Transformation of human induced pluripotent stem cells into hepatocytes in 3D culture system

doi:10.3969/j.issn.2095-4344.0627

Abstract

Abstract:

BACKGROUND: The construction of bioartificial liver is expected to be an effective method for the treatment of acute liver failure. However, there are still many problems in seed cell source, culture mode and nutrient acquisition, which restrict the development and clinical application of blood purification-artificial liver.
OBJECTIVE: To investigate the feasibility of inducing the differentiation of induced pluripotent stem cells from human skin into hepatocyte-like cells without addition of exogenous sources such as exogenous serum.
METHODS: A 3D culture system was constructed by using a three-dimensional culture system containing 6-well plates of the Transwell chamber. The induction and differentiation of human induced pluripotent stem cells were performed by addition of valproic acid and nicotinamide. The differentiated hepatocyte-like cells were co-cultured with biological patches. There were five groups in the experiment: human induced pluripotent stem cells were set as group A, normal hepatocytes as group B, hepatocyte-like cells without induction by nicotinamide as group C, hepatocyte-like cells with induction by nicotinamide as group D, and hepatocyte-like cells cultured on the biological patch as group E. The morphology of hepatocyte-like cells in group D was observed under inverted phase contrast microscope. The expression of nuclear factor 4α and alpha-fetoprotein in hepatocytes of group D was detected by immunofluorescence. Real-time quantitative PCR and western blot were used to detect mRNA and protein expressions of alpha-fetoprotein and albumin in the cells of groups A, B, C and D. Flow cytometry was used to detect the differentiation efficiency of cells in groups A, C and D. Immunocytochemistry detection was used to detect the protein expression of bile salt export pump in groups B and E. ELISA assay was used to detect lactate dehydrogenase activity, albumin and urea nitrogen contents in the supernatants of the groups D and E.
RESULTS AND CONCLUSION: (1) The cells of group D changed from fusiform to polygon in shape. (2) Positive expression of hepatocyte nuclear factor 4 alpha and alpha fetoprotein was found in the cells of group D. (3) The gene and protein expressions of alpha fetoprotein and albumin in group D was significantly higher than those in groups A and C (P < 0.01). (4) The protein expression of bile salt export pump in the cells was remarkably positive in groups B and E. (4) The activity of lactate dehydrogenase and the contents of albumin and urea nitrogen in cultured cells of group E were significantly higher than those in group D (P < 0.01). To conclude, the combination of 3D culture system with exogenous small molecules and biological surgical patch helps to induce induced pluripotent stem cells to differentiate into functional hepatocyte-like cells in vitro.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Induced Pluripotent Stem Cells, Liver, Artificial, Niacinamide, Valproic Acid, Hepatocytes, Tissue Engineering

CLC Number:

R394.2

Li Jia-jin, Wang Rong-li, Li Ting-ting, He Dong, Shi Wei. Transformation of human induced pluripotent stem cells into hepatocytes in 3D culture system[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(29): 4681-4686.

Figures/Tables 2

2.1 倒置相差显微镜观察诱导性多能干细胞和在三维培养体系中诱导分化为肝细胞样细胞的形态倒置相差显微镜下，D组诱导分化1 d时细胞聚集生长，多呈梭形，见图1A；分化21 d时细胞突起减少，呈多边形，见图1B。 2.2 免疫荧光检测肝细胞样细胞中肝细胞核因子4α和甲胎蛋白的表达荧光显微镜下，D组肝细胞样细胞中肝细胞核因子4α的阳性表达呈红色，甲胎蛋白的阳性表达呈绿色，见图2。 2.3 流式细胞术检测细胞转化率 A组细胞转化率为0%，C组细胞转化率为(10.89±2.54)%，D组细胞转化率为(24.67±3.71)%，差异有显著性意义(P < 0.01)，D组转化率高于A组和C组。 2.4 荧光实时定量PCR检测各组细胞中白蛋白和甲胎蛋白的mRNA表达 A组细胞内未见白蛋白与甲胎蛋白的mRNA表达，B组、C组、D组细胞内白蛋白的mRNA表达率分别为(77.59±4.22)%，(15.12±1.55)%，(43.59±2.17)%，甲胎蛋白的mRNA表达率分别为(81.39±3.86)%，(23.69±2.43)%，(56.78±1.52)%，4组间比较差异有显著性意义(P < 0.01)，B、C、D组白蛋白和甲胎蛋白的mRNA表达高于A组，B组白蛋白和甲胎蛋白的mRNA表达高于C组和D组，D组白蛋白和甲胎蛋白的mRNA表达高于C组。 2.5 Western blot检测各组细胞中白蛋白和甲胎蛋白的表达 A组细胞内未见白蛋白和甲胎蛋白的表达，B组、C组、D组细胞内白蛋白表达率分别为(65.24±6.38)%，(5.01±0.46)%，(21.78±3.25)%，甲胎蛋白表达率分别为(69.22±5.93)%，(6.52±0.60)%，(39.94±4.66)%，4组间比较差异有显著性意义(F=78.397，P=0.000)，B、C、D组白蛋白和甲胎蛋白的表达高于A组，B组白蛋白和甲胎蛋白的表达高于C组和D组，D组白蛋白和甲胎蛋白的表达高于C组，见图3。 2.6 免疫细胞化学法检测构建的人工肝组织细胞中胆盐输出泵蛋白的表达 B组和E组细胞胞膜上均可见棕黄色颗粒的胆盐输出泵蛋白阳性表达，见图4。 2.7 ELISA法检测构建的人工肝组织上清液中乳酸脱氢酶活性、白蛋白含量和尿素氮含量培养10 d，E组培养上清液中乳酸脱氢酶活性、白蛋白和尿素氮水平均高于D组，差异有显著性意义(P < 0.01)，见表1。"

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