Retaining mTeSR1 Medium during Hepatic Differentiation Facilitates Hepatocyte-Like Cell Survival by Decreasing Apoptosis
Retaining mTeSR1 Medium during Hepatic Differentiation Facilitates Hepatocyte-Like Cell Survival by Decreasing Apoptosis
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在肝分化过程中保留 mTeSR1 培养基可通过减少细胞凋亡促进肝细胞样细胞存活
DOI：
10.1159/000495644
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发表时间：
2018
影响因子：
--
通讯作者：
Wu Zhongkai
中科院分区：
文献类型：
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作者：
Hou Jian;Long Yan;Hu Bo;Huang Shaojie;Xu Guangtao;Gao Tesheng;Wu Fenfang;Li Yinxiong;Wu Zhongkai
背景/目的人多能干细胞来源的肝细胞样细胞可能成为肝细胞移植的重要细胞来源。本研究探讨了保留mTeSR 1培养基在肝细胞分化过程中对肝细胞样cells in vitro.Methodshuman embryonic stem cell lines H1的影响激活素A和骨形态发生蛋白4（BMP 4）处理的定形内胚层（DE）细胞诱导，随后处理与BMP 2和成纤维细胞生长因子4（FGF 4）的早期肝细胞诱导。加入肝细胞生长因子（HGF）和成纤维细胞生长因子（KGF）用于早期肝细胞扩增，然后与抑瘤素-M混合用于成熟。在DE诱导过程中，分别加入0%、25%、50%和75%浓度的mTeSR 1培养基用于早期肝诱导和扩增。为了优化，通过qRT-PCR定量了分化细胞中第4天SRY相关HMG盒17（SOX 17）和叉头盒A2（FOXA 2）、第15天甲胎蛋白（AFP）和肝细胞核因子4α（HNF 4 α）以及第25天白蛋白（ALB）的表达水平。流式细胞术检测ALB阳性细胞比例。通过ELISA检测ALB分泌和吲哚菁绿色（ICG）摄取和释放，通过尿素测定试剂盒检测尿素生成，通过过碘酸Schif反应（PAS）染色检测糖原储存能力。以诱导性多能干细胞（iPS）为材料，研究优化的诱导分化方法是否适用于iPS细胞的诱导分化。通过免疫染色检测DE和肝标志物，并如上所述进行功能测试。结果H1细胞分化后，在保留0%、25%、50%和75%mTeSR1的情况下，所有分化细胞均表达SOX 17、FOXA 2、AFP、HNF 4 α和ALB。而在0%和25%组的分化细胞中观察到更高的表达水平。流式细胞术结果显示，25%mTeSR1组中来自H1细胞的ALB阳性分化细胞的比例高于其他组。但25%组与0%组之间的ALB分泌、尿素生成、ICG摄取和释放以及糖原储存能力无显著差异。iPS细胞可分化为肝细胞样细胞，mTeSR 1保留率为25%。25%mTeSR1组的分化细胞的凋亡率低于0%mTeSR1 group.ConclusionRetaining 25%mTeSR1培养基在肝分化过程中已提出增加ALB阳性细胞的百分比和细胞存活减少细胞凋亡。
Background/AimsHepatocyte-like cells derived from human pluripotent stem cells could be an important cell source for hepatocyte transplantation. The present study investigated the effect of retaining mTeSR1 medium during hepatic differentiation on hepatocyte-like cells in vitro.MethodsHuman embryonic stem cell line H1 were treated with activin A and bone morphogenetic protein 4 (BMP4) for definitive endoderm (DE) cell induction and subsequently treated with BMP2 and fibroblast growth factor 4 (FGF4) for early hepatic cell induction. Hepatocyte growth factor (HGF) and fibroblast growth factor (KGF) were added for early hepatic cell expansion and then mixed with oncostatin-M for maturation. During DE induction, 0%, 25%, 50% and 75% concentrations of mTeSR1 medium were separately added for early hepatic induction and expansion. For optimization, the expression levels of SRY-related HMG-box 17 (SOX17) and forkhead box A2 (FOXA2) at day 4, alpha fetoprotein (AFP) and hepatocyte nuclear factor 4α (HNF4α) at day 15, and albumin (ALB) at day 25 were quantified in differentiated cells by qRT-PCR. The ALB-positive cell proportion was measured by flow cytometry. Functional tests including ALB secretion and indocyanine green (ICG) angiography uptake and release by ELISA, urea production by urea assay kit, and glycogen storage ability by periodic acid Schif reaction (PAS) staining were performed in the differentiated cells. The induced pluripotent stem (iPS) cells were used to examine whether the optimized method was suitable for differentiating iPS cells. DE and hepatic markers were detected by immunostaining, and functional testing was performed as described above. Flow cytometry with an Annexin V-FITC apoptosis detection kit and fluorescence microscopy with Hoechst 33258 were used to analyze apoptosis in differentiated cells derived from H1 cells.ResultsAll differentiated cells with retention of 0%, 25%, 50% and 75% mTeSR1 expressed SOX17, FOXA2, AFP, HNF4α, and ALB, while higher expression levels were observed in differentiated cells in the 0% and 25% groups. The flow cytometry results showed that the proportion of ALB-positive differentiated cells derived from H1 cells was higher in the 25% mTeSR1 group than in other groups. However, no significant difference in ALB secretion, urea production, ICG uptake and release and glycogen storage ability was detected between the 25% and 0% groups. The iPS cells could differentiate into hepatocyte-like cells with 25% mTeSR1 retention. The apoptosis ratio of differentiated cells was lower in the 25% mTeSR1 group than in the 0% mTeSR1 group.ConclusionRetaining 25% mTeSR1 medium during hepatic differentiation has been proposed to increase the percentage of ALB-positive cells and cell survival by decreasing cell apoptosis.