Isolation and characterization of human hepatic stem/progenitor cells and reconstruction of liver tissue

为从人肝脏中分离和鉴定人肝干/祖细胞，并用于肝组织重建，本研究采用细胞聚集法建立了从胎鼠和成年鼠肝脏中分离肝干/祖细胞的方法：用特异性细胞表面标志物Thy-1阳性的间充质细胞鉴定胎鼠肝脏细胞聚集体中的3个细胞群; CD 49 f阳性肝祖细胞和CD 45阳性血细胞，Thy-1阳性细胞促进CD 49 f阳性细胞向成熟肝细胞分化。从绿色荧光蛋白（GFP）转基因成年小鼠的肝脏中也通过荧光激活细胞分选分离肝祖细胞。我们选择了30个基因。利用GFP阳性肝祖细胞与正常成熟肝细胞之间的cDNA差减法筛选肝干/祖细胞表面标志物候选物。30个基因中有3个在胎鼠肝前体细胞中特异性表达。小鼠骨髓细胞具有多向分化能力，在肝再生过程中定向分化为肝窦内皮细胞和枯否细胞，在肝纤维化过程中定向分化为肝星状细胞。采用肝细胞和肝非实质细胞在I型胶原片上共培养的方法重建三维肝类器官。

The aim of this study was to isolate and characterize human hepatic stem/progenitor cells from human liver and to reconstruct the liver tissue.Isolation method of hepatic stem progenitor cells from fetal and adult mise liver was established using cell aggregation method: We characterized three populations in the cell aggregations from the liver of fetal mice using specific cell surface markers Thy-1 positive mesenchymal cells; CD49f positive hepatic progenitor cells and CD45-positive blood cells, Thy-1 positive cells promoted (differentiation of C D49f positive cells to mature hepatocytes. Hepatic progenitor cells from the liver of green fluorescent protein (GFP) transgenic adult mice were also isolated by fluorescent activated cell sorting. We select 30 genes. for candidates of surface markers of hepatic stem/progenitor cells using cDNA subtraction between GFP positive hepatic progenitor cells and normal mature hepatocyte. Three of 30 genes were expressed in the fetal mouse hepatic progenitor cells specifically. Bone marrow cells in mice, which have multi-lineage differentiation activity, were committed to hepatic sinusoidal endothelial cell and Kupffer cells during liver regeneration and to hepatic stellate cells during liver fibrosis.We developed a suitable method for human hepatocytes isolated from resected specimens to maintain both proliferative capacity and differentiated functions. Three-dimensional hepatic organoid was reconstructed using co-culture of hepatocytes and hepatic non-parenchymal cells isolated from resected specimens on collagen type I sheet.