Nakamura Naoko, Saeki Kumiko, Mitsumoto Masami, Matsuyama Satoko, Nishio Miwako, Saeki Koichi, Hasegawa Mamoru, Miyagawa Yoshiyuki, Ohkita Hajime, Kiyokawa Nobutaka, Toyoda Masashi, Akutsu Hidenori, Umezawa Akihiro, Yuo Akira
Department of Disease Control, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan.
Cell Reprogram. 2012 Apr;14(2):171-85. doi: 10.1089/cell.2011.0064. Epub 2012 Mar 2.
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 α-fetoprotein, albumin, α1 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: indocyanine green dye uptake (≈ 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 E1 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.
我们建立了一种无血清和饲养层的培养系统,可从人胚胎干细胞(ES细胞)和三种完全不同的诱导多能干细胞(iPS细胞,包括使用仙台病毒载体产生的无载体/转基因iPS细胞)高效分化出多功能肝细胞,无需细胞分选和基因操作。分化诱导方案包括三个阶段:第一阶段为内胚层诱导,第二阶段为肝脏起始,第三阶段为肝脏成熟。在分化培养结束时,与三种对照人肝细胞或肝细胞系相比,人多能干细胞诱导产生的肝细胞表达肝细胞特异性蛋白,如甲胎蛋白、白蛋白、α1抗胰蛋白酶和细胞色素P450(CYP3A4),且表达水平相似或更高。这些源自人iPS/ES细胞的肝细胞还表现出成熟的肝细胞功能:吲哚菁绿染料摄取(约30%)、糖原储存(>80%)和CYP3A4的代谢活性。此外,它们构建了一个高度敏感的D-半乳糖胺肝毒性检测系统,该系统通过肝细胞特异性酶的细胞外释放来测定。具有肝脏保护作用的前列腺素E1可减轻这种毒性。有趣的是,药物处理后还检测到了胆管特异性酶,这表明我们的培养系统中存在胆管上皮细胞(胆管细胞)。电子显微镜研究证实了胆管细胞的存在,免疫染色研究证明了具有高增殖潜能的双能肝母细胞的存在。分化后的细胞可转移到新的培养皿上,在新培养皿上出现并扩增出带有肝细胞标志物的小尺寸增殖细胞。因此,我们的分化培养系统可从多种人多能干细胞中提供具有增殖潜能的成熟功能性肝细胞、胆管细胞及其祖细胞。