人源多能干细胞体外定向诱导分化为肝细胞

目的: 探索人源诱导多能干细胞（human induced pluripotent stem cells,hiPSCs）在体外二维条件下诱导分化生成肝细胞的条件，并初步探讨三维条件下 hiPSCs 的肝细胞诱导分化。方法: 通过测定4种诱导培养基中白蛋白和甲胎蛋白含量，筛选诱导分化的最佳方案。用筛选出的最优培养基对hiPSCs进行二维培养，采用免疫荧光法、蛋白质印迹法检测诱导后的细胞中肝脏标志蛋白的表达，采用酶联免疫吸附法检测肝细胞功能相关蛋白的含量；对hiPSCs进行三维培养，用酶联免疫吸附法对比二维与三维条件下肝细胞功能，并用 HE 染色法观察三维条件下形成细胞的形态。结果： 二维条件下诱导分化23 d，倒置显微镜下细胞呈典型的肝细胞形态；免疫荧光法、蛋白质印迹法检测到诱导分化后的细胞阳性表达肝脏特征蛋白（甲胎蛋白、白蛋白、角蛋白CK8、角蛋白CK18、SOX17），且与胚胎干细胞来源肝细胞（hESCs HEPs）内含量相近；酶联免疫吸附法测定结果显示，诱导形成的肝细胞具有正常肝细胞功能，表明hiPSCs已分化为肝细胞；HE染色结果显示三维条件下诱导形成的细胞具有明显双核，呈现典型肝细胞形态；酶联免疫法测定结果显示，三维条件下，诱导形成的肝细胞功能没有受到损伤，仍具有正常肝细胞功能。 结论： 在二维与三维培养体系下，采用最佳诱导分化方案，hiPSCs 能够被诱导分化为肝细胞，且表达肝脏相关蛋白，并具有正常肝细胞功能。

Optimal conditions of human induced pluripotent stem cells committed differentiation into hepatocytes in vitro

Objective: Exploring the optimal conditions of human induced pluripotent stem cells(hiPSCs) committed differentiation into hepatocytes under two dimensional condition in vitro, and differentiation under three dimensional condition. Methods: The hepatocyte induced differentiation medium was screened and optimized, and the optimal scheme was obtained by measuring the expression of albumin and AFP under four different differentiation medium.The hiPSCs were cultured under two dimensional condition.The expressions of liver marker protein were detected by immunofluorescence and Western blotting.ELISA was used to investigate the function of hepatocytes.The hiPSCs were cultured under three dimensional condition.Cell morphological changes were observed by HE staining, and compared the function of hepatocytes by ELISA under two and three dimensional condition. Results: After 23 days of differentiation under two dimensional condition, the cells observed in inverted microscope showed typical hepatocyte like morphology, and positive expression of AFP, albumin, CK8, CK18 and SOX17 by immunofluorescence and Western blotting, which was similar to that of embryonic stem derived hepatocytes(hESCs HEPs).The result of ELISA showed that cells differentiated from hiPSCs had normal function, indicating that hiPSCs had differentiated into hepatocytes. The results of HE staining showed that cells differentiated from hiPSCs had obvious dual nuclei under three dimensional condition, and it was the morphology of typical hepatocyte.The results of ELISA showed that the function of cells were not abnormal. Conclusion: In the two and three dimensional conditions,hiPSCs can be induced to differentiate into hepatocytes, expressing liver related proteins, and had normal function.