Occupational Disease Research Center, Peking University Third Hospital, Haidian District, Beijing, People's Republic of China.
Cell Biol Int. 2011 Sep;35(9):927-38. doi: 10.1042/CBI20110031.
hESCs (human embryonic stem cells) can differentiate into tissue derivatives of all three germ layers in vitro and mimic the development of the embryo in vivo. In this study, we have investigated the potential of an hESC-based assay for the detection of toxicity to cardiac differentiation in embryonic development. First of all, we developed the protocol of cardiac induction from hESCs according to our previous work and distinguished cardiac precursor cells and late mature cardiomyocytes from differentiated cells, demonstrated by the Q-PCR (quantitative real-time PCR), immunocytochemistry and flow cytometry analysis. In order to test whether CPA (cyclophosphamide) induces developmental and cellular toxicity in the human embryo, we exposed the differentiating cells from hESCs to CPA (a well-known proteratogen) at different stages. We have found that a high concentration of CPA could inhibit cardiac differentiation of hESCs. Two separate exposure intervals were used to determine the effects of CPA on cardiac precursor cells and late mature cardiomyocytes respectively. The cardiac precursor cells were sensitive to CPA in non-cytotoxic concentrations for the expression of the cardiac-specific mRNA markers Nkx2.5 (NK2 transcription factor related, locus 5), GATA-4 (GATA binding protein 4 transcription factor) and TNNT2 (troponin T type 2). Non-cytotoxic CPA concentrations did not affect the mRNA markers' expression in late mature cardiomyocytes, indicating that cardiac precursors were more sensitive to CPA than late cardiomyocytes in cardiogenesis. We set up the in vitro developmental toxicity test model so as to reduce the number of test animals and expenses without compromising the safety of consumers and patients. Furthermore, such in vitro methods may be possibly suited to test a large number of chemicals than the classical employed in vivo tests.
人类胚胎干细胞（hESCs）在体外可以分化为三个胚层的组织衍生物，并模拟体内胚胎的发育。在这项研究中，我们研究了基于 hESC 的检测方法在检测胚胎发育中心肌分化毒性方面的潜力。首先，我们根据以前的工作开发了 hESC 向心脏诱导的方案，并通过 Q-PCR（实时定量 PCR）、免疫细胞化学和流式细胞术分析，从分化细胞中区分出心脏前体细胞和晚期成熟心肌细胞。为了测试环磷酰胺（CPA）是否在人类胚胎中引起发育和细胞毒性，我们将 hESC 分化细胞暴露于不同阶段的环磷酰胺（一种已知的促畸形剂）中。我们发现高浓度的环磷酰胺可以抑制 hESC 的心脏分化。使用两个单独的暴露间隔来分别确定环磷酰胺对心脏前体细胞和晚期成熟心肌细胞的影响。心脏前体细胞对环磷酰胺在非细胞毒性浓度下的表达敏感，其心脏特异性 mRNA 标志物 Nkx2.5（NK2 转录因子相关，基因座 5）、GATA-4（GATA 结合蛋白 4 转录因子）和 TNNT2（肌钙蛋白 T 型 2）。非细胞毒性浓度的环磷酰胺不影响晚期成熟心肌细胞的 mRNA 标志物表达，表明心脏前体细胞在心肌发生中比晚期心肌细胞对环磷酰胺更敏感。我们建立了体外发育毒性测试模型，以减少测试动物的数量和费用，同时不影响消费者和患者的安全性。此外，与经典的体内试验相比，此类体外方法可能更适合测试大量的化学物质。
