Personalized regenerative medicine and biomedical research have been galvanized and revolutionized by human pluripotent stem cells in combination with recent advances in genomics, artificial intelligence, and genome engineering. More recently, we have witnessed the unprecedented breakthrough life-saving translation of mRNA-based vaccines for COVID-19 to contain the global pandemic and the investment in billions of US dollars in space exploration projects and the blooming space-tourism industry fueled by the latest reusable space vessels. Now, it is time to examine where the translation of pluripotent stem cell research stands currently, which has been touted for more than the last two decades to cure and treat millions of patients with severe debilitating degenerative diseases and tissue injuries. This review attempts to highlight the accomplishments of pluripotent stem cell research together with cutting-edge genomics and genome editing tools and, also, the promises that have still not been transformed into clinical applications, with cardiovascular research as a case example. This review also brings to our attention the scientific and socioeconomic challenges that need to be effectively addressed to see the full potential of pluripotent stem cells at the clinical bedside.

Induced pluripotent stem cell (iPSC)-based disease modelling and the cell replacement therapy approach have proven to be very powerful and instrumental in biomedical research and personalized regenerative medicine as evidenced in the past decade by unraveling novel pathological mechanisms of a multitude of monogenic diseases at the cellular level and the ongoing and emerging clinical trials with iPSC-derived cell products. iPSC-based disease modelling has sparked widespread enthusiasm and has presented an unprecedented opportunity in high throughput drug discovery platforms and safety pharmacology in association with three-dimensional multicellular organoids such as personalized organs-on-chips, gene/base editing, artificial intelligence and high throughput omics methodologies. This critical review summarizes the progress made in the past decade with the advent of iPSC discovery in biomedical applications and regenerative medicine with case examples and the current major challenges that need to be addressed to unleash the full potential of iPSCs in clinical settings and pharmacology for more effective and safer regenerative therapy.

BACKGROUND:Heart failure (HF) is a leading cause of mortality and is associated with cardiac remodeling. Vulnerability to atrial fibrillation (AF) has been shown to be greater in the early stages of HF, whereas ventricular tachycardia/fibrillation develop during late stages. Here, we explore changes in gene expression that underlie the differential development of fibrosis and structural alterations that predispose to atrial and ventricular arrhythmias.OBJECTIVE:To study transcriptomic changes associated with the development of cardiac arrhythmias in early and late stages of heart failure.METHODS:Dogs were tachy-paced from right ventricle (RV) for 2-3 or 5-6 weeks (early and late HF). We performed transcriptomic analysis of right atria (RA) and RV isolated from control dogs and those in early and late HF. Transcripts with mean relative log2-fold change ≥2 were included in the differential analysis with significance threshold adjusted to p<0.05.RESULTS:Early HF remodeling was more prominent in RA with enrichment of extracellular matrix, circulatory system, wound healing and immune response pathways; many of these processes were not present in RA in late HF. RV showed no signs of remodeling in early HF but enrichment of extracellular matrix and wound healing in late HF.CONCLUSION:Our transcriptomic data indicate significant fibrosis-associated transcriptional changes in RA in early HF and in RV in late HF, with strong atrial predominance. These alterations in gene expression are consistent with the development of arrhythmogenesis in atria in early but not late HF and in the ventricle in late but not early HF.

In vivo, cardiomyocytes comprise a heterogeneous population of contractile cells defined by unique electrophysiologies, molecular markers and morphologies. The mechanisms directing myocardial cells to specific sub-lineages remain poorly understood. Here we report that overexpression of TGFβ-Activated Kinase (TAK1/Map3k7) in mouse embryonic stem (ES) cells faithfully directs myocardial differentiation of embryoid body (EB)-derived cardiac cells toward the sinoatrial node (SAN) lineage. Most cardiac cells in Map3k7-overexpressing EBs adopt markers, cellular morphologies, and electrophysiological behaviors characteristic of the SAN. These data, in addition to the fact that Map3k7 is upregulated in the sinus venous—the source of cells for the SAN—suggest that Map3k7 may be an endogenous regulator of the SAN fate.

The Melanoma-associated Antigen gene family (MAGE) generally encodes for tumour antigens. We had identified that one of the MAGE gene members, Mageb16 was highly expressed in undifferentiated murine embryonic stem cells (ESCs). While the role of Mageb16 in stemness and differentiation of pluripotent stem cells is completely unknown, here, in our current study, we have demonstrated that Mageb16 (41 kDa) is distributed in cytosol and/or in surface membrane in undifferentiated ESCs. A transcriptome study performed at differentiated short hairpin RNA (shRNA)-mediated Mageb16 knockdown (KD) ESCs and scrambled control (SCR) ESCs until a period of 22 days, revealed that Mageb16 KD ESCs mainly differentiated towards cells expressing mesodermal and cardiovascular lineage - gene markers. Gene markers of other mesoderm-oriented biological processes such as adipogenesis, osteogenesis, limb morphogenesis and spermatogenesis were also significantly enriched in the differentiated Mageb16 KD ESCs. The expression levels of contractile genes were higher in differentiated Mageb16 KD ESCs when compared to differentiated SCR and wild ESCs, suggesting a higher cardiomyogenic potential of Mageb16 depleted ESCs. Further analysis indicates that regulative epigenetic networks and nucleocytoplasmic modifications induced by the depletion of Mageb16, may play a probable role in differentiation.

The role of striatin interacting protein 2 (Strip2) in differentiation of embryonic stem cells (ESCs) is still under debate. Strip2-silenced murine (KD) ESCs were differentiated for 4, 8, 12, and 16 days. We show that Strip2 is distributed in the perinucleus or nuclei of wild-type (WT) undifferentiated ESCs, but is localized in high-density nuclear bodies in differentiated cells. CellNet analysis of microarray gene expression data for the KD and scrambled control (SCR) embryoid bodies (EBs), as well as immunostainings of key pluripotent factors, demonstrated that differentiation of KD ESCs is repressed. This occurs even in 16-day-old EBs, which possessed a high tumorigenic potential. Correlated with very high expression levels of epigenetic regulator genes, Hat1 and Dnmt3, enzymatic activities of the histone acetyltransferase type B (Hat1) and DNA (cytosine-5)-methyltransferase 3 beta (Dnmt3b) were higher in differentiated 16-day-old KD EBs than in SCR or WT EBs. The expression levels of let-7, 290, and 302 microRNA families were opposed in KD ESCs, while KD EBs had levels comparable to WT and SCR ESCs during differentiation. Strip2 is critical for the regular differentiation of ESCs. Moreover, Strip2 deficient ESCs showed a dysregulation of epigenetic regulators and microRNAs regulating pluripotency.

Background: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs) requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs) of different murine ESC lines. Methods: Two wild-type (D3 and R1) and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7) were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP) and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC) promoter. Action potentials (APs) were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. Results: Spontaneous AP frequency and AP duration (APD) as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. Conclusion: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.

We performed a systematic analysis of gene expression features in early (10-21 days) development of human vs mouse embryonic cells (hESCs vs mESCs). Many development features were found to be conserved, and a majority of differentially regulated genes have similar expression change in both organisms. The similarity is especially evident, when gene expression profiles are clustered together and properties of clustered groups of genes are compared. First 10 days of mESC development match the features of hESC development within 21 days, in accordance with the differences in population doubling time in human and mouse ESCs. At the same time, several important differences are seen. There is a clear difference in initial expression change of transcription factors and stimulus responsive genes, which may be caused by the difference in experimental procedures. However, we also found that some biological processes develop differently; this can clearly be shown, for example, for neuron and sensory organ development. Some groups of genes show peaks of the expression levels during the development and these peaks cannot be claimed to happen at the same time points in the two organisms, as well as for the same groups of (orthologous) genes. We also detected a larger number of upregulated genes during development of mESCs as compared to hESCs. The differences were quantified by comparing promoters of related genes. Most of gene groups behave similarly and have similar transcription factor (TF) binding sites on their promoters. A few groups of genes have similar promoters, but are expressed differently in two species. Interestingly, there are groups of genes expressed similarly, although they have different promoters, which can be shown by comparing their TF binding sites. Namely, a large group of similarly expressed cell cycle-related genes is found to have discrepant TF binding properties in mouse vs human.

Recently, growing attention has been directed toward stem cell metabolism, with the key observation that the plasticity of stem cells also reflects the plasticity of their energy substrate metabolism. There seems to be a clear link between the self-renewal state of stem cells, in which cells proliferate without differentiation, and the activity of specific metabolic pathways. Differentiation is accompanied by a shift from anaerobic glycolysis to mitochondrial respiration. This metabolic switch of differentiating stem cells is required to cover the energy demands of the different organ-specific cell types. Among other metabolic signatures, amino acid and carbohydrate metabolism is most prominent in undifferentiated embryonic stem cells, whereas the fatty acid metabolic signature is unique in cardiomyocytes derived from embryonic stem cells. Identifying the specific metabolic pathways involved in pluripotency and differentiation is critical for further progress in the field of developmental biology and regenerative medicine. The recently generated knowledge on metabolic key processes may help to generate mature stem cell–derived somatic cells for therapeutic applications without the requirement of genetic manipulation. In the present review, the literature about metabolic features of stem cells and their cardiovascular cell derivatives as well as the specific metabolic gene signatures differentiating between stem and differentiated cells are summarized and discussed.

FAM40B (STRIP2) is a member of the striatin-interacting phosphatase and kinase (STRIPAK) complex that is involved in the regulation of various processes such as cell proliferation and differentiation. Its role for differentiation processes in embryonic stem cells (ESCs) is till now completely unknown. Short hairpin RNA (shRNA)-mediated silencing of Fam40b expression in ESCs and differentiating embryoid bodies (EBs) led to perturbed differentiation to embryonic germ layers and their derivatives including a complete abrogation of cardiomyogenesis. Pluripotency factors such as Nanog, Oct4 and Sox2 as well as epigenetic factors such as histone acetyltransferase type B (HAT1) and DNA (cytosine-5)-methyltransferase 3-β (Dnmt3b) were highly upregulated in Fam40b knockdown EBs as compared with control and scrambled EBs. To examine the relevance of Fam40b for development in vivo, Fam40b was knocked down in developing zebrafish. Morpholino-mediated knockdown of Fam40b led to severe abnormalities of the cardiovascular system, including an impaired expression of ventricular myosin heavy chain (vmhc) and of cardiac myosin light chain 2 (cmlc2) in the heart. We identified the gene product of Fam40b in ESCs as a perinuclear and nucleolar protein with a molecular weight of 96 kDa. We conclude that the expression of Fam40b is essential for the lineage commitment of murine embryonic stem cells (mESCs) into differentiated somatic cells via mechanisms involving pluripotency and epigenetic networks.

AimsHeart failure (HF) is associated with development of AF and life‐threatening ventricular tachycardia and fibrillation (VT/VF). Vulnerability to development of AF and VT/VF at different stages of HF and the underlying pathophysiological mechanisms are poorly defined. The present study was designed to determine the time‐course of development of electrical and structural remodelling of the atria and ventricles, and their contribution to induction of AF and VT/VF in a canine model of HF.Methods and resultsDogs were ventricular tachypaced (VTP) for 2–3 weeks or 5–6 weeks (‘early’ and ‘late’ HF, respectively). Electrophysiological studies were performed in isolated atrial and ventricular preparations and correlated with cardiac dimensions and haemodynamic parameters recorded in vivo. Vulnerability to programmed electrical stimulation‐induced AF was greater in early vs. late stages of HF (78% vs. 38%). In contrast, VT/VF was inducible in late but not in early stages of HF (38% vs. 0%). The temporal distinction in atrial and ventricular arrhythmia susceptibility was associated with a much more rapid development of electrical and structural remodelling in atria. Vulnerability to AF developed following moderate electro‐structural remodelling and waned with further progression to severe remodelling, which averted rapid atrial activation.ConclusionsA temporal window of vulnerability for AF appears relatively early during development of VTP‐induced HF in dogs, whereas VT/VF vulnerability is observed at more advanced stages of HF. These findings, if confirmed in humans, may have clinical implications with regard to prognosis and approach to therapy of patients with HF.

Krüppel-like factors (Klf) 4 and 5 are two closely related members of the Klf family, known to play key roles in cell cycle regulation, somatic cell reprogramming and pluripotency. Here we focus on the functional divergence between Klf4 and Klf5 in the inhibition of mouse embryonic stem (ES) cell differentiation. Using microarrays and chromatin immunoprecipitation coupled to ultra-high-throughput DNA sequencing, we show that Klf4 negatively regulates the expression of endodermal markers in the undifferentiated ES cells, including transcription factors involved in the commitment of pluripotent stem cells to endoderm differentiation. Knockdown of Klf4 enhances differentiation towards visceral and definitive endoderm. In contrast, Klf5 negatively regulates the expression of mesodermal markers, some of which control commitment to the mesoderm lineage, and knockdown of Klf5 specifically enhances differentiation towards mesoderm. We conclude that Klf4 and Klf5 differentially inhibit mesoderm and endoderm differentiation in murine ES cells.

Background The transient outward potassium current (Ito) plays a prominent role in the development of cardiac arrhythmias associated with the J-wave syndromes, including Brugada and early repolarization syndromes. The J-wave syndromes are strongly male-dominated, presumably due to higher expression of Ito in the right ventricular epicardium of male vs female dogs. In this study we tested the hypothesis that the male androgen testosterone underlies these gender differences by examining its influence on expression of Ito in hiPSC-derived cardiomyocytes. Methods and Results Embryoid bodies (EBs) were made from a human iPS cell line reprogrammed with Oct4, Nanog, Lin28, and Sox2 using a serum-free differentiation protocol supplemented with growth factors for selective cardiac differentiation. The beating clusters were microdissected from the entire EBs, enzymatically dissociated, and plated on fibronectin-coated dishes. Cells were treated with 1 µM testosterone for 4 weeks. Ito was measured using whole-cell patch-clamp techniques at 36ºC. Ito density was significantly greater in testosterone-treated hiPSC CMs compared to controls (18.7 ± 3.2 pA/pF vs 11.8 ± 1.6 pA/pF, 1 μM testosterone, P <.05). Recovery from inactivation was very slow and slightly accelerated by testosterone (τ = 2682.7 ± 226 ms vs 1772 ± 328 ms at –80 mV, P = .036). Steady state (SS) inactivation was unchanged. Expression of the Ito-associated gene transcript (KCND3, Kv4.3) was 547.51% greater (P <.05) in hiPSC-beating clusters exposed to 1 µM testosterone compared with controls. Testosterone-treated hiPSC CM beating clusters did not display an action potential notch because of the very slow recovery of Ito from inactivation and a depolarized MDP (–65.4 ± 10.3 mV) due to a deficient IK1. Conclusions Our results suggest that testosterone is capable of increasing expression of Ito, at least in part by upregulating expression of Kv4.3 mRNA. These results provide insights into the gender-dependent expression of Ito and may explain the marked gender differences observed in the manifestation of life-threatening arrhythmias associated with the J-wave syndromes. [Supported by NYSTEM contract # C026424.] The transient outward potassium current (Ito) plays a prominent role in the development of cardiac arrhythmias associated with the J-wave syndromes, including Brugada and early repolarization syndromes. The J-wave syndromes are strongly male-dominated, presumably due to higher expression of Ito in the right ventricular epicardium of male vs female dogs. In this study we tested the hypothesis that the male androgen testosterone underlies these gender differences by examining its influence on expression of Ito in hiPSC-derived cardiomyocytes. Embryoid bodies (EBs) were made from a human iPS cell line reprogrammed with Oct4, Nanog, Lin28, and Sox2 using a serum-free differentiation protocol supplemented with growth factors for selective cardiac differentiation. The beating clusters were microdissected from the entire EBs, enzymatically dissociated, and plated on fibronectin-coated dishes. Cells were treated with 1 µM testosterone for 4 weeks. Ito was measured using whole-cell patch-clamp techniques at 36ºC. Ito density was significantly greater in testosterone-treated hiPSC CMs compared to controls (18.7 ± 3.2 pA/pF vs 11.8 ± 1.6 pA/pF, 1 μM testosterone, P <.05). Recovery from inactivation was very slow and slightly accelerated by testosterone (τ = 2682.7 ± 226 ms vs 1772 ± 328 ms at –80 mV, P = .036). Steady state (SS) inactivation was unchanged. Expression of the Ito-associated gene transcript (KCND3, Kv4.3) was 547.51% greater (P <.05) in hiPSC-beating clusters exposed to 1 µM testosterone compared with controls. Testosterone-treated hiPSC CM beating clusters did not display an action potential notch because of the very slow recovery of Ito from inactivation and a depolarized MDP (–65.4 ± 10.3 mV) due to a deficient IK1. Our results suggest that testosterone is capable of increasing expression of Ito, at least in part by upregulating expression of Kv4.3 mRNA. These results provide insights into the gender-dependent expression of Ito and may explain the marked gender differences observed in the manifestation of life-threatening arrhythmias associated with the J-wave syndromes. [Supported by NYSTEM contract # C026424.]

Background: Human induced pluripotent stem cell (hiPS)-cardiomyocytes can be used to create in vitro models of genetic disease such as Brugada Syndrome (BrS). Central to the development of BrS is the Ca2+-independent transient outward K+ current (Ito). In this study, we characterized Ito in single hiPS -cardiomyocytes and determined its functional role in beating clusters. Methods: Embryoid bodies (EBs) were made from a hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Whole cell patch clamp was used to record Ito in single hiPS cardiomyocytes. Action potential (AP) recordings from spontaneously beating clusters (BCs) were made using sharp microelectrodes. All recordings were done at 36°C. Results: BCs exhibited spontaneous APs with an average rate of 54.9±30.1 bpm and maximum diastolic potential (MDP) of −65.6±9.3 mV (n=122). A small phase 1 repolarization which could be blocked by 4-AP (1 mM) was observed in 6/149 hiPS BCs suggesting the presence of Ito. Interestingly, in single dissociated hiPS cardiomyocytes, patch clamp analysis revealed a robust Ito (13.4±1.79 pA/pF at +40 mV, n=14) in the majority of cells studied. Recovery of Ito (at −80 mV) showed a fast and slow phase as follows: i) 1=271±93 ms and 2= 2697±103 (n=8 cells). These observations demonstrate that Ito is present but the slow recovery suggests minimal contribution during the course of an action potential. Mathematical modeling of APs from hiPSC-CMs confirmed these observations. Conclusion: There is a disconnect between the presence of Ito in cells and the absence of phase 1 repolarization in BCs. In BCs the depolarized MDP and fast spontaneous AP rate suggests negligible contribution of Ito to phase 1 repolarization. Our results point to an important deficiency of hiPSC-CMs in recapitulating the phenotype of adult native myocytes.

Background The ability to recapitulate mature adult phenotypes is critical to the development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) as models of disease. The present study examines the characteristics of the transient outward current (Ito) and its contribution to the hiPSC-CM action potential (AP). Method Embryoid bodies were made from a hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record APs from beating-clusters (BC) and patch-clamp techniques were used to record Ito in single hiPSC-CM. mRNA levels of Kv1.4, KChIP2 and Kv4.3 were quantified from BCs. Results BCs exhibited spontaneous beating (60.5±2.6bpm) and maximum-diastolic-potential (MDP) of 67.8±0.8mV (n=155). A small 4-aminopyridine-sensitive phase-1-repolarization was observed in only 6/155 BCs. A robust Ito was recorded in the majority of cells (13.7±1.9 pA/pF at +40mV; n=14). Recovery of Ito from inactivation (at −80mV) showed slow kinetics (τ1=200±110ms (12%) and τ2=2380±240ms (80%)) accounting for its minimal contribution to the AP. Transcript data revealed relatively high expression of Kv1.4 and low expression of KChIP2 compared to human native ventricular tissues. Mathematical modeling predicted that restoration of IK1 to normal levels would result in a more negative MDP and a prominent phase-1-repolarization. Conclusion The slow recovery kinetics of Ito coupled with a depolarized MDP account for the lack of an AP notch in the majority of hiPSC-CM. These characteristics reveal a deficiency for the development of in vitro models of inherited cardiac arrhythmia syndromes in which Ito-induced AP notch is central to the disease phenotype.

Intracellular calcium overload has been linked to arrhythmias in conditions such as ischemia reperfusion, heart failure, catecholaminergic polymorphic ventricular tachycardia and digitalis intoxication. While this link has been extensively studied at the cellular level using animal models, there is a paucity of information on the causes and consequences of Ca2+ overload in healthy human myocytes. With the advent of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), human myocytes are becoming more readily available. However, it remains unclear if these myocytes faithfully recapitulate all aspects of adult cardiomyocyte physiology. In order to determine if iPSC-CM will be a useful platform to study the causes and consequences of Ca2+ overload, we developed methodology to examine Ca2+ overload in beating clusters of iPSC-CM and in isolated myocytes. Spontaneous and field stimulated action potentials were measured with high resistance microelectrodes in spontaneously beating clusters of iPSC-CM while measuring cluster contraction with simultaneous video edge detection. Free intracellular Ca2+ was measured with fluo-4 and confocal microscopy in beating clusters and in isolated iPSC-CM. Ca2+ overload was induced in both preparations by treatment with ouabain (2.5-5 uM or with isoproterenol (1 uM) plus 5.4 mM extracellular Ca2+. Both maneuvers produced a rise in diastolic Ca2+ as well as the appearance of oscillatory action potentials, putative delayed afterdepolarizations and triggered activityies in beating clusters. These results indicate that iPS-derived cardiomyocytes provide a useful platform to study the mechanisms of Ca2+ overload-induced arrhythmias as well as possible treatments. However, given the spontaneous nature of iPS-derived cardiomyocytes, these cells may more readily recapitulate the effects of Ca2+ overload on sinoatrial or latent pacemaker cells.