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（hESCs）的建立从人胚胎干细胞基因组中克隆人甲胎蛋白（AFP）的杂交增强子/启动子。将增强型绿色荧光蛋白（EGFP）序列连接到人甲胎蛋白（AFP）杂合启动子下，构建表达质粒，在细胞中表达与人甲胎蛋白（AFP）表达量相对应的EGFP。用脂质体转染该质粒，获得转基因人胚胎干细胞系，其表达作为AFP表达标志物的EGFP. hESC来源的内胚层细胞的分离和向肝细胞的分化在几种条件下分化后，使用流式细胞术（FACS Vantage SE）比较先前描述的转基因hESC中EGFP的表达，以确定理想的分化方案。因此，我们确定了有效性的改善（约为10%）。21%）和内胚层分化率f ...更多信息 当它们在含有激活素和肝细胞生长因子的培养基的Matrigel上培养时，来自胚胎干细胞。用流式细胞术分离与转基因hESC分化的AFP表达细胞相对应的EGFP表达细胞，并通过RT-PCR研究基因表达。表达EGFP的内胚层细胞表达中内胚层细胞的标记基因，这表明在我们系统中从hESC分化和分离的细胞群中含有定形内胚层细胞的事实。为了促进hESC来源的内胚层细胞向肝细胞的分化，根据我们先前的研究证据，分离的EGFP阳性的AFP表达的hESC来源的内胚层细胞与来自小鼠胚胎肝组织的间充质细胞共培养。只有当人胚胎干细胞来源的内胚细胞与间充质细胞共培养时，才能检测到肝脏标志物基因的表达。这些结果表明，我们可以产生从hESC分化的肝细胞，具有足够的功能，用于细胞移植治疗严重的肝脏疾病在可预见的未来。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