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.     

Applications of patient-specific induced pluripotent stem cells; focused on disease modeling, drug screening and therapeutic potentials for liver disease

The recent advances in the induced pluripotent stem cell (iPSC) research have significantly changed our perspectives on regenerative medicine by providing researchers with a unique tool to derive disease-specific stem cells for study. In this review, we describe the human iPSC generation from developmentally diverse origins (i.e. endoderm-, mesoderm-, and ectoderm- tissue derived human iPSCs) and multistage hepatic differentiation protocols, and discuss both basic and clinical applications of these cells including disease modeling, drug toxicity screening/drug discovery, gene therapy and cell replacement therapy.     

A cell surfaceome map for immunophenotyping and sorting pluripotent stem cells

Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N-glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations.     

iPS cells: a game changer for future medicine

The induced pluripotent stem cell (iPSC) technology is instrumental in advancing the fields of disease modeling and cell transplantation. We herein discuss the various issues regarding disease modeling and cell transplantation presented in previous reports, and also describe new iPSC‐based medicine including iPSC clinical trials. In such trials, iPSCs from patients can be used to predict drug responders/non‐responders by analyzing the efficacy of the drug on iPSC‐derived cells. They could also be used to stratify patients after actual clinical trials, including those with sporadic diseases, based on the drug responsiveness of each patient in the clinical trials. iPSC‐derived cells can be used for the identification of response markers, leading to increased success rates in such trials. Since iPSCs can be used in micromedicine for drug discovery, and in macromedicine for actual clinical trials, their use would tightly connect both micro‐ and macromedicine. The use of iPSCs in disease modeling, cell transplantation, and clinical trials could therefore lead to significant changes in the future of medicine.     

Impact of induced pluripotent stem cells on the study of central nervous system disease

The derivation of pluripotent stem cells from somatic tissues has provided researchers with a source of patient-specific stem cells. The potential applications of this technology are truly momentous, and include cellular modeling of disease processes, drug discovery, and cell-based therapy. Here, we review the use of induced pluripotent stem cells (iPSCs) to study CNS disease. Since the iPSC field is still in its infancy, we also discuss some of the challenges that will need to be overcome before the potential of this technology to study and to treat neurological and psychiatric disorders can be fully harnessed.     

The Potential for Immunogenicity of Autologous Induced Pluripotent Stem Cell-derived Therapies

Induced pluripotent stem cell (iPSC) technology offers the promise of immune-matched cell therapies for a wide range of diseases and injuries. It is generally assumed that cells derived from autologous iPSCs will be immune-privileged. However, there are reasons to question this assumption, including recent studies that have tested iPSC immunogenicity in various ways with conflicting results. Understanding the risk of an immune response and developing strategies to minimize it will be important steps before clinical testing. Here, we review the evidence for autologous iPSC immunogenicity, its potential causes, and approaches for assessment and mitigation.     

Biomaterial aided differentiation and maturation of induced pluripotent stem cells

Engineering/reprogramming differentiated adult somatic cells to gain the ability to differentiate into any type of cell lineage are called as induced pluripotent stem cells (iPSCs). Offering unlimited self-renewal and differentiation potential, these iPSC are aspired to meet the growing demands in the field of regenerative medicine, tissue engineering, disease modeling, nanotechnology, and drug discovery. Biomaterial fabrication with the rapid evolution of technology increased their versatility and utility in regenerative medicine and tissue engineering, revolutionizing the stem cell biology research with the property to guide the process of proliferation, differentiation, and morphogenesis. Combining traditional culture platforms of iPSC with biomaterials aids to overcome the limitations associated with derivation, proliferation, and maturation, thereby could improve the clinical translation of iPSC. The present review discusses in brief about the reprogramming techniques for the derivation iPSC and details on several biomaterial guided differentiation of iPSC to different cell types with specific relevance to tissue engineering/regenerative medicine.     

Epigenetic characterization of the FMR1 gene and aberrant neurodevelopment in human induced pluripotent stem cell models of fragile X syndrome

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. In addition to cognitive deficits, FXS patients exhibit hyperactivity, attention deficits, social difficulties, anxiety, and other autistic-like behaviors. FXS is caused by an expanded CGG trinucleotide repeat in the 5' untranslated region of the Fragile X Mental Retardation (FMR1) gene leading to epigenetic silencing and loss of expression of the Fragile X Mental Retardation protein (FMRP). Despite the known relationship between FMR1 CGG repeat expansion and FMR1 silencing, the epigenetic modifications observed at the FMR1 locus, and the consequences of the loss of FMRP on human neurodevelopment and neuronal function remain poorly understood. To address these limitations, we report on the generation of induced pluripotent stem cell (iPSC) lines from multiple patients with FXS and the characterization of their differentiation into post-mitotic neurons and glia. We show that clones from reprogrammed FXS patient fibroblast lines exhibit variation with respect to the predominant CGG-repeat length in the FMR1 gene. In two cases, iPSC clones contained predominant CGG-repeat lengths shorter than measured in corresponding input population of fibroblasts. In another instance, reprogramming a mosaic patient having both normal and pre-mutation length CGG repeats resulted in genetically matched iPSC clonal lines differing in FMR1 promoter CpG methylation and FMRP expression. Using this panel of patient-specific, FXS iPSC models, we demonstrate aberrant neuronal differentiation from FXS iPSCs that is directly correlated with epigenetic modification of the FMR1 gene and a loss of FMRP expression. Overall, these findings provide evidence for a key role for FMRP early in human neurodevelopment prior to synaptogenesis and have implications for modeling of FXS using iPSC technology. By revealing disease-associated cellular phenotypes in human neurons, these iPSC models will aid in the discovery of novel therapeutics for FXS and other autism-spectrum disorders sharing common pathophysiology.     

Ten years of iPSC: clinical potential and advances in vitro hematopoietic differentiation

Ten years have passed since the first publication announcing the generation of induced pluripotent stem cells (iPSCs). Issues related to ethics, immune rejection, and cell availability seemed to be solved following this breakthrough. The development of iPSC technology allows advances in in vitro cell differentiation for cell therapy purpose and other clinical applications. This review provides a perspective on the iPSC potential for cell therapies, particularly for hematological applications. We discuss the advances in in vitro hematopoietic differentiation, the possibilities to employ iPSC in hematology studies, and their potential clinical application in hematologic diseases. The generation of red blood cells and functional T cells and the genome editing technology applied to mutation correction are also covered. We highlight some of the requirements and obstacles to be overcome before translating these cells from research to the clinic, for instance, iPSC variability, genotoxicity, the differentiation process, and engraftment. Also, we evaluate the patent landscape and compile the clinical trials in the field of pluripotent stem cells. Currently, we know much more about iPSC than in 2006, but there are still challenges that must be solved. A greater understanding of molecular mechanisms underlying the generation of hematopoietic stem cells is necessary to produce suitable and transplantable hematopoietic stem progenitor cells from iPSC.     

In vitro models of multiple system atrophy from primary cells to induced pluripotent stem cells

Multiple system atrophy (MSA) is a rare neurodegenerative disease with a fatal outcome. Nowadays, only symptomatic treatment is available for MSA patients. The hallmarks of the disease are glial cytoplasmic inclusions (GCIs), proteinaceous aggregates mainly composed of alpha‐synuclein, which accumulate in oligodendrocytes. However, despite the extensive research efforts, little is known about the pathogenesis of MSA. Early myelin dysfunction and alpha‐synuclein deposition are thought to play a major role, but the origin of the aggregates and the causes of misfolding are obscure. One of the reasons for this is the lack of a reliable model of the disease. Recently, the development of induced pluripotent stem cell (iPSC) technology opened up the possibility of elucidating disease mechanisms in neurodegenerative diseases including MSA. Patient specific iPSC can be differentiated in glia and neurons, the cells involved in MSA, providing a useful human disease model. Here, we firstly review the progress made in MSA modelling with primary cell cultures. Subsequently, we focus on the first iPSC‐based model of MSA, which showed that alpha‐synuclein is expressed in oligodendrocyte progenitors, whereas its production decreases in mature oligodendrocytes. We then highlight the opportunities offered by iPSC in studying disease mechanisms and providing innovative models for testing therapeutic strategies, and we discuss the challenges connected with this technique.     

Reprogramming somatic cells to pluripotency: A fresh look at Yamanaka’s model

In 2006, Dr Shinya Yamanaka succeeded to reprogram somatic cells into pluripotent stem cells (iPSC) by delivering the genes encoding Oct4, Sox2, Klf4, and c-Myc. This achievement represents a fundamental breakthrough in stem cell biology and opens up a new era in regenerative medicine. However, the molecular processes by which somatic cells are reprogrammed into iPSC remain poorly understood. In 2009, Yamanaka proposed the elite and stochastic models for reprogramming mechanisms. To date, many investigators in the field of iPSC research support the concept of stochastic model, i.e., somatic cell reprogramming is an event of epigenetic transformation. A mathematical model, f (C_d, k), has also been proposed to predict the stochastic process. Here we wish to revisit the Yamanaka model and summarize the recent advances in this research field.     

Bioluminescent Imaging of Genetically Selected Induced Pluripotent Stem Cell-Derived Cardiomyocytes after Transplantation into Infarcted Heart of Syngeneic Recipients

Cell loss after transplantation is a major limitation for cell replacement approaches in regenerative medicine. To assess the survival kinetics of induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) we generated transgenic murine iPSC lines which, in addition to CM-specific expression of puromycin N-acetyl-transferase and enhanced green fluorescent protein (EGFP), also constitutively express firefly luciferase (FLuc) for bioluminescence (BL) in vivo imaging. While undifferentiated iPSC lines generated by random integration of the transgene into the genome retained stable FLuc activity over many passages, the BL signal intensity was strongly decreased in purified iPS-CM compared to undifferentiated iPSC. Targeted integration of FLuc-expression cassette into the ROSA26 genomic locus using zinc finger nuclease (ZFN) technology strongly reduced transgene silencing in iPS-CM, leading to a several-fold higher BL compared to iPS-CM expressing FLuc from random genomic loci. To investigate the survival kinetics of iPS-CM in vivo, purified CM obtained from iPSC lines expressing FLuc from a random or the ROSA26 locus were transplanted into cryoinfarcted hearts of syngeneic mice. Engraftment of viable cells was monitored by BL imaging over 4 weeks. Transplanted iPS-CM were poorly retained in the myocardium independently of the cell line used. However, up to 8% of cells survived for 28 days at the site of injection, which was confirmed by immunohistological detection of EGFP-positive iPS-CM in the host tissue. Transplantation of iPS-CM did not affect the scar formation or capillary density in the periinfarct region of host myocardium. This report is the first to determine the survival kinetics of drug-selected iPS-CM in the infarcted heart using BL imaging and demonstrates that transgene silencing in the course of iPSC differentiation can be greatly reduced by employing genome editing technology. FLuc-expressing iPS-CM generated in this study will enable further studies to reduce their loss, increase long-term survival and functional integration upon transplantation.     

Mechanism and methods to induce pluripotency

Pluripotent stem cells are able to self-renew indefinitely and differentiate into all types of cells in the body. They can thus be an inexhaustible source for future cell transplantation therapy to treat degenerative diseases which currently have no cure. However, non-autologous cells will cause immune rejection. Induced pluripotent stem cell (iPSC) technology can convert somatic cells to the pluripotent state, and therefore offers a solution to this problem. Since the first generation of iPSCs, there has been an explosion of relevant research, from which we have learned much about the genetic networks and epigenetic landscape of pluripotency, as well as how to manipulate genes, epigenetics, and microRNAs to obtain iPSCs. In this review, we focus on the mechanism of cellular reprogramming and current methods to induce pluripotency. We also highlight new problems emerging from iPSCs. Better understanding of the fundamental mechanisms underlying pluripotenty and refining the methodology of iPSC generation will have a significant impact on future development of regenerative medicine.     

A defined, feeder-free, serum-free system to generate in vitro hematopoietic progenitors and differentiated blood cells from hESCs and hiPSCs

Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes, for drug discovery, and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimensional, defined and highly efficient protocol that avoids the use of feeder cells, serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells.     

Rise of iPSCs as a cell source for adoptive immunotherapy

Adoptive T cell transfer is a potentially effective strategy for treating cancer and viral infections. However, previous studies of cancer immunotherapy have shown that T cells expanded in vitro fall into an exhausted state and, consequently, have limited therapeutic effect. One way to overcome this obstacle is to use induced pluripotent stem cells (iPSCs) as a cell source for making effector T cells. In recent years, there have been several reports on generating effector T cells suitable for adoptive immunotherapy. The reported findings suggest that using iPSC technology, it may be possible to stably derive large numbers of juvenile memory T cells targeted to cancers or viruses. In this review, we describe a strategy for applying iPSC technology to immunotherapy and the characteristics of T cells derived from iPSCs. We also discuss how these technologies can be applied clinically in the future.     

Human induced pluripotent stem cells derived hepatocytes: rising promise for disease modeling,drug development and cell therapy

Recent advances in the study of human hepatocytes derived from induced pluripotent stem cells (iPSC) represent new promises for liver disease study and drug discovery. Human hepatocytes or hepatocyte-like cells differentiated from iPSC recapitulate many functional properties of primary human hepatocytes and have been demonstrated as a powerful and efficient tool to model human liver metabolic diseases and facilitate drug development process. In this review, we summarize the recent progress in this field and discuss the future perspective of the application of human iPSC derived hepatocytes.     

Inducing pluripotency in vitro: recent advances and highlights in induced pluripotent stem cells generation and pluripotency reprogramming

Induced pluripotent stem cells (iPSCs) are considered patient‐specific counterparts of embryonic stem cells as they originate from somatic cells after forced expression of pluripotency reprogramming factors Oct4, Sox2, Klf4 and c‐Myc. iPSCs offer unprecedented opportunity for personalized cell therapies in regenerative medicine. In recent years, iPSC technology has undergone substantial improvement to overcome slow and inefficient reprogramming protocols, and to ensure clinical‐grade iPSCs and their functional derivatives. Recent developments in iPSC technology include better reprogramming methods employing novel delivery systems such as non‐integrating viral and non‐viral vectors, and characterization of alternative reprogramming factors. Concurrently, small chemical molecules (inhibitors of specific signalling or epigenetic regulators) have become crucial to iPSC reprogramming; they have the ability to replace putative reprogramming factors and boost reprogramming processes. Moreover, common dietary supplements, such as vitamin C and antioxidants, when introduced into reprogramming media, have been found to improve genomic and epigenomic profiles of iPSCs. In this article, we review the most recent advances in the iPSC field and potent application of iPSCs, in terms of cell therapy and tissue engineering.     

Application of induced pluripotent stem cells to hematologic disease

Induced pluripotent stem cells (iPSC) were first generated from somatic cells via the transduction of four ‘Yamanaka’ factors, Oct4, Sox2, Klf4 and c-Myc. Because iPSC are similar to embryonic stem cells (ESC) and can be differentiated into any cell type of choice, iPSC have the potential to become a platform for personalized medicine by allowing a patient's own cells to become a source of therapeutic tissue. This review describes the main challenges in iPSC technology by focusing on its application to hematologic diseases. The explosive interest in improving iPSC technology has generated numerous genetic and chemical methods for iPSC derivation, but these methods must be evaluated comparatively for their safety and efficacy because there are risks of genetic abnormalities and oncogenesis. Competent iPSC will need to be selected carefully based on physical, genetic and functional criteria, and differentiated efficiently into hematopoietic stem cells via modulation of several signaling pathways before they prove valuable in the clinic.