Hepatocyte-like cells derived from induced pluripotent stem cells inhibit liver fibrosis in rats

doi:10.3969/j.issn.2095-4344.1607

Abstract

Abstract:

BACKGROUND: Induced pluripotent stem cells have similar self-renewal, proliferation and differentiation abilities to embryonic stem cells. They have no source limitations, no ethical problems, and no current problems of cell xenogenesis, which are expected to be the source of cells for the treatment of liver diseases.
OBJECTIVE: To observe the effect of induced pluripotent stem cell-derived hepatocyte-like cells on liver fibrosis.
METHODS: The three-step method in vitro was used to induce the differentiation of induced pluripotent stem cells into hepatocyte-like cells. Glycogen staining, immunohistochemistry and low-density lipoprotein uptake assay were used to detect the ability of induced cells to synthesize glycogen, alpha-fetoprotein, albumin, CK18 protein and low-density lipoprotein uptake. Forty-five Sprague-Dawley rats (provided by the Experimental Animal Center, the Affiliated Haikou Hospital, Xiangya School of Medicine, Central South University) were randomized into three groups: normal control group, model group and cell transplantation group (n=15 per group). The rats in the latter two groups were intraperitoneally injected with carbon tetrachloride to establish liver fibrosis models. Cell transplantation group was given intravenous injectin of hepatocyte-like cells (induced for 21 days), 0.5 mL, 2×10⁹/L, at 3 days after modeling. Four weeks after cell transplantation, venous blood and liver tissue samples were taken to analyze the changes of liver function, liver fibrosis index and liver pathology.
RESULTS AND CONCLUSION: (1) After 21 days of induction, human induced pluripotent stem cell clonal clusters became loose, mainly round or polygonal in shape, and presented with a dense paving stone-like arrangement. A large amount of pink glycogens could be seen in the cytoplasm, indicating that induced pluripotent stem cells have the ability to synthesize glycogen. Low-density lipoprotein uptake test showed that induced pluripotent stem cells had the ability to uptake low-density lipoprotein. Immunohistochemistry detection showed that the cells were positive for alpha fetoprotein, albumin and CK18. (2) At 4 weeks after cell transplantation, the level of albumin in the model group was significantly lower than that in the normal control group (P < 0.05), while the levels of direct bilirubin, indirect bilirubin, aspartate aminotransferase, alanine aminotransferase, type IV collagen, serum hyaluronidase, serum type III procollagen in the model group were significantly higher than those in the normal control group (P < 0.05). Compared with the model group, the level of albumin in the cell transplantation group was significantly increased (P < 0.05), while the levels of direct bilirubin, indirect bilirubin, aspartate aminotransferase, alanine aminotransferase, type IV collagen, serum hyaluronidase, serum type III procollagen in the cell transplantation group were significantly decreased (P < 0.05). (3) Four weeks after cell transplantation, inflammatory cell infiltration, hepatocyte degeneration and necrosis in the cell transplantation group were improved to different extents. Therefore, hepatocyte-like cells derived from induced pluripotent stem cells could significantly improve liver fibrosis in rats.

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: Liver Cirrhosis, Induced Pluripotent Stem Cells, Hepatocytes, Tissue Engineering

CLC Number:

Cheng Gang, Huang Denggao, Liang Ying. Hepatocyte-like cells derived from induced pluripotent stem cells inhibit liver fibrosis in rats[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(9): 1384-1389.

Figures/Tables 5

References

[1] Zoubek ME, Trautwein C, Strnad P. Reversal of liver fibrosis: From fiction to reality. Best Pract Res Clin Gastroenterol. 2017;31(2):129-141.

[2] Pellicoro A, Ramachandran P, Iredale JP. Reversibility of liver fibrosis. Fibrogenesis Tissue Repair. 2012;5(Suppl 1):S26.

[3] Pinzani M, Romanelli RG, Magli S. Progression of fibrosis in chronic liver diseases: time to tally the score. J Hepatol. 2001; 34(5):764-767.

[4] 唐卫平,胡琦,戴小明,等.脂肪间充质干细胞移植对肝纤维化的治疗作用及其机制[J].中华肝脏病杂志,2018,26(1):28-33.

[5] 薛改.脐带间充质干细胞对肝损伤的治疗作用及其机制研究[D].石家庄:河北医科大学,2017.

[6] 刘卫,余森源,严和中,等. 骨髓间质干细胞移植对肝纤维化模型大鼠治疗效果[J].临床军医杂志,2017,45(9):903-907.

[7] Kim JH, Jang YJ, An SY, et al. Enhanced Metabolizing Activity of Human ES Cell-Derived Hepatocytes Using a 3D Culture System with Repeated Exposures to Xenobiotics. Toxicol Sci. 2015;147(1):190-206.

[8] Imai K,Tanoue A,Nakamura K,et al．An attempt to in vitro embryotoxicity test using mouse ES cells and human hepatocytes. Aatex.2013;18(1):33-38.

[9] 杜盼,余江南,徐希明.人源多能干细胞体外定向诱导分化为肝细胞[J]. 江苏大学学报(医学版),2018,28(1):39-46.

[10] 史德宝,吕礼应. 血清白蛋白检测方法对糖化白蛋白的影响[J].安徽医科大学学报,2015,50(12):1805-1808.

[11] 王杨,谷冬梅,陈京山,等. 三种常规方法检测血清胆红素结果的可比性和偏倚评估[J]. 中国实验诊断学, 2017,21(10): 1744-1747.

[12] 饶雅琪,张丽,曾金祥,等. 禾叶风毛菊醇提物对四氯化碳致小鼠肝损伤的保护作用[J]. 中国临床药理学杂志, 2017,33(20): 2058-2061.

[13] 易梦秋,余旻,王鹏.多器官功能障碍综合征大鼠肺组织MMP-9、TIMP-1和Ⅳ型胶原的表达[J]. 中国病理生理杂志, 2018,34(1): 123-129.

[14] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663-676.

[15] Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861-872.

[16] Song Z, Cai J, Liu Y, et al. Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Res. 2009;19(11):1233-1242.

[17] Twaroski K, Mallanna SK, Jing R, et al. FGF2 mediates hepatic progenitor cell formation during human pluripotent stem cell differentiation by inducing the WNT antagonist NKD1. Genes Dev. 2015;29(23):2463-2474.

[18] Takagi C, Yagi H, Hieda M, et al. Mesenchymal Stem Cells Contribute to Hepatic Maturation of Human Induced Pluripotent Stem Cells. Eur Surg Res. 2017;58(1-2):27-39.

[19] Mallanna SK, Cayo MA, Twaroski K, et al. Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes. Stem Cell Reports. 2016;7(3): 543-556.

[20] 刘国菊,李丛丛,李睿坤,等.转化生长因子β1在纤维化疾病发病中作用的研究进展[J].山东医药, 2018,58(30):106-109.

[21] 李梓豪,尹大龙,崔逸峰,等.肝硬化肝癌肝部分切除术后影响肝再生的抗纤维化因子研究[J].国际外科学杂志, 2018,45(2): 134-137.

[22] 段佳悦,史清峰,王明光,等. 血管内皮生长因子、碱性成纤维细胞生长因子和内皮抑素在肝硬化中的表达及意义[J].临床荟萃, 2018,33(5):427-430.

[23] Sulzbacher S, Schroeder IS, Truong TT, et al. Activin A-induced differentiation of embryonic stem cells into endoderm and pancreatic progenitors-the influence of differentiation factors and culture conditions. Stem Cell Rev. 2009;5(2):159-173.

[24] 李毅,李欣,孙涛. CDX2基因修饰的脐带间充质干细胞向杯状细胞分化的可行性研究[J].生物技术通讯,2014,25(4):515-519.

[25] Kang SJ, Lee HM, Park YI, et al. Chemically induced hepatotoxicity in human stem cell-induced hepatocytes compared with primary hepatocytes and HepG2. Cell Biol Toxicol. 2016;32(5):403-417.

[26] 张文成.细胞重编程与诱导内胚层多能干细胞的获取与鉴定[D].北京:中国人民解放军军事医学科学院,解放军军事医学科学院, 2012.

[27] Boulter L, Govaere O, Bird TG, et al. Macrophage-derived Wnt opposes Notch signaling to specify hepatic progenitor cell fate in chronic liver disease. Nat Med. 2012;18(4): 572-579.

[28] Gieseck RL 3rd, Hannan NR, Bort R, et al. Maturation of induced pluripotent stem cell derived hepatocytes by 3D-culture. PLoS One. 2014;9(1):e86372.

[29] Chen Y, Li Y, Wang X, et al. Amelioration of Hyperbilirubinemia in Gunn Rats after Transplantation of Human Induced Pluripotent Stem Cell-Derived Hepatocytes. Stem Cell Reports. 2015;5(1):22-30.

[30] Roy-Chowdhury N, Wang X, Guha C, et al. Hepatocyte-like cells derived from induced pluripotent stem cells. Hepatol Int. 2017;11(1):54-69.

[31] Sakurai F, Mitani S, Yamamoto T, et al. Human induced-pluripotent stem cell-derived hepatocyte-like cells as an in vitro model of human hepatitis B virus infection. Sci Rep. 2017;7:45698.

[32] Huang CS, Lin HC, Lu KH, et al. Generation of high quality of hepatocyte-like cells from induced pluripotent stem cells with Parp1 but lacking c-Myc. J Chin Med Assoc. 2018;81(10): 871-877.

[33] Cao YY, Zeng X. Research advances in hepatocyte-like cells from human induced pluripotent stem cells and their application. Zhonghua Gan Zang Bing Za Zhi. 2018;26(1):69-72.

[34] Starokozhko V, Hemmingsen M, Larsen L, et al. Differentiation of human-induced pluripotent stem cell under flow conditions to mature hepatocytes for liver tissue engineering. J Tissue Eng Regen Med. 2018;12(5): 1273-1284.

[35] Takayama K, Akita N, Mimura N, et al. Generation of safe and therapeutically effective human induced pluripotent stem cell-derived hepatocyte-like cells for regenerative medicine. Hepatol Commun. 2017;1(10):1058-1069.

[36] Lorvellec M, Scottoni F, Crowley C, et al. Mouse decellularised liver scaffold improves human embryonic and induced pluripotent stem cells differentiation into hepatocyte-like cells. PLoS One. 2017;12(12):e0189586.

[37] Kotaka M, Toyoda T, Yasuda K, et al. Adrenergic receptor agonists induce the differentiation of pluripotent stem cell-derived hepatoblasts into hepatocyte-like cells. Sci Rep. 2017;7(1):16734.

[38] Asgari S, Moslem M, Bagheri-Lankarani K, et al. Differentiation and transplantation of human induced pluripotent stem cell-derived hepatocyte-like cells. Stem Cell Rev. 2013;9(4):493-504.