Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.
Biotechnol Bioeng. 2011 Mar;108(3):704-19. doi: 10.1002/bit.22987. Epub 2010 Nov 30.
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.
胚胎干细胞（ESC）衍生物是心脏细胞治疗有前途的细胞来源。在传统动物模型中进行细胞注射的机制研究受到传递效率低和细胞存活率差的限制。作为替代方法，我们使用基于电刺激培养的新生大鼠心肌细胞（CM）的工程化心脏组织（EHT）作为体外模型来研究细胞注射。我们注射（0.001、0.01 和 0.1 百万个）并追踪（通过 qPCR 和组织学）未分化的黄色荧光蛋白转基因小鼠 ESC 和 Flk1+/PDGFRα+ 心脏祖细胞（CP）细胞，以研究心脏环境对细胞分化的影响，以及测试我们的体外模型系统是否可以重现体内 ESC 注射后通常观察到的类畸胎瘤结构的形成。注射后 8 天，ESC 与心脏细胞群体在空间上分离；然而，ESC 注射增加了 CM 的存活率。ESC 的存在阻止了组织的电传导，导致兴奋阈值增加了 46%。与 ESC 注射的构建体相比，CP 注射的构建体中鼠心肌肌钙蛋白 I 的表达在所有时间点和测试的细胞剂量下均明显增加。早在 2 周时，就观察到 ESC 注射的构建体中出现上皮和神经节样结构。ESC 注射 4 周后，在构建体中观察到含有神经、上皮和结缔组织的类畸胎瘤样结构。仅在延长培养（4 周）和仅在高细胞剂量下，才在 CP 注射的构建体中观察到非心脏结构，这表明这些细胞在移植前需要进一步富集或分化。我们的数据表明，宿主组织的心脏环境和电场刺激并没有优先将 ESC 向心脏谱系分化。在相同的环境中，CP 的注射导致比 ESC 注射更强烈的心脏分化。我们的数据表明，本文开发的模型系统可用于研究候选干细胞对宿主心肌的功能影响，以及测量混合物中未分化细胞的残留活性。
