Basic Study for Regenerative Medicine : Differentiate of Embryonic Stem Cells to Hepatocytes and Expressions of Cytochrome P450 in the Differentiating Cells

为了将小鼠胚胎干细胞分化为肝细胞，对拟胚体进行了36天的培养。在ES细胞分化的细胞中有肝细胞分化标志的表达。细胞色素P450(CYP)基因在细胞中的表达模式可分为四类：早、中期高表达，仅在晚期高表达，在所有阶段均有表达，在任何阶段都不表达。培养的细胞在整个培养过程中均检测到芳香烃受体、芳香烃受体核转位蛋白和糖皮质激素受体。只有培养24天以上的细胞才能检测到孕烷X受体。甲胎蛋白和白蛋白分别与细胞色素P3A2和细胞色素P1A1共表达。猴ES细胞在体外也可分化为肝细胞。细胞表达甲胎蛋白、白蛋白、细胞色素P3A8、细胞色素P3A66和细胞色素P7A1。我们已经从分化中的猴ES细胞中分离到一个cDNA克隆，并鉴定出一个新的CYP亚型，命名为CYP3A7m。其推导的氨基酸序列与细胞色素P3A4、细胞色素3A5、细胞色素P3A7和细胞色素P3A43的同源性分别为89%、81%、91%和72%。这些结果表明，在ES细胞分化的肝细胞中有CYP和转录因子基因的表达，并且在分化过程中各个酶的表达水平和模式发生了很大的变化。

To differentiate mouse embryonic stem (ES) cells to hepatocytes, embryoid bodies were cultured for 36 days. Hepatocyte differentiation markers were expressed in the cells differentiated from ES cells. The expression pattern of cytochrome P450 (CYP) genes in the cells could be grouped into four different categories, such as highly expressed at early to mid stage, only expressed at late stage ; expressed throughout at all stages and not detectable at any stages. Aryl hydrocarbon receptor, aryl hydrocarbon receptor nuclear translocator and glucocorticoid receptor were detected in the cells cultured throughout the time course. Pregnane X receptor was detected only the cells cultured for more than 24 days. The alpha-fetoprotein and albumin were co-expressed with CYP3A2 and CYP1A1, respectively. Monkey ES cells were also differentiated into hepatocytes in vitro. The cells expressed alpha-fetoprotein, albumin, CYP3A8, CYP3A66 and CYP7A1. We have isolated a cDNA clone from the differentiating monkey ES cells and identified a novel CYP isoform, named CYP3A7m. Deduced amino acid sequence of CYP3A7m shows 89, 81, 91 and 72% homology to CYP3A4, CYP3A5, CYP3A7 and CYP3A43, respectively. These results suggested that CYP and transcription factor genes were expressed in hepatocytes differentiated from ES cells and those expression levels and patterns were considerably changed in individual enzymes during the course of the differentiation.