University of Massachusetts Medical School, Worcester, MA, USA.
Liver Int. 2012 May;32(5):732-41. doi: 10.1111/j.1478-3231.2011.02743.x. Epub 2012 Jan 31.
BACKGROUND/AIMS: Liver diseases are common in the United States and often require liver transplantation; however, donated organs are limited and thus alternative sources for liver cells are in high demand. Embryonic stem cells (ESC) can provide a continuous and readily available source of liver cells. ESC differentiation to liver cells is yet to be fully understood and comprehensive differentiation protocols are yet to be defined. Here, we aimed to achieve human (h)ESC differentiation into mature hepatocytes using defined recombinant differentiation factors and metabolites.
Embryonic stem cell H1 line was sub-cultured on feeder layer. We induced hESCs into endodermal differentiation succeeded by early/late hepatic specification and finally into hepatocyte maturation using step combinations of Activin A and fibroblast growth factor (FGF)-2 for 7 days; followed by FGF-4 and bone morphogenic protein 2 (BMP2) for 7 days, succeeded by FGF-10 + hepatocyte growth factor 4 + epidermal growth factor for 14 days. Specific inhibitors/stimulators were added sequentially throughout differentiation. Cells were analysed by PCR, flow cytometry, microscopy or functional assays.
Our hESC differentiation protocol resulted in viable cells with hepatocyte shape and morphology. We observed gradual changes in cell transcriptome, including up-regulation of differentiation-promoting GATA4, GATA6, POU5F1 and HNF4 transcription factors, steady levels of stemness-promoting SOX-2 and low levels of Nanog, as defined by PCR. The hESC-derived hepatocytes expressed alpha-antitrypsin, CD81, cytokeratin 8 and low density lipoprotein (LDL) receptor. The levels of alpha-fetoprotein and proliferation marker Ki-67 in hESC-derived hepatocytes remained elevated. Unlike stem cells, the hESC-derived hepatocytes performed LDL uptake, produced albumin and alanine aminotransferase and had functional alcohol dehydrogenase.
We report a novel protocol for hESC differentiation into morphological and functional yet immature hepatocytes as an alternative method for hepatocyte generation.
背景/目的：肝脏疾病在美国很常见，通常需要进行肝移植；然而，可供捐赠的器官有限，因此对肝细胞的替代来源的需求很高。胚胎干细胞（ESC）可以提供源源不断且易于获取的肝细胞来源。ESC 向肝细胞的分化尚未被完全理解，全面的分化方案尚未被定义。在这里，我们旨在使用定义明确的重组分化因子和代谢物将人（h）ESC 分化为成熟的肝细胞。
胚胎干细胞 H1 系在饲养层上进行传代培养。我们通过激活素 A 和成纤维细胞生长因子（FGF）-2 的序贯组合将 hESC 诱导为内胚层分化，随后进行早期/晚期肝特异性分化，最后通过 FGF-4 和骨形态发生蛋白 2（BMP2）诱导分化 7 天，然后再用 FGF-10+肝细胞生长因子 4+表皮生长因子诱导分化 14 天。在整个分化过程中，我们依次添加了特定的抑制剂/刺激剂。通过 PCR、流式细胞术、显微镜或功能测定分析细胞。
我们的 hESC 分化方案导致具有肝细胞形状和形态的存活细胞。我们观察到细胞转录组逐渐发生变化，包括分化促进因子 GATA4、GATA6、POU5F1 和 HNF4 转录因子的上调，干细胞促进因子 SOX-2 的稳定水平和 Nanog 的低水平，如通过 PCR 定义的。hESC 衍生的肝细胞表达α-抗胰蛋白酶、CD81、细胞角蛋白 8 和低密度脂蛋白（LDL）受体。hESC 衍生的肝细胞中α-胎蛋白和增殖标志物 Ki-67 的水平仍然升高。与干细胞不同，hESC 衍生的肝细胞摄取 LDL、产生白蛋白和丙氨酸氨基转移酶，并具有功能性的醇脱氢酶。
我们报告了一种将 hESC 分化为形态和功能但不成熟的肝细胞的新方案，这是生成肝细胞的替代方法。
