Chinese Journal of Tissue Engineering Research ›› 2018, Vol. 22 ›› Issue (29): 4681-4686.doi: 10.3969/j.issn.2095-4344.0627

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

Li Jia-jin1, 2, Wang Rong-li1, Li Ting-ting2, He Dong3, Shi Wei2   

  1. 1School of Clinical Medicine, Southwest Medical University, Luzhou 646000, Sichuan Province, China; 2Department of Critical Care Medicine, 3Department of Nephropathy, Mianyang People’s Hospital, Mianyang 621000, Sichuan Province, China
  • Revised:2018-05-31 Online:2018-10-18 Published:2018-10-18
  • Contact: Wang Rong-li, Master, Professor, School of Clinical Medicine, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • About author:Li Jia-jin, Attending physician, School of Clinical Medicine, Southwest Medical University, Luzhou 646000, Sichuan Province, China; Department of Critical Care Medicine, Mianyang People’s Hospital, Mianyang 621000, Sichuan Province, China

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

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