Abstract

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Disease Modeling Using Human iPS Cells

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

2024 Wiley Periodicals LLC.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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iPSC Therapy: Advances and Clinical Potential - BiologyInsights

IPS Cell Therapy Comments Off on iPSC Therapy: Advances and Clinical Potential – BiologyInsights | February 11th, 2025

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

IPS Cell Therapy Comments Off on I Peace establishes and offers low immunoreaction risk GMP iPS Cells – PR Newswire | February 11th, 2025

Abstract

Despite considerable advances in medicine, cardiovascular disease is still rising; with ischemic heart disease being the leading cause of death and disability worldwide. Thus extensive efforts are continuing to establish effective therapeutic modalities that would improve both quality of life and survival in this patient population. Novel therapies are now being investigated not only to protect the myocardium against ischemia-reperfusion injury but also regenerate the heart. Stem cell therapy, such as potential use of human mesenchymal stem cells, induced pluripotent stem cells and their exosomes will make it possible not only to address molecular mechanisms of cardiac conditioning, but also develop new therapies for ischemic heart disease.

Despite all the studies and progress made over the last 15 years on the use of stem cell therapy for cardiovascular disease, the efforts are still in their infancy. While the expectations have been high, the findings indicate that most of the clinical trials are generally small and the results are inconclusive. Because of many negative findings, there is certain pessimism that cardiac cell therapy is likely to yield any meaningful results over the next decade or so. Similar to other new technologies, early failures are not unusual and they may be followed by impressive success. Nevertheless, there has been considerable attention to safety by the clinical investigators since the adverse events of stem cell therapy have been impressively rare. In summary, while the regenerative biology might not help the cardiovascular patient in the near term, it is destined to do so over the next several decades.

Cardiovascular disease is the leading global cause of death, accounting for over 17 million deaths per year. The number of cardiovascular deaths is expected to grow to more than 23 million by 2030, according to a report from the American Heart Association.1 In 2011 nearly 787,000 people died from heart disease, stroke and other cardiovascular diseases in the United States. Two new approaches have been identified that have the potential of added benefits to the current therapeutic strategies. The first focuses on enhancing the heart/myocardiums tolerance to ischemia-reperfusion injury using cardiac conditioning that will be covered here only briefly as a historical background. The second approach is to create an environment within the heart muscle that will result in repair of the damaged myocardium; a topic of this review.

Considerable experimental evidence obtained in multiple models and species has demonstrated that all forms of myocardial ischemic conditioning (pre-conditioning, per-conditioning, post-conditioning and remote preconditioning) induce very potent cardioprotection in animal models.25 In healthy, young hearts, many of these conditioning methods can significantly increase the hearts resistance against ischemia and reperfusion injury. However, essentially none of these forms of myocardial ischemic conditioning have been effective in patients. Remote ischemic pre-conditioning using transient arm ischemiareperfusion did not improve clinical outcomes in the ERICCA study, with 1,612 patients undergoing elective on-pump coronary artery bypass grafting.6 Additionally, upper-limb remote ischemic preconditioning performed in 1,385 patients did not show any significant benefit among patients undergoing elective cardiac surgery.7 Therefore, these large multicenter trials have not only proved that ischemic conditioning was unsuccessful in cardiac surgeries; they also failed to confirm the presence of initial cardioprotection by ischemic conditioning-induced reduction of cardiac troponin release,8, 9 which is a standard diagnostic indicator of myocardial injury. The lack of clinical success most likely is due to underlying risk factors that interfere with cardiac conditioning, along with the use of cardioprotective agents that activate the endogenous cardioprotective mechanisms. Future preclinical validation of drug targets and cardiac conditioning will need to focus more on comorbid animal models (such as age, diabetes, and hypertension) and choosing the relevant endpoints for assessing the efficacy of cardioprotective procedures to have a successful, clinical translation.

While the existing therapies for the ischemic heart disease lower the early mortality rates, prevent additional damage to the heart muscle, and reduce the risk of further heart attacks, most of the patients are likely to have worse quality of life including frequent hospitalizations. Therefore, there is an ultimate need for a treatment to improve the clinical conditions by either replacing the damaged heart cells and/or improve cardiac performance. Thus, the cardiac tissue regeneration with the application of stem cells, or their exosomes, may be an effective therapeutic option.10 Stem cells, both adult and embryonic stem cells (ESCs) have the ability to self-replicate and transform into an array of specialized cells. Stem cells are becoming the most important tool in regenerative medicine since these cells have the potential to differentiate into cardiomyocytes. It would, therefore, be useful to find out if the differentiated cells can restore and improve cardiac function safely and effectively.

The purpose of this review is to present the current state of knowledge of potential use of human stem cells, induced human pluripotent stem cells (hiPSCs), and stem cell-derived exosomes as a cell based therapeutic strategy for the treatment of the damaged heart. These stem cells also provide feasibility to address fundamental research questions directly relevant to human health, including their challenges, limitations, and potential, along with future prospects. Human induced pluripotent stem cell technology, in particular, patient-specific hiPSC-derived cardiomyocytes (hiPSC-CMs) recently has enabled modeling of human diseases, offering a unique opportunity to investigate potential disease-causing genetic variants in their natural environment.

Although there are many different kinds of stem cells, in this review we will include only those that have been used for most current cardiac regeneration studies.

Embryonic stem cells are obtained from the inner cell mass of the blastocyst that forms three to five days after an egg cell is fertilized by a sperm. They can give rise to every cell type in the fully formed body, but not the placenta and umbilical cord.

Tissue-specific stem cells (also referred to as somatic or adult stem cells) are more specialized than embryonic stem cells. The primary roles of adult stem cells in a living organism are to maintain and repair the tissue in which they are found.

Mesenchymal stem cells are multipotent stromal cells which can be isolated from the bone marrow. They are non-hematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as bone cells, cartilage cells, muscle cells and fat cells.

Induced pluripotent stem cells, or iPS cells, are cells taken from any tissue (usually skin or blood) and are genetically modified to behave like an embryonic stem cell. They are pluripotent, which means that they have the ability to form all adult cell types.

Umbilical cord blood stem cells are collected from the umbilical cord at birth and they can produce all of the blood cells in the body.

There is great potential with the recent advances in stem cell research and hiPSC-CMs, as these cells express the same ion channels and signaling pathways as primary human cardiomyocytes, can be cultured for a long time and are available in sufficient quantity. In addition, hiPSCs derived from diseased patients may be able to provide new forms of treatment of ischemic heart disease due to their potential for repairing damaged cardiac tissue, as shown in the Wus laboratory.11 Apart from their more direct role of tissue regeneration, stem cells may also have a clinical impact by secreting multiple growth factors and cytokines. Trophic mediators secreted by stem cells improve cardiac function by a combination of various mechanisms such as attenuating tissue injury, inhibiting fibrotic remodeling, promoting angiogenesis, mobilizing host tissue stem cells, and reducing inflammation. The cardioprotective panel of stem cell secreted factors are considerable and include, but not limited to bFGF/FGF-2, IL-1, IL-10, PDGF, VEGF, HGF, IGF-1, SDF-1, thymosin-4, Wnt5a, Ang-1 and Ang-2, MIP-1, EPO and PDGF.1218 FGF-2 reduces ischemia-induced myocardial apoptosis, cell death and arrhythmias, and stimulates increased expression of anti-apoptotic Bcl-2.19, 20 HGF, bFGF, Ang-1 and -2, and VEGF secreted by BMMSCs lead to augmented vascular density and blood flow in the ischemic heart2123, whereas SDF-1, IGF-1, HGF facilitate circulating progenitor cell recruitment to injury sites thereby promoting repair and regeneration.2427 Stem cells also secrete ECM components including collagens, TGF-, matrix metalloproteinases (MMPs) and tissue-derived inhibitors (TIMPs) that inhibit fibrosis.2830

Therefore, the use of the right mediator may contribute to a better outcome in cell therapy. Many stem cell types have been used in regenerative cardiac research, including bone marrow-derived cells, myoblasts, endogenous cardiac stem cells, umbilical cord-derived mesenchymal stem cells and embryonic cells. However, an exciting new milestone in the field of regenerative and precision medicine was the development of hiPSCs. The therapeutic potential of hiPSCs is considerable, as they are patient-specific stem cells that do not face the immunologic barrier, in contrast to embryonic stem cells. Furthermore, there are sources of tissue to be reprogrammed into hiPSCs that are easily accessible, such as the donors skin, fat, or blood. Their use may avoid common legal and ethical problems that arise from the use of embryonic stem cells; they can differentiate into functional cardiomyocytes and they are now one of the most promising cell sources for cardiac regenerative therapy.

Pluripotent stem cells (PSCs) have been derived by explanting cells from embryos at different stages of development under various growth conditions. PSCs can be classified into two distinct states, naive and primed, which are believed to represent successive snapshots of pluripotency as embryonic development proceeds.31, 32 Nave pluripotent stem cells can be maintained in vitro by supplying leukocyte inhibitory factor combined with inhibition of mitogen-activated protein kinase/extracellular regulated kinase and glycogen synthase kinase 3 signaling, and are characterized by two active X chromosomes. Primed pluripotent stem cells are dependent on fibroblast growth factor 2 signaling and transforming growth factor- signaling, and display inactivation of one X chromosome.31 Human embryonic stem cells and induced PSCs (iPSCs) are considered to share some properties of nave mouse embryonic stem cells.33 Nave human iPSCs can be derived by reversion of primed iPSCs into a state that resembles nave mouse ESCs.34

Fibroblasts are the most commonly used primary somatic cell type for the generation of iPSCs. Fibroblasts can be reprogrammed to stable self-renewing iPSCs which resemble ESCs by enforced expression of a cocktail of transcription factors consisting of octamer-binding protein (Oct4), SRY-box containing gene 2 (Sox2), Kruppel-like factor 4 (Klf4), c-myelocytomatosis oncogene (c-Myc), Lin28, and Nanog gene.35, 36 iPSCs can be generated, expanded, and then differentiated into any cell types including endothelial cells (ECs) and cardiomyocytes for in vitro studies or, ultimately, cell therapy.37, 38

In recent years, it has been shown that somatic cells can be directly converted to cardiomyocytes, although the efficacy is extremely low. Transgenic expression of three cardiac-specific transcription factors (Gata4, Mef2c, and Tbx5) resulted in the trans-differentiation of fibroblasts into contracting cardiomyocytes referred to as induced cardiomyocytes (iCMs). In addition, other reports have also shown that direct reprogramming of somatic cells to iCMs is also feasible using various small molecules and microRNAs (miRNAs), such as Hand2, Mesp1, Myocardin, ESRRG, miR-1, and miR-133.3942 Subsequently, alternative approaches have succeeded in generating human iCMs with gene expression profiles and functional characteristics similar to those detected in ESC-CMs.43

Due to the aforementioned advances in iPSC-derived CMs, it is now possible to generate an unlimited quantity of a patients own heart cells. This new model allows researchers to study and understand the molecular and cellular mechanisms of inherited cardiomyopathies, channelopathies, as well as model acquired heart diseases. Although additional studies are needed to test their safety and efficacy, these heart cells may be also used for regenerative medicine applications following myocardial infarction.

It was shown that hiPSCs may lose their pluripotency when transplanted into a border zone of infarcted cardiac tissue, and engraft into native myocardium where they only partially differentiate into cardiac myocytes. In Yans study, they reported that iPS cell transplantation in the infarcted diabetic db/db and nondiabetic mice resulted in an increase in vascular smooth muscle and endothelial cells in the infarcted heart, leading to a significantly improved cardiac function (Figure 1).44

iPS cell transplantation in the infarcted diabetic db/db and nondiabetic mice resulted in an increase in vascular smooth muscle and endothelial cells in the infarcted heart, leading to a significantly improved cardiac function. Photomicrographs show anti-CD-31 in red (A, panels a, e, i), anti-red fluorescence protein (RFP) in green (A, panels b, f, j) and total nuclei stained with diamidino-phenylindole (DAPI) in blue (A, panels c, g, k). Merged images are shown in A, panels d, h, i. Scale bar=100 m. Panel B shows quantitative analysis of total endothelial cells generation from transplanted iPS cells in both C57BL/6 and db/db mouse hearts two weeks post-MI,*P < 0.001 vs MI.44

Another study demonstrated that iPSC derived progenitor cells differentiated into a cardiomyocyte phenotype and developed contracting areas in mice heart tissue. Beneficial remodeling and improved ventricular function were observed despite the lack of well-aligned mature donor cardiomyocytes.45

In regards to safety, an important obstacle to the clinical use of hiPSCs for the regenerative purposes is their great heterogeneity in terms of plasticity and epigenetic landscape. There is a potential that allogeneic hiPSC transplantation into the heart may cause in situ tumorigenesis.46 In addition, the heterogeneity of the cardiac cells produced from pluripotent hiPSCs administration and their random implantation is likely to cause cardiac arrhythmias. One of the main limitations of the hiPSC-derived cardiomyocytes is that they are embryonic in nature as compared to adult cells. Many laboratories are still trying to make these myocytes more mature and to make lineage-specific cells so as to obtain a pure population of atrial cells, nodal cells, or ventricular cells. iPSC-derived cardiomyocytes exhibit an immaturity of the sarcoplasmic reticulum, and a -adrenergic response that is significantly different from native ventricular tissue of a comparable age. Once the cells are mature, it is also likely that investigators will be able to test the effects of various drugs using hiPSC-CMs from a diverse population of patients with different sexes, ethnicities, and cardiovascular diseases.

We are utilizing a model of the patient-specific hiPSCs differentiated into cardiac lineage in order to delineate the environmental and cellular mechanisms responsible for impaired cardioprotection in diabetes. The advantage of this approach is that the effect of cardioprotection can be evaluated in human cells, thereby capturing the complex physiologic interactions at the patient-specific myocyte level. Our results indicate that iPSC-derived cardiomyocytes are not only a viable model to investigate the underlying mechanisms of anesthetic cardioprotection,47 but they also respond similarly to human myocytes48 and human embryonic stem-cell-derived cardiomyocytes.49 Isoflurane preconditioning protected hiPSC-derived cardiomyocytes from oxidative stress-induced lactate dehydrogenase release and mitochondrial permeability transition pore opening at normal glucose concentrations (Figure 2).50

Isoflurane delayed mitochondrial permeability transition pore (mPTP) opening and protected induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from oxidative stress in 5 mM and 11 mM glucose. mPTP opening was induced by photoexcitation-generated oxidative stress. Isoflurane delayed mPTP opening in iPSC-CMs in the presence of 5 mM and 11 mM glucose concentrations (A). Isoflurane did not delay mPTP opening in the presence of 25 mM glucose concentrations (A). *P < 0.001 versus control, n=18 cells/group. H2O2-induced oxidative stress increased lactate dehydrogenase (LDH) release from iPSC-CMs in 5 mM, 11 mM and 25 mM glucose concentrations (B). In iPSC-CMs, isoflurane reduced stress-induced LDH release in 5 mM and 11 mM glucose, but not in 25 mM glucose (B). *P < 0.05 versus stress, n=3 experiments/group. Ctrl = Control; Iso = Isoflurane treatment; and Stress = H2O2 + 2-Deoxyglucose.50

Anesthetic preconditioning also protects cardiomyocytes indirectly through its action on other cell types in the heart, such as endothelial cells,51 or by modulation of inflammatory response. However, hyperglycemia undermines the effectiveness of anesthetic-induced cardioprotection by dysregulating cellular signaling.47 In addition, this study demonstrated that the cardioprotective effects of isoflurane in elevated glucose conditions can be restored by scavenging reactive oxygen species or inhibiting mitochondrial fission. These findings may contribute to further understanding and guidance for restoring pharmacological cardioprotection in hyperglycemic patients. Cardiomyocytes derived from healthy donors and patients with a particular disease (such as diabetes) open new possibilities of studying genotype and phenotype related pathologies in a human-relevant model. Such diseases were nearly impossible to investigate in the past due to the lack of human cardiac cells available for experimental investigation.

Some preclinical studies provide evidence that bone marrow stem cells contribute to cardiac function and reverse remodeling after ischemic damage52 acting both locally53 and remotely.54 In studies to date, bone marrow stem cells have been either infused55 or injected56 in areas that were undergoing revascularization. In preclinical studies, Bollis group reported that multiple treatments are necessary to properly evaluate the full therapeutic potential of cell therapy.57, 58 In their study on mice with a myocardial infarction received one or three doses of cardiac mesenchymal cells through the percutaneous infusion into the left ventricular cavity, 14 days apart. The single-dose group showed improved left ventricular ejection fraction after the 1st infusion but not after the 2nd and 3rd-vehicle infusion. In contrast, in the multiple-dose group, left ventricular ejection fraction improved after each cardiac mesenchymal cell-infusion. The multiple-dose group also exhibited less collagen in the non-infarcted region vs. the single-dose group. Engraftment and differentiation of cardiac mesenchymal cells were negligible in both groups, indicating paracrine effects.58 There appear to be at least three dominant mechanisms that underlie the cardiac reparative response: reduction in tissue fibrosis, neovascularization, and neomyogenesis.54, 59

Human ESC-CMs isolated from embryoid bodies have also been used for replacing myocardial scar tissue with new, functional cardiac cells, and therefore achieving actual myocardial regeneration. The ESC-CMs behave structurally and functionally like cardiomyocytes, expressing characteristic morphology, cell marker and transcription factor expression, sarcomeric organization and electrophysiological properties, including spontaneous action potentials and beating activity.60 Mouse and human cardiac-committed ESCs have been transplanted into small and large animal models of acute and old MI. Although these studies have demonstrated durable in vivo engraftment, proliferation and differentiation of ESC-CMs, as well as electromechanical integration with host cardiomyocytes61, they have not universally shown improvement in myocardial remodeling and function. While the reported benefits can thus be attributed to a potential synergy62 between the favorable environment created by the revascularization of the region and the mesenchymal stem cells, the precise delineation of each contribution, however, remains unknown. In addition, so far there is no evidence that critical number of new cells is regenerated or injected stem cells survive following the transplant. Animal studies have shown that only 1% of the stem cells injected into the heart tissue are detectable after 1 month. Nevertheless, in one of the recent systemic reviews and meta-analysis studies of preclinical work, cardiac stem cells treatment resulted in significant improvement of ejection fraction compared with placebo.63 In addition, there was a reduction in the magnitude of effect in large compared with small animal models.

A cell-based therapy could offer additional clinical benefits for post-ischemic heart by improving revascularization along with structural and functional properties.12, 64, 65 There are several limitations for effective stem cell therapy, but the major problems deal with their delivery, type of cells to be used, limited retention of the cells in the heart and the risk of immune rejection. Direct injection of stem cells into the heart muscle results in significant cell death and washout resulting in majority of cells being removed from the heart soon after the injection. Many preclinical studies have reported that intravenously administered MSCs for acute myocardial infarction attenuate the progressive deterioration in LV function and adverse remodeling in mice with large infarcts, and in ischemic cardiomyopathy, they improve LV function.63 Moreover, the cardiac phenotype of human embryonic stem cell-derived cardiac myocytes and human induced pluripotent stem cell-derived cardiac myocytes salvages the injured myocardium better than undifferentiated stem cells through their differential paracrine effects.66

In the clinical studies, the investigators have used mainly two approaches of cell administration: intramyocardial delivery and intracoronary injection. Direct cardiac muscle injections can be performed either surgically or using percutaneous endocardial injection catheters, while coronary injection of stem cells can be done using an antegrade intracoronary artery injection or a retrograde sinus injection. The antegrade intracoronary artery injection is more attractive because it is the least invasive but some microvascular plugging can occur as a result of stem cell injection leading to microinfactions when the cells injected are too large for the capillary bed. Since the stem cells also need to cross the capillary wall, this approach has been found to be less effective as compared to intramyocardial delivery. Although the cell type, dosage, concentration, and delivery modalities are important considerations for regenerative cell therapy clinical trials, the available data are inconclusive and additional early phase studies will be needed before proceeding to pivotal clinical trials.67

The stem cells derived from the bone marrow of the healthy donors have been used in majority of clinical trials as briefly summarized in Table 1. So far, the clinical trials for cardiac regeneration have mainly used cell-based therapies, including bone marrow-derived cells, mesenchymal stem cells and cardiac progenitor cells. While the listed studies have met safety end points either with autologous or allogeneic cell sources68, the effect on cardiac function has been somewhat disappointing. One of the largest multicenter clinical trials using bone-marrow cells given via intracoronary injection for myocardial infarct patients, failed to reinforce the notion that these therapies are efficacious since it did not meet its primary goal.69 A recently published Cochrane review of bone-marrow trials for heart attack patients also found no benefits for various primary goals such as mortality, morbidity, life quality and LVEF.70 An additional Cochrane review using bone-marrow-derived stem/progenitor cells as a treatment for chronic ischemic heart disease and congestive heart failure identified low-quality evidence of reduction in mortality and improvement of LVEF.71

Selected list of landmark clinical trials using mostly bone marrow-derived mesenchymal stem cells conducted to treat acute myocardial infarction and heart failure.

The limited clinical success of stem-cell injections for the treatment of myocardial infarction or heart failure has been mainly attributed to the low retention and survival of injected cells. One of the clinical trials for treatment of heart failure resulting from ischemic heart disease used autologous c-kit(+) cardiac stem cells and produced a significant improvement in both global (Figure 3) and regional LV function (Figure 4), a reduction in infarct size, and an increase in viable tissue that persisted at least 1 year after cardiac infusion (SCIPIO trial).72 Another study, also involving small number of patients, used intracoronary administration of autologous cardiosphere-derived cells and the treatment led to a decreased scar size, increased viable myocardium, and improved regional function of infarcted myocardium at 1 year post-injection (CADUCEUS trial) (Figure 5).73

Administration of Cardiac Stem Cells (CSC) in Patients with Ischemic Cardiomyopathy. Panel A: The mean baseline LVEF in the eight treated patients who were included in the cardiac magnetic resonance analysis was 27.5% at baseline (4 months after CABG surgery and before CSC infusion), and increased markedly to 35.1% (P=0.004, n=8) at 4 months and 41.2% (P=0.013, n=5) at 12 months after CSC infusion. Panel B: Change in LVEF at 4 months and 12 months after CSC infusion (absolute EF units). Data are means SEMs. 72

Panel A: Regional EF at baseline and 4 and 12 months after CSC infusion in the infarct-related regions. Panel B: Change in regional EF in the infarct-related regions at 4 and 12 months after CSC infusion (absolute EF units). Panel C: Regional EF in the dyskinetic segments of the infarct-related regions at baseline and 4 and 12 months after CSC infusion. Panel D: Change in regional EF in the dyskinetic segments of the infarct-related regions at 4 and 12 months after CSC infusion (absolute EF units). Panel E: Regional EF in the least functional segment of the infarct-related regions at baseline and 4 and 12 months after CSC infusion. Panel F: Change in regional EF in the least functional segment of the infarct-related regions at 4 and 12 months after CSC infusion (absolute EF units). Data are means SEMs. 72

(A) Representative matched, delayed contrast-enhanced magnetic resonance images and their corresponding cine short-axis images (at end-diastole [ED] and end-systole [ES]) at baseline and 1 year. In the pseudocolored, delayed contrast-enhanced images, infarct scar tissue, as determined by the full width half maximum method, appears pink. Each cardiac slice was divided into 6 segments and the infarcted segments were visually identified from delayed contrast enhanced images. Scar size (percentage of infarcted tissue per segment) and systolic thickening were calculated for each individual infarcted segment at baseline and 1 year. Endocardial (red) and epicardial (green) contours of the left ventricle are shown. In the CDC-treated patient (top row), scar decreased, viable mass increased and regional systolic function improved over the period of 1 year in the treated infarcted segments (highlighted by arrows). In contrast, no major changes in scar mass, viable myocardial mass or regional systolic function were observed in the control patient (bottom row). (B) Scatterplots of the changes in the percentage of infarcted tissue and the changes in systolic thickening for every infarcted segment of treated and control patients. ED = end-diastole. 73

Umbilical cord blood has been demonstrated as a very useful and rich source of stem and progenitor cells, capable of restoring blood formation and immunological functions after transplantation. Cord blood stem cells are currently used to treat a range of blood disorders and immune system conditions such as leukemia, anemia and autoimmune diseases. These stem cells are used largely in the treatment of children but have also started being used in adults following chemotherapy treatment. Another type of cell that can also be collected from umbilical cord blood is mesenchymal stromal cells. These cells can grow into bone, cartilage and other types of tissues and are being used in many research studies to see if patients could benefit from these cells too. The fact that cord blood can be frozen and stored for later use led, in 1991, to the establishment of the first cord blood bank from voluntary donors in New York. To date, there are over 54 public cord blood banks in different parts of the world with more than 350,000 units frozen and ready to be used.74 Indeed cord blood transplantation is being used as an alternative to bone marrow transplantation, and more than 14,000 transplants have been documented. Cord blood stem cell treatments differ from bone marrow stem cell treatments in three key areas: increased tolerance of the human leukocyte antigen-mismatching, decreased risk of graft-versus-host disease, and enhanced proliferation ability.75 Recent results of the RIMECARD study by Bartolucci et al. in human subjects using umbilical cord-derived MSCs as potential heart failure therapy are quite encouraging.76 The patients had stable heart failure (HF), with reduced ejection fraction of less than 40. Although the sample size was small (15 controls and 15 HF patients treated with UC-MSCs) to establish either safety or efficacy, the echocardiographic and cardiac MRI evaluations demonstrated improvements in ejection fraction, starting at 3 months, and persisting through 12 months. The patients treated with placebo did not improve in either left ventricular ejection fraction or clinical functional class. As indicated by the authors, it is tempting to speculate that the robust paracrine secretion of various factors, including hepatocyte growth factor, might play an important role in mediating the therapeutic effects of the UC-MSCs.

The main disadvantage of cord blood use is that the volume collected is fixed and relatively small. Therefore, the number of stem cells available for transplantation is low compared to the number of cells that can be collected in customizable bone marrow or peripheral blood stem cell harvests. Nevertheless, there are many opportunities for further development of this technology such as the cord blood selection algorithms that are currently heavily weighted toward maximizing cell doses at the expense of the human leukocyte antigen-matching.77

Beside the stem cell injection therapy, currently there are non-cardiogenic and cardiogenic stem-cell tissue patches, for the repair of myocardial infarction. Recent studies have utilized non-cardiogenic tissue patches made of skeletal myoblasts7881, bone marrow-derived stem cells82, 83, or endothelial progenitor cells84 for the repair of damaged heart. Compared with the injection of a cell suspension, the implantation of tissue sheets composed of skeletal myoblasts has been proven more advantageous for the treatment of myocardial infarction in rats78, 85, and dilated cardiomyopathy in hamsters.86 Moreover several cardiogenic cardiac tissue-engineering methodologies have been developed for use with primary neonatal cardiomyocytes. These include: injection of a mixture of bioactive hydrogels and cells followed by cell-hydrogel polymerization in situ87 and the epicardial implantation of a tissue-engineered cardiac patch.88, 89

Generally, implantation of the engineered myoblast sheets over an infarction site yielded improved neovascularization, attenuated left ventricular dilatation, decreased fibrosis, improved fractional shortening, and prolonged animal survival compared to the delivery of the same number of myoblasts by cell injection.85 In addition, the bone marrow-derived, spatially arranged SMC-endothelial progenitor bi-level cell sheet interactions between SMCs and endothelial progenitor cells augment mature neovascularization, limit adverse remodeling, and improve ventricular function after myocardial infarction.90 In diabetic patients treatment of diabetes mellitus-induced cardiomyopathy with tissue-engineered smooth muscle cell-endothelial progenitor cell bi-level cell sheets prevented cardiac dysfunction and microvascular disease associated with diabetes mellitus-induced cardiomyopathy.91 As indicated before, the main disadvantage of injecting the cell-suspensions directly into the heart muscle compare to engineered heart tissue technique is that most of the injected cells are washed out of the heart or do not survive the injection. This is inefficient and can also be dangerous if some cells have not yet fully developed into myocardial cells and are therefore still pluripotent. These cells could survive in other parts of the body and form tumors. The advantage of the tissue patches is that significantly fewer of the stem cell-derived heart cells are required and fewer cells undergo apoptosis. Some of the major drawbacks currently encountered with regeneration using tissue patches, include the problems with electrical continuity and patch vascularization. Using a similar tissue-engineering strategy, Shimko et al. formed cardiac constructs using pure differentiated cardiomyocytes derived by genetic selection from D3 mouse ESCs with a neomycin-resistance gene driven by the -MHC promoter.92 They found that 10% cyclic stretch at rate of 13 Hz for 3 days increased the expression of cardiac markers such as -cardiac actin, -MHC and Mef-2c, but the resulting cardiac tissues were noncontractile. Immunostaining showed that pure cardiomyocytes were present, but they had disorganized sarcomeres and a relatively rounded appearance.92

Recently, in a study published by Nummi et al., they reported that during on-pump coronary artery bypass graft surgery, part of the right atrial appendage can be excised upon insertion of the right atrial cannula of the heart-lung machine and the removed tissue can be easily cut into micrografts for transplantation.93 Appendage tissue is harvested during cannulation of the right atrium, and therefore, no additional procedure is needed. Isolation of the cells and preparing the matrix for transplantation is done simultaneously with the coronary artery bypass graft operation in the operating room, so the perfusion time and the aorta clamp time are not increased. After the bypass anastomoses, the atrial appendage sheet is placed on the myocardium with three to four sutures allowing the myocardium to contract without interference. They believe that atrial appendage-derived cells therapy administered during CABG surgery will have an impact on patient treatment in the future.93

While some of the outcomes of these trials have been modest at best, it is now evident that the success of future cardiac cell therapies will be highly dependent on the ability to overcome the problem of low retention and survival of implanted cells.94 Potential approaches to address this issue include: coinjecting cells with bioactive, in situ polymerizable hydrogels87, preconditioning cells with hypoxia or prosurvival factors95, genetic engineering of cells to enhance their angiogenic and/or antiapoptotic action96 and the epicardial implantation of a preassembled tissue-engineered patches.27, 85, 97 In particular, tissue patch implantation, although surgically more complex than cell or cell/hydrogel injections, may support long-term survival of transplanted cells and exert a more efficient structural and functional cardiac tissue reconstruction at the infarct site.98

The adult human heart lacks sufficient ability to replenish the damaged cardiac muscles since the rate of cardiomyocyte renewal activity is less than 1% per year. The mechanical and electrical engraftment of injected cardiomyocytes is largely not feasible at the scale that would be necessary for cardiac improvement. On the other hand, the human heart contains large population of fibroblasts that could be used for direct reprograming. As such, direct fibroblasts reprogramming in vivo has emerged as a possible approach for cardiac regeneration. With considerably better understanding of the various molecular mechanisms, direct fibroblast reprogramming has improved considerably but still lacks sufficient efficacy using human cells (Figure 6).

There are various novel treatment options that have been tested for the heart failure due to ischemic heart disease or genetic disorders. Previous clinical trials have employed various adult stem cell and progenitor cell populations to test their efficacy for therapeutic applications. Additional approaches are being explored, including implantation of in vitro constructed cell sheets of engineered heart muscles (EHMs) as well as direct in vivo reprogramming of cardiac fibroblasts in the scar region to cardiomyocytes. The regenerative capacity of adult stem and progenitor cell populations is also being evaluated along with administration of exosomes and small vesicles secreted by the stem cells.36

As indicated earlier, the survival of transplanted stem cells is dismal and the beneficial effects of stem cell therapies is not due to their differentiation into new cardiomyocytes but instead because they are the temporary source of the exosomal growth factors. Therefore, despite the stem cells early demise, there are some limited cardiac benefits from this treatment, including decreased cardiomyocyte apoptosis, reduced fibrosis, enhanced neovascularization and improved left ventricular ejection fraction. It is for that reason why the exosome therapy recapitulates the benefits of stem cell therapy,99 and many studies have shown that the activation of cardioprotective pathways obtained by stem cell therapy can be reproduced by the injection of exosomes produced by the stem cells.100 An additional benefit of using exosomes for cardioprotection and regeneration is the lack of tumor-forming potential of exosomes. However, the underlying mechanisms of stem cells or hiPSC-derived exosome therapy are still unclear. Numerous scientific investigations have identified recent applications of exosomes in the development of molecular diagnostics, drug delivery systems and therapeutic agents.

Exosomes are small membrane vesicles (30100 nm) of endocytic origin that are secreted by most cells after being formed in the cellular multivesicular bodies. The fusion of multivesicular bodies into the plasma membrane leads to the release of their intraluminal vesicles as exosomes. Once released in the extracellular environment, their cargo of functional molecules can be taken up by recipient cells via several mechanisms including fusion with the plasma membrane, phagocytosis and endocytosis. The formation and release can be upregulated through different steps based on environmental stimuli such as stress or hypoxia. There are two main mechanisms responsible for exosome release. First, there is a constitutive mechanism that is mediated by specific proteins involved in membrane trafficking, such as RAB heterotrimeric G-proteins and protein kinase D. Second, there exists an inducible mechanism that can be activated by several stimuli including increased intracellular Ca2+ and DNA damage. Studies have used different approaches to also increase the angiogenic potential of exosomes released by the stem cells.101 Exosome release with a basal angiogenic potential can be substantially increased in vitro using stress conditions that mimic organ injury, such as hypoxia, irradiation, or drug treatments. Changes in exosomal composition facilitate angiogenesis and tissue repair most likely via enhanced level of growth factors and cytokines.

Exosomes contain various molecular constituents of their cell of origin, including proteins and RNA. The cargo of mRNA and miRNA in exosomes was first discovered at the University of Gothenburg in Sweden.102 In that study, the differences in cellular and exosomal mRNA and miRNA content were described, as well as the functionality of the exosomal mRNA cargo. Exosomes facilitate cell-cell communication to the recipient cell membrane and deliver effectors including transcription factors, oncogenes, small and large non-coding regulatory RNAs (such as microRNAs) and mRNAs into recipient cells and can be used for cardiac protection and repair. Exosomes have also been shown to carry double-stranded DNA. Exosomes can be derived from many different types of stem cells including umbilical cord, cardiosphere-derived cells, cardiac stem cells, embryonic, induced pluripotent, mesenchymal and endothelial progenitor cells. They can carry and deliver mRNAs, miRNAs and proteins to the injured heart muscle and play a significant role in resident cardiac stem cell activity, cardiomyocyte proliferation, beneficial cardiac remodeling, apoptosis reduction, angiogenesis, anti-inflammatory response and a decrease in infarct size. The advantages for effective exosome therapy include the cell free component, log-term stability and low or no immune response. Some of the limitations include the necessity of repeated injections, target cell selection and the random packing of the exosome cargo.

Some preliminary reports have demonstrated that exosomes released from cardiac progenitor cells can improve cardiac function in the damaged heart.103, 104 It has been proposed that exosomes released from transplanted cardiomyocytes are involved in metabolic events in target cells by facilitating an array of metabolism-related processes, including modulation of gene expression. Moreover, exosomes secreted from the hiPSC-derived cardiomyocytes exert protective effects by transferring endogenous molecules to salvage injured neighboring cells by regulating angiogenesis, apoptosis, fibrosis, and inflammation. It has been shown that ischemic preconditioned hearts promote exosome release and help spread cardioprotective signals within the myocardium.105, 106 Also, the administration of mesenchymal stromal cell-secreted exosomes demonstrated improved cardiac function in the acute myocardial infarction mouse model. Mesenchymal stem cell-derived exosomes increased adenosine triphosphate levels, reduced oxidative stress, and activated the PI3K/Akt pathway to enhance cardiomyocyte viability after ischemia-reperfusion (I/R) injury.107 Recently, it was shown that ischemic preconditioning of mesenchymal stromal cells increased levels of miR-21, miR-22, miR-199a-3p, miR-210, and miR-24 in exosomes released by the cells, and the administration of mesenchymal stromal cell-ischemic preconditioning exosomes resulted in a reduction of cardiac fibrosis and apoptosis compared with the hearts treated with control exosomes. Stem cell-derived exosomes possess the ability to modulate cardiomyocyte survival and confer protection against angiotensin II-induced hypertrophy by activating PI3K/Akt/eNOS pathways via RNA enriched within the exosomes. Additionally, it has been shown that exosome treatment increased levels of adenosine triphosphate and NADH, decreased oxidative stress, increased phosphorylated-Akt and phosphorylated- glycogen synthase kinase 3, and reduced phosphorylated-c-JNK in hearts after I/R. Subsequently, both local and systemic inflammations were significantly reduced 24 hours after reperfusion.107 Intact exosomes restore bioenergetics, reduce oxidative stress, and activate pro-survival signaling, thereby enhancing cardiac function and geometry after myocardial I/R injury.107 Clearly, stem cell-derived exosomes may be a potential adjuvant to reperfusion therapy for myocardial infarction and heart failure.

The existing therapies for the ischemic heart disease have many limitations and efforts are underway for new treatments using the stem cell therapy to improve the clinical conditions by either replacing the damaged heart cells and/or improve cardiac performance. The cardiac tissue regeneration with stem cells, their exosomes or small vesicles and tissue engineering may be effective therapeutic options. Although the expectations have been high, the results from majority of clinical trials are negative. Due to very low engraftment and survival of stem cells injected into a cardiac muscle, there is convincing evidence that the release of paracrine factors from the stem cells contributes to myocardial cardioprotection and regeneration. It is likely that the future research will be focused on the biology of these endogenous signaling pathways, and will lead the way for different applications of exosomes and small vesicles in regenerative medicine. In the future there might be more successful approaches that would utilize stem cell technology with various bioengineering constructs having not only cardiomyocytes but other cardiac cells.

Regarding the regulatory agencies, one would need a significant efficacy/safety data and meaningful end points when compared with standard-of-care drugs that are used today for heart attacks and heart failures. So where are we today since most of the clinical trials did not achieve their primary efficacy end points? Because the cell-therapy studies for heart disease did not achieve this so far, more preclinical work might be necessary using current and other approaches in order to demonstrate compelling rationale for new clinical trials. Although the regenerative biology might not be very helpful to the cardiovascular patient in the near term, it is most likely that we will witness very impressive and exciting results over the next several decades.

This work was supported in part by grants P01GM066730 and T32 GM089586 from the National Institutes of Health, Bethesda, MD.

calcium ion

endothelial cells

endothelial nitric oxide synthase

endothelial nitric oxide synthase/protein kinase G

Extracellular signal-regulated protein kinases 1 and 2

human stem cells, induced pluripotent stem cells

human stem cells, induced pluripotent stem cells-derived cardiomyocytes

induced pluripotent stem cells

Nicotinamide adenine dinucleotide

Janus kinase and Signal Transducer and Activator of Transcription pathways

specific isoforms of mitogen-activated protein kinase and extracellular regulated kinase

Phosphatidylinositol 3-kinase, serine/threonine kinase also known as protein kinase B, and mammalian target of rapamycin pathway

protein kinase C

cGMP protein kinase G pathway

Disclosure

The authors declare that they have no disclosures.

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Maia Terashvili, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin, 53226, USA.

Zeljko J. Bosnjak, Departments of Medicine and Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin, 53226, USA.

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Scientists trial implant to patch up the heart  BBC.com

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Engineered heart muscle allografts for heart repair in primates and humans  Nature.com

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The cure for a broken heart could be stem cell patches  The Times

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Revolutionary Stem-Cell Patches Offer Hope For Heart Repair  Evrim Aac

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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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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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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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Nanoparticle that cuts middlemen could improve stem cell therapy  Futurity: Research News

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Nanoparticle that cuts middlemen could improve stem cell therapy - Futurity: Research News

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Coordinated immune networks in leukemia bone marrow microenvironments distinguish response to cellular therapy  Science

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Coordinated immune networks in leukemia bone marrow microenvironments distinguish response to cellular therapy - Science

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