Naama Zeevi-Levin

Functional Properties of Human Embryonic Stem Cell–Derived Cardiomyocytes: Intracellular Ca2+ Handling and the Role of Sarcoplasmic Reticulum in the Contraction

Since cardiac transplantation is limited by the small availability of donor organs, regeneration of the diseased myocardium by cell transplantation is an attractive therapeutic modality. To determine the compatibility of human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs) (7 to 55 days old) with the myocardium, we investigated their functional properties regarding intracellular Ca2+ handling and the role of the sarcoplasmic reticulum in the contraction. The functional properties of hESC‐CMs were investigated by recording simultaneously [Ca2+]i transients and contractions. Additionally, we performed Western blot analysis of the Ca2+‐handling proteins SERCA2, calsequestrin, phospholamban, and Na+/Ca2+ exchanger (NCX). Our major findings are, first, that hESC‐CMs displayed temporally related [Ca2+]i transients and contractions, negative force‐frequency relations, and lack of post‐rest potentiation. Second, ryanodine, thapsigargin, and caffeine did not affect the [Ca2+]i transient and contraction, indicating that at this developmental stage, contraction depends on transsarcolemmal Ca2+ influx rather than on sarcoplasmic reticulum Ca2+ release. Third, in agreement with the notion that a voltage‐dependent Ca2+ current is present in hESC‐CMs and contributes to the mechanical function, verapamil completely blocked contraction. Fourth, whereas hESC‐CMs expressed SERCA2 and NCX at levels comparable to those of the adult porcine myocardium, calsequestrin and phospholamban were not expressed. Our study shows for the first time that functional properties related to intracellular Ca2+ handling of hESC‐CMs differ markedly from the adult myocardium, probably due to immature sarcoplasmic reticulum capacity.

Multipotent Vasculogenic Pericytes From Human Pluripotent Stem Cells Promote Recovery of Murine Ischemic Limb

Background— Pericytes represent a unique subtype of microvessel-residing perivascular cells with diverse angiogenic functions and multilineage developmental features of mesenchymal stem cells. Although various protocols for derivation of endothelial and/or smooth muscle cells from human pluripotent stem cells (hPSC, either embryonic or induced) have been described, the emergence of pericytes in the course of hPSC maturation has not yet been elucidated. Methods and Results— We found that during hPSC development, spontaneously differentiating embryoid bodies give rise to CD105+CD90+CD73+CD31− multipotent clonogenic mesodermal precursors, which can be isolated and efficiently expanded. Isolated and propagated cells expressed characteristic pericytic markers, including CD146, NG2, and platelet-derived growth factor receptor &bgr;, but not the smooth muscle cell marker &agr;-smooth muscle actin. Coimplantation of hPSC-derived endothelial cells with pericytes resulted in functional and rapid anastomosis to the murine vasculature. Administration of pericytes into immunodeficient mice with limb ischemia promoted significant vascular and muscle regeneration. At day 21 after transplantation, recruited hPSC pericytes were found incorporated into recovered muscle and vasculature. Conclusions— Derivation of vasculogenic and multipotent pericytes from hPSC can be used for the development of vasculogenic models using multiple vasculogenic cell types for basic research and drug screening and can contribute to angiogenic regenerative medicine.

Cardiomyocytes generated from CPVTD307H patients are arrhythmogenic in response to β-adrenergic stimulation.

Sudden cardiac death caused by ventricular arrhythmias is a disastrous event, especially when it occurs in young individuals. Among the five major arrhythmogenic disorders occurring in the absence of a structural heart disease is catecholaminergic polymorphic ventricular tachycardia (CPVT), which is a highly lethal form of inherited arrhythmias. Our study focuses on the autosomal recessive form of the disease caused by the missense mutation D307H in the cardiac calsequestrin gene, CASQ2. Because CASQ2 is a key player in excitation contraction coupling, the derangements in intracellular Ca2+ handling may cause delayed afterdepolarizations (DADs), which constitute the mechanism underlying CPVT. To investigate catecholamine‐induced arrhythmias in the CASQ2 mutated cells, we generated for the first time CPVT‐derived induced pluripotent stem cells (iPSCs) by reprogramming fibroblasts from skin biopsies of two patients, and demonstrated that the iPSCs carry the CASQ2 mutation. Next, iPSCs were differentiated to cardiomyocytes (iPSCs‐CMs), which expressed the mutant CASQ2 protein. The major findings were that the β‐adrenergic agonist isoproterenol caused in CPVT iPSCs‐CMs (but not in the control cardiomyocytes) DADs, oscillatory arrhythmic prepotentials, after‐contractions and diastolic [Ca2+]i rise. Electron microscopy analysis revealed that compared with control iPSCs‐CMs, CPVT iPSCs‐CMs displayed a more immature phenotype with less organized myofibrils, enlarged sarcoplasmic reticulum cisternae and reduced number of caveolae. In summary, our results demonstrate that the patient‐specific mutated cardiomyocytes can be used to study the electrophysiological mechanisms underlying CPVT.

Molecular characterization and functional properties of cardiomyocytes derived from human inducible pluripotent stem cells

In view of the therapeutic potential of cardiomyocytes derived from induced pluripotent stem (iPS) cells (iPS‐derived cardiomyocytes), in the present study we investigated in iPS‐derived cardiomyocytes, the functional properties related to [Ca2+]i handling and contraction, the contribution of the sarcoplasmic reticulum (SR) Ca2+ release to contraction and the b‐adrenergic inotropic responsiveness. The two iPS clones investigated here were generated through infection of human foreskin fibroblasts (HFF) with retroviruses containing the four human genes: OCT4, Sox2, Klf4 and C‐Myc. Our major findings showed that iPS‐derived cardiomyocytes: (i) express cardiac specific RNA and proteins; (ii) exhibit negative force–frequency relations and mild (compared to adult) post‐rest potentiation; (iii) respond to ryanodine and caffeine, albeit less than adult cardiomyocytes, and express the SR‐Ca2+ handling proteins ryanodine receptor and calsequestrin. Hence, this study demonstrates that in our cardiomyocytes clones differentiated from HFF‐derived iPS, the functional properties related to excitation–contraction coupling, resemble in part those of adult cardiomyocytes.

Enhanced reprogramming and cardiac differentiation of human keratinocytes derived from plucked hair follicles, using a single excisable lentivirus.

Induced pluripotent stem cells (iPSCs) represent an ideal cell source for future cell therapy and regenerative medicine. However, most iPSC lines described to date have been isolated from skin fibroblasts or other cell types that require harvesting by surgical intervention. Because it is desirable to avoid such intervention, an alternative cell source that can be readily and noninvasively isolated from patients and efficiently reprogrammed, is required. Here we describe a detailed and reproducible method to derive iPSCs from plucked human hair follicle keratinocytes (HFKTs). HFKTs were isolated from single plucked hair, then expanded and reprogrammed by a single polycistronic excisable lentiviral vector. The reprogrammed HFKTs were found to be very sensitive to human embryonic stem cell (hESC) growth conditions, generating a built-in selection with easily obtainable and very stable iPSCs. All emerging colonies were true iPSCs, with characteristics typical of human embryonic stem cells, differentiated into derivatives of all three germ layers in vitro and in vivo. Spontenaeouly differentiating functional cardiomyocytes (CMs) were successfully derived and characterized from these HFKT-iPSCs. The contracting CMs exhibited well-coordinated intracellular Ca²+ transients and contractions that were readily responsive to β-adrenergic stimulation with isoproterenol. The introduction of Cre-recombinase to HFKT-iPSC clones was able to successfully excise the integrated vector and generate transgene-free HFKT-iPSC clone that could be better differentiated into contracting CMs, thereby revealing the desired cells for modeling human diseases. Thus, HFKTs are easily obtainable, and highly reprogrammed human cell source for all iPSC applications.

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.

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.

Impulse conduction and gap junctional remodelling by endothelin-1 in cultured neonatal rat ventricular myocytes

Endothelin‐1 (ET‐1) is an important contributor to ventricular hypertrophy and failure, which are associated with arrhythmogenesis and sudden death. To elucidate the mechanism(s) underlying the arrhythmogenic effects of ET‐1 we tested the hypothesis that long‐term (24 hrs) exposure to ET‐1 impairs impulse conduction in cultures of neonatal rat ventricular myocytes (NRVM). NRVM were seeded on micro‐electrode‐arrays (MEAs, Multi Channel Systems, Reutlingen, Germany) and exposed to 50 nM ET‐1 for 24 hrs. Hypertrophy was assessed by morphological and molecular methods. Consecutive recordings of paced activation times from the same cultures were conducted at baseline and after 3, 6 and 24 hrs, and activation maps for each time period constructed. Gap junctional Cx43 expression was assessed using Western blot and confocal microscopy of immunofluorescence staining using anti‐Cx43 antibodies. ET‐1 caused hypertrophy as indicated by a 70% increase in mRNA for atrial natriuretic peptide (P < 0.05), and increased cell areas (P < 0.05) compared to control. ET‐1 also caused a time‐dependent decrease in conduction velocity that was evident after 3 hrs of exposure to ET‐1, and was augmented at 24 hrs, compared to controls (P < 0.01). ET‐1 increased total Cx43 protein by ∼40% (P < 0.05) without affecting non‐ phosphorylated Cx43 (NP‐Cx43) protein expression. Quantitative confocal microscopy showed a ∼30% decrease in the Cx43 immunofluorescence per field in the ET‐1 group (P < 0.05) and a reduced field stain intensity (P < 0.05), compared to controls. ET‐1‐induced hypertrophy was accompanied by reduction in conduction velocity and gap junctional remodelling. The reduction in conduction velocity may play a role in ET‐1 induced susceptibility to arrhythmogenesis.

Human Embryonic Stem Cells vs Human Induced Pluripotent Stem Cells for Cardiac Repair

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have the capacity to differentiate into any specialized cell type, including cardiomyocytes. Therefore, hESC-derived and hiPSC-derived cardiomyocytes (hESC-CMs and hiPSC-CMs, respectively) offer great potential for cardiac regenerative medicine. Unlike some organs, the heart has a limited ability to regenerate, and dysfunction resulting from significant cardiomyocyte loss under pathophysiological conditions, such as myocardial infarction (MI), can lead to heart failure. Unfortunately, for patients with end-stage heart failure, heart transplantation remains the main alternative, and it is insufficient, mainly because of the limited availability of donor organs. Although left ventricular assist devices are progressively entering clinical practice as a bridge to transplantation and even as an optional therapy, cell replacement therapy presents a plausible alternative to donor organ transplantation. During the past decade, multiple candidate cells were proposed for cardiac regeneration, and their mechanisms of action in the myocardium have been explored. The purpose of this article is to critically review the comprehensive research involving the use of hESCs and hiPSCs in MI models and to discuss current controversies, unresolved issues, challenges, and future directions.

Developmental changes in electrophysiological characteristics of human-induced pluripotent stem cell–derived cardiomyocytes

BACKGROUND Previous studies proposed that throughout differentiation of human induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs), only 3 types of action potentials (APs) exist: nodal-, atrial-, and ventricular-like. OBJECTIVES To investigate whether there are precisely 3 phenotypes or a continuum exists among them, we tested 2 hypotheses: (1) During culture development a cardiac precursor cell is present that-depending on age-can evolve into the 3 phenotypes. (2) The predominant pattern is early prevalence of a nodal phenotype, transient appearance of an atrial phenotype, evolution to a ventricular phenotype, and persistence of transitional phenotypes. METHODS To test these hypotheses, we (1) performed fluorescence-activated cell sorting analysis of nodal, atrial, and ventricular markers; (2) recorded APs from 280 7- to 95-day-old iPSC-CMs; and (3) analyzed AP characteristics. RESULTS The major findings were as follows: (1) fluorescence-activated cell sorting analysis of 30- and 60-day-old cultures showed that an iPSC-CMs population shifts from the nodal to the atrial/ventricular phenotype while including significant transitional populations; (2) the AP population did not consist of 3 phenotypes; (3) culture aging was associated with a shift from nodal to ventricular dominance, with a transient (57-70 days) appearance of the atrial phenotype; and (4) beat rate variability was more prominent in nodal than in ventricular cardiomyocytes, while pacemaker current density increased in older cultures. CONCLUSION From the onset of development in culture, the iPSC-CMs population includes nodal, atrial, and ventricular APs and a broad spectrum of transitional phenotypes. The most readily distinguishable phenotype is atrial, which appears only transiently yet dominates at 57-70 days of evolution.