Feeder-Free and Serum-Free Production of Hepatocytes, Cholangiocytes, and Their Proliferating Progenitors from Human Pluripotent Stem Cells: Application to Liver-Specific Functional and Cytotoxic Assays
Feeder-Free and Serum-Free Production of Hepatocytes, Cholangiocytes, and Their Proliferating Progenitors from Human Pluripotent Stem Cells: Application to Liver-Specific Functional and Cytotoxic Assays
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DOI：
10.1089/cell.2011.0064
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发表时间：
2012-04-01
影响因子：
1.6
通讯作者：
Yuo, Akira
中科院分区：
文献类型：
--
作者：
Nakamura, Naoko;Saeki, Kumiko;Yuo, Akira
我们建立了一个无血清和无饲料的培养系统，用于从人胚胎干细胞（ES）细胞和三种完全不同的诱导多能干细胞（包括使用仙台病毒载体产生的无载体/无转基因的iPS细胞）高效分化多功能肝细胞，而无需细胞分选和基因操作。诱导分化方案包括第一阶段；内胚层诱导，第二阶段；肝脏起始阶段和第三阶段；肝成熟。在分化培养结束时，由人多能干细胞诱导的肝细胞表达肝细胞特异性蛋白，如甲胎蛋白、白蛋白、α 1抗胰蛋白酶和细胞色素P450 (CYP3A4)，与三种对照人肝细胞或肝细胞系相比，表达水平相似或更高。这些人类iPS/ES细胞衍生的肝细胞也显示出成熟的肝细胞功能：吲哚青绿染料摄取（类似于30%），糖原储存（>80%）和CYP3A4的代谢活性。此外，他们还通过肝细胞特异性酶的细胞外释放来测定d -半乳糖胺的高灵敏度肝毒性测定系统。保肝前列腺素El减轻了这种毒性。有趣的是，药物治疗后也检测到胆管特异性酶，表明在我们的培养系统中存在胆管上皮细胞（胆管细胞）。电镜研究证实了胆管细胞的存在，免疫染色研究证实了具有高增殖潜力的双电位肝母细胞的存在。分化的细胞可转移到新的培养皿中，在培养皿上出现并扩增具有肝细胞标记的小型增殖细胞。因此，我们的分化培养系统提供了成熟的功能性肝细胞、胆管细胞及其祖细胞，这些细胞来自各种各样的人类多能干细胞，具有增殖潜力。
We have established a serum- and feeder-free culture system for the efficient differentiation of multifunctional hepatocytes from human embryonic stem (ES) cells and three entirely different induced pluripotent stem (iPS) cells (including vector/transgene-free iPS cells generated using Sendai virus vector) without cell sorting and gene manipulation. The differentiation-inducing protocol consisted of a first stage; endoderm induction, second stage; hepatic initiation, and third stage; hepatic maturation. At the end of differentiation culture, hepatocytes induced from human pluripotent stem cells expressed hepatocyte-specific proteins, such as alpha-fetoprotein, albumin, alpha 1 antitrypsin and cytochrome P450 (CYP3A4), at similar or higher levels compared with three control human hepatocyte or hepatic cell lines. These human iPS/ES cell-derived hepatocytes also showed mature hepatocyte functions: inclocyanine green dye uptake (similar to 30%), storage of glycogen (>80%) and metabolic activity of CYP3A4. Furthermore, they produced a highly sensitive hepatotoxicity assay system for D-galactosamine as determined by the extracellular release of hepatocyte-specific enzymes. Hepatoprotective prostaglandin El attenuated this toxicity. Interestingly, bile duct-specific enzymes were also detected after drug treatment, suggesting the presence of bile-duct epithelial cells (cholangiocytes) in our culture system. Electron microscopic studies confirmed the existence of cholangiocytes, and an immunostaining study proved the presence of bipotential hepatoblasts with high potential for proliferation. Differentiated cells were transferrable onto new dishes, on which small-sized proliferating cells with hepatocyte markers emerged and expanded. Thus, our differentiation culture system provides mature functional hepatocytes, cholangiocytes, and their progenitors with proliferative potential from a wide variety of human pluripotent stem cells.