Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA 16802, USA.
Chem Biol Interact. 2011 Mar 15;190(1):62-72. doi: 10.1016/j.cbi.2011.01.009. Epub 2011 Jan 15.
The limited availability of hepatic tissue suitable for the treatment of liver disease and drug discovery research advances the generation of hepatic-like cells from alternative sources as a valuable approach. In this investigation we exploited a unique hepatic differentiation approach to generate hepatocyte-like cells from human embryonic stem cells (hESCs). hESCs were cultured for 10-20 days on collagen substrate in highly defined and serum free hepatocyte media. The resulting cell populations exhibited hepatic cell-like morphology and were characterized with a variety of biological endpoint analyses. Real-time PCR analysis demonstrated that mRNA expression of the 'stemness' marker genes NANOG and alkaline phosphatase in the differentiated cells was significantly reduced, findings that were functionally validated using alkaline phosphatase activity detection measures. Immunofluorescence studies revealed attenuated levels of the 'stemness' markers OCT4, SOX2, SSEA-3, TRA-1-60, and TRA-1-81 in the hepatic-like cell population. The hepatic character of the cells was evaluated additionally by real-time PCR analyses that demonstrated increased mRNA expression of the hepatic transcription factors FOXA1, C/EBPα, and HNF1α, the nuclear receptors CAR, RXRα, PPARα, and HNF4α, the liver-generated plasma proteins α-fetoprotein, transthyretin, transferrin, and albumin, the protease inhibitor α-1-antitrypsin, metabolic enzymes HMGCS2, PEPCK, and biotransformation enzymes CYP3A7, CYP3A4, CYP3A5, and CYP2E1. Indocyanine green uptake albumin secretion and glycogen storage capacity further confirmed acquisition of hepatic function. These studies define an expeditious methodology that facilitates the differentiation of hESCs along a hepatic lineage and provide a framework for their subsequent use in pharmacological and toxicological research applications requiring a renewable supply of human hepatocytes.
有限的适合治疗肝病的肝组织和药物发现研究进展推动了从替代来源生成肝样细胞的方法，这是一种很有价值的方法。在这项研究中，我们利用一种独特的肝分化方法，从人胚胎干细胞（hESC）中生成肝样细胞。hESC 在胶原基质上，在高度定义的、无血清的肝细胞培养基中培养 10-20 天。所得细胞群体表现出肝细胞样形态，并通过各种生物学终点分析进行了特征描述。实时 PCR 分析表明，分化细胞中“干性”标记基因 NANOG 和碱性磷酸酶的 mRNA 表达显著降低，这一发现通过碱性磷酸酶活性检测措施得到了功能验证。免疫荧光研究显示，肝样细胞群体中“干性”标记物 OCT4、SOX2、SSEA-3、TRA-1-60 和 TRA-1-81 的水平降低。通过实时 PCR 分析进一步评估了细胞的肝特性，结果表明肝转录因子 FOXA1、C/EBPα 和 HNF1α、核受体 CAR、RXRα、PPARα 和 HNF4α、肝源性血浆蛋白α-胎蛋白、转甲状腺素蛋白、转铁蛋白和白蛋白、蛋白酶抑制剂α-1-抗胰蛋白酶、代谢酶 HMGCS2、PEPCK 和生物转化酶 CYP3A7、CYP3A4、CYP3A5 和 CYP2E1 的 mRNA 表达增加。靛氰绿摄取、白蛋白分泌和糖原储存能力进一步证实了肝功能的获得。这些研究定义了一种快速的方法，可促进 hESC 沿着肝谱系分化，并为它们随后在需要可再生的人类肝细胞供应的药理学和毒理学研究应用中提供了一个框架。
