Generation of Functional Hepatocytes from Human Embryonic Stem Cells Under Chemically Defined Conditions that Recapitulate Liver Development
Generation of Functional Hepatocytes from Human Embryonic Stem Cells Under Chemically Defined Conditions that Recapitulate Liver Development
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DOI：
10.1002/hep.23506
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发表时间：
2010-05-01
期刊：
影响因子：
13.5
通讯作者：
Vallier, Ludovic
中科院分区：
文献类型：
--
作者：
Touboul, Thomas;Hannan, Nicholas R. F.;Vallier, Ludovic
从人胚胎干细胞（hESC）产生肝细胞可以代表用于细胞治疗方法的有利的细胞来源，作为原位肝移植的替代方案。然而，从hESC产生分化的肝细胞仍然是一个重大挑战，特别是使用与临床应用兼容的方法。我们报告了一种新的方法，使用完全定义的培养条件，概括了肝脏发育的基本阶段，将hESC分化为功能性肝细胞。首先使用激活素、成纤维细胞生长因子2和骨形态发生蛋白4的组合以及磷酸肌醇3-激酶抑制将hFSC分化成内胚层细胞的同质群体。然后使用成纤维细胞生长因子10、视黄酸和激活素/nodal受体抑制剂诱导内胚层细胞进一步分化为肝祖细胞。进一步成熟后，这些细胞表达成熟肝细胞的标志物，包括脱唾液酸糖蛋白受体、酪氨酸氨基转移酶、α 1-抗胰蛋白酶、Cyp 7A 1和肝转录因子如肝细胞核因子4 α和6。此外，在这些条件下产生的细胞在体外表现出肝功能，包括糖原储存，细胞色素活性和低密度脂蛋白摄取。在用表达绿色荧光蛋白的慢病毒载体转导并移植到免疫缺陷uPA转基因小鼠中后，分化的细胞移植到肝脏中，生长并表达人白蛋白和α 1-抗胰蛋白酶以及绿色荧光蛋白至少8周。此外，我们发现肝细胞可以从来自重编程成纤维细胞的人诱导多能细胞中产生，证明了这种方法对不同来源的多能干细胞的有效性。结论：我们已经开发了一种稳健有效的方法来将多能干细胞分化为肝细胞，其表现出人肝细胞的特征。我们的方法应该促进临床级肝细胞移植和药物发现的研究的发展。（《肝脏学》2010年;51：1754-1765）
Generation of hepatocytes from human embryonic stem cells (hESCs) could represent an advantageous source of cells for cell therapy approaches as an alternative to orthotopic liver transplantation. However, the generation of differentiated hepatocytes from hESCs remains a major challenge, especially using a method compatible with clinical applications. We report a novel approach to differentiate hESCs into functional hepatic cells using fully defined culture conditions, which recapitulate essential stages of liver development. hFSCs were first differentiated into a homogenous population of endoderm cells using a combination of activin, fibroblast growth factor 2, and bone morphogenetic protein 4 together with phosphoinositide 3-kinase inhibition. The endoderm cells were then induced to differentiate further into hepatic progenitors using fibroblast growth factor 10, retinoic acid, and an inhibitor of activin/nodal receptor. After further maturation, these cells expressed markers of mature hepatocytes, including asialoglycoprotein receptor, tyrosine aminotransferase, alpha 1-antitrypsin, Cyp7A1, and hepatic transcription factors such as hepatocyte nuclear factors 4 alpha and 6. Furthermore, the cells generated under these conditions exhibited hepatic functions in vitro, including glycogen storage, cytochrome activity, and low-density lipoprotein uptake. After transduction with a green fluorescent protein-expressing lentivector and transplantation into immunodeficient uPA transgenic mice, differentiated cells engrafted into the liver, grew, and expressed human albumin and alpha 1-antitrypsin as well as green fluorescent protein for at least 8 weeks. In addition, we showed that hepatic cells could be generated from human-induced pluripotent cells derived from reprogrammed fibroblasts, demonstrating the efficacy of this approach with pluripotent stem cells of diverse origins. Conclusion: We have developed a robust and efficient method to differentiate pluripotent stem cells into hepatic cells, which exhibit characteristics of human hepatocytes. Our approach should facilitate the development of clinical grade hepatocytes for transplantation and for research on drug discovery. (HEPATOLOGY 2010;51:1754-1765.)