Drobinskaya Irina, Linn Thomas, Saric Tomo, Bretzel Reinhard G, Bohlen Heribert, Hescheler Jürgen, Kolossov Eugen
Institute for Neurophysiology, Center of Physiology and Pathophysiology, University of Cologne, Robert-Koch Str. 39, D-50931 Cologne, Germany.
Stem Cells. 2008 Sep;26(9):2245-56. doi: 10.1634/stemcells.2008-0387. Epub 2008 Jun 12.
Potential therapeutic applications of embryonic stem cell (ESC)-derived hepatocytes are limited by their relatively low output in differentiating ESC cultures, as well as by the danger of contamination with tumorigenic undifferentiated ESCs. To address these problems, we developed transgenic murine ESC clones possessing bicistronic expression vector that contains the alpha-fetoprotein gene promoter driving a cassette for the enhanced green "live" fluorescent reporter protein (eGFP) and a puromycin resistance gene. Under established culture conditions these clones allowed for both monitoring of differentiation and for puromycin selection of hepatocyte-committed cells in a suspension mass culture of transgenic ESC aggregates ("embryoid bodies" [EBs]). When plated on fibronectin, the selected eGFP-positive cells formed colonies, in which intensely proliferating hepatocyte precursor-like cells gave rise to morphologically differentiated cells expressing alpha-1-antitrypsin, alpha-fetoprotein, and albumin. A number of cells synthesized glycogen and in some of the cells cytokeratin 18 microfilaments were detected. Major hepatocyte marker genes were expressed in the culture, along with the gene and protein expression of stem/progenitor markers, suggesting the features of both hepatocyte precursors and more advanced differentiated cells. When cultured in suspension, the EB-derived puromycin-selected cells formed spheroids capable of outgrowing on an adhesive substrate, resembling the behavior of fetal mouse hepatic progenitor cells. The established system based on the highly efficient selection/purification procedure could be suitable for scalable generation of ESC-derived hepatocyte- and hepatocyte precursor-like cells and offers a potential in vitro source of cells for transplantation therapy of liver diseases, tissue engineering, and drug and toxicology screening.
胚胎干细胞（ESC）来源的肝细胞的潜在治疗应用受到分化的ESC培养物中相对较低产量的限制，以及与致瘤性未分化ESC污染的风险的限制。为了解决这些问题，我们开发了具有双顺反子表达载体的转基因小鼠ESC克隆，该载体包含甲胎蛋白基因启动子，驱动用于增强绿色“活”荧光报告蛋白（eGFP）的盒式结构和嘌呤霉素抗性基因。在既定的培养条件下，这些克隆既允许监测分化，也允许在转基因ESC聚集体（“胚状体”[EBs]）的悬浮大规模培养中对肝细胞定向细胞进行嘌呤霉素选择。当接种在纤连蛋白上时，所选的eGFP阳性细胞形成集落，其中强烈增殖的肝细胞前体样细胞产生形态学上分化的细胞，表达α-1-抗胰蛋白酶、甲胎蛋白和白蛋白。许多细胞合成了糖原，并且在一些细胞中检测到细胞角蛋白18微丝。主要的肝细胞标记基因在培养物中表达，同时还有干/祖细胞标记物的基因和蛋白表达，表明具有肝细胞前体和更高级分化细胞的特征。当在悬浮培养时，EB来源的经嘌呤霉素选择的细胞形成能够在粘附底物上生长的球体，类似于胎鼠肝祖细胞的行为。基于高效选择/纯化程序建立的系统可能适用于可扩展地生成ESC来源的肝细胞和肝细胞前体样细胞，并为肝病的移植治疗、组织工程以及药物和毒理学筛选提供潜在的体外细胞来源。
