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

为了使小鼠胚胎干细胞向肝细胞分化，胚胎样体培养36天。肝细胞分化标志物在胚胎干细胞分化的细胞中表达。细胞色素P450 （CYP）基因在细胞中的表达模式可分为早中期高表达、晚期只表达的4类；在所有阶段都表达，但在任何阶段都检测不到。在培养的细胞中检测到芳烃受体、芳烃受体核转运体和糖皮质激素受体。仅培养24 d以上的细胞检测到孕烷X受体。甲胎蛋白和白蛋白分别与CYP3A2和CYP1A1共表达。猴胚胎干细胞也在体外分化为肝细胞。这些细胞表达甲胎蛋白、白蛋白、CYP3A8、CYP3A66和CYP7A1。我们从分化的猴胚胎干细胞中分离出一个cDNA克隆，并鉴定出一个新的CYP亚型，命名为CYP3A7m。CYP3A7m氨基酸序列与CYP3A4、CYP3A5、CYP3A7和CYP3A43的同源性分别为89%、81%、91%和72%。这些结果表明，CYP和转录因子基因在胚胎干细胞分化的肝细胞中有表达，且在分化过程中，各酶的表达水平和表达模式发生了较大的变化。

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.