Joseph Itskovitz-Eldor

Cardiomyocytes derived from human embryonic and induced pluripotent stem cells: comparative ultrastructure

Induced pluripotent stem cells (iPSC) are generated from fully differentiated somatic cells that were reprogrammed into a pluripotent state. Human iPSC which can be obtained from various types of somatic cells such as fibroblasts or keratinocytes can differentiate into cardiomyocytes (iPSC‐CM), which exhibit cardiac‐like transmembrane action potentials, intracellular Ca2+ transients and contractions. While major features of the excitation‐contraction coupling of iPSC‐CM have been well‐described, very little is known on the ultrastructure of these cardiomyocytes. The ultrastructural features of 31‐day‐old (post‐plating) iPSC‐CM generated from human hair follicle keratinocytes (HFKT‐iPSC‐CM) were analysed by electron microscopy, and compared with those of human embryonic stem‐cell‐derived cardiomyocytes (hESC‐CM). The comparison showed that cardiomyocytes from the two sources share similar proprieties. Specifically, HFKT‐iPSC‐CM and hESC‐CM, displayed ultrastructural features of early and immature phenotype: myofibrils with sarcomeric pattern, large glycogen deposits, lipid droplets, long and slender mitochondria, free ribosomes, rough endoplasmic reticulum, sarcoplasmic reticulum and caveolae. Noteworthy, the SR is less developed in HFKT‐iPSC‐CM. We also found in both cell types: (1) ‘Ca2+‐release units’, which connect the peripheral sarcoplasmic reticulum with plasmalemma; and (2) intercellular junctions, which mimic intercalated disks (desmosomes and fascia adherens). In conclusion, iPSC and hESC differentiate into cardiomyocytes of comparable ultrastructure, thus supporting the notion that iPSC offer a viable option for an autologous cell source for cardiac regenerative therapy.

Pluripotent Stem Cell Model Reveals Essential Roles for miR‐450b‐5p and miR‐184 in Embryonic Corneal Lineage Specification

Approximately 6 million people worldwide are suffering from severe visual impairments or blindness due to corneal diseases. Corneal allogeneic transplantation is often required to restore vision; however, shortage in corneal grafts and immunorejections remain major challenges. The molecular basis of corneal diseases is poorly understood largely due to lack of appropriate cellular models. Here, we described a robust differentiation of human‐induced pluripotent stem cells (hiPSCs) derived from hair follicles or skin fibroblasts into corneal epithelial‐like cells. We found that BMP4, coupled with corneal fibroblast‐derived conditioned medium and collagen IV allowed efficient corneal epithelial commitment of hiPSCs in a manner that recapitulated corneal epithelial lineage development with high purity. Organotypic reconstitution assays suggested the ability of these cells to stratify into a corneal‐like epithelium. This model allowed us identifying miR‐450b‐5p as a molecular switch of Pax6, a major regulator of eye development. miR‐450b‐5p and Pax6 were reciprocally distributed at the presumptive epidermis and ocular surface, respectively. miR‐450b‐5p inhibited Pax6 expression and corneal epithelial fate in vitro, altogether, suggesting that by repressing Pax6, miR‐450b‐5p triggers epidermal specification of the ectoderm, while its absence allows ocular epithelial development. Additionally, miR‐184 was detectable in early eye development and corneal epithelial differentiation of hiPSCs. The knockdown of miR‐184 resulted in a decrease in Pax6 and K3, in line with recent findings showing that a point mutation in miR‐184 leads to corneal dystrophy. Altogether, these data indicate that hiPSCs are valuable for modeling corneal development and may pave the way for future cell‐based therapy. STEM CELLS 2012;30:898–909

Functional Properties of Human Embryonic Stem Cell-Derived Cardiomyocytes

Abstract: Regeneration of the diseased myocardium by cardiac cell transplantation is an attractive therapeutic modality. Yet, because the transplanted cardiomyocytes should functionally integrate within the diseased myocardium, it is preferable that their properties resemble those of the host. To determine the functional adaptability of human embryonic stem cell‐derived cardiomyocytes (hESC‐CM) to the host myocardium, the authors investigated the excitation‐contraction (E‐C) coupling and the responsiveness to common physiological stimuli. The main findings are: (1) hESC‐CM readily respond to electrical pacing and generate corresponding [Ca2+]i transients (measured by fura‐2 fluorescence) and contractions (measured by video edge detector). (2) In contrast to the mature myocardium, hESC‐CM display negative force‐frequency relations. (3) The hESC‐CM contraction is dependent on [Ca2+]o and blocked by verapamil. (4) Surprisingly, ryanodine, the sarcoplasmic‐endoplasmic reticulum Ca2+‐ATPase inhibitor thapsigargin, and caffeine do not affect the [Ca2+]i transient or contraction. Collectively, these results indicate that at the developmental stage of 45 to 60 days, the contraction is largely dependent on [Ca2+]o rather than on sarcoplasmic reticulum (SR) Ca2+ stores. The results show for the first time that the E‐C coupling properties of hESC‐CM differ from the adult myocardium, probably due to immature SR function. Based on these findings, genetic manipulation of hESC‐CM toward the adult myocardium should be considered.

Human Embryonic Stem Cells Exhibit Increased Propensity to Differentiate During the G1 Phase Prior to Phosphorylation of Retinoblastoma Protein

While experimentally induced arrest of human embryonic stem cells (hESCs) in G1 has been shown to stimulate differentiation, it remains unclear whether the unperturbed G1 phase in hESCs is causally related to differentiation. Here, we use centrifugal elutriation to isolate and investigate differentiation propensities of hESCs in different phases of their cell cycle. We found that isolated G1 cells exhibit higher differentiation propensity compared with S and G2 cells, and they differentiate at low cell densities even under self‐renewing conditions. This differentiation of G1 cells was partially prevented in dense cultures of these cells and completely abrogated in coculture with S and G2 cells. However, coculturing without cell‐to‐cell contact did not rescue the differentiation of G1 cells. Finally, we show that the subset of G1 hESCs with reduced phosphorylation of retinoblastoma has the highest propensity to differentiate and that the differentiation is preceded by cell cycle arrest. These results provide direct evidence for increased propensity of hESCs to differentiate in G1 and suggest a role for neighboring cells in preventing differentiation of hESCs as they pass through a differentiation sensitive, G1 phase. STEM CELLS2012;30:1097–1108

1,4,5‐Inositol Trisphosphate‐Operated Intracellular Ca2+ Stores and Angiotensin‐II/Endothelin‐1 Signaling Pathway Are Functional in Human Embryonic Stem Cell‐Derived Cardiomyocytes

On the basis of previous findings suggesting that in human embryonic stem cell‐derived cardiomyocytes (hESC‐CM) the sarcoplasmic reticulum Ca2+‐induced release of calcium machinery is either absent or immature, in the present study we tested the hypothesis that hESC‐CM contain fully functional 1,4,5‐inositol trisphosphate (1,4,5‐IP3)‐operated intracellular Ca2+ ([Ca2+]i) stores that can be mobilized upon appropriate physiological stimuli. To test this hypothesis we investigated the effects of angiotensin‐II (AT‐II) and endothelin‐1 (ET‐1), which activate the 1,4,5‐IP3 pathway, on [Ca2+]i transients and contractions in beating clusters of hESC‐CM. Our major findings were that in paced hESC‐CM both AT‐II and ET‐1 (10−9 to 10−7 M) increased the contraction amplitude and the maximal rates of contraction and relaxation. In addition, AT‐II (10−9 to 10−7 M) increased the [Ca2+]i transient amplitude. The involvement of 1,4,5‐IP3‐dependent intracellular Ca2+ release in the inotropic effect of AT‐II was supported by the findings that (a) hESC‐CM express AT‐II, ET‐1, and 1,4,5‐IP3 receptors determined by immunofluorescence staining, and (b) the effects of AT‐II were blocked by 2 μM 2‐aminoethoxyphenyl borate (a 1,4,5‐IP3 receptor blocker) and U73122 (a phospholipase C blocker). In conclusion, these findings demonstrate for the first time that hESC‐CM exhibit functional AT‐II and ET‐1 signaling pathways, as well as 1,4,5‐IP3‐operated releasable Ca2+ stores.

GnRH agonist ovulation trigger and hCG-based, progesterone-free luteal support: a proof of concept study

BACKGROUND It is now well established that a GnRH agonist (GnRHa) ovulation trigger completely prevents ovarian hyperstimulation syndrome. However, early studies, using conventional luteal support, showed inferior clinical results following a GnRHa trigger compared with a conventional hCG trigger in normal responder IVF patients. We here present a novel approach for luteal support after a GnRHa trigger. METHODS Normal responder patients who failed at least one previous IVF attempt, during which a conventional hCG trigger was used, were consecutively enrolled in the study. A GnRH antagonist-based ovarian stimulation protocol was used in combination with a GnRHa trigger (Triptorelin 0.2 mg). The luteal phase was supported with a total of two boluses of 1500 IU hCG: on the day of oocyte retrieval and 4 days later. Neither progesterone nor estradiol was administered for luteal support. RESULTS The mean age was 33.8 years. The mean (± SD) numbers of oocytes and fertilized oocytes were 6.7 (± 2.5) and 3.6 (± 1.7), respectively. All 15 patients had embryo transfers and 11 patients conceived. On the day of pregnancy test (14 days after retrieval), the mean serum E(2) and progesterone levels were 6607 (± 3789) and 182 (± 50) nmol/l, respectively. Of the pregnancies, seven are ongoing, while four ended as miscarriages. CONCLUSIONS These preliminary results suggest that two boluses of 1500 IU hCG revert the luteolysis after a GnRHa trigger in the normo-responder patient. Importantly, no additional luteal support is needed. The novel concept combines the potential advantages of a physiological dual trigger (LH and FSH) with a simple, patient friendly, luteal support.

SK4 Ca2+ activated K+ channel is a critical player in cardiac pacemaker derived from human embryonic stem cells

Significance The contractions of the heart are initiated and coordinated by pacemaker tissues, responsible for cardiac automaticity. Although the cardiac pacemaker was discovered more than a hundred years ago, the pacemaker mechanisms remain controversial. We used human embryonic stem cell-derived cardiomyocytes to study the embryonic cardiac automaticity of the human heart. We identified a previously unrecognized Ca2+-activated K+ channel (SK4), which appears to play a pivotal role in cardiac automaticity. Our results suggest that SK4 Ca2+-activated K+ channels represent an important target for the management of cardiac rhythm disorders and open challenging horizons for developing biological pacemakers. Proper expression and function of the cardiac pacemaker is a critical feature of heart physiology. Two main mechanisms have been proposed: (i) the “voltage-clock,” where the hyperpolarization-activated funny current If causes diastolic depolarization that triggers action potential cycling; and (ii) the “Ca2+ clock,” where cyclical release of Ca2+ from Ca2+ stores depolarizes the membrane during diastole via activation of the Na+–Ca2+ exchanger. Nonetheless, these mechanisms remain controversial. Here, we used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to study their autonomous beating mechanisms. Combined current- and voltage-clamp recordings from the same cell showed the so-called “voltage and Ca2+ clock” pacemaker mechanisms to operate in a mutually exclusive fashion in different cell populations, but also to coexist in other cells. Blocking the “voltage or Ca2+ clock” produced a similar depolarization of the maximal diastolic potential (MDP) that culminated by cessation of action potentials, suggesting that they converge to a common pacemaker component. Using patch-clamp recording, real-time PCR, Western blotting, and immunocytochemistry, we identified a previously unrecognized Ca2+-activated intermediate K+ conductance (IKCa, KCa3.1, or SK4) in young and old stage-derived hESC-CMs. IKCa inhibition produced MDP depolarization and pacemaker suppression. By shaping the MDP driving force and exquisitely balancing inward currents during diastolic depolarization, IKCa appears to play a crucial role in human embryonic cardiac automaticity.

Immunoevasive Pericytes From Human Pluripotent Stem Cells Preferentially Modulate Induction of Allogeneic Regulatory T Cells

Isolated microvessel‐residing pericytes and pericytes from human pluripotent stem cells (hPSCs) exhibit mesenchymal stem cell‐like characteristics and therapeutic properties. Despite growing interest in pericyte‐based stem cell therapy, their immunogenicity and immunomodulatory effects on nonactivated T cells are still poorly defined, in particular those of vasculogenic hPSC pericytes. We found that tissue‐embedded and unstimulated cultured hPSC‐ or tissue‐derived pericytes constitutively expressed major histocompatibility complex (MHC) class I and the inhibitory programmed cell death‐ligand 1/2 (PD‐L1/2) molecules but not MHC class II or CD80/CD86 costimulatory molecules. Pretreatment with inflammatory mediators failed to induce an antigen‐presenting cell‐like phenotype in stimulated pericytes. CD146+ pericytes from hPSCs did not induce activation and proliferation of allogeneic resting T cells independent of interferon (IFN)‐γ prestimulation, similarly to pericytes from human brain or placenta. Instead, pericytes mediated a significant increase in the frequency of allogeneic CD25highFoxP3+ regulatory T cells when cocultured with nonactivated peripheral blood T cells. Furthermore, when peripheral blood CD25high regulatory T cells (Tregs) were depleted from isolated CD3+ T cells, pericytes preferentially induced de novo formation of CD4+CD25highFoxP3+CD127−, suppressive regulatory T cells. Constitutive expression of PD‐L1/2 and secretion of transforming growth factor‐β by hPSC pericytes directly regulated generation of pericyte‐induced Tregs. Pericytes cotransplanted into immunodeficient mice with allogeneic CD25− T cells maintained a nonimmunogenic phenotype and mediated the development of functional regulatory T cells. Together, these findings reveal a novel feature of pericyte‐mediated immunomodulation distinguished from immunosuppression, shared by native tissue pericytes and hPSC pericytes, and support the notion that pericytes can be applied for allogeneic cell therapy.

From beat rate variability in induced pluripotent stem cell-derived pacemaker cells to heart rate variability in human subjects.

BACKGROUND We previously reported that induced pluripotent stem cell-derived cardiomyocytes manifest beat rate variability (BRV) resembling heart rate variability (HRV) in the human sinoatrial node. We now hypothesized the BRV-HRV continuum originates in pacemaker cells. OBJECTIVE To investigate whether cellular BRV is a source of HRV dynamics, we hypothesized 3 levels of interaction among different cardiomyocyte entities: (1) single pacemaker cells, (2) networks of electrically coupled pacemaker cells, and (3) the in situ sinoatrial node. METHODS We measured BRV/HRV properties in single pacemaker cells, induced pluripotent stem cell-derived contracting embryoid bodies (EBs), and electrocardiograms from the same individual. RESULTS Pronounced BRV/HRV was present at all 3 levels. The coefficient of variance of interbeat intervals and Poincaré plot indices SD1 and SD2 for single cells were 20 times greater than those for EBs (P < .05) and the in situ heart (the latter two were similar; P > .05). We also compared BRV magnitude among single cells, small EBs (~5-10 cells), and larger EBs (>10 cells): BRV indices progressively increased with the decrease in the cell number (P < .05). Disrupting intracellular Ca(2+) handling markedly augmented BRV magnitude, revealing a unique bimodal firing pattern, suggesting that intracellular mechanisms contribute to BRV/HRV and the fractal behavior of heart rhythm. CONCLUSION The decreased BRV magnitude in transitioning from the single cell to the EB suggests that the HRV of in situ hearts originates from the summation and integration of multiple cell-based oscillators. Hence, complex interactions among multiple pacemaker cells and intracellular Ca(2+) handling determine HRV in humans and cardiomyocyte networks.

Morphology of Human Embryonic and Induced Pluripotent Stem Cell Colonies Cultured with Feeders

To prolong the stage of undifferentiation, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have traditionally been isolated and cultured using feeder layers, such as mouse embryonic fibroblasts (MEFs) or foreskin fibroblasts, with medium supplemented by fetal bovine serum (FBS). For research purposes, these conditions are preferable and are often referred to as the gold standard. This chapter describes the colony morphology of undifferentiated hESCs and iPSCs cultured with MEFs or human foreskin fibroblasts.