturation, and lineage specificity, develop new high-throughput assays for cellular phenotyping, and promote clinical implementation of patient-specific genetic models. A study by Wu J.C. et al. [48], utilized patient iPSC-derived cardiomyocytes (iPSC-CMs) and devised various strategies to reduce heterogeneity. These include derivation of chamber-specific cardiomyocytes, cultivation for extended period, 3-dimensional and mechanical conditioning, rapid electric stimulation, and hormonal stimulation; use of multicellular preparations to reduce intercellular variability; and development of high-throughput cellular phenotyping using optogenetic sensors including genetically coded voltage and calcium indicators. Further, this study also established the utility of iPSC-CMs to distinguish between pathogenic and benign variants to improve diagnosis and management of LQTS using CRISPR genome editing. This study, using iPSC-CMs, also identified factors causative for prolonged QT including upregulation of genes; DLG2, KCNE4, PTRF, and HTR2C and downregulation of CAMKV gene. Thus iPSC-based model platforms aid in developing a better understanding around intractable clinical problems associated with diseases like LQTS.

In case of DCM, characterized by ventricular chamber enlargement, and dilation as well as systolic dysfunction, human derived iPSCs have been used to investigate the excitation-contraction-coupling machinery, response to positive inotropic interventions, and study the proteome profile. This study utilized DCM patient specific-iPSC derived from skin fibroblasts and identified defects in assembly and maintenance of sarcomeric structure in the mutated iPSC-CM, as well as lower response to -adrenergic stimulation with isoproterenol, and increased [Ca2+] out and angiotensin-II. This indicates mutated CM from DCM patients to express blunted inotropic response [49]. In case of HCM which is the most common cause of sudden death among the young, iPSC models have been used to identify pathogenesis of the condition. Once such study involving iPSC-CM derived from patients in a maternally inherited HCM family positive for the mitochondrial 16s rRNA gene (MT-RNR2) mutation m.2336T > C identified mitochondrial dysfunction, and ultrastructure defects among the carriers. Further, reduction in levels of mitochondrial proteins, the ATP/ADP ratio, and mitochondrial potential was also found. These lead to increase in intracellular Ca2+ levels, that becomes causative for HCM-specific electrophysiological abnormalities [50]. Recent studies have also generated peripheral blood mononuclear cells-derived iPSC from HCM patient positive for the myosin binding protein C (MYBPC3) pathogenic mutation c.33693370 insC by the episomal method, which underwent successful differentiation to triblast cells with normal male karyotype, and expression of pluripotent markers indicating its usefulness as a tool to study HCM [51].

The iPSC models around FPAH have identified modification of BMPR2 signaling causing reduced endothelial cell adhesion, migration, survival, as well as angiogenesis. The autosomal dominant BMPR2 disease causing mutation has been found to be only 20% penetrant and the use of iPSC identified increased BIRC3 to be related to improved survival, indicating the potential to use protective modifiers of FPAH for developing treatment strategies in the future [52]. The iPSC model around WBS with haploinsufficiency found deficiency of elastin and the patient-derived smooth muscle cell to be immature and highly proliferative with defects in function and contractile properties. The rescue was done by upregulating elastin signaling and use of anti-proliferative drug rapamycin [53]. In case of MFS, disease pathogenesis investigation using iPSCs identified defects in fibrillin-1 accumulation, degradation of extracellular matrix, abnormal activation of transforming growth factor-, and cellular apoptosis [54].

The iPSC technology is also largely viewed to promote pre-clinical drug trials and screening over animal models to overcome differences in electrophysiological properties between human and animal cardiomyocytes. Studies have shown patient-derived iPSCs to exhibit higher sensitivity towards cardiotoxic drugs that could be the cause for change in action potential and arrhythmia [55]. Studies which have analyzed the beat characteristics of 3D engineered cardiac tissues have proven the occurrence of physiologically relevant changes in cardiac contraction in response to increasing concentrations of drugs like verapamil (multi-ion channel blocker) and metoprolol (-adrenergic antagonist) [56].

Thus, iPSC has been successfully used to model and understand pathogenesis of different cardiac diseases, providing insights on pathways around progression as well as for assessment of drug toxicity. These highlight the potential to use iPSC-based models for precision medicine in clinical use.

Theoretically iPSC has the potential to be programmed to form any cell in the human body, and coupled with improvements in reprogramming techniques, this technology has advanced our knowledge on disease pathology, developing precise therapeutics, as well as fuel advances in regenerative medicine [57]. In case of neurodegenerative conditions, and psychiatric disorders, the genetic predisposition and its relation to the disease pathophysiology is complex, and often there is alteration at structural as well as functional levels. In case of schizophrenia, which is aptly termed the disease of the synapses, studies have generated iPSC from family members positive for a frameshift mutation in schizophrenia 1 (DISC1) and used gene editing to generate isogenic iPS cell lines. This study found depletion of DISC1 protein among the mutation carriers, along with dysregulation of genes associated with synapses and psychiatric disorders in the forebrain. This mutation causes deficit of synaptic vesicles among the iPS-cell derived forebrain neurons. This identification of transcriptional dysregulation in human neurons, highlights a new facet involving synaptic dysregulation in mental disorders [58]. The technology of stem cell therapy has also been used to restore the functionality in many degenerative conditions including that of the retina that leads to loss of vision. Studies have evaluated the use iPSC to overcome challenges posed by use of stem cell therapy. The proposed strategy revolves around transplantation of photoreceptors with or without the retinal pigment epithelium cells for treating retinal degradation, with minimal risk using iPSC [59].

Degenerative disease generally progresses through multiple differentiation stages, and using iPSC models, these pathways of transition can be easily identified to assess cause as well as etiopathology better. Amyotrophic lateral sclerosis (ALS) involves loss of neurons from the spinal cord and motor cortex causing paralysis and death. The research around advancement of therapeutics, requires supply of human motor neurons positive for the causative genetic mutations that will also aid in understanding the root cause of motor neuron death. One study documented the production of iPS from ALS patient specific-skin fibroblasts from two sisters. Both were identified to be positive for the L144F (Leu144 Phe) mutation of the superoxide dismutase (SOD1) gene that is associated with a slowly progressing form of ALS. This study found successful reprogramming to be possible with only four factors; KLF4, SOX2, OCT4, and c-MYC. Further, the severe disability state of the patients used for harvesting in this case did not seem to block the transformation process or efficiency [60]. Fanconi anemia (FA) is an inherited bone marrow failure syndrome and is a chromosomal instability disorder needing transplantation of hematopoietic grafts from HLA-identical sibling donors. The reduced quality of the hematopoietic stem cells from the bone marrow of the affected limits the benefit of gene therapy trials. Studies have worked upon formation of genetically corrected FA-specific iPSCs through non-hematopoietic somatic cells reprogramming to generate large number of genetically-stable autologous hematopoietic stem cells for treating bone marrow failure in FA. The reprogramming was done on dermal fibroblasts involving two rounds of infection with mouse-stem-cell-virus-based retrovirus encoding amino-terminal flag-tagged version of the four transcription factors; OCT4, SOX2, KLF4, c-MYC. A batch of genetically corrected somatic cells using lentiviral vectors encoding FANCA or FANCD2 was also used for reprogramming to overcome the predisposition to apoptosis found in FA cells. The FANCA involved fibroblasts also underwent successful transformation to generate iPSCs. This study also found restoration of the FA pathway as a necessity to generate iPS from somatic cells of FA patients. The persistent FANCA expression in the FA-iPS cells indicated successful generation of genetically corrected FA-iPSCs with functional FA pathway, and disease-free status [61].

Parkinsons disease (PD) is a common chronic progressive disorder due to loss of nigrostriatal dopaminergic neurons. The pathophysiology of the disease is complex and research till date lacks complete understanding. Further, sporadic cases are not linked to any genetic variation. Development of patient-specific invitro iPSC models have been attempted to understand disease etiology better. Studies have worked upon generating iPSCs from sporadic cases of PD, which have been successfully reprogrammed to form dopaminergic neurons free of the reprogramming factors. This study utilized doxycycline-inducible lentiviral vectors that were excised with Cre-/lox-recombinase, resulting in generation of iPSC free of programming factors, and which retained all the pluripotent characteristics after removal of transgenes. This removal of promoter and transgene sequences from the vector reduced risk of oncogenic transformation and re-expression of the transduced transcription factors. This study highlighted the possibility of generating stable iPS-cell line in PD for better disease modelling [62]. Another study worked on improving the safety of human and non-human primate iPSC derived dopaminergic neurons for cell transplantation treatment in PD. This study found the protocol of NCAM(+)/CD29(low) sorting to result in enriching ventral midbrain dopaminergic neurons from the pluripotent stem cell-derived neural cell populations. Further, these neurons also exhibited increased expression of FOXA2, LMX1A, TH, GIRK2, PITX3, EN1, and NURR1 mRNA. These neurons were also found to bear the potential to restore motor function among the 6-hydroxydopamine lesioned rats, 16 weeks after transplantation. Further, the primate iPSC-derived neural cell was found to have survived without any immunosuppression after one year of autologous transplant, highlighting the proof-of-concept around feasibility and safety of iPSC-derived transplantation for PD [10].

Type 1 diabetes is an autoimmune condition involving destruction of the -cells of the pancreas wherein transplantation with -cells as islet tissues or the entire pancreas is suggested as an alternative over the traditional exogenous insulin supplementation. However, these come with risk of rejection, need of immunosuppression, apart from difficulty in the physiological control on blood glucose levels. To circumvent this block, generation of -cells or islet tissues from human pluripotent stem cells like iPSCs has been attempted. Many studies have generated pancreatic -like cells which secrete insulin in response to stimuli like potassium chloride [63]. However, co-excretion of glucagon, and somatostatin, apart from releasing unsuitable amounts of insulin; make these clinically inferior. iPSC-derived pancreatic endoderm cells have been shown to retain the potential to differentiate and are functionally comparable with adult -cells. Further, the shortage of donor islet has been overcome using iPSCs, as pancreatic cells generated from these have been evaluated in clinical trials as a new source for transplantation therapy. The differentiation of iPSCs through mimicking the natural in vivo process was facilitated using a combination of growth factors including Nodal-activin, Wnt, retinoic acid, hedgehog, epidermal and fibroblast growth factor, bone morphogenetic protein, and Notch to activate as well as inhibit the key signaling pathway. This study thus highlighted the possibility of generating patient-specific fully functional pancreatic tissue for transplantation over donor islet for diabetes treatment [64].

These studies highlight the development around iPSCs and transplantation technology for treatment of degenerative diseases as well as use them as disease models. The ability to generate patient-specific iPSC from skin biopsies, increases safety of autologous transplants without risk of immunorejection.

The treatment for blood disorders involves need for mature red blood cells/erythrocytes from the bone marrow or umbilical cord blood, for blood transfusion, and is limited due to incompatibility in blood group and Rh antigens, and risk of infections [65]. Erythropoiesis is a complex process for generation of mature erythrocytes from the precursor erythroblasts that are difficult to culture in vitro, as the entire process occurs in the bone marrow mediated by complex interaction between cellular and extracellular environment involving hormones, cytokines, and growth factors [66]. Further, the fully differentiated red blood cells (RBCs) are not proliferative, and setting up a system for erythropoiesis-like maturation in precursor cells is a challenge. Further, recruitment of donors, need for rare blood group types, as well as safety in sensitive population groups, add to the roadblock [67]. Studies have investigated human pluripotent stem cells, including iPSCs as an alternative source for unlimited supply of functional erythrocytes. Studies have discussed different methods devised for RBC production, including using PSCs by repeating the developmental haematopoiesis; reprogramming somatic cells through transcription factors including OCT4, SOX2, c-MYC, KLF4, NANOG, LIN28; and stimulating the maturation of hematopoietic stem cells isolated from peripheral or umbilical cord blood [67,68]. The advantage of using iPSCs is their ability to differentiate into any cell type, and can be maintained indefinitely, thus becoming a potential source for cell replacement therapies. The potential of iPSc becomes highlighted by the fact that the French National Registry of People with a Rare Blood Phenotype/Genotype claims a single iPSc clone from their database could meet 73% of the needs of sickle cell disease patients [69]. This highlights that a limited number or RBC clones have the potential to supply to the majority needs of alloimmunized patients with rare blood groups.

Studies have also worked on developing iPSC models for blood malignancies including myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and myeloproliferative neoplasms (MPN). A study worked on generating iPSC clones from bone marrow and blood of patients by integrating mutational analysis with cell programming to generate different iPSC clones which represent different disease stage as well as spectrum of the diseases including predisposition, low- and high-risk conditions. Additionally, the researchers also utilized the CRISPR/Cas9 system to introduce as well as correct mutations in the iPSCs. This study found iPSC from AML patients upon differentiation exhibited the leukemic phenotype, and the derived hematopoietic stem cells contained two immunophenotypically distinct cell populations; an adherent and non-adherent fraction, wherein the adherent fraction cells continuously renewed and generated the non-adherent cells. The AML-iPSC thus generated was found to exhibit characteristics of the leukemia stem cell model thus becoming an efficient model for molecular analysis and studying key functional aspects to be utilized for developing better therapeutics [70]. In case of chronic myeloid leukemia (CML), the BCR-ABL gene fusion is the major disease driver, and treatment involves use of tyrosine kinase inhibitor (TKI), causing remission in the vast majority of the cases. Studies have shown the CML-iPSCs to not be affected by TKI even in presence of BCR-ABL expression, indicating absence of dependency in this state of differentiation. The CML-iPSCs factors essential for maintenance of BCR-ABL positive and iPSCs including phosphorylation of AKT, JNK, ERK1/2 remained unchanged while the expression of STAT5 and CRKL was decreased. Further, the hematopoietic cells derived from CML-iPSC regained TKI sensitivity thus facilitating understanding on the disease pathogenesis better [71,72]. In case of MDS, reprogramming to generate iPSCs has been done from patients with del7q mutation, which is the signature for the disease. The iPSCs with the mutation upon hematopoietic differentiation were found to generate low quantities of CD34+/CD45+ myeloid progenitor cells. Further, studying genetically engineered clones as well as the MDS-iPSC-del7q clone from the patient, the researchers functionally mapped MDS phenotype to regions 7q32.37q36.1, which is linked to loss of hematopoietic differentiation potential [73]. To highlight the efficiency of iPSC-technology in precision oncology, studies have also created isogenic iPSCs with del7q and mutation SRSF2 P95L, each of these connected to a specific phenotype and drug response [74].

Human iPSC preclinical models also exist for monogenic blood disorders including thalassemia, and hemoglobinopathies for gene and cell therapy. Pilot trial investigations have explored the safety and effectiveness of mobilizing CD34+ hematopoietic progenitor cells in beta-thalassemia major adults. Further, the CD34+ were transduced with globin lentiviral vector, wherein the vector-encoded beta-chain was found to be expressed at normal hemizygous protein output levels in NSG mice. This trial thus validated an effective protocol for beta-globin gene transfer among thalassemia major CD34+ hematopoietic progenitor cells [75]. The risk of insertional mutagenesis using hematopoietic stem cells can be overcome through iPSCs which can be cloned and the clones with vector integration in the safe harbor sites become possible. The genomic safe harbors (GSHs) ensure that the inserted new genetic material functions as predicted, and do not cause any alterations to the host genome [76]. Studies have shown the use of gene editing tools in case of beta-thalassemia to not be successful in expression of beta-globin in the corrected locus, because of the developmental immaturity of the iPSCs. In such cases, insertion of globin gene copy in the GSH site like AAVS1 has been recommended as an alternative approach [77]. Human iPSC models for gene therapy have also been developed and studied for primary immunodeficiency syndromes, including chronic granulomatous disease (CGD) caused by mutations in genes which code for the phagocyte NADPH oxidase that produces reactive oxygen species (ROS) that kill bacteria. Studies have shown genetically corrected CGD-iPSCs from macrophages and neutrophils using CRISPR/Cas9 system in the single intronic mutation of the CYBB gene to exhibit antimicrobial activity through generation of ROS and phagocytosis [78].

Thus, the potential of iPSCs to study etiology of complex diseases which manifest late in life, as well as to identify markers for precision therapeutics, is worth exploring in the arena of clinical biomedical research. Human iPSC-based models are a true success in our understanding of disease pathogenesis away from the animal models.

Organ donations are a key clinical need to treat end-stage organ failure conditions, and in often cases, patients are left to fight the acute shortage for the same. This apart, from identifying HLA-matched donors, handling risk of infections and rejection, as well as life-long immunosuppression, to a great extent damages quality of life for the affected as well as leads to loss of crucial time. Human iPSCs are being evaluated as a potential source for generating organs that can overcome roadblocks of shortage as well as risk of rejection. Studies have explored the possibility of generating a three-dimensional vascularized and functional liver organ from human iPSCs [79,80,81]. Generation of hepatocyte-like cells using iPSC technology has been reviewed to be fundamentally beneficial for treatment of severe liver disease, screening for drug toxicities, in liver transplantation, as well as to facilitate basic research [21]. Liver organogenesis involves delamination of specific hepatic cells from the foregut endodermal sheet to form a liver bud, which is then vascularized. One study prepared hepatic endoderm cells from human iPSCs through direct differentiation, wherein 80% of the treated cells were found to be positive for the cell fate determining hepatic marker; HNF4A. Further, to stimulate early organogenesis, the iPSCs were cocultured with stromal cells, human umbilical vein endothelial cells, and human mesenchymal stem cells, and after 48h of seeding, the human iPSCs were found to be self-organized into three-dimensional cell clusters visible macroscopically. This iPSC-derived liver bud, when further assessed by quantitative polymerase chain reaction (PCR) and microarray assay for expression analysis, highlighted the pattern to be similar to human fetal liver cell-derived liver buds. Hemodynamic stimulation to form organ was done by cranial window model, and the iPSC-derived tissue was found to perform liver-specific functions including protein synthesis and human-drug specific metabolism actions. This proof-of-concept study highlights the potential to use organ-bud transplantation for organ regeneration [82]. Figure 3 highlights the process of liver development and hepatic differentiation from hiPSCs.

Process of liver development and hepatic differentiation from hiPSCs. The process of isolated cells from patients can be cultured and reprogrammed into patient-specific hiPSCs and quick comparison from natural liver development.

Hepatocytes represent 80% of the liver mass and are the specialized epithelial cells crucial for maintaining homeostasis. The hepatic differentiation involves induction of endoderm differentiation by activin A, fibroblast growth factor 2 (FGF2), and bone morphogenetic protein 4 (BMP4), and such generated hepatocytes have been found to retain features of human liver including lipid and glycogen storage, urea synthesis, etc. Cholangiocytes in the inner space of the bile duct tree have also been generated from the common progenitor hepatoblast, through downregulation of signaling factors including epidermal growth factor (EGF), interleukin 6 (IL-6), Jagged 1, sodium taurocholate, and the generated cholangiocytes have been detected to express mature markers including SOX9 (SRY-Box Transcription Factor 9), OPN (Osteopontin), CK7 (Cytokeratin 7), CK19 (Cytokeratin 19), etc. The kupffer cells are the largest population of resident macrophages in the human body and also facilitate liver regeneration after an ischemic injury. Studies have demonstrated generation of iPSC-derived kupffer cells from macrophage precursors by adding a hepatic stimulus [83,84].

Another study evaluated lung regeneration by endogenous and exogenous stem cell mediated therapeutic approaches. Physiologically the tissue turnover rate in lung is slow and any insult to the regeneration process can lead to development of chronic obstructive pulmonary disease (COPD) as well as idiopathic pulmonary fibrosis. Bone marrow stem cells, embryonic stem cells, as well as iPSCs have shown excellent regenerative capacity to repair injured lung by generating whole lung in the lab using de-cellularized tissue scaffold and stem cells [85]. Lung organogenesis involves proximodistal patterning, branching morphogenesis, alveolarization, and cellular differentiation [86]. A study by Mou et al. [87], described generation of multipotent lung and airway progenitors from mouse ESCs and patient-specific cystic fibrosis (CF) iPSCs. The definitive endoderm from mouse ESCs were converted to foregut endoderm and then into replicating lung endoderm+Nkx2.1 (earliest marker of lung endoderm), which further transformed to a multipotent embryonic lung progenitor and airway progenitor cells. This study further highlighted that precise timing of the BMP, WNT, FGF signaling pathways are crucial for induction of NKX2.1. This study also utilized the same strategy to develop disease-specific lung progenitor cells from CF-iPSCs to make a model platform to study lung diseases. Further, the disease-specific lung progenitors were also engrafted in immunodeficient mice. One study derived lung progenitor cells with ~80% efficiency from iPSCs which differentiated onto alveolar epithelium both in vitro and in vivo. This study used Activin/BMP-4/bFGF treatment to obtain definitive endoderm from iPSC, which was further exposed to a series of pathway inhibitors (BMP, TGF-, WNT), followed by longer exposure to FGF-19, KGF, BMP-4 and a small molecule CHIR99021 to mimic Wnt pathway to generate anterior foregut endoderm. The generated lung progenitors were further differentiated to many pulmonary progenitor cells including basal cells, goblet cells, ciliated cells, in vitro as well as in immunodeficient mice [88].

Studies have also utilized iPSC-derived organ models to study pathogenesis of the coronavirus disease-2019 (COVID-19). One study established a screening strategy to identify drugs that reduce angiotensin converting enzyme 2 (ACE2) using human ESCs-derived cardiac cells and lung organoids, as the infection occurs due to binding of the virus to ACE2 on the cell membrane. Target analysis revealed treatment with antiandrogenic drugs to reduce ACE2 expression, thus protecting the lung organoids from the SARS-CoV-2 infection. Clinical studies on COVID-19 identified patients with prostate disease, with elevated levels of circulating androgen to pose increased risk for high disease severity [89]. Another study utilized human lung stem-cell based alveolospheres to generate insights on SARS-CoV-2 mediated interferon response and pneumocyte dysfunction. This study described a chemically defined modular alveolosphere culture system for propagation and differentiation of the human alveolar type 2 (AT2) derived from primary lung tissue. The cultured cells were found to express ACE2 and transcriptome analysis of the infected alveolospheres were found to mirror features of the COVID-19 infected human lung, together with the interferon-mediated inflammatory response, loss of surfactant proteins, and apoptosis. Further, infected alveolospheres when treated with low dose interferons, a reduction in viral replication was noted. Thus, human stem-cell based models have also added insight to COVID-19 pathogenesis [90]. In case of use of iPSC three-dimensional model, a study by Huang et al. [91] found the derived AT2 to be susceptible to SARS-CoV-2 with decreased expression of surfactant proteins, and cell death, exhibiting delayed type I interferon response with multiplicities of infection of 5 and interferon-stimulated genes. Another study assessed inhibitor of SARS-CoV-2 infection using lung and colonic organoids from the gut. The derived iPSCs in three-dimensional, were positive for SARS-CoV-2 infection. In case of immune response, the tumor necrosis factor (TNF) and interleukin-17 (IL-17) signatures were noted after 24 h with multiplicities of infection of 0.1. This study also screened US Food and Drug Administration (USFDA) approved entry inhibitors including imatinib, mycophenolic acid, and quinacrine dihydrochloride; wherein treatment at physiologically relevant levels highlighted inhibition of SARS-CoV-2 infection both in iPSC-lung organoids and colonoids, indicating that iPSC models also prove to be a valuable source for safe drug screening [92].

Development of organ-specific progenitor cells which progress into the complete three-dimensional organ in a lab highlights the potential of iPSCs in regenerative medicine. Further, the impact of organ-system models to study infection pathology, highlights the wide clinical arena in which iPSC-technology can be used.

The iPSCs have been generated for modelling pathogenesis of many diseases, and one of the most notable additions to the same is cancer, including models for familial cancer syndromes. One such study reports on the successful establishment of Li-Fraumeni Syndrome (LFS) patient-derived iPSC to study role of p53 in development of osteosarcoma. LFS being a heterogenous cancer condition, osteosarcoma is one of the types wherein relevance of germline p53 mutations have been highly reported. The pre-existing murine LFS models have been insufficient in charting the entire tumor landscape and patient-derived iPSCs in this regard have demonstrated the feasibility to effectively study human cancer syndromes. Studies have found the LFS-derived mesenchymal stem cells to exhibit low expression of targets of p53 including p21 and MDM2; highlighting their ability to retain the defective p53 function from the parental fibroblasts. Further, p53 knockdown was found to cause upregulation of osteogenic markers in LFS osteoblasts, and the possibility to attain osteosarcoma-related phenotypes in LFS iPSC-derived osteoblasts was found. Further, gene expression analysis in LFS-derived osteoblasts was found to correlate with poor patient survival, and decreased time for recurrence. The impaired H19 restoration was also found to repress tumorigenic potential [36]. Another study involving modelling of osteosarcoma from LFS derived-iPSC identified the LFS osteoblasts to recapitulate oncogenic properties of osteosarcoma proving to be an excellent model to study disease pathogenesis [93]. In case of Noonan syndrome (NS) characterized by germline PTPN11 mutations, studies which have derived hiPSCs from hematopoietic cells and which harbor the PTPN11 mutations were found to successfully recapitulate features of NS. The iPSC-derived NS myeloid cells were found to exhibit increased STAT5 signaling and enhanced expression of micro-RNAs viz. miR-223 and miR-15a. Further, reducing miR-223 function was found to normalize myelogenesis, highlighting the role of micro-RNA dysregulation in early oncogenesis [94]. Human iPSC-derived hereditary cancer models have also aided in identifying BRCA1-deleted tumor niche to be the cause for disease progression [95].

The iPSC models around cancer aid in overcoming the hurdles posed by traditional cancer cell line systems, which may lose the characteristics of the original tumor with time, and further harnessing primary cancer cells at different stages of carcinogenesis is not feasible. The established iPSC reprogramming strategies can aid in differentiation of cancer cells to target cell lineages which can aid in studying each of the different stages in cancer progression [96]. The iPSCs developed from primary tumors, as well as cancer cell lines are invaluable tools to study genetic alterations early-on in familial cancer syndromes which is crucial in understand disease pathogenesis. Apart from cancer cell lines, patient-derived xenograft models have also been proven to be efficacious for understanding tumor heterogeneity, genetic alterations, and testing efficacy of cytotoxic drugs. However, the need for successful engraftment, technical challenges, and variable growth rates, are the key limitations. Even in case of animal models, high rate of mortality, and absence of metastasis are the limitations [97,98,99]. Advancements in iPSC models have also led researchers to be able to design autologous iPSC-based vaccine which presents a broad spectrum of tumor antigens to the immune system of the mice, and also found success in eliciting a prophylactic reaction against multiple cancer types. These studies highlight the great promise iPSC-based autologous vaccines present towards cancer prevention as well as therapy [100].

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January 31st, 2025

Induced pluripotent stem cells | UCLA BSCRC – University of California …

By daniellenierenberg

Induced pluripotent stem cells are derived from skin or blood cells that have been reprogrammed back into an embryonic-like pluripotent state that enables the development of an unlimited source of any type of human cell needed for therapeutic purposes. For example, iPSC can be prodded into becoming beta islet cells to treat diabetes, blood cells to create new blood free of cancer cells for a leukemia patient or neurons to treat neurological disorders.

Using iPSC technology, center researchers have reprogrammed skin cells into active motor neurons, egg and sperm precursors, liver cells, bone precursors, and blood cells. In addition, patients with untreatable diseases such as, ALS, Rett syndrome, Lesch-Nyhan syndrome, and Duchenne muscular dystrophy donate skin cells to our center for iPSC reprogramming In stem cell research, scientists can reprogram cells that have undergone differentiation, such as skin or blood cells, to revert back into an embryonic-like state. The resulting cells are called induced pluripotent stem cells. reprogramming In stem cell research, scientists can reprogram cells that have undergone differentiation, such as skin or blood cells, to revert back into an embryonic-like state. The resulting cells are called induced pluripotent stem cells. research. The generous participation of patients and their families in this research enables our scientists to study these diseases in the laboratory in the hope of developing new treatment technologies.

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January 31st, 2025

iPS cells and reprogramming: turn any cell of the body into a stem cell

By daniellenierenberg

An important step in developing a therapy for a given disease is understanding exactly how the disease works: what exactly goes wrong in the body? To do this, researchers need to study the cells or tissues affected by the disease, but this is not always as simple as it sounds. For example, its almost impossible to obtain genuine brain cells from patients with Parkinsons disease, especially in the early stages of the disease before the patient is aware of any symptoms. Reprogramming means scientists can now get access to large numbers of the particular type of neurons (brain cells) that are affected by Parkinsons disease. Researchers first make iPS cells from, for example, skin biopsies from Parkinsons patients. They then use these iPS cells to produce neurons in the laboratory. The neurons have the same genetic background (the same basic genetic make-up) as the patients own cells. Thus scientist can directly work with neurons affected by Parkinsons disease in a dish. They can use these cells to learn more about what goes wrong inside the cells and why. Cellular disease models like these can also be used to search for and test new drugs to treat or protect patients against the disease.

iPS cells - derivation and applications:Certain genes can be introduced into adult cells to reprogramme them. The resulting iPS cells resemble embryonic stem cells and can be differentiated into any type of cell to study disease, test drugs or-after gene correction-develop future cell therapies

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January 31st, 2025

FDA Grants Orphan Drug Designation to IPS HEARTs GIVI-MPC Stem Cell Therapy for Becker Muscular Dystrophy – Business Wire

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FDA Grants Orphan Drug Designation to IPS HEARTs GIVI-MPC Stem Cell Therapy for Becker Muscular Dystrophy Business Wire

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FDA Grants Orphan Drug Designation to IPS HEARTs GIVI-MPC Stem Cell Therapy for Becker Muscular Dystrophy - Business Wire

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January 14th, 2025

GMP-compliant iPS cell lines show widespread plasticity in a new set of differentiation workflows for cell replacement and cancer immunotherapy -…

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GMP-compliant iPS cell lines show widespread plasticity in a new set of differentiation workflows for cell replacement and cancer immunotherapy RegMedNet

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January 14th, 2025

Stem cells head to the clinic: treatments for cancer, diabetes and Parkinsons disease could soon be here – Nature.com

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Stem cells head to the clinic: treatments for cancer, diabetes and Parkinsons disease could soon be here Nature.com

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December 27th, 2024

Exclusive: Cell therapy startup Shinobi adds Borges as science chief, Katz as top medical officer – Endpoints News

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Exclusive: Cell therapy startup Shinobi adds Borges as science chief, Katz as top medical officer Endpoints News

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December 18th, 2024

Sumitomo Chemical and Sumitomo Pharma to Establish Regenerative Medicine and Cell Therapy Joint Venture –

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Sumitomo Chemical and Sumitomo Pharma to Establish Regenerative Medicine and Cell Therapy Joint Venture

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The Future of Medicine: Japan Forges Ahead in iPS Cell Research – nippon.com

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Japans big bet on stem-cell therapies might soon pay off with medical breakthroughs – Nature

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Clinical trials test the safety of stem-cell therapy for Parkinsons disease – Nature

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Dont rush promising stem-cell therapies – Nature

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Pioneering a Cure for Parkinson’s Disease: First Allogeneic iPS Cell-Based Therapy Demonstrates Safety and Efficacy – cira.kyoto-u.ac.jp

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Kyoto University Team Achieves Breakthrough In Parkinson’s Treatment – Evrim Aac

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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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Induced Pluripotent Stem Cells and Their Potential for Basic and …

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iPS cell therapy 2.0: Preparing for next-generation regenerative …

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iPSC Therapy: Advances and Clinical Potential – BiologyInsights

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I Peace establishes and offers low immunoreaction risk GMP iPS Cells – PR Newswire

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Induced Pluripotent Stem Cells (iPSCs)Roles in Regenerative Therapies …

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Induced pluripotent stem cells | UCLA BSCRC – University of California …

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iPS cells and reprogramming: turn any cell of the body into a stem cell

By daniellenierenberg

FDA Grants Orphan Drug Designation to IPS HEARTs GIVI-MPC Stem Cell Therapy for Becker Muscular Dystrophy – Business Wire

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GMP-compliant iPS cell lines show widespread plasticity in a new set of differentiation workflows for cell replacement and cancer immunotherapy -…

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Stem cells head to the clinic: treatments for cancer, diabetes and Parkinsons disease could soon be here – Nature.com

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Exclusive: Cell therapy startup Shinobi adds Borges as science chief, Katz as top medical officer – Endpoints News

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Sumitomo Chemical and Sumitomo Pharma to Establish Regenerative Medicine and Cell Therapy Joint Venture –

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