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

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

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

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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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Shinobi Strengthens Leadership to Propel Scalable Immune-Evasive Cell Therapies to the Clinic  The Eastern Progress Online

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BrightPath Bio and Cellistic Announces Process Development and Manufacturing Collaboration for Phase 1 Clinical Trial of iPSC-derived BCMA CAR-iNKT cell  Via Ritzau

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Abstract

Induced pluripotent stem cells (iPSCs) are a new type of pluripotent cellsthat can be obtained by reprogramming animal and human differentiated cells. In this review,issues related to the nature of iPSCs are discussed and different methods ofiPSC production are described. We particularly focused on methods of iPSC production withoutthe genetic modification of the cell genome and with means for increasing the iPSC productionefficiency. The possibility and issues related to the safety of iPSC use in cell replacementtherapy of human diseases and a study of new medicines are considered.

Keywords: induced pluripotent stem cells, directed stem cell differentiation, cell replacement therapy

Pluripotent stem cells are a unique model for studying a variety of processes that occur inthe early development of mammals and a promising tool in cell therapy of human diseases. Theunique nature of these cells lies in their capability, when cultured, for unlimitedselfrenewal and reproduction of all adult cell types in the course of theirdifferentiation [1]. Pluripotency is supported by acomplex system of signaling molecules and gene network that is specific for pluripotent cells.The pivotal position in the hierarchy of genes implicated in the maintenance of pluripotency isoccupied by Oct4, Sox2 , and Nanog genes encodingtranscription factors [2, 3]. The mutual effect of outer signaling molecules and inner factors leads tothe formation of a specific expression pattern, as well as to the epigenome statecharacteristic of stem cells. Both spontaneous and directed differentiations are associatedwith changes in the expression pattern and massive epigenetic transformations, leading totranscriptome and epigenome adjustment to a distinct cell type.

Until recently, embryonic stem cells (ESCs) were the only wellstudied source ofpluripotent stem cells. ESCs are obtained from either the inner cell mass or epiblast ofblastocysts [46]. A series of protocols has been developed for the preparation of variouscell derivatives from human ESCs. However, there are constraints for ESC usein cell replacement therapy. The first constraint is the immune incompatibility between thedonor cells and the recipient, which can result in the rejection of transplanted cells. Thesecond constraint is ethical, because the embryo dies during the isolation of ESCs. The firstproblem can be solved by the somatic cell nuclear transfer into the egg cell and then obtainingthe embryo and ESCs. The nuclear transfer leads to genome reprogramming, in which ovariancytoplasmic factors are implicated. This way of preparing pluripotent cells from certainindividuals was called therapeutic cloning. However, this method is technologyintensive,and the reprogramming yield is very low. Moreover, this approach encounters theabovementioned ethic problem that, in this case, is associated with the generation ofmany human ovarian cells [7].

In 2006, the preparation of pluripotent cells by the ectopic expression of four genes Oct4 , Sox2 , Klf4 , and cMyc in both embryonic and adult murine fibroblasts was first reported[8]. The pluripotent cells derived from somatic ones werecalled induced pluripotent stem cells (iPSCs). Using this set of factors(Oct4, Sox2, Klf4, and cMyc), iPSCs were prepared later from variousdifferentiated mouse [914] and human [1517] cell types. Human iPSCs were obtainedwith a somewhat altered gene set: Oct4 , Sox2 , Nanog , and Lin28 [18].Induced PSCs closely resemble ESCs in a broad spectrum of features. They possess similarmorphologies and growth manners and are equally sensitive to growth factors and signalingmolecules. Like ESCs, iPSCs can differentiate in vitro intoderivatives of all three primary germ layers (ectoderm, mesoderm, and endoderm) and formteratomas following their subcutaneous injection into immunodeficient mice. MurineiPSCs injected into blastocysts are normally included in the development toyield animals with a high degree of chimerism. Moreover, murine iPSCs, wheninjected into tetraploid blastocycts, can develop into a whole organism [19, 20]. Thus, an excellent method thatallows the preparation of pluripotent stem cells from various somatic cell types whilebypassing ethical problems has been uncovered by researchers.

In the first works on murine and human iPSC production, either retro or lentiviralvectors were used for the delivery of Oct4 , Sox2 , Klf4 , and cMyc genes into somatic cells. Theefficiency of transduction with retroviruses is high enough, although it is not the same fordifferent cell types. Retroviral integration into the host genome requires a comparatively highdivision rate, which is characteristic of the relatively narrow spectrum of cultured cells.Moreover, the transcription of retroviral construct under the control of a promoter localizedin 5LTR (long terminal repeat) is terminated when the somatic celltransform switches to the pluripotent state [21]. Thisfeature makes retroviruses attractive in iPSC production. Nevertheless, retroviruses possesssome properties that make iPSCs that are produced using them improper for celltherapy of human diseases. First, retroviral DNA is integrated into the host cell genome. Theintegration occurs randomly; i.e., there are no specific sequences or apparent logic forretroviral integration. The copy number of the exogenous retroviral DNA that is integrated intoa genome may vary to a great extent [15]. Retrovirusesbeing integrated into the cell genome can introduce promoter elements and polyadenylationsignals; they can also interpose coding sequences, thus affecting transcription. Second, sincethe transcription level of exogenous Oct4 , Sox2 , Klf4 , and cMyc in the retroviral constructdecreases with cell transition into the pluripotent state, this can result in a decrease in theefficiency of the stable iPSC line production, because the switch from the exogenous expressionof pluripotency genes to their endogenous expression may not occur. Third, some studies showthat the transcription of transgenes can resume in the cells derived fromiPSCs [22]. The high probability thatthe ectopic Oct4 , Sox2 , Klf4 , and cMyc gene expression will resume makes it impossible to applyiPSCs produced with the use of retroviruses in clinical trials; moreover,these iPSCs are hardly applicable even for fundamental studies onreprogramming and pluripotency principles. Lentiviruses used for iPSC production can also beintegrated into the genome and maintain their transcriptional activity in pluripotent cells.One way to avoid this situation is to use promoters controlled by exogenous substances added tothe culture medium, such as tetracycline and doxycycline, which allows the transgenetranscription to be regulated. iPSCs are already being produced using suchsystems [23].

Another serious problem is the gene set itself that is used for the induction of pluripotency[22]. The ectopic transcription of Oct4 , Sox2 , Klf4 , and cMyc can lead to neoplastic development from cells derived from iPSCs,because the expression of Oct4 , Sox2 , Klf4, and cMyc genes is associated with the development ofmultiple tumors known in oncogenetics [22, 24]. In particular, the overexpression of Oct4 causes murine epithelial cell dysplasia [25],the aberrant expression of Sox2 causes the development of serrated polypsand mucinous colon carcinomas [26], breast tumors arecharacterized by elevated expression of Klf4 [27] , and the improper expression of cMyc is observed in 70% of human cancers [28].Tumor development is oberved in ~50% of murine chimeras obtained through the injection ofretroviral iPSCs into blastocysts, which is very likely associated with thereactivation of exogenous cMyc [29, 30].

Several possible strategies exist for resolving the above-mentioned problems:

The search for a less carcinogenic gene set that is necessary and sufficient for reprogramming;

The minimization of the number of genes required for reprogramming and searching for the nongenetic factors facilitating it;

The search for systems allowing the elimination of the exogenous DNA from the host cell genome after the reprogramming;

The development of delivery protocols for nonintegrated genetic constructs;

The search for ways to reprogram somatic cells using recombinant proteins.

The ectopic expression of cMyc and Klf4 genes isthe most dangerous because of the high probability that malignant tumors will develop [22]. Hence the necessity to find other genes that couldsubstitute cMyc and Klf4 in iPSC production. Ithas been reported that these genes can be successfully substituted by Nanog and Lin28 for reprogramming human somatic cells [18;] . iPSCs were prepared from murine embryonic fibroblastsby the overexpression of Oct4 and Sox2 , as well as the Esrrb gene encoding the murine orphan nuclear receptor beta. It has alreadybeen shown that Esrrb , which acts as a transcription activator of Oct4 , Sox2 , and Nanog , is necessary for theselfrenewal and maintenance of the pluripotency of murine ESCs. Moreover, Esrrb can exert a positive control over Klf4 . Thus, the genes causingelevated carcinogenicity of both iPSCs and their derivatives can besuccessfully replaced with less dangerous ones [31].

The Most Effectively Reprogrammed Cell Lines . Murine and humaniPSCs can be obtained from fibroblasts using the factors Oct4, Sox2, and Klf4,but without cMyc . However, in this case, reprogramming deceleratesand an essential shortcoming of stable iPSC clones is observed [32, 33]. The reduction of a number ofnecessary factors without any decrease in efficiency is possible when iPSCsare produced from murine and human neural stem cells (NSCs) [12, 34, 35]. For instance, iPSCs were produced fromNSCs isolated from adult murine brain using two factors, Oct4 and Klf4, aswell as even Oct4 by itself [12, 34]. Later, human iPSCs were produced by the reprogramming offetal NSCs transduced with a retroviral vector only carrying Oct4 [35] . It is most likely that the irrelevanceof Sox2, Klf4, and cMyc is due to the high endogenous expression level of these genes inNSCs.

Successful reprogramming was also achieved in experiments withother cell lines, in particular, melanocytes of neuroectodermal genesis [36]. Both murine and human melanocytes are characterized by a considerableexpression level of the Sox2 gene, especially at early passages.iPSCs from murine and human melanocytes were produced without the use of Sox2or cMyc. However, the yield of iPSC clones produced from murine melanocytes was lower(0.03% without Sox2 and 0.02% without cMyc) in comparison with that achieved when allfour factors were applied to melanocytes (0.19%) and fibroblasts (0.056%). A decreasedefficiency without Sox2 or cMyc was observed in human melanocyte reprogramming (0.05%with all four factors and 0.01% without either Sox2 or cMyc ). All attempts to obtain stable iPSC clones in the absence of both Sox2 andcMyc were unsuccessful [36]. Thus, theminimization of the number of factors required for iPSC preparation can be achieved by choosingthe proper somatic cell type that most effectively undergoes reprogramming under the action offewer factors, for example, due to the endogenous expression of pluripotencygenes. However, if human iPSCs are necessary, these somatic cellsshould be easily accessible and wellcultured and their method of isolation should be asnoninvasive as possible.

One of these cell types can be adipose stem cells (ASCs). This is aheterogeneous group of multipotent cells which can be relatively easily isolated in largeamounts from adipose tissue following liposuction. Human iPSCs weresuccessfully produced from ASCs with a twofold reprogramming rate and20fold efficiency (0.2%), exceeding those of fibroblasts [37].

However, more accessible resources for the effective production of humaniPSCs are keratinocytes. When compared with fibroblasts, human iPSC productionfrom keratinocytes demonstrated a 100fold greater efficiency and a twofold higherreprogramming rate [38].

It has recently been found that the reprogramming of murine papillary dermal fibroblasts(PDFs) into iPSCs can be highly effective with theoverexpression of only two genes, Oct4 and Klf4 ,inserted into retroviral vectors [39;].PDFs are specialized cells of mesodermal genesis surrounding the stem cells ofhair follicles . One characteristic feature of these cells is the endogenous expression of Sox2 , Klf4 , and cMyc genes,as well as the geneencoding alkaline phosphatase, one of the murine and humanESC markers. PDFs can be easily separated from other celltypes by FACS (fluorescenceactivated cell sorting) using life staining with antibodiesagainst the surface antigens characteristic of one or another cell type. The PDF reprogrammingefficiency with the use of four factors (Oct4, Sox2, Klf4, and cMyc) retroviral vectorsis 1.38%, which is 1,000fold higher than the skin fibroblast reprogramming efficiency inthe same system. Reprogramming PDFs with two factors, Oct4 and Klf4 , yields 0.024%, which is comparable to the efficiency of skinfibroblast reprogramming using all four factors. The efficiency of PDF reprogramming iscomparable with that of NSCs, but PDF isolation is steady and far lessinvasive [39]. It seems likely that human PDF lines arealso usable, and this cell type may appear to be one of the most promising for human iPSCproduction in terms of pharmacological studies and cell replacement therapy. The use of suchcell types undergoing more effective reprogramming, together with methods providing thedelivery of pluripotency genes without the integration of foreign DNA into thehost genome and chemical compounds increasing the reprogramming efficiency and substitutingsome factors required for reprogramming, is particularly relevant.

Chemical Compounds Increasing Cell Reprogramming Efficiency. As was noted above,the minimization of the factors used for reprogramming decreases the efficiency of iPSCproduction. Nonetheless, several recent studies have shown that the use of genetic mechanisms,namely, the initiation of ectopic gene expression, can be substituted by chemical compounds,most of them operating at the epigenetic level. For instance, BIX01294 inhibitinghistone methyltransferase G9a allows murine fibroblast reprogramming using only two factors,Oct4 and Klf4, with a fivefold increased yield of iPSC clones in comparison with the controlexperiment without BIX01294 [40]. BIX01294taken in combination with another compound can increase the reprogramming efficiency even more.In particular, BIX01294 plus BayK8644 elevated the yield of iPCSs 15 times, andBIX01294 plus RG108 elevated it 30 times when only two reprogramming factors, Oct4 andKlf4, were used. RG108 is an inhibitor of DNA methyltransferases, and its role in reprogrammingis apparently in initiating the more rapid and effective demethylation of promoters ofpluripotent cellspecific genes, whereas BayK8644 is an antagonist of Ltypecalcium channels, and its role in reprogramming is not understood very well [40]. However, more considerable results were obtained inreprogramming murine NSCs. The use of BIX01294 allowed a 1.5foldincrease in iPSC production efficiency with two factors, Oct4 and Klf4, in comparison withreprogramming with all four factors. Moreover, BIX01294 can even substitute Oct4 in thereprogramming of NSCs, although the yield is very low [41]. Valproic (2propylvaleric) acid inhibiting histone deacetylases canalso substitute cMyc in reprogramming murine and human fibroblasts. Valproic acid (VPA)increases the reprogramming efficiency of murine fibroblasts 50 times, and human fibroblastsincreases it 1020 times when three factors are used [42, 43]. Other deacetylase inhibitors,such as TSA (trichostatin A) and SAHA (suberoylanilide hyroxamic acid), also increase thereprogramming efficiency. TSA increases the murine fibroblast reprogramming efficiency 15times, and SAHA doubles it when all four factors are used [42]. Besides epigenetic regulators, the substances inhibiting the proteincomponents of signaling pathways implicated in the differentiation of pluripotent cells arealso applicable in the substitution of reprogramming factors. In particular, inhibitors of MEKand GSK3 kinases (PD0325901 and CHIR99021, respectively) benefit the establishment of thecomplete and stable pluripotency of iPSCs produced from murineNSCs using two factors, Oct4 and Klf4 [41, 44].

It has recently been shown that antioxidants can considerably increase the efficiency ofsomatic cell reprogramming. Ascorbic acid (vitamin C) can essentially influence the efficiencyof iPSC production from various murine and human somatic cell types [45]. The transduction of murine embryonic fibroblasts (mEFs) with retrovirusescarrying the Oct4 , Sox2 , and Klf4 genes results in a significant increase in the production level of reactive oxygen species(ROS) compared with that of both control and Efs tranduced with Oct4 , Sox2 , cMyc , and Klf4 . Inturn, the increase in the ROS level causes accelerated aging and apoptosis of the cell, whichshould influence the efficiency of cell reprogramming. By testing several substances possessingantioxidant activity such as vitamin B1, sodium selenite, reduced glutathione, and ascorbicacid, the authors have found that combining these substances increases the yield ofGFPpositive cells in EF reprogramming (the Gfp genewas under the control of the Oct4 gene promoter). The use of individualsubstances has shown that only ascorbate possesses a pronounced capability to increase thelevel of GFPpositive cells, although other substances keep theirROSdecreasing ability. In all likelihood, this feature of ascorbates is not directlyassociated with its antioxidant activity [45]. The scoreof GFPpositive iPSC colonies expressing an alkaline phosphatase hasshown that the efficiency of iPSC production from mEFs with three factors (Oct4, Sox2, andKlf4) can reach 3.8% in the presence of ascorbate. When all four factors (Oct4, Sox2, Klf4, andcMyc) are used together with ascorbate, the efficiency of iPSC production may reach8.75%. A similar increase in the iPSC yield was also observed in the reprogramming of murinebreast fibroblasts; i.e., the effect of vitamin C is not limited by one cell type. Moreover,the effect of vitamin C on the reprogramming efficiency is more profound than that of thedeacetylase inhibitor valproic (2propylvaleric) acid. The mutual effect of ascorbate andvalproate is additive; i.e., these substances have different action mechanisms. Moreover,vitamin C facilitates the transition from preiPSCs to stablepluripotent cells. This feature is akin to the effects of PD0325901 and CHIR99021, which areinhibitors of MEK and GSK3 kinases, respectively. This effect of vitamin C expands to humancells as well [45]. Following the transduction of humanfibroblasts with retroviruses carrying Oct4 , Sox2 , Klf4 , and cMyc and treatment with ascorbate, theauthors prepared iPSCs with efficiencies reaching 6.2%. The reprogrammingefficiency of ASCs under the same conditions reached 7.06%. The mechanism ofthe effect that vitamin C has on the reprogramming efficiency is not known in detail.Nevertheless, the acceleration of cell proliferation was observed at the transitional stage ofreprogramming. The levels of the p53 and p21 proteins decreased in cells treated withascorbate, whereas the DNA repair machinery worked properly [45]. It is interesting that an essential decrease in the efficiency of iPSCproduction has been shown under the action of processes initiated by p53 and p21 [4650].

As was mentioned above, for murine and human iPSC production, both retro andlentiviruses were initially used as delivery vectors for the genes required for cellreprogramming. The main drawback of this method is the uncontrolled integration of viral DNAinto the host cells genome. Several research groups have introduced methods fordelivering pluripotency genes into the recipient cell which either do notintegrate allogenic DNA into the host genome or eliminate exogenous genetic constructs from thegenome.

CreloxP Mediated Recombination. To prepareiPSCs from patients with Parkinsons disease, lentiviruses were used,the proviruses of which can be removed from the genome by Cre recombinase. To do this, the loxP site was introduced into thelentiviral 3LTRregions containing separate reprogramming genesunder the control of the doxycyclineinducible promoter. During viral replication, loxP was duplicated in the 5LTR of the vector. As aresult, the provirus integrated into the genome was flanked with two loxP sites. The inserts were eliminated using the temporary transfection ofiPSCs with a vector expressing Cre recombinase[51].

In another study, murine iPSCs were produced using a plasmid carrying the Oct4 , Sox2 , Klf4I, and cMyc genes in the same reading frame in which individual cDNAs were separatedby sequences encoding 2 peptides, and practically the whole construct was flanked with loxP sites [52]. The use ofthis vector allowed a notable decrease in the number of exogenous DNA inserts in the hostcells genome and, hence, the simplification of their following excision [52]. It has been shown using lentiviruses carrying similarpolycistronic constructs that one copy of transgene providing a high expression level of theexogenous factors Oct4, Sox2, Klf4, and cMyc is sufficient for the reprogramming ofdifferentiated cells into the pluripotent state [53,54].

The drawback of the CreloxP system is the incomplete excisionof integrated sequences; at least the loxP site remains in thegenome, so the risk of insertion mutations remains.

Plasmid Vectors . The application of lentiviruses and plasmids carrying the loxP sites required for the elimination of transgene constructsmodifies, although insignificantly, the host cells genome. One way to avoid this is touse vector systems that generally do not provide for the integration of the whole vector orparts of it into the cells genome. One such system providing a temporary transfectionwith polycistronic plasmid vectors was used for iPSC production from mEFs [29]. A polycistronic plasmid carrying the Oct4 , Sox2 , and Klf4 gene cDNAs, as well as aplasmid expressing cMyc , was transfected into mEFs one, three, five,and seven days after their primary seeding. Fibroblasts were passaged on the ninth day, and theiPSC colonies were selected on the 25th day. Seven out of ten experiments succeeded inproducing GFPpositive colonies (the Gfp gene wasunder the control of the Nanog gene promoter). The iPSCsthat were obtained were similar in their features to murine ESCs and did not contain inserts ofthe used DNA constructs in their genomes. Therefore, it was shown that wholesome murineiPSCs that do not carry transgenes can be reproducibly produced, and that thetemporary overexpression of Oct4 , Sox2 , Klf4 , and cMyc is sufficient for reprogramming. The maindrawback of this method is its low yield. In ten experiments the yield varied from 1 to 29 iPSCcolonies per ten million fibroblasts, whereas up to 1,000 colonies per ten millions wereobtained in the same study using retroviral constructs [29].

Episomal Vectors . Human iPSCs were successfully produced fromskin fibroblasts using single transfection with polycistronic episomal constructs carryingvarious combinations of Oct4 , Sox2 , Nanog , Klf4 , cMyc , Lin28 , and SV40LT genes. These constructs were designed on the basis of theoriP/EBNA1 (EpsteinBarr nuclear antigen1) vector [55]. The oriP/EBNA1 vector contains the IRES2 linker sequence allowing theexpression of several individual cDNAs (encoding the genes required for successfulreprogramming in this case) into one polycistronic mRNA from which several proteins aretranslated. The oriP/EBNA1 vector is also characterized by lowcopy representation in thecells of primates and can be replicated once per cell cycle (hence, it is not rapidlyeliminated, the way common plasmids are). Under nonselective conditions, the plasmid iseliminated at a rate of about 5% per cell cycle [56]. Inthis work, the broad spectrum of the reprogramming factor combinations was tested, resulting inthe best reprogramming efficiency with cotransfection with three episomes containing thefollowing gene sets: Oct4 + Sox2 + Nanog + Klf4 , Oct4 + Sox2 + SV40LT + Klf4 , and cMyc + Lin28 . SV40LT ( SV40 large T gene )neutralizes the possible toxic effect of overexpression [57]. The authors have shown thatwholesome iPSCs possessing all features of pluripotent cells can be producedfollowing the temporary expression of a certain gene combination in human somatic cells withoutthe integration of episomal DNA into the genome. However, as in the case when plasmid vectorsare being used, this way of reprogramming is characterized by low efficiency. In separateexperiments the authors obtained from 3 to 6 stable iPSC colonies per 106transfected fibroblasts [55]. Despite the fact that skinfibroblasts are wellcultured and accessible, the search for other cell types which arerelatively better cultured and more effectively subject themselves to reprogramming throughthis method is very likely required. Another drawback of the given system is that this type ofepisome is unequally maintained in different cell types.

PiggyBacTransposition . One promising system used foriPSC production without any modification of the host genome is based on DNA transposons.Socalled PiggyBac transposons containing2linkered reprogramming genes localized between the 5 and3terminal repeats were used for iPSC production from fibroblasts. The integrationof the given constructs into the genome occurs due to mutual transfection with a plasmidencoding transposase. Following reprogramming due to the temporary expression of transposase,the elimination of inserts from the genome took place [58, 59]. One advantage of the PiggyBac system on CreloxP is that the exogenous DNA iscompletely removed [60].

However, despite the relatively high efficiency of exogenous DNA excision from the genome by PiggyBac transposition, the removal of a large number of transposoncopies is hardly achievable.

Nonintegrating Viral Vectors . Murine iPSCs were successfullyproduced from hepatocytes and fibroblasts using four adenoviral vectors nonintegrating into thegenome and carrying the Oct4 , Sox2 , Klf4 , and cMyc genes. An analysis of the obtainediPSCs has shown that they are similar to murine ESCs in their properties(teratoma formation, gene promoter DNA methylation, and the expression of pluripotent markers),but they do not carry insertions of viral DNA in their genomes [61]. Later, human fibroblastderived iPSCs wereproduced using this method [62].

The authors of this paper cited the postulate that the use of adenoviral vectors allows theproduction of iPSCs, which are suitable for use without the risk of viral oroncogenic activity. Its very low yield (0.00010.001%), the deceleration ofreprogramming, and the probability of tetraploid cell formation are the drawbacks of themethod. Not all cell types are equally sensitive to transduction with adenoviruses.

Another method of gene delivery based on viral vectors was recently employed for theproduction of human iPSCs. The sendaivirus (SeV)based vector wasused in this case [63]. SeV is a singlestrandedRNA virus which does not modify the genome of recipient cells; it seems to be a good vector forthe expression of reprogramming factors. Vectors containing either all pluripotencyfactors or three of them (without ) were used for reprogramming the human fibroblast. The construct based on SeV is eliminatedlater in the course of cell proliferation. It is possible to remove cells with the integratedprovirus via negative selection against the surface HN antigen exposed on the infected cells.The authors postulate that reprogramming technology based on SeV will enable the production ofclinically applicable human iPSCs [63].

Cell Transduction with Recombinant Proteins . Although the methods for iPSCproduction without gene modification of the cells genome (adenoviral vectors, plasmidgene transfer, etc.) are elaborated, the theoretical possibility for exogenous DNA integrationinto the host cells genome still exists. The mutagenic potential of the substances usedpresently for enhancing iPSC production efficiency has not been studied in detail. Fullychecking iPSC genomes for exogenous DNA inserts and other mutations is a difficult task, whichbecomes impossible to solve in bulk culturing of multiple lines. The use of protein factorsdelivered into a differentiated cell instead of exogenous DNA may solve this problem. Tworeports have been published to date in which murine and human iPSCs wereproduced using the recombinant Oct4, Sox2, Klf4, and cMyc proteins [64, 65] . T he methodused to deliver the protein into the cell is based on the ability of peptides enriched withbasic residues (such as arginine and lysine) to penetrate the cells membrane. MurineiPSCs were produced using the recombinant Oct4, Sox2, Klf4, and cMycproteins containing eleven Cterminal arginine residues and expressed in E. coli . The authors succeeded in producing murine iPSCs during four roundsof protein transduction into embryonic fibroblasts [65].However, iPSCs were only produced when the cells were additionally treatedwith 2propylvalerate (the deacetylase inhibitor). The same principle was used for theproduction of human iPSCs, but protein expression was carried out in humanHEK293 cells, and the proteins were expressed with a fragment of nine arginins at the proteinCend. Researchers have succeeded in producing human iPSCs after sixtransduction rounds without any additional treatment [64]. The efficiency of producing human iPSC in this way was 0.001%, which isone order lower than the reprogramming efficiency with retroviruses. Despite some drawbacks,this method is very promising for the production of patientspecificiPSCs.

The first lines of human pluripotent ESCs were produced in 1998 [6]. In line with the obvious fundamental importance of embryonic stem cellstudies with regard to the multiple processes taking place in early embryogenesis, much of theinterest of investigators is associated with the possibility of using ESCs and theirderivatives as models for the pathogenesis of human diseases, new drugs testing, and cellreplacement therapy. Substantial progress is being achieved in studies on directed humanESC differentiation and the possibility of using them to correct degenerativedisorders. Functional cell types, such as motor dopaminergic neurons, cardiomyocytes, andhematopoietic cell progenitors, can be produced as a result of ESCdifferentiation. These cell derivatives, judging from their biochemical and physiologicalproperties, are potentially applicable for the therapy of cardiovascular disorders, nervoussystem diseases, and human hematological disorders [66].Moreover, derivatives produced from ESCs have been successfully used for treating diseasesmodeled on animals. Therefore, bloodcell progenitors produced from ESCs weresuccessfully used for correcting immune deficiency in mice. Visual functions were restored inblind mice using photoreceptors produced from human ESCs, and the normal functioning of thenervous system was restored in rats modeling Parkinsons disease using the dopaminergicneurons produced from human ESCs [6770]. Despite obvious success, the fullscale applicationof ESCs in therapy and the modeling of disorders still carry difficulties, because of thenecessity to create ESC banks corresponding to all HLAhaplotypes, whichis practically unrealistic and hindered by technical and ethical problems.

Induced pluripotent stem cells can become an alternative for ESCs in the area of clinicalapplication of cell replacement therapy and screening for new pharmaceuticals.iPSCs closely resemble ESCs and, at the same time, can be produced in almostunlimited amounts from the differentiated cells of each patient. Despite the fact that thefirst iPSCs were produced relatively recently, work on directed iPSCdifferentiation and the production of patientspecific iPSCs isintensive, and progress in this field is obvious.

Dopamine and motor neurons were produced from human iPSCs by directeddifferentiation in vitro [71, 72]. These types of neurons are damaged in many inherited oracquired human diseases, such as spinal cord injury, Parkinsons disease, spinal muscularatrophy, and amyotrophic lateral sclerosis. Some investigators have succeeded in producingvarious retinal cells from murine and human iPSCs [7375]. HumaniPSCs have been shown to be spontaneously differentiated in vitro into the cells of retinal pigment epithelium [76]. Another group of investigators has demonstrated that treating human andmurine iPSCs with Wnt and Nodal antagonists in a suspended culture induces theappearance of markers of cell progenitors and pigment epithelium cells. Further treating thecells with retinoic acid and taurine activates the appearance of cells expressing photoreceptormarkers [75].

Several research groups have produced functional cardiomyocytes (CMs) in vitro from murine and human iPSCs [7781]. Cardiomyocytes producedfrom iPSC are very similar in characteristics (morphology, marker expression,electrophysiological features, and sensitivity to chemicals) to the CMs ofcardiac muscle and to CMs produced from differentiated ESCs. Moreover, murineiPSCs, when injected, can repair muscle and endothelial cardiac tissuesdamaged by cardiac infarction [77].

Hepatocytelike cell derivatives, dendritic cells, macrophages, insulinproducingcell clusters similar to the duodenal islets of Langerhans, and hematopoietic and endothelialcells are currently produced from murine and human iPSCs, in addition to thealreadylisted types of differentiated cells [8285].

In addition to directed differentiation in vitro , investigators apply mucheffort at producing patientspecific iPSCs. The availability ofpluripotent cells from individual patients makes it possible to study pathogenesis and carryout experiments on the therapy of inherited diseases, the development of which is associatedwith distinct cell types that are hard to obtain by biopsy: so the use ofiPSCs provides almost an unlimited resource for these investigations.Recently, the possibility of treating diseases using iPSCs was successfullydemonstrated, and the design of the experiment is presented in the figure. A mutant allele wassubstituted with a normal allele via homologous recombination in murine fibroblastsrepresenting a model of human sickle cell anemia. iPSCs were produced fromrepaired fibroblasts and then differentiated into hematopoietic cell precursors.The hematopoietic precursors were then injected into a mouse from which the skin fibroblastswere initially isolated (Fig. 1). As a result, the initialpathological phenotype was substantially corrected [86].A similar approach was applied to the fibroblasts and keratinocytes of a patient withFanconis anemia. The normal allele of the mutant gene producing anemia was introducedinto a somatic cell genome using a lentivirus, and then iPSCs were obtainedfrom these cells. iPSCs carrying the normal allele were differentiated intohematopoietic cells maintaining a normal phenotype [87].The use of lentiviruses is unambiguously impossible when producing cells to be introduced intothe human body due to their oncogenic potential. However, new relatively safe methods of genomemanipulation are currently being developed; for instance, the use of synthetic nucleasescontaining zinc finger domains allowing the effective correction of genetic defects invitro [88].

Design of an experiment on repairing the mutant phenotype in mice modeling sickle cell anemia development [2]. Fibroblasts isolatedfrom the tail of a mouse (1) carrying a mutant allele of the gene encoding the human hemoglobin -chain (hs) were used for iPSCproduction (2). The mutation was then repaired in iPSCs by means of homological recombination (3) followed by cell differentiationvia the embryoid body formation (4). The directed differentiation of the embryoid body cells led to hematopoietic precursor cells (5)that were subsequently introduced into a mouse exposed to ionizing radiation (6).

The induced pluripotent stem cells are an excellent model for pathogenetic studies at the celllevel and testing compounds possessing a possible therapeutic effect.

The induced pluripotent stem cells were produced from the fibroblasts of a patient with spinalmuscular atrophy (SMA) (SMAiPSCs). SMA is an autosomalrecessive disease caused by a mutation in the SMN1 ( survival motorneuron 1 ) gene, which is manifested as the selective nonviability of lower motor neurons. Patients with this disorder usually die at the age of about two years.Existing experimental models of this disorder based on the use of flatworms, drosophila, andmice are not satisfactory. The available fibroblast lines from patients withSMA cannot provide the necessary data on the pathogenesis of this disordereither. It was shown that motor neurons produced from SMAiPSCs canretain the features of SMA development, selective neuronal death, and the lackof SMN1 transcription. Moreover, the authors succeeded in elevating the SMNprotein level and aggregation (encoded by the SMN2 gene, whose expressioncan compensate for the shortage in the SMN1 protein) in response to the treatment of motorneurons and astrocytes produced from SMAiPSCs with valproate andtorbomycin [89;]. iPSCs and theirderivatives can serve as objects for pharmacological studies, as has been demonstrated oniPSCs from patients with familial dysautonomia (FDA) [90]. FDA is an inherited autosomal recessive disorder manifested as thedegeneration of sensor and autonomous neurons. This is due to a mutation causing thetissuespecific splicing of the IKBKAP gene, resulting in a decreasein the level of the fulllength IKAP protein. iPSCs were produced fromfibroblasts of patients with FDA. They possessed all features of pluripotent cells. Neuralderivatives produced from these cells had signs of FDA pathogenesis and low levels of thefulllength IKBKAP transcript. The authors studied the effect of threesubstances, kinetin, epigallocatechin gallate, and tocotrienol, on the parameters associatedwith FDA pathogenesis. Only kinetin has been shown to induce an increase in the level offulllength IKBKAP transcript. Prolonged treatment with kinetininduces an increase in the level of neuronal differentiation and expression of peripheralneuronal markers.

Currently, a broad spectrum of iPSCs is produced from patients with variousinherited pathologies and multifactorial disorders, such as Parkinsons disease, Downsyndrome, type 1 diabetes, Duchenne muscular dystrophy, talassemia, etc., whichare often lethal and can scarcely be treated with routine therapy [51, 87, 89, 9194]. The data on iPSCs produced by reprogramming somaticcells from patients with various pathologies are given in the Table 1.

Functional categories of M. tuberculosis genes with changed expression level during transition to the NC state

One can confidently state that both iPSCs themselves and their derivativesare potent instruments applicable in biomedicine, cell replacement therapy, pharmacology, andtoxicology. However, the safe application of iPSCbased technologies requires the use ofmethods of iPSCs production and their directed differentiation which minimizeboth the possibility of mutations in cell genomes under in vitro culturingand the probability of malignant transformation of the injected cells. The development ofmethods for human iPSC culturing without the use of animal cells (for instance, the feederlayer of murine fibroblasts) is necessary; they make a viralorigin pathogen transferfrom animals to humans impossible. There is a need for the maximum standardization ofconditions for cell culturing and differentiation.

This study was supported by the Russian Academy of Sciences Presidium ProgramMolecular and Cell Biology.

embryonic stem cells

induced pluripotent stem cells

neural stem cells

adipose stem cells

papillary dermal fibroblasts

cardiomyocytes

spinal muscular atrophy

iPCSs derived from fibroblasts of SMA patients

green fluorescent protein

long terminal repeat

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How Minaris is Tackling the Scalability Challenge in Cell and Gene Therapy: A Conversation with CEO, Dr. Hiroto Bando  geneonline

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Toward Personalized Cell Therapies by Using Stem Cells 2013: BioMed Research International  Wiley Online Library

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Cell therapy for heart disease and therapeutic cloning: will embryos re-enter the stem cell race?  Genethique

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It's official: The first use of induced pluripotent stem (iPS) cells in a human has proved safe, if not clearly effective. Japanese researchers reported in this week's issue of The New England Journal of Medicine (NEJM) that using the cells to replace eye tissue damaged by age-related macular degeneration (AMD) did not improve a patient's ...

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A scheme of the generation of induced pluripotent stem (IPS) cells. (1) Isolate and culture donor cells. (2) Transduce stem cell-associated genes into the cells by viral vectors. Red cells indicate the cells expressing the exogenous genes. (3) Harvest and culture the cells according to ES cell culture, using mitotically inactivated feeder cells (lightgray). (4) A small subset of the transfected cells become iPS cells and generate ES-like colonies.

iPSCs are typically derived by introducing products of specific sets of pluripotency-associated genes, or "reprogramming factors", into a given cell type. The original set of reprogramming factors (also dubbed Yamanaka factors) are the transcription factors Oct4 (Pou5f1), Sox2, Klf4 and cMyc. While this combination is most conventional in producing iPSCs, each of the factors can be functionally replaced by related transcription factors, miRNAs, small molecules, or even non-related genes such as lineage specifiers.[11] It is also clear that pro-mitotic factors such as C-MYC/L-MYC or repression of cell cycle checkpoints, such as p53, are conduits to creating a compliant cellular state for iPSC reprogramming.[12]

iPSC derivation is typically a slow and inefficient process, taking onetwo weeks for mouse cells and threefour weeks for human cells, with efficiencies around 0.010.1%. However, considerable advances have been made in improving the efficiency and the time it takes to obtain iPSCs. Upon introduction of reprogramming factors, cells begin to form colonies that resemble pluripotent stem cells, which can be isolated based on their morphology, conditions that select for their growth, or through expression of surface markers or reporter genes.

Induced pluripotent stem cells were first generated by Shinya Yamanaka and Kazutoshi Takahashi at Kyoto University, Japan, in 2006.[1] They hypothesized that genes important to embryonic stem cell (ESC) function might be able to induce an embryonic state in adult cells. They chose twenty-four genes previously identified as important in ESCs and used retroviruses to deliver these genes to mouse fibroblasts. The fibroblasts were engineered so that any cells reactivating the ESC-specific gene, Fbx15, could be isolated using antibiotic selection.

Upon delivery of all twenty-four factors, ESC-like colonies emerged that reactivated the Fbx15 reporter and could propagate indefinitely. To identify the genes necessary for reprogramming, the researchers removed one factor at a time from the pool of twenty-four. By this process, they identified four factors, Oct4, Sox2, cMyc, and Klf4, which were each necessary and together sufficient to generate ESC-like colonies under selection for reactivation of Fbx15.

In June 2007, three separate research groups, including that of Yamanaka's, a Harvard/University of California, Los Angeles collaboration, and a group at MIT, published studies that substantially improved on the reprogramming approach, giving rise to iPSCs that were indistinguishable from ESCs. Unlike the first generation of iPSCs, these second generation iPSCs produced viable chimeric mice and contributed to the mouse germline, thereby achieving the 'gold standard' for pluripotent stem cells.

These second-generation iPSCs were derived from mouse fibroblasts by retroviral-mediated expression of the same four transcription factors (Oct4, Sox2, cMyc, Klf4). However, instead of using Fbx15 to select for pluripotent cells, the researchers used Nanog, a gene that is functionally important in ESCs. By using this different strategy, the researchers created iPSCs that were functionally identical to ESCs.[13][14][15][16]

Reprogramming of human cells to iPSCs was reported in November 2007 by two independent research groups: Shinya Yamanaka of Kyoto University, Japan, who pioneered the original iPSC method, and James Thomson of University of Wisconsin-Madison who was the first to derive human embryonic stem cells. With the same principle used in mouse reprogramming, Yamanaka's group successfully transformed human fibroblasts into iPSCs with the same four pivotal genes, Oct4, Sox2, Klf4, and cMyc, using a retroviral system,[17] while Thomson and colleagues used a different set of factors, Oct4, Sox2, Nanog, and Lin28, using a lentiviral system.[18]

Obtaining fibroblasts to produce iPSCs involves a skin biopsy, and there has been a push towards identifying cell types that are more easily accessible.[19][20] In 2008, iPSCs were derived from human keratinocytes, which could be obtained from a single hair pluck.[21][22] In 2010, iPSCs were derived from peripheral blood cells,[23][24] and in 2012, iPSCs were made from renal epithelial cells in the urine.[25]

Other considerations for starting cell type include mutational load (for example, skin cells may harbor more mutations due to UV exposure),[19][20] time it takes to expand the population of starting cells,[19] and the ability to differentiate into a given cell type.[26]

[citation needed]

The generation of induced pluripotent cells is crucially dependent on the transcription factors used for the induction.

Oct-3/4 and certain products of the Sox gene family (Sox1, Sox2, Sox3, and Sox15) have been identified as crucial transcriptional regulators involved in the induction process whose absence makes induction impossible. Additional genes, however, including certain members of the Klf family (Klf1, Klf2, Klf4, and Klf5), the Myc family (c-myc, L-myc, and N-myc), Nanog, and LIN28, have been identified to increase the induction efficiency.

Although the methods pioneered by Yamanaka and others have demonstrated that adult cells can be reprogrammed to iPS cells, there are still challenges associated with this technology:

The table on the right summarizes the key strategies and techniques used to develop iPS cells in the first five years after Yamanaka et al.'s 2006 breakthrough. Rows of similar colors represent studies that used similar strategies for reprogramming.

One of the main strategies for avoiding problems (1) and (2) has been to use small molecules that can mimic the effects of transcription factors. These compounds can compensate for a reprogramming factor that does not effectively target the genome or fails at reprogramming for another reason; thus they raise reprogramming efficiency. They also avoid the problem of genomic integration, which in some cases contributes to tumor genesis. Key studies using such strategy were conducted in 2008. Melton et al. studied the effects of histone deacetylase (HDAC) inhibitor valproic acid. They found that it increased reprogramming efficiency 100-fold (compared to Yamanaka's traditional transcription factor method).[42] The researchers proposed that this compound was mimicking the signaling that is usually caused by the transcription factor c-Myc. A similar type of compensation mechanism was proposed to mimic the effects of Sox2. In 2008, Ding et al. used the inhibition of histone methyl transferase (HMT) with BIX-01294 in combination with the activation of calcium channels in the plasma membrane in order to increase reprogramming efficiency.[43] Deng et al. of Beijing University reported in July 2013 that induced pluripotent stem cells can be created without any genetic modification. They used a cocktail of seven small-molecule compounds including DZNep to induce the mouse somatic cells into stem cells which they called CiPS cells with the efficiency at 0.2% comparable to those using standard iPSC production techniques. The CiPS cells were introduced into developing mouse embryos and were found to contribute to all major cells types, proving its pluripotency.[44][45]

Ding et al. demonstrated an alternative to transcription factor reprogramming through the use of drug-like chemicals. By studying the mesenchymal-epithelial transition (MET) process in which fibroblasts are pushed to a stem-cell like state, Ding's group identified two chemicals ALK5 inhibitor SB431412 and MEK (mitogen-activated protein kinase) inhibitor PD0325901 which was found to increase the efficiency of the classical genetic method by 100 fold. Adding a third compound known to be involved in the cell survival pathway, thiazovivin further increases the efficiency by 200 fold. Using the combination of these three compounds also decreased the reprogramming process of the human fibroblasts from four weeks to two weeks.[46][47]

In April 2009, it was demonstrated that generation of iPS cells is possible without any genetic alteration of the adult cell: a repeated treatment of the cells with certain proteins channeled into the cells via poly-arginine anchors was sufficient to induce pluripotency.[48] The acronym given for those iPSCs is piPSCs (protein-induced pluripotent stem cells).

Another key strategy for avoiding problems such as tumorgenesis and low throughput has been to use alternate forms of vectors: adenoviruses, plasmids, and naked DNA or protein compounds.

In 2008, Hochedlinger et al. used an adenovirus to transport the requisite four transcription factors into the DNA of skin and liver cells of mice, resulting in cells identical to ESCs. The adenovirus is unique from other vectors like viruses and retroviruses because it does not incorporate any of its own genes into the targeted host and avoids the potential for insertional mutagenesis.[43] In 2009, Freed et al. demonstrated successful reprogramming of human fibroblasts to iPS cells.[49] Another advantage of using adenoviruses is that they only need to present for a brief amount of time in order for effective reprogramming to take place.

Also in 2008, Yamanaka et al. found that they could transfer the four necessary genes with a plasmid.[35] The Yamanaka group successfully reprogrammed mouse cells by transfection with two plasmid constructs carrying the reprogramming factors; the first plasmid expressed c-Myc, while the second expressed the other three factors (Oct4, Klf4, and Sox2). Although the plasmid methods avoid viruses, they still require cancer-promoting genes to accomplish reprogramming. The other main issue with these methods is that they tend to be much less efficient compared to retroviral methods. Furthermore, transfected plasmids have been shown to integrate into the host genome and therefore they still pose the risk of insertional mutagenesis. Because non-retroviral approaches have demonstrated such low efficiency levels, researchers have attempted to effectively rescue the technique with what is known as the PiggyBac Transposon System. Several studies have demonstrated that this system can effectively deliver the key reprogramming factors without leaving footprint mutations in the host cell genome. The PiggyBac Transposon System involves the re-excision of exogenous genes, which eliminates the issue of insertional mutagenesis.[citation needed]

In January 2014, two articles were published claiming that a type of pluripotent stem cell can be generated by subjecting the cells to certain types of stress (bacterial toxin, a low pH of 5.7, or physical squeezing); the resulting cells were called STAP cells, for stimulus-triggered acquisition of pluripotency.[50]

In light of difficulties that other labs had replicating the results of the surprising study, in March 2014, one of the co-authors has called for the articles to be retracted.[51] On 4 June 2014, the lead author, Obokata agreed to retract both the papers[52] after she was found to have committed 'research misconduct' as concluded in an investigation by RIKEN on 1 April 2014.[53]

MicroRNAs are short RNA molecules that bind to complementary sequences on messenger RNA and block expression of a gene. Measuring variations in microRNA expression in iPS cells can be used to predict their differentiation potential.[54] Addition of microRNAs can also be used to enhance iPS potential. Several mechanisms have been proposed.[54] ES cell-specific microRNA molecules (such as miR-291, miR-294 and miR-295) enhance the efficiency of induced pluripotency by acting downstream of c-Myc.[55] MicroRNAs can also block expression of repressors of Yamanaka's four transcription factors, and there may be additional mechanisms induce reprogramming even in the absence of added exogenous transcription factors.[54]

The task of producing iPS cells continues to be challenging due to the six problems mentioned above. A key tradeoff to overcome is that between efficiency and genomic integration. Most methods that do not rely on the integration of transgenes are inefficient, while those that do rely on the integration of transgenes face the problems of incomplete reprogramming and tumor genesis, although a vast number of techniques and methods have been attempted. Another large set of strategies is to perform a proteomic characterization of iPS cells.[58] Further studies and new strategies should generate optimal solutions to the five main challenges. One approach might attempt to combine the positive attributes of these strategies into an ultimately effective technique for reprogramming cells to iPS cells.

Another approach is the use of iPS cells derived from patients to identify therapeutic drugs able to rescue a phenotype. For instance, iPS cell lines derived from patients affected by ectodermal dysplasia syndrome (EEC), in which the p63 gene is mutated, display abnormal epithelial commitment that could be partially rescued by a small compound.[67]

An attractive feature of human iPS cells is the ability to derive them from adult patients to study the cellular basis of human disease. Since iPS cells are self-renewing and pluripotent, they represent a theoretically unlimited source of patient-derived cells which can be turned into any type of cell in the body. This is particularly important because many other types of human cells derived from patients tend to stop growing after a few passages in laboratory culture. iPS cells have been generated for a wide variety of human genetic diseases, including common disorders such as Down syndrome and polycystic kidney disease.[68][69][70] In many instances, the patient-derived iPS cells exhibit cellular defects not observed in iPS cells from healthy subjects, providing insight into the pathophysiology of the disease.[71][72] An international collaborated project, StemBANCC, was formed in 2012 to build a collection of iPS cell lines for drug screening for a variety of diseases. Managed by the University of Oxford, the effort pooled funds and resources from 10 pharmaceutical companies and 23 universities. The goal is to generate a library of 1,500 iPS cell lines which will be used in early drug testing by providing a simulated human disease environment.[73] Furthermore, combining hiPSC technology and small molecule or genetically encoded voltage and calcium indicators provided a large-scale and high-throughput platform for cardiovascular drug safety screening.[74][75][76][77][78]

A proof-of-concept of using induced pluripotent stem cells (iPSCs) to generate human organ for transplantation was reported by researchers from Japan. Human 'liver buds' (iPSC-LBs) were grown from a mixture of three different kinds of stem cells: hepatocyte (for liver function) coaxed from iPSCs; endothelial stem cells (to form lining of blood vessels) from umbilical cord blood; and mesenchymal stem cells (to form connective tissue). This new approach allows different cell types to self-organize into a complex organ, mimicking the process in fetal development. After growing in vitro for a few days, the liver buds were transplanted into mice where the 'liver' quickly connected with the host blood vessels and continued to grow. Most importantly, it performed regular liver functions including metabolizing drugs and producing liver-specific proteins. Further studies will monitor the longevity of the transplanted organ in the host body (ability to integrate or avoid rejection) and whether it will transform into tumors.[79][80]

In 2021, a switchable Yamanaka factors-reprogramming-based approach for regeneration of damaged heart without tumor-formation was demonstrated in mice and was successful if the intervention was carried out immediately before or after a heart attack.[81]

Embryonic cord-blood cells were induced into pluripotent stem cells using plasmid DNA. Using cell surface endothelial/pericytic markers CD31 and CD146, researchers identified 'vascular progenitor', the high-quality, multipotent vascular stem cells. After the iPS cells were injected directly into the vitreous of the damaged retina of mice, the stem cells engrafted into the retina, grew and repaired the vascular vessels.[82][83]

Labelled iPSCs-derived NSCs injected into laboratory animals with brain lesions were shown to migrate to the lesions and some motor function improvement was observed.[84]

Beating cardiac muscle cells, iPSC-derived cardiomyocytes, can be mass-produced using chemically defined differentiation protocols.[85][86] These protocols typically modulate the same developmental signaling pathways required for heart development.[87] These iPSC-cardiomyocytes can recapitulate genetic arrhythmias and cardiac drug responses, since they exhibit the same genetic background as the patient from which they were derived.[88][89][90][91]

In June 2014, Takara Bio received technology transfer from iHeart Japan, a venture company from Kyoto University's iPS Cell Research Institute, to make it possible to exclusively use technologies and patents that induce differentiation of iPS cells into cardiomyocytes in Asia. The company announced the idea of selling cardiomyocytes to pharmaceutical companies and universities to help develop new drugs for heart disease.[92]

On March 9, 2018, the Specified Regenerative Medicine Committee of Osaka University officially approved the world's first clinical research plan to transplant a "myocardial sheet" made from iPS cells into the heart of patients with severe heart failure. Osaka University announced that it had filed an application with the Ministry of Health, Labor and Welfare on the same day.

On May 16, 2018, the clinical research plan was approved by the Ministry of Health, Labor and Welfare's expert group with a condition.[93][94]

In October 2019, a group at Okayama University developed a model of ischemic heart disease using cardiomyocytes differentiated from iPS cells.[95]

Although a pint of donated blood contains about two trillion red blood cells and over 107 million blood donations are collected globally, there is still a critical need for blood for transfusion. In 2014, type O red blood cells were synthesized at the Scottish National Blood Transfusion Service from iPSC. The cells were induced to become a mesoderm and then blood cells and then red blood cells. The final step was to make them eject their nuclei and mature properly. Type O can be transfused into all patients. Human clinical trials were not expected to begin before 2016.[96]

The first human clinical trial using autologous iPSCs was approved by the Japan Ministry Health and was to be conducted in 2014 at the Riken Center for Developmental Biology in Kobe. However the trial was suspended after Japan's new regenerative medicine laws came into effect in November 2015.[97] More specifically, an existing set of guidelines was strengthened to have the force of law (previously mere recommendations).[98] iPSCs derived from skin cells from six patients with wet age-related macular degeneration were reprogrammed to differentiate into retinal pigment epithelial (RPE) cells. The cell sheet would be transplanted into the affected retina where the degenerated RPE tissue was excised. Safety and vision restoration monitoring were to last one to three years.[99][100]

In March 2017, a team led by Masayo Takahashi completed the first successful transplant of iPS-derived retinal cells from a donor into the eye of a person with advanced macular degeneration.[101] However it was reported that they are now having complications.[102] The benefits of using autologous iPSCs are that there is theoretically no risk of rejection and that it eliminates the need to use embryonic stem cells. However, these iPSCs were derived from another person.[100]

New clinical trials involving iPSCs are now ongoing not only in Japan, but also in the US and Europe.[103] Research in 2021 on the trial registry Clinicaltrials.gov identified 129 trial listings mentioning iPSCs, but most were non-interventional.[104]

To make iPSC-based regenerative medicine technologies available to more patients, it is necessary to create universal iPSCs that can be transplanted independently of haplotypes of HLA. The current strategy for the creation of universal iPSCs has two main goals: to remove HLA expression and to prevent NK cells attacks due to deletion of HLA. Deletion of the B2M and CIITA genes using the CRISPR/Cas9 system has been reported to suppress the expression of HLA class I and class II, respectively. To avoid NK cell attacks. transduction of ligands inhibiting NK-cells, such as HLA-E and CD47 has been used.[105] HLA-C is left unchanged, since the 12 common HLA-C alleles are enough to cover 95% of the world's population.[105]

A multipotent mesenchymal stem cell, when induced into pluripotence, holds great promise to slow or reverse aging phenotypes. Such anti-aging properties were demonstrated in early clinical trials in 2017.[106] In 2020, Stanford University researchers concluded after studying elderly mice that old human cells when subjected to the Yamanaka factors, might rejuvenate and become nearly indistinguishable from their younger counterparts.[107]

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Research is ongoing in Japan and overseas with the aim of realizing cell transplantation therapy using iPS cells. One safety issue of concern is the risk of tumor formation. CiRA in particular has focused its resources on this issue.

Broadly speaking, there are two main theories as to the mechanism whereby iPS cells may form tumors. One theory is that iPS cells form tumors in response either to reactivation of the reprogramming factors inserted into the cell or through damage caused to the original cell genome through the artificial insertion of the reprogramming factors. In response, a search was launched for optimal reprogramming factors which do not cause reactivation, and a method of generating iPS cells was developed in which reprogramming factors are not incorporated into the cell chromosomes and damage to the host genome is therefore avoided.

The other theory is that residues of undifferentiated cells - cells which have not successfully completed differentiation to the target cell type - or other factors lead to the formation of teratomas, a kind of benign tumor. This theory requires research on iPS cell proliferation and differentiation.

1. Search for optimal reprogramming factorsWhen Professor Shinya Yamanaka and his research team announced the successful generation of mouse iPS cells, one of the reprogramming factors they used was c-Myc, which is known to be an oncogene, that is a cancer-causing gene. There have been suggestions that this gene may be activated within the cell and cause a tumor to form. However, in 2010, CiRA Lecturer Masato Nakagawa and his team reported that L-Myc was a promising replacement factor for c-Myc. iPS cells created using L-Myc not only display almost no tumor formation, they also have a high rate of successful generation and a high degree of pluripotency.

2. Search for optimal vectorsWhen the reprogramming factors required to generate iPS cells were inserted into the cells of the skin or other body tissues, early methods employed a retrovirus or lentivirus as a "vector," or carrier. In these methods, the target genes are inserted into the viruses with the which the cells were then infected in order to deliver the target genes. When a retrovirus or lentivirus is used as a vector, however, the viruses are incorporated into the cells genomic DNA in a random fashion. This may cause some of the cells original genes to be lost, or in other cases activated, resulting in a risk of cancerous changes.

In 2008, to remedy this risk, CiRA Lecturer Keisuke Okita and his team explored the use of a circular DNA fragment known as a plasmid, which is not incorporated into the cell chromosome, as a substitute to the retrovirus or lentivirus methods. In this way, they developed a method of generating iPS cells in which the reprogramming factors are not incorporated into the cell chromosome. In 2011, Okita and his team further improved the efficiency generation by introducing into a self-replicating episomal plasmid six factors - OCT3/4, SOX2, KLF4, LIN28, L-MYC, and p53shRNA.

3. Establishing a method for generating and screening safe cellsOnce iPS cells have been induced to differentiate into the target somatic cells using the appropriate genes and gene insertion methods as explained above, the differentiated cells can be relied upon not to revert to the undifferentiated state. However, there may sometimes be a residue of undifferentiated cells which have not completed the process of differentiation into the target cells, and it is possible that these cells, however few, may form a tumor. Scientists had already established that different iPS cell lines, even if generated from the same individual using the same method, might nevertheless display differences in proliferation and differentiation potentials. This meant that, if iPS cells with low differentiation potential were used, there was a risk that a residue of cells in the cell group might fail to fully differentiate and result in the formation of a teratoma. In 2013, a team led by CiRA Lecturer Kazutoshi Takahashi and Dr. Michiyo Aoi, now an assistant professor at Kobe University, developed a simple method to screen for iPS cell lines that have high potential to differentiate into nerve cells. There is also a risk of tumorigenesis from genomic or other damage arising at the iPS cell generation stage or at the subsequent culture stage. CiRA Assistant Professor Akira Watanabe and his team have developed a sensitive method to detect genomic and other damage in iPS cells using the latest equipment.

4. Developing a reliable method of differentiation into the target cell typeIn cell transplantation therapy, iPS cells are not transplanted directly into the human body. Instead, cells are transplanted after first being differentiated into the target cell type. It is therefore important to develop a reliable method of inducing iPS cells to differentiate into the target cell type. CiRA is currently working to develop technology for differentiation into a range of different cell types from iPS cells. CiRA Professor Jun Takahashi and his team have developed a highly efficient method of inducing iPS cells to differentiate into dopamine-producing nerve cells. In 2014, CiRA Professor Koji Eto and his team reported a method of producing platelets from iPS cells that is both reliable and can yield high volumes. These findings represent a major step toward iPS cell-based regenerative medicine for nerve diseases such as Parkinsons disease and blood diseases such as aplastic anemia.

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