Establishment of hepatocyte differentiation from human embryonic stem cells in the realizaion of cell transplantation therapy for liver disease.

1. 转基因人胚胎干细胞系（hESCs）的建立利用人胚胎干细胞基因组克隆制备了人甲胎蛋白（AFP）的杂交增强子/启动子。将增强型绿色荧光蛋白（EGFP）序列连接在人AFP杂交启动子下构建表达质粒，在细胞中表达与人AFP表达相对应的EGFP。该质粒通过脂质转染获得了表达EGFP作为AFP表达标记的转基因人胚胎干细胞。分化后，利用流式细胞术（FACS Vantage SE）比较了上述转基因hESCs中EGFP的表达，以确定理想的分化方案。结果，我们确定了疗效的改善(大约。在含有激活素和肝细胞生长因子的基质上培养胚胎干细胞时，胚胎干细胞的内胚层分化率为21%。用流式细胞术分离转基因hESCs中表达afp的细胞对应的表达egfp的细胞，用RT-PCR检测基因表达。表达egfp的内胚层细胞表达了中胚层细胞的标记基因，这表明在我们的系统中，hESCs分化分离的细胞群中含有确定的内胚层细胞。为了促进hesc来源的内胚层细胞向肝细胞的分化，根据我们前期研究的证据，将分离的表达egfp阳性afp的hesc来源的内胚层细胞与小鼠胚胎肝组织来源的间充质细胞共培养。肝标记基因的表达只有在hesc来源的内胚层细胞与间充质细胞共培养时才能确定。这些结果表明，在可预见的未来，我们可以从hESCs中分化出具有足够功能的肝细胞，用于严重肝脏疾病的细胞移植。少

1. Establishment of transgenic human embryonic stem cell line (hESCs)The hybrid enhancer/promoter of human alpha-fetoprotein (AFP) was produced after cloning from the genome of human embryonic stem cells. The sequence of enhanced green fluorescent protein (EGFP) was ligated under the hybrid promoter of human AFP to construct the expression plasmid, which express EGFP corresponding to the expression of human AFP in the cells. This plasmid was transfected using lipofection to produce transgenic human embryonic stem cell lines, which express EGFP as a marker of AFP expression.2. Isolation of hESCs derived endodermal cells and differentiation to hepatocytesThe expression of EGFP in the transgenic hESCs described previously was compared using flow cytometry (FACS Vantage SE) after the differentiation among several conditions to determine the ideal differentiation protocol. As a result, we identified the improvement of the efficacy (approx. 21%) and the yields of endodermal differentiation f … More rom embryonic stem cells when they were cultured on Matrigel with culture media containing activin and hepatocyte growth factor. The EGFP-expressing cells corresponding to the AFP-expressing cells differentiated from the transgenic hESCs were isolated using flow cytometry and investigated of the gene expression by RT-PCR. The EGFP-expressing endodermal cells expressed the marker genes of mesoendodermal cells, which indicated the fact that definitive endodermal cells were contained in the cell population differentiated and isolated from hESCs in our system. To promote the differentiation of hESC-derived endodermal cells into hepatocytes, the isolated EGFP-positive AFP-expressing endodermal cells derived from hESCs were co-cultured with mesenchymal cells derived from mouse embryonic liver tissue according to the evidence of our previous research. The expression of hepatic marker gene was identified only when hESC-derived endodermal cells were co-cultured with mesenchymal cells. These results suggested that we could produce hepatocytes differentiated from hESCs with enough function to be used for cell transplantation for severe liver diseases in the foreseeable future. Less