Faculty of Pharmacy, Research Institute for Medicines (iMed.ULisboa), Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisbon, Portugal.
Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
Arch Toxicol. 2017 Jun;91(6):2469-2489. doi: 10.1007/s00204-016-1901-x. Epub 2016 Dec 1.
The development of predictive in vitro stem cell-derived hepatic models for toxicological drug screening is an increasingly important topic. Herein, umbilical cord tissue-derived mesenchymal stem cells (hnMSCs) underwent hepatic differentiation using an optimized three-step core protocol of 24 days that mimicked liver embryogenesis with further exposure to epigenetic markers, namely the histone deacetylase inhibitor trichostatin A (TSA), the cytidine analogue 5-azacytidine (5-AZA) and dimethyl sulfoxide (DMSO). FGF-2 and FGF-4 were also tested to improve endoderm commitment and foregut induction during Step 1 of the differentiation protocol, being HHEX expression increased with FGF-2 (4 ng/mL). DMSO (1%, v/v) when added at day 10 enhanced cell morphology, glycogen storage ability, enzymatic activity and induction capacity. Moreover, the stability of the hepatic phenotype under the optimized differentiation conditions was examined up to day 34. Our findings showed that hepatocyte-like cells (HLCs) acquired the ability to metabolize glucose, produce albumin and detoxify ammonia. Global transcriptional analysis of the HLCs showed a partial hepatic differentiation degree. Global analysis of gene expression in the different cells revealed shared expression of gene groups between HLCs and human primary hepatocytes (hpHeps) that were not observed between HepG2 and hpHeps. In addition, bioinformatics analysis of gene expression data placed HLCs between the HepG2 cell line and hpHeps and distant from hnMSCs. The enhanced hepatic differentiation observed was supported by the presence of the hepatic drug transporters OATP-C and MRP-2 and gene expression of the hepatic markers CK18, TAT, AFP, ALB, HNF4A and CEBPA; and by their ability to display stable UGT-, EROD-, ECOD-, CYP1A1-, CYP2C9- and CYP3A4-dependent activities at levels either comparable with or even higher than those observed in primary hepatocytes and HepG2 cells. Overall, an improvement of the hepatocyte-like phenotype was achieved for an extended culture time suggesting a role of the epigenetic modifiers in hepatic differentiation and maturation and presenting hnMSC-HLCs as an advantageous alternative for drug discovery and in vitro toxicology testing.
脐带组织来源间充质干细胞（hnMSCs）通过优化的三步核心方案进行肝分化，该方案为期 24 天，模拟了肝胚胎发生过程，并进一步暴露于表观遗传标记物，即组蛋白去乙酰化酶抑制剂曲古抑菌素 A（TSA）、胞嘧啶类似物 5-氮杂胞苷（5-AZA）和二甲基亚砜（DMSO）。还测试了 FGF-2 和 FGF-4 以改善内胚层的承诺和前肠诱导在分化方案的第 1 步，HHEX 表达增加与 FGF-2（4ng/ml）。当在第 10 天添加 1%（v/v）的 DMSO 时，增强了细胞形态、糖原储存能力、酶活性和诱导能力。此外，在优化的分化条件下，还检查了肝表型的稳定性，直至第 34 天。我们的研究结果表明，类肝细胞（HLCs）获得了代谢葡萄糖、产生白蛋白和解毒氨的能力。HLCs 的全转录组分析显示出部分肝分化程度。不同细胞中基因表达的全基因组分析显示，HLCs 与原代人肝细胞（hpHeps）之间存在基因表达的共享群组，而在 HepG2 和 hpHeps 之间没有观察到。此外，基因表达数据的生物信息学分析将 HLCs 置于 HepG2 细胞系和 hpHeps 之间，并且远离 hnMSCs。观察到的肝分化增强得到了肝药物转运蛋白 OATP-C 和 MRP-2 的存在以及肝标记物 CK18、TAT、AFP、ALB、HNF4A 和 CEBPA 的基因表达的支持；并且它们能够显示稳定的 UGT-、EROD-、ECOD-、CYP1A1-、CYP2C9- 和 CYP3A4 依赖性活性，其水平与原代肝细胞和 HepG2 细胞观察到的水平相当，甚至更高。总体而言，延长培养时间可改善类肝细胞表型，提示表观遗传修饰物在肝分化和成熟中的作用，并将 hnMSC-HLCs 作为药物发现和体外毒理学测试的有利替代方法。
