Benjamin Van Biber

Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts

Transplantation studies in mice and rats have shown that human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts, but two critical issues related to their electrophysiological behaviour in vivo remain unresolved. First, the risk of arrhythmias following hESC-CM transplantation in injured hearts has not been determined. Second, the electromechanical integration of hESC-CMs in injured hearts has not been demonstrated, so it is unclear whether these cells improve contractile function directly through addition of new force-generating units. Here we use a guinea-pig model to show that hESC-CM grafts in injured hearts protect against arrhythmias and can contract synchronously with host muscle. Injured hearts with hESC-CM grafts show improved mechanical function and a significantly reduced incidence of both spontaneous and induced ventricular tachycardia. To assess the activity of hESC-CM grafts in vivo, we transplanted hESC-CMs expressing the genetically encoded calcium sensor, GCaMP3 (refs 4, 5). By correlating the GCaMP3 fluorescent signal with the host ECG, we found that grafts in uninjured hearts have consistent 1:1 host–graft coupling. Grafts in injured hearts are more heterogeneous and typically include both coupled and uncoupled regions. Thus, human myocardial grafts meet physiological criteria for true heart regeneration, providing support for the continued development of hESC-based cardiac therapies for both mechanical and electrical repair.

Efficient generation and cryopreservation of cardiomyocytes derived from human embryonic stem cells

AIM Human embryonic stem cells (hESCs) represent a novel cell source to treat diseases such as heart failure and for use in drug screening. In this study, we aim to promote efficient generation of cardiomyocytes from hESCs by combining the current optimal techniques of controlled growth of undifferentiated cells and specific induction for cardiac differentiation. We also aim to examine whether these methods are scalable and whether the differentiated cells can be cryopreserved. METHODS & RESULTS hESCs were maintained without conditioned medium or feeders and were sequentially treated with activin A and bone morphogenetic protein-4 in a serum-free medium. This led to differentiation into cell populations containing high percentages of cardiomyocytes. The differentiated cells expressed appropriate cardiomyocyte markers and maintained contractility in culture, and the majority of the cells displayed working chamber (atrial and ventricular) type electrophysiological properties. In addition, the cell growth and differentiation process was adaptable to large culture formats. Moreover, the cardiomyocytes survived following cryopreservation, and viable cardiac grafts were detected after transplantation of cryopreserved cells into rat hearts following myocardial infarctions. CONCLUSION These results demonstrate that cardiomyocytes of high quality can be efficiently generated and cryopreserved using hESCs maintained in serum-free medium, a step forward towards the application of these cells to human clinical use or drug discovery.

Methods for the Derivation and Use of Cardiomyocytes from Human Pluripotent Stem Cells

The availability of human cardiomyocytes derived from embryonic stem cells (ESCs) has generated -considerable excitement, as these cells are an excellent model system for studying myocardial development and may have eventual application in cell-based cardiac repair. Cardiomyocytes derived from the related induced pluripotent stem cells (iPSCs) have similar properties, but also offer the prospects of patient-specific disease modeling and cell therapies. Unfortunately, the methods by which cardiomyocytes have been historically generated from pluripotent stem cells are unreliable and typically result in preparations of low cardiac purity (typically <1% cardiomyocytes). We detail here the methods for a recently reported directed cardiac differentiation protocol, which involves the serial application of two growth factors known to be involved in early embryonic heart development, activin A, and bone morphogenetic protein-4 (BMP-4). This protocol reliably yields preparations of 30-60% cardiomyocytes, which can then be further enriched to >90% cardiomyocytes using straightforward physical methods.

Electrical integration of human embryonic stem cell-derived cardiomyocytes in a guinea pig chronic infarct model

Background: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were recently shown to be capable of electromechanical integration following direct injection into intact or recently injured guinea pig hearts, and hESC-CM transplantation in recently injured hearts correlated with improvements in contractile function and a reduction in the incidence of arrhythmias. The present study was aimed at determining the ability of hESC-CMs to integrate and modulate electrical stability following transplantation in a chronic model of cardiac injury. Methods and Results: At 28 days following cardiac cryoinjury, guinea pigs underwent intracardiac injection of hESC-CMs, noncardiac hESC derivatives (non-CMs), or vehicle. Histology confirmed partial remuscularization of the infarct zone in hESC-CM recipients while non-CM recipients showed heterogeneous xenografts. The 3 experimental groups showed no significant difference in the left ventricular dimensions or fractional shortening by echocardiography or in the incidence of spontaneous arrhythmias by telemetric monitoring. Although recipients of hESC-CMs and vehicle showed a similar incidence of arrhythmias induced by programmed electrical stimulation at 4 weeks posttransplantation, non-CM recipients proved to be highly inducible, with a ∼3-fold greater incidence of induced arrhythmias. In parallel studies, we investigated the ability of hESC-CMs to couple with host myocardium in chronically injured hearts by the intravital imaging of hESC-CM grafts that stably expressed a fluorescent reporter of graft activation, the genetically encoded calcium sensor GCaMP3. In this work, we found that only ∼38% (5 of 13) of recipients of GCaMP3+ hESC-CMs showed fluorescent transients that were coupled to the host electrocardiogram. Conclusions: Human embryonic stem cell-derived cardiomyocytes engraft in chronically injured hearts without increasing the incidence of arrhythmias, but their electromechanical integration is more limited than previously reported following their transplantation in a subacute injury model. Moreover, non-CM grafts may promote arrhythmias under certain conditions, a finding that underscores the need for input preparations of high cardiac purity.

Differentiation of cardiomyocytes from human embryonic stem cells is accompanied by changes in the extracellular matrix production of versican and hyaluronan

Proteoglycans and hyaluronan play critical roles in heart development. In this study, human embryonic stem cells (hESC) were used as a model to quantify the synthesis of proteoglycans and hyaluronan in hESC in the early stages of differentiation, and after directed differentiation into cardiomyocytes. We demonstrated that both hESC and cardiomyocyte cultures synthesize an extracellular matrix (ECM) enriched in proteoglycans and hyaluronan. During cardiomyocyte differentiation, total proteoglycan and hyaluronan decreased and the proportion of proteoglycans bearing heparan sulfate chains was reduced. Versican, a chondroitin sulfate proteoglycan, accumulated in hESC and cardiomyocyte cultures. Furthermore, versican synthesized by hESC contained more N‐ and O‐linked oligosaccharide than versican from cardiomyocytes. Transcripts for the versican variants, V0, V1, V2, and V3, increased in cardiomyocytes compared to hESC, with V1 most abundant. Hyaluronan in hESC had lower molecular weight than hyaluronan from cardiomyocyte cultures. These changes were accompanied by an increase in HAS‐1 and HAS‐2 mRNA in cardiomyocyte cultures, with HAS‐2 most abundant. Interestingly, HAS‐3 was absent from the cardiomyocyte cultures, but expressed by hESC. These results indicate that human cardiomyocyte differentiation is accompanied by specific changes in the expression and accumulation of ECM components and suggest a role for versican and hyaluronan in this process. J. Cell. Biochem. 111: 585–596, 2010.

Roles of DNA polymerase I in leading and lagging-strand replication defined by a high-resolution mutation footprint of ColE1 plasmid replication

DNA polymerase I (pol I) processes RNA primers during lagging-strand synthesis and fills small gaps during DNA repair reactions. However, it is unclear how pol I and pol III work together during replication and repair or how extensive pol I processing of Okazaki fragments is in vivo. Here, we address these questions by analyzing pol I mutations generated through error-prone replication of ColE1 plasmids. The data were obtained by direct sequencing, allowing an accurate determination of the mutation spectrum and distribution. Pol I’s mutational footprint suggests: (i) during leading-strand replication pol I is gradually replaced by pol III over at least 1.3 kb; (ii) pol I processing of Okazaki fragments is limited to ∼20 nt and (iii) the size of Okazaki fragments is short (∼250 nt). While based on ColE1 plasmid replication, our findings are likely relevant to other pol I replicative processes such as chromosomal replication and DNA repair, which differ from ColE1 replication mostly at the recruitment steps. This mutation footprinting approach should help establish the role of other prokaryotic or eukaryotic polymerases in vivo, and provides a tool to investigate how sequence topology, DNA damage, or interactions with protein partners may affect the function of individual DNA polymerases.

Cobalt Protoporphyrin Pretreatment Protects Human Embryonic Stem Cell-Derived Cardiomyocytes From Hypoxia/Reoxygenation Injury In Vitro and Increases Graft Size and Vascularization In Vivo

Human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs) can regenerate infarcted myocardium. However, when implanted into acutely infarcted hearts, few cells survive the first week postimplant. To improve early graft survival, hESC‐CMs were pretreated with cobalt protoporphyrin (CoPP), a transcriptional activator of cytoprotective heme oxygenase‐1 (HO‐1). When hESC‐CMs were challenged with an in vitro hypoxia/reoxygenation injury, mimicking cell transplantation into an ischemic site, survival was significantly greater among cells pretreated with CoPP versus phosphate‐buffered saline (PBS)‐pretreated controls. Compared with PBS‐pretreated cells, CoPP‐pretreated hESC‐CM preparations exhibited higher levels of HO‐1 expression, Akt phosphorylation, and vascular endothelial growth factor production, with reduced apoptosis, and a 30% decrease in intracellular reactive oxygen species. For in vivo translation, 1 × 107 hESC‐CMs were pretreated ex vivo with CoPP or PBS and then injected intramyocardially into rat hearts immediately following acute infarction (permanent coronary ligation). At 1 week, hESC‐CM content, assessed by quantitative polymerase chain reaction for human Alu sequences, was 17‐fold higher in hearts receiving CoPP‐ than PBS‐pretreated cells. On histomorphometry, cardiomyocyte graft size was 2.6‐fold larger in hearts receiving CoPP‐ than PBS‐pretreated cells, occupying up to 12% of the ventricular area. Vascular density of host‐perfused human‐derived capillaries was significantly greater in grafts composed of CoPP‐ than PBS‐pretreated cells. Taken together, these experiments demonstrate that ex vivo pretreatment of hESC‐CMs with a single dose of CoPP before intramyocardial implantation more than doubled resulting graft size and improved early graft vascularization in acutely infarcted hearts. These findings open the door for delivery of these, or other, stem cells during acute interventional therapy following myocardial infarction or ischemia.