Human induced pluripotent stem cell-derived therapies for regeneration after central nervous system injury

In recent years,the progression of stem cell therapies has shown great promise in advancing the nascent field of regenerative medicine.Considering the non-regenerative nature of the mature central nervous system,the concept that“blank”cells could be reprogrammed and functionally integrated into host neural networks remained intriguing.Previous work has also demonstrated the ability of such cells to stimulate intrinsic growth programs in post-mitotic cells,such as neurons.While embryonic stem cells demonstrated great potential in treating central nervous system pathologies,ethical and technical concerns remained.These barriers,along with the clear necessity for this type of treatment,ultimately prompted the advent of induced pluripotent stem cells.The advantage of pluripotent cells in central nervous system regeneration is multifaceted,permitting differentiation into neural stem cells,neural progenitor cells,glia,and various neuronal subpopulations.The precise spatiotemporal application of extrinsic growth factors in vitro,in addition to microenvironmental signaling in vivo,influences the efficiency of this directed differentiation.While the pluri-or multipotency of these cells is appealing,it also poses the risk of unregulated differentiation and teratoma formation.Cells of the neuroectodermal lineage,such as neuronal subpopulations and glia,have been explored with varying degrees of success.Although the risk of cancer or teratoma formation is greatly reduced,each subpopulation varies in effectiveness and is influenced by a myriad of factors,such as the timing of the transplant,pathology type,and the ratio of accompanying progenitor cells.Furthermore,successful transplantation requires innovative approaches to develop delivery vectors that can mitigate cell death and support integration.Lastly,host immune responses to allogeneic grafts must be thoroughly characterized and further developed to reduce the need for immunosuppression.Translation to a clinical setting will involve careful consideration when assessing both physiologic and functional outcomes.This review will highlight both successes and challenges faced when using human induced pluripotent stem cell-derived cell transplantation therapies to promote endogenous regeneration.

Induced pluripotent stem cell-related approaches to generate dopaminergic neurons for Parkinson's disease

The progressive loss of dopaminergic neurons in affected patient brains is one of the pathological features of Parkinson's disease,the second most common human neurodegenerative disease.Although the detailed pathogenesis accounting for dopaminergic neuron degeneration in Parkinson's disease is still unclear,the advancement of stem cell approaches has shown promise for Parkinson's disease research and therapy.The induced pluripotent stem cells have been commonly used to generate dopaminergic neurons,which has provided valuable insights to improve our understanding of Parkinson's disease pathogenesis and contributed to anti-Parkinson's disease therapies.The current review discusses the practical approaches and potential applications of induced pluripotent stem cell techniques for generating and differentiating dopaminergic neurons from induced pluripotent stem cells.The benefits of induced pluripotent stem cell-based research are highlighted.Various dopaminergic neuron differentiation protocols from induced pluripotent stem cells are compared.The emerging three-dimension-based brain organoid models compared with conventional two-dimensional cell culture are evaluated.Finally,limitations,challenges,and future directions of induced pluripotent stem cell–based approaches are analyzed and proposed,which will be significant to the future application of induced pluripotent stem cell-related techniques for Parkinson's disease.

Multiple factors to assist human-derived induced pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neurons

Midbrain dopaminergic neurons play an important role in the etiology of neurodevelopmental and neurodegenerative diseases.They also represent a potential source of transplanted cells for therapeutic applications.In vitro differentiation of functional midbrain dopaminergic neurons provides an accessible platform to study midbrain neuronal dysfunction and can be used to examine obstacles to dopaminergic neuronal development.Emerging evidence and impressive advances in human induced pluripotent stem cells,with tuned neural induction and differentiation protocols,makes the production of induced pluripotent stem cell-derived dopaminergic neurons feasible.Using SB431542 and dorsomorphin dual inhibitor in an induced pluripotent stem cell-derived neural induction protocol,we obtained multiple subtypes of neurons,including 20%tyrosine hydroxylase-positive dopaminergic neurons.To obtain more dopaminergic neurons,we next added sonic hedgehog(SHH)and fibroblast growth factor 8(FGF8)on day 8 of induction.This increased the proportion of dopaminergic neurons,up to 75%tyrosine hydroxylase-positive neurons,with 15%tyrosine hydroxylase and forkhead box protein A2(FOXA2)co-expressing neurons.We further optimized the induction protocol by applying the small molecule inhibitor,CHIR99021(CHIR).This helped facilitate the generation of midbrain dopaminergic neurons,and we obtained 31-74%midbrain dopaminergic neurons based on tyrosine hydroxylase and FOXA2 staining.Thus,we have established three induction protocols for dopaminergic neurons.Based on tyrosine hydroxylase and FOXA2 immunostaining analysis,the CHIR,SHH,and FGF8 combined protocol produces a much higher proportion of midbrain dopaminergic neurons,which could be an ideal resource for tackling midbrain-related diseases.

Patient-derived induced pluripotent stem cells with a MERTK mutation exhibit cell junction abnormalities and aberrant cellular differentiation potential

BACKGROUND Human induced pluripotent stem cell(hiPSC)technology is a valuable tool for generating patient-specific stem cells,facilitating disease modeling,and invest-igating disease mechanisms.However,iPSCs carrying specific mutations may limit their clinical applications due to certain inherent characteristics.AIM To investigate the impact of MERTK mutations on hiPSCs and determine whether hiPSC-derived extracellular vesicles(EVs)influence anomalous cell junction and differentiation potential.METHODS We employed a non-integrating reprogramming technique to generate peripheral blood-derived hiPSCs with and hiPSCs without a MERTK mutation.Chromo-somal karyotype analysis,flow cytometry,and immunofluorescent staining were utilized for hiPSC identification.Transcriptomics and proteomics were employed to elucidate the expression patterns associated with cell junction abnormalities and cellular differentiation potential.Additionally,EVs were isolated from the supernatant,and their RNA and protein cargos were examined to investigate the involvement of hiPSC-derived EVs in stem cell junction and differentiation.RESULTS The generated hiPSCs,both with and without a MERTK mutation,exhibited normal karyotype and expressed pluripotency markers;however,hiPSCs with a MERTK mutation demonstrated anomalous adhesion capability and differentiation potential,as confirmed by transcriptomic and proteomic profiling.Furthermore,hiPSC-derived EVs were involved in various biological processes,including cell junction and differentiation.CONCLUSION HiPSCs with a MERTK mutation displayed altered junction characteristics and aberrant differentiation potential.Furthermore,hiPSC-derived EVs played a regulatory role in various biological processes,including cell junction and differentiation.

Modulation of the Nogo signaling pathway to overcome amyloid-β-mediated neurite inhibition in human pluripotent stem cell-derived neurites

Neuronal cell death and the loss of connectivity are two of the primary pathological mechanisms underlying Alzheimer's disease.The accumulation of amyloid-βpeptides,a key hallmark of Alzheimer's disease,is believed to induce neuritic abnormalities,including reduced growth,extension,and abnormal growth cone morphology,all of which contribute to decreased connectivity.However,the precise cellular and molecular mechanisms governing this response remain unknown.In this study,we used an innovative approach to demonstrate the effect of amyloid-βon neurite dynamics in both two-dimensional and three-dimensional cultu re systems,in order to provide more physiologically relevant culture geometry.We utilized various methodologies,including the addition of exogenous amyloid-βpeptides to the culture medium,growth substrate coating,and the utilization of human-induced pluripotent stem cell technology,to investigate the effect of endogenous amyloid-βsecretion on neurite outgrowth,thus paving the way for potential future applications in personalized medicine.Additionally,we also explore the involvement of the Nogo signaling cascade in amyloid-β-induced neurite inhibition.We demonstrate that inhibition of downstream ROCK and RhoA components of the Nogo signaling pathway,achieved through modulation with Y-27632(a ROCK inhibitor)and Ibuprofen(a Rho A inhibitor),respectively,can restore and even enhance neuronal connectivity in the presence of amyloid-β.In summary,this study not only presents a novel culture approach that offers insights into the biological process of neurite growth and inhibition,but also proposes a specific mechanism for reduced neural connectivity in the presence of amyloid-βpeptides,along with potential intervention points to restore neurite growth.Thereby,we aim to establish a culture system that has the potential to serve as an assay for measuring preclinical,predictive outcomes of drugs and their ability to promote neurite outgrowth,both generally and in a patient-specific manner.

Generation of male germ cells from induced pluripotent stem cells （iPS cells）： an in vitro and in vivo study

Recent studies have reported that induced pluripotent stem （iPS） cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitrodifferentiation and in vivotransplantation. Embryoid bodies （EBs） were obtained from iPS cells using leukaemia inhibitor factor （LIF）-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in StraSand Vasa mRNA in the EBs derived from iPS cells, iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells （SSCs）, as evidenced by their expression of VASA, as well as CDH1 and GFRal, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.

Genetic Correction and Hepatic Differentiation of Hemophilia B-specific Human Induced Pluripotent Stem Cells

Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.

The MORC2 p.S87L mutation reduces proliferation of pluripotent stem cells derived from a patient with the spinal muscular atrophy-like phenotype by inhibiting proliferation-related signaling pathways

Mutations in the microrchidia CW-type zinc finger protein 2(MORC2)gene are the causative agent of Charcot-Marie-Tooth disease type 2Z(CMT2Z),and the hotspot mutation p.S87L is associated with a more seve re spinal muscular atrophy-like clinical phenotype.The aims of this study were to determine the mechanism of the severe phenotype caused by the MORC2 p.S87L mutation and to explore potential treatment strategies.Epithelial cells were isolated from urine samples from a spinal muscular atrophy(SMA)-like patient[MORC2 p.S87L),a CMT2Z patient[MORC2 p.Q400R),and a healthy control and induced to generate pluripotent stem cells,which were then differentiated into motor neuron precursor cells.Next-generation RNA sequencing followed by KEGG pathway enrichment analysis revealed that differentially expressed genes involved in the PI3K/Akt and MAP K/ERK signaling pathways were enriched in the p.S87L SMA-like patient group and were significantly downregulated in induced pluripotent stem cells.Reduced proliferation was observed in the induced pluripotent stem cells and motor neuron precursor cells derived from the p.S87L SMA-like patient group compared with the CMT2Z patient group and the healthy control.G0/G1 phase cell cycle arrest was observed in induced pluripotent stem cells derived from the p.S87L SMA-like patient.MORC2 p.S87Lspecific antisense oligonucleotides(p.S87L-ASO-targeting)showed significant efficacy in improving cell prolife ration and activating the PI3K/Akt and MAP K/ERK pathways in induced pluripotent stem cells.Howeve r,p.S87L-ASO-ta rgeting did not rescue prolife ration of motor neuron precursor cells.These findings suggest that downregulation of the PI3K/Akt and MAP K/ERK signaling pathways leading to reduced cell proliferation and G0/G1 phase cell cycle arrest in induced pluripotent stem cells might be the underlying mechanism of the severe p.S87L SMA-like phenotype.p.S87L-ASO-targeting treatment can alleviate disordered cell proliferation in the early stage of pluripotent stem cell induction.

Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells

Human induced pluripotent stem （iPS） cells are similar to embryonic stem （ES） cells, and can proliferate intensively and differentiate into a variety of cell types. However, the hepatic differentiation of human iPS cells has not yet been reported. In this report, human iPS cells were induced to differentiate into hepatic cells by a stepwise protocol. The expression of liver cell markers and liver-related functions of the human iPS cell-derived cells were monitored and compared with that of differentiated human ES cells and primary human hepatocytes. Approximately 60% of the differentiated human iPS cells at day 7 expressed hepatic markers alpha fetoprotein and Alb. The differentiated cells at day 21 exhibited liver cell functions including albumin Asecretion, glycogen synthesis, urea production and inducible cytochrome P450 activity. The expression of hepatic markers and fiver-related functions of the iPS cellderived hepatic ceils were comparable to that of the human ES cell-derived hepatic cells. These results show that human iPS cells, which are similar to human ES cells, can be efficiently induced to differentiate into hepatocyte-like cells.

Transfer of mitochondria from mesenchymal stem cells derived from induced pluripotent stem cells attenuates hypoxia-ischemia-induced mitochondrial dysfunction in PC12 cells

Mitochondrial dysfunction in neurons has been implicated in hypoxia-ischemia-induced brain injury.Although mesenchymal stem cell therapy has emerged as a novel treatment for this pathology,the mechanisms are not fully understood.To address this issue,we first co-cultured 1.5×10^5 PC12 cells with mesenchymal stem cells that were derived from induced pluripotent stem cells at a ratio of 1:1,and then intervened with cobalt chloride(CoCl2)for 24 hours.Reactive oxygen species in PC12 cells was measured by Mito-sox.Mitochondrial membrane potential(ΔΨm)in PC12 cells was determined by JC-1 staining.Apoptosis of PC12 cells was detected by terminal deoxynucleotidal transferase-mediated dUTP nick end-labeling staining.Mitochondrial morphology in PC12 cells was examined by transmission electron microscopy.Transfer of mitochondria from the mesenchymal stem cells derived from induced pluripotent stem cells to damaged PC12 cells was measured by flow cytometry.Mesenchymal stem cells were induced from pluripotent stem cells by lentivirus infection containing green fluorescent protein in mitochondria.Then they were co-cultured with PC12 cells in Transwell chambers and treated with CoCl2 for 24 hours to detect adenosine triphosphate level in PC12 cells.CoCl2-induced PC12 cell damage was dose-dependent.Co-culture with mesenchymal stem cells significantly reduced apoptosis and restoredΔΨm in the injured PC12 cells under CoCl2 challenge.Co-culture with mesenchymal stem cells ameliorated mitochondrial swelling,the disappearance of cristae,and chromatin margination in the injured PC12 cells.After direct co-culture,mitochondrial transfer from the mesenchymal stem cells stem cells to PC12 cells was detected via formed tunneling nanotubes between these two types of cells.The transfer efficiency was greatly enhanced in the presence of CoCl2.More importantly,inhibition of tunneling nanotubes partially abrogated the beneficial effects of mesenchymal stem cells on CoCl2-induced PC12 cell injury.Mesenchymal stem cells reduced CoCl2-induced PC12 cell injury and these effects were in part due to efficacious mitochondrial transfer.

Methods of induced pluripotent stem cells for clinical application

Reprograming somatic cells using exogenetic gene expression represents a groundbreaking step in regenerative medicine. Induced pluripotent stem cells(i PSCs) are expected to yield novel therapies with the potential to solve many issues involving incurable diseases. In particular, applying i PSCs clinically holds the promise of addressing the problems of immune rejection and ethics that have hampered the clinical applications of embryonic stem cells. However, as i PSC research has progressed, new problems have emerged that need to be solved before the routine clinical application of i PSCs can become established. In this review, we discuss the current technologies and future problems of human i PSC generation methods for clinical use.

Modelling mitochondrial dysfunction in Alzheimer’s disease using human induced pluripotent stem cells

Alzheimer’s disease (AD) is the most common form of dementia. To date, only five pharmacological agents have been approved by the Food and Drug Administration for clinical use in AD, all of which target the symptoms of the disease rather than the cause. Increasing our understanding of the underlying pathophysiology of AD will facilitate the development of new therapeutic strategies. Over the years, the major hypotheses of AD etiology have focused on deposition of amyloid beta and mitochondrial dysfunction. In this review we highlight the potential of experimental model systems based on human induced pluripotent stem cells (iPSCs) to provide novel insights into the cellular pathophysiology underlying neurodegeneration in AD. Whilst Down syndrome and familial AD iPSC models faithfully reproduce features of AD such as accumulation of Aβ and tau, oxidative stress and mitochondrial dysfunction, sporadic AD is much more difficult to model in this way due to its complex etiology. Nevertheless, iPSC-based modelling of AD has provided invaluable insights into the underlying pathophysiology of the disease, and has a huge potential for use as a platform for drug discovery.

Using induced pluripotent stem cells as a tool for modelling carcinogenesis

Cancer is a highly heterogeneous group of diseases that despite improved treatments remain prevalent accounting for over 14 million new cases and 8.2 million deaths per year. Studies into the process of carcinogenesis are limited by lack of appropriate models for the development and pathogenesis of the disease based on human tissues. Primary culture of patient samples can help but is difficult to grow for a number of tissues. A potential opportunity to overcome these barriers is based on the landmark study by Yamanaka which demonstrated the ability of four factors;Oct4, Sox2, Klf4, and c-Myc to reprogram human somatic cells in to pluripotency. These cells were termed induced pluripotent stem cells(i PSCs) and display characteristic properties of embryonic stem cells. This technique has a wide range of potential uses including disease modelling, drug testing and transplantation studies. Interestingly i PSCs also share a number of characteristics with cancer cells including self-renewal and proliferation, expression of stem cell markers and altered metabolism. Recently, i PSCs have been generated from a number of human cancer cell lines and primary tumour samples from a range of cancers in an attempt to recapitulate the development of cancer and interrogate the underlying mechanisms involved. This review will outline the similarities between the reprogramming process and carcinogenesis, and how these similarities have been exploited to generate i PSC models for a number of cancers.

Thinking outside the liver: Induced pluripotent stem cells for hepatic applications

The discovery of induced pluripotent stem cells (iPSCs) unraveled a mystery in stem cell research, after identification of four re-programming factors for generating pluripotent stem cells without the need of embryos. This breakthrough in generating iPSCs from somatic cells has overcome the ethical issues and immune rejection involved in the use of human embryonic stem cells. Hence, iPSCs form a great potential source for developing disease models, drug toxicity screening and cell-based therapies. These cells have the potential to differentiate into desired cell types, including hepatocytes, under in vitro as well as under in vivo conditions given the proper microenvironment. iPSC-derived hepatocytes could be useful as an unlimited source, which can be utilized in disease modeling, drug toxicity testing and producing autologous cell therapies that would avoid immune rejection and enable correction of gene defects prior to cell transplantation. In this review, we discuss the induction methods, role of reprogramming factors, and characterization of iPSCs, along with hepatocyte differentiation from iPSCs and potential applications. Further, we discuss the location and detection of liver stem cells and their role in liver regeneration. Although tumor formation and genetic mutations are a cause of concern, iPSCs still form a promising source for clinical applications.

Differentiation of retinal ganglion cells from induced pluripotent stem cells: a review

Glaucoma is a common optic neuropathy that is characterized by the progressive degeneration of axons and the loss of retinal ganglion cells(RGCs). Glaucoma is one of the leading causes of irreversible blindness worldwide. Current glaucoma treatments only slow the progression of RGCs loss. Induced pluripotent stem cells(iPSCs) are capable of differentiating into all three germ layer cell lineages. iPSCs can be patient-specific,making iPSC-derived RGCs a promising candidate for cell replacement. In this review, we focus on discussing the detailed approaches used to differentiate iPSCs into RGCs.

Small extracellular vesicles secreted by induced pluripotent stem cell-derived mesenchymal stem cells improve postoperative cognitive dysfunction in mice with diabetes

Postoperative cognitive dysfunction(POCD)is a common surgical complication.Diabetes mellitus(DM)increases risk of developing POCD after surgery.DM patients with POCD seriously threaten the quality of patients’life,however,the intrinsic mechanism is unclear,and the effective treatment is deficiency.Previous studies have demonstrated neuronal loss and reduced neurogenesis in the hippocampus in mouse models of POCD.In this study,we constructed a mouse model of DM by intraperitoneal injection of streptozotocin,and then induced postoperative cognitive dysfunction by transient bilateral common carotid artery occlusion.We found that mouse models of DM-POCD exhibited the most serious cognitive impairment,as well as the most hippocampal neural stem cells(H-NSCs)loss and neurogenesis decline.Subsequently,we hypothesized that small extracellular vesicles secreted by induced pluripotent stem cell-derived mesenchymal stem cells(iMSC-sEVs)might promote neurogenesis and restore cognitive function in patients with DM-POCD.iMSC-sEVs were administered via the tail vein beginning on day 2 after surgery,and then once every 3 days for 1 month thereafter.Our results showed that iMSC-sEVs treatment significantly recovered compromised proliferation and neuronal-differentiation capacity in H-NSCs,and reversed cognitive impairment in mouse models of DM-POCD.Furthermore,miRNA sequencing and qPCR showed miR-21-5p and miR-486-5p were the highest expression in iMSC-sEVs.We found iMSC-sEVs mainly transferred miR-21-5p and miR-486-5p to promote H-NSCs proliferation and neurogenesis.As miR-21-5p was demonstrated to directly targete Epha4 and CDKN2C,while miR-486-5p can inhibit FoxO1 in NSCs.We then demonstrated iMSC-sEVs can transfer miR-21-5p and miR-486-5p to inhibit EphA4,CDKN2C,and FoxO1 expression in H-NSCs.Collectively,these results indicate significant H-NSC loss and neurogenesis reduction lead to DM-POCD,the application of iMSC-sEVs may represent a novel cell-free therapeutic tool for diabetic patients with postoperative cognitive dysfunction.

Induced pluripotent stem cells for therapy personalization in pediatric patients:Focus on drug-induced adverse events

Adverse drug reactions(ADRs)are major clinical problems,particularly in special populations such as pediatric patients.Indeed,ADRs may be caused by a plethora of different drugs leading,in some cases,to hospitalization,disability or even death.In addition,pediatric patients may respond differently to drugs with respect to adults and may be prone to developing different kinds of ADRs,leading,in some cases,to more severe consequences.To improve the comprehension,and thus the prevention,of ADRs,the set-up of sensitive and personalized assays is urgently needed.Important progress is represented by the possibility of setting up groundbreaking patient-specific assays.This goal has been powerfully achieved using induced pluripotent stem cells(iPSCs).Due to their genetic and physiological species-specific differences and their ability to be differentiated ideally into all tissues of the human body,this model may be accurate in predicting drug toxicity,especially when this toxicity is related to individual genetic differences.This review is an up-to-date summary of the employment of iPSCs as a model to study ADRs,with particular attention to drugs used in the pediatric field.We especially focused on the intestinal,hepatic,pancreatic,renal,cardiac,and neuronal levels,also discussing progress in organoids creation.The latter are three-dimensional in vitro culture systems derived from pluripotent or adult stem cells simulating the architecture and functionality of native organs such as the intestine,liver,pancreas,kidney,heart,and brain.Based on the existing knowledge,these models are powerful and promising tools in multiple clinical applications including toxicity screening,disease modeling,personalized and regenerative medicine.

Induced pluripotent stem cells, a giant leap for mankind therapeutic applications

Induced pluripotent stem cells(iPSC)technology has propelled the field of stem cells biology,providing new cells to explore the molecular mechanisms of pluripotency,cancer biology and aging.A major advantage of human iPSC,compared to the pluripotent embryonic stem cells,is that they can be generated from virtually any embryonic or adult somatic cell type without destruction of human blastocysts.In addition,iPSC can be generated from somatic cells harvested from normal individuals or patients,and used as a cellular tool to unravel mechanisms of human development and to model diseases in a manner not possible before.Besides these fundamental aspects of human biology and physiology that are revealed using iPSC or iPSC-derived cells,these cells hold an immense potential for cell-based therapies,and for the discovery of new or personalized pharmacological treatments for many disorders.Here,we review some of the current challenges and concerns about iPSC technology.We introduce the potential held by iPSC for research and development of novel health-related applications.We briefly present the efforts made by the scientific and clinical communities to create the necessary guidelines and regulations to achieve the highest quality standards in the procedures for iPSC generation,characterization and long-term preservation.Finally,we present some of the audacious and pioneer clinical trials in progress with iPSC-derived cells.

Induced pluripotent stem cells throughout the animal kingdom:Availability and applications

Up until the mid 2000s, the capacity to generate every cell of an organism was exclusive to embryonic stem cells. In 2006, researchers Takahashi and Yamanaka developed an alternative method of generating embryonic-like stem cells from adult cells, which they coined induced pluripotent stem cells (iPSCs). Such iPSCs possess most of the advantages of embryonic stem cells without the ethical stigma associated with derivation of the latter. The possibility of generating “custom-made” pluripotent cells, ideal for patient-specific disease models, alongside their possible applications in regenerative medicine and reproduction, has drawn a lot of attention to the field with numbers of iPSC studies published growing exponentially. IPSCs have now been generated for a wide variety of species, including but not limited to, mouse, human, primate, wild felines, bovines, equines, birds and rodents, some of which still lack well-established embryonic stem cell lines. The paucity of robust characterization of some of these iPSC lines as well as the residual expression of transgenes involved in the reprogramming process still hampers the use of such cells in species preservation or medical research, underscoring the requirement for further investigations. Here, we provide an extensive overview of iPSC generated from a broad range of animal species including their potential applications and limitations.

Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells

Mesenchymal stem cells(MSCs)have received significant attention in recent years due to their large potential for cell therapy.Indeed,they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases.MSCs can be extracted from multiple tissues of the human body.However,several factors may restrict their use for clinical applications:the requirement of invasive procedures for their isolation,their limited numbers,and their heterogeneity according to the tissue of origin or donor.In addition,MSCs often present early signs of replicative senescence limiting their expansion in vitro,and their therapeutic capacity in vivo.Due to the clinical potential of MSCs,a considerable number of methods to differentiate induced pluripotent stem cells(iPSCs)into MSCs have emerged.iPSCs represent a new reliable,unlimited source to generate MSCs(MSCs derived from iPSC,iMSCs)from homogeneous and well-characterized cell lines,which would relieve many of the above mentioned technical and biological limitations.Additionally,the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells.In this review,we analyze the main current protocols used to differentiate human iPSCs into MSCs,which we classify into five different categories:MSC Switch,Embryoid Body Formation,Specific Differentiation,Pathway Inhibitor,and Platelet Lysate.We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization.Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added.The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.