Engineered heart tissue enables study of residual undifferentiated embryonic stem cell activity in a cardiac environment

Abstract

Embryonic stem cell (ESC) derivatives are a promising cell source for cardiac cell therapy. Mechanistic studies upon cell injection in conventional animal models are limited by inefficient delivery and poor cell survival. As an alternative, we have used an engineered heart tissue (EHT) based on neonatal rat cardiomyocytes (CMs) cultivated with electrical field stimulation as an in vitro model to study cell injection. We injected (0.001, 0.01, and 0.1 million) and tracked (by qPCR and histology) undifferentiated yellow-fluorescent protein transgenic mouse ESCs and Flk1 + /PDGFRα+ cardiac progenitor (CPs) cells, to investigate the effect of the cardiac environment on cell differentiation, as well as to test whether our in vitro model system could recapitulate the formation of teratoma-like structures commonly observed upon in vivo ESC injection. By 8 days post-injection, ESCs were spatially segregated from the cardiac cell population; however, ESC injection increased survival of CMs. The presence of ESCs blocked electrical conduction through the tissue, resulting in a 46% increase in the excitation threshold. Expression of mouse cardiac troponin I, was markedly increased in CP injected constructs compared to ESC injected constructs at all time points and cell doses tested. As early as 2 weeks, epithelial and ganglion-like structures were observed in ESC injected constructs. By 4 weeks of ESC injection, teratoma-like structures containing neural, epithelial, and connective tissue were observed in the constructs. Non-cardiac structures were observed in the CP injected constructs only after extended culture (4 weeks) and only at high cell doses, suggesting that these cells require further enrichment or differentiation prior to transplantation. Our data indicate that the cardiac environment of host tissue and electrical field stimulation did not preferentially guide the differentiation of ESCs towards the cardiac lineage. In the same environment, injection of CP resulted in a more robust cardiac differentiation than injection of ESC. Our data demonstrate that the model-system developed herein can be used to study the functional effects of candidate stem cells on the host myocardium, as well as to measure the residual activity of undifferentiated cells present in the mixture.

Design and formulation of functional pluripotent stem cell-derived cardiac microtissues

“…Rat neonatal cardiomyocytes were isolated as previously reported (10). Briefly, hearts were isolated from 1-to 2-d-old neonatal Sprague-Dawley rats using protocol approved by the University of Toronto Committee on Animal Care.…”

“…Although differentiation of hPSCs into contracting CMs is well-established, their maturation into adult-equivalent cells, and their formulation into functional adult-like tissue, remains an unmet challenge (5). We and others have previously developed in vitro heart tissue models (10)(11)(12); however, these recently developed systems require further definition in terms of their underlying design criteria and the impact of key design parameters such as tissue geometry and input-cell composition on microtissue properties.…”

“…Our previous engineered heart tissue design, which used centimeter-scale collagen scaffolds, had an isotropic matrix architecture (that induced limited CM alignment) and heterogeneity in spatial cell distribution within the volume of the matrix (10). Eschenhagen and coworkers have advanced the field by developing aligned tissue models suitable for some multiwell-plate analytics and that are capable of measuring forces of contraction (22).…”

Cardiac Tissue Engineering

“…Others injected ESCs or ESCderived progenitor cells into 3D engineered tissues and observed both beneficial, paracrine, and unwanted effects, such as conduction block and teratoma formation with undifferentiated ESC. 84 3. Disease modeling is an exciting new avenue for biomedical research that has been opened by the availability of patient-specific hiPSC lines to model genetic and other cardiac diseases on an individual basis.…”

Stem Cell-Based Cardiac Tissue Engineering

“…In vitro cardiac test systems have been developed previously [23,24]. Such systems can account for some of the heterogeneities inherent in the in vivo system (Fig.…”