EFFECT OF BLOOD MICROENVIRONMENT OF RATS WITH HEPATIC FIBROSIS ON DIFFERENTIATION OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS INTO HEPATOCYTES AND ITS MECHANISMS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YANCheng ¹ , XUEGai ² , ZHANGWei ² , HANXiaolei ² , LIUJianfang ² ,  HOUYanning ¹

1. Medical School of Chinese PLA, Beijing, 100853, P.R.China;
2. Department of Pharmacology, Bethune International Peace Hospital;

Corresponding author：

HOUYanning, Email: biph2014@163.com

Keywords：

Liver fibrosis; Human umbilical cord mesenchymal stem cells; Cell differentiation; Serum; Rat

DOI：

10.7507/1002-1892.20160154

Video：

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Objective To investigate the effect of blood microenvironment of rats with hepatic fibrosis on differentiation of human umbilical cord mesenchymal stem cells (HUCMSCs) into hepatocytes and its mechanisms. Methods Eighteen male adult Sprague Dawley rats [weighing, (200±20) g] were used, liver fibrosis was induced in 12 rats by repeated intraperitoneal injections of thioacetamide. The serum was separated after successful model preparation, and the serum of 6 normal rats was collected. ELISA assay was used to detect the concentrations of epidermal growth factor (EGF), hepatocyte growth factor (HGF), oncostatin M (OSM), and basic fibroblastic growth factor (bFGF). Passage 3 HUCMSCs were divided into 3 groups: cells were cultured for 7 days in DMEM/F12 containing 10% fetal bovine serum and 5 mL/ L serum from rats with hepatic fibrosis (group A), in DMEM/F12 containing 10% fetal bovine serum and 5 mL/ L serum from normal rats (group B), and in DMEM/F12 containing 10% fetal bovine serum (group C). The morphological changes of the cells were observed. The expressions of α-fetoprotein (AFP) and cytokeratin 18 (CK18) were detected by immunofluorescence. The protein levels of albumin (ALB), tryptophan 2, 3-dioxygenase (TPH2), and CYP3A4 and MAPK/ERK signal pathway protein (P-ERK) were detected using Western blot. The content of blood urea nitrogen (BUN) was measured by diacetyl m onoxime method. Results HE staining showed that the liver tissue of rats was in accordance with the change of fibrosis, indicating successful model preparation. In serum of normal rats and rats with hepatic fibrosis, the concentrations of EGF were (21.42±0.32) pg/mL and (17.57±0.31) pg/mL respectively, showing significant difference (t=14.989, P=0.000); the concentrations of OSM were (129.96±0.65) pg/mL and (98.44±1.32) pg/mL respectively, showing significant difference (t=37.172, P=0.000); the concentrations of HGF were below the detection limit and (1.03±0.12) ng/ mL respectively; and the concentrations of bFGF were lower than the detection limit in both groups. No morphological changes of cells were observed in both groups at 7 days, and there was no significant difference between groups. At 7 days after culture, the cells in group A could express human hepatocyte biomarkers of AFP, CK18 and hepatocyte-specific-function proteins of ALB, TPH2, and CYP3 A4 while cells in groups B and C did not. Western blot showed that cells in each group could express P-ERK protein. The relative level of P-ERK protein in group A was significantly higher than that in groups B and C (P < 0.05), but no significant difference was found between groups B and C (P > 0.05). The BUN concentration of group A [(0.74±0.07) mmol/ L] was significantly higher than that of groups B [(0.40±0.04) mmol/ L] and C [(0.38±0.04) mmol/L] (P < 0.05), but no significant difference was shown between groups B and C (P > 0.05). Conclusion Under the condition of hepatic fibrosis, the level of HGF will increase while EGF and OSM will decrease. The formed blood microenvironment will activate MAPK/ERK signal pathway in HUCMSCs, induce them differentiate into hepatocytes.

Citation： YANCheng, XUEGai, ZHANGWei, HANXiaolei, LIUJianfang, HOUYanning. EFFECT OF BLOOD MICROENVIRONMENT OF RATS WITH HEPATIC FIBROSIS ON DIFFERENTIATION OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS INTO HEPATOCYTES AND ITS MECHANISMS. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(6): 754-760. doi: 10.7507/1002-1892.20160154 Copy

1.	Tachi Y, Hirai T, Ishizu Y, et al. α-fetoprotein levels after interferon therapy predict regression of liver fibrosis in patients with sustained virological response. J Gastroenterol Hepatol, 2016, 31(5): 1001-1008.
2.	Baiocchini A, Montaldo C, Conigliaro A, et al. Extracellular matrix molecular remodeling in human liver fibrosis evolution. PLoS One, 2016,11(3): e0151736.
3.	Nicolas CT, Wang Y, Nyberg SL. Cell therapy in chronic liver disease. Curr Opin Gastroenterol, 2016, 32(3): 189-194.
4.	Matsumoto T, Takami T, Sakaida I. Cell transplantation as a non-invasive strategy for treating liver fibrosis. Expert Rev Gastroenterol Hepatol, 2016, 10(5): 639-648.
5.	Lin SZ, Chang YJ, Liu JW, et al. Transplantation of human Wharton’s Jelly-derived stem cells alleviates chemically induced liver fibrosis in rats. Cell Transplant, 2010, 19(11): 1451-1463.
6.	Ren H, Zhao Q, Cheng T, et al. No contribution of umbilical cord mesenchymal stromal cells to capillarization and venularization of hepatic sinusoids accompanied by hepatic differentiation in carbon tetrachloride-induced mouse liver fibrosis. Cytotherapy, 2010, 12(3): 371-383.
7.	Chen JZ, Hong H, Xiang J, et al. A selective tropism of transfused oval cells for liver. World J Gastroenterol, 2003, 9(3): 544-546.
8.	马欣, 薛改, 刘建芳, 等. 肝匀浆上清液诱导人脐带间充质干细胞分化为肝样细胞. 中国组织工程研究, 2013, 17(45): 7877-7884.
9.	闫成, 薛改, 吴丽颖, 等. 大鼠纤维化肝组织匀浆上清液诱导人脐带间充质干细胞向肝细胞分化的实验研究. 中国修复重建外科杂志, 2015, 29(7): 878-882.
10.	Kobolak J, Dinnyes A, Memic A, et al. Mesenchymal stem cells: Identification, phenotypic characterization, biological properties and potential for regenerative medicine through biomaterial micro-engineering of their niche. Methods, 2016, 99: 62-68.
11.	Khunvirojpanich M, Showpittaporchai U, Moongkamdi P, et al. Alpha-mangostin partially preserves expression of ammonia-metabolizing enzymes in thioacetamide-induced fibrotic and cirrhotic rats. J Med Assoc Thai, 2015, 98(10): S53-S60.
12.	胡志文, 柳建华, 古维立, 等. 制备适用影像学研究的各期肝纤维化及肝硬化模型. 广州医药, 2013, 44(5): 45-47.
13.	Xue G, Han X, Ma X, et al. Effect of microenvironment on differentiation of human umbilical cord mesenchymal stem cells into hepatocytes in vitro and in vivo. Biomed Res Int, 2016, (2016): 8916534.
14.	Zheng G, Liu Y, Jing Q, et al. Differentiation of human umbilical cord-derived mesenchymal stem cells into hepatocytes in vitro. Bio-Medical Materials and Engineering, 2015, 25(1 Suppl): S145-S157.
15.	Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature, 2001, 414(6859): 98-104.
16.	王韫芳, 南雪, 尉承泽, 等. 丙烯醇致肝损伤微环境定向诱导骨髓干细胞向肝细胞分化. 中华肝脏病杂志, 2005, 13(4): 274-277.
17.	Forte G, Minieri M, Cossa P, et al. Hepatocyte growth factor effects on mesenchymal stem cells: proliferation, migration, and differentiation. Stem Cells, 2006, 24(1): 23-33.
18.	Lowes KN, Croager EJ, Olynyk JK, et al. Oval cell-mediated liver regeneration: role of cytokines and growth factors. J Gastroenterol Hepatol, 2003, 18(1): 4-12.
19.	Rustad KC, Gurtner GC. Mesenchymal stem cells home to sites of injury and inflammation. Adv Wound Care (New Rochelle), 2012, 1(4): 147-152.
20.	陈甫寰, 岳晓彤, 宋慧锋, 等. 表皮干细胞向汗腺细胞分化的表型改变及其通路机制研究. 中国修复重建外科杂志, 2016, 30(2): 245-250.
21.	Ciccarelli C, Vulcano F, Milazzo L, et al. Key role of MEK/ERK pathway in sustaining tumorigenicity and in vitro radioresistance of embryonal rhabdomyosarcoma stem-like cell population. Mol Cancer, 2016, 15: 16.
22.	Yan Y, Zhu Y, Sun F, et al. Extracellular regulated protein kinases 1/2 phosphorylation is required for hepatic differentiation of human umbilical cord-derived mesenchymal stem cells. Exp Biol Med (Maywood), 2015, 240(4): 534-545.
23.	Ye JS, Su XS, Stoltz JF, et al. Signalling pathways involved in the process of mesenchymal stem cells differentiating into hepatocytes. Cell Prolif, 2015, 48(2): 157-165.
24.	Patcharee P, Wilairat L. Developing a new two-step protocol to generate functional hepatocytes from Wharton’s jelly-derived mesenchymal stem cells under hypoxic condition. Stem Cells Int, 2013, (2013): 762196.
25.	Tan Y, Xiao EH, Xiao LZ, et al. VEGF(165) expressing bone marrow mesenchymal stem cells differentiate into hepatocytes under HGF and EGF induction in vitro. Cytotechnology, 2012, 64(6): 635-647.
26.	Sherif SA, Moharib MN, El-Lakkany NM, et al. The use of microencapsulated hepatocytes transplantation reduces mortality and liver alterations in Schistosoma mansoni infected hamsters. J Egypt Soc Parasitol, 2014, 44(1): 229-241.
27.	Shi XL, Gao Y, Yan Y, et al. Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes. Cell Res, 2016, 26(2): 206-216.

1. Tachi Y, Hirai T, Ishizu Y, et al. α-fetoprotein levels after interferon therapy predict regression of liver fibrosis in patients with sustained virological response. J Gastroenterol Hepatol, 2016, 31(5): 1001-1008.
2. Baiocchini A, Montaldo C, Conigliaro A, et al. Extracellular matrix molecular remodeling in human liver fibrosis evolution. PLoS One, 2016,11(3): e0151736.
3. Nicolas CT, Wang Y, Nyberg SL. Cell therapy in chronic liver disease. Curr Opin Gastroenterol, 2016, 32(3): 189-194.
4. Matsumoto T, Takami T, Sakaida I. Cell transplantation as a non-invasive strategy for treating liver fibrosis. Expert Rev Gastroenterol Hepatol, 2016, 10(5): 639-648.
5. Lin SZ, Chang YJ, Liu JW, et al. Transplantation of human Wharton’s Jelly-derived stem cells alleviates chemically induced liver fibrosis in rats. Cell Transplant, 2010, 19(11): 1451-1463.
6. Ren H, Zhao Q, Cheng T, et al. No contribution of umbilical cord mesenchymal stromal cells to capillarization and venularization of hepatic sinusoids accompanied by hepatic differentiation in carbon tetrachloride-induced mouse liver fibrosis. Cytotherapy, 2010, 12(3): 371-383.
7. Chen JZ, Hong H, Xiang J, et al. A selective tropism of transfused oval cells for liver. World J Gastroenterol, 2003, 9(3): 544-546.
8. 马欣, 薛改, 刘建芳, 等. 肝匀浆上清液诱导人脐带间充质干细胞分化为肝样细胞. 中国组织工程研究, 2013, 17(45): 7877-7884.
9. 闫成, 薛改, 吴丽颖, 等. 大鼠纤维化肝组织匀浆上清液诱导人脐带间充质干细胞向肝细胞分化的实验研究. 中国修复重建外科杂志, 2015, 29(7): 878-882.
10. Kobolak J, Dinnyes A, Memic A, et al. Mesenchymal stem cells: Identification, phenotypic characterization, biological properties and potential for regenerative medicine through biomaterial micro-engineering of their niche. Methods, 2016, 99: 62-68.
11. Khunvirojpanich M, Showpittaporchai U, Moongkamdi P, et al. Alpha-mangostin partially preserves expression of ammonia-metabolizing enzymes in thioacetamide-induced fibrotic and cirrhotic rats. J Med Assoc Thai, 2015, 98(10): S53-S60.
12. 胡志文, 柳建华, 古维立, 等. 制备适用影像学研究的各期肝纤维化及肝硬化模型. 广州医药, 2013, 44(5): 45-47.
13. Xue G, Han X, Ma X, et al. Effect of microenvironment on differentiation of human umbilical cord mesenchymal stem cells into hepatocytes in vitro and in vivo. Biomed Res Int, 2016, (2016): 8916534.
14. Zheng G, Liu Y, Jing Q, et al. Differentiation of human umbilical cord-derived mesenchymal stem cells into hepatocytes in vitro. Bio-Medical Materials and Engineering, 2015, 25(1 Suppl): S145-S157.
15. Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature, 2001, 414(6859): 98-104.
16. 王韫芳, 南雪, 尉承泽, 等. 丙烯醇致肝损伤微环境定向诱导骨髓干细胞向肝细胞分化. 中华肝脏病杂志, 2005, 13(4): 274-277.
17. Forte G, Minieri M, Cossa P, et al. Hepatocyte growth factor effects on mesenchymal stem cells: proliferation, migration, and differentiation. Stem Cells, 2006, 24(1): 23-33.
18. Lowes KN, Croager EJ, Olynyk JK, et al. Oval cell-mediated liver regeneration: role of cytokines and growth factors. J Gastroenterol Hepatol, 2003, 18(1): 4-12.
19. Rustad KC, Gurtner GC. Mesenchymal stem cells home to sites of injury and inflammation. Adv Wound Care (New Rochelle), 2012, 1(4): 147-152.
20. 陈甫寰, 岳晓彤, 宋慧锋, 等. 表皮干细胞向汗腺细胞分化的表型改变及其通路机制研究. 中国修复重建外科杂志, 2016, 30(2): 245-250.
21. Ciccarelli C, Vulcano F, Milazzo L, et al. Key role of MEK/ERK pathway in sustaining tumorigenicity and in vitro radioresistance of embryonal rhabdomyosarcoma stem-like cell population. Mol Cancer, 2016, 15: 16.
22. Yan Y, Zhu Y, Sun F, et al. Extracellular regulated protein kinases 1/2 phosphorylation is required for hepatic differentiation of human umbilical cord-derived mesenchymal stem cells. Exp Biol Med (Maywood), 2015, 240(4): 534-545.
23. Ye JS, Su XS, Stoltz JF, et al. Signalling pathways involved in the process of mesenchymal stem cells differentiating into hepatocytes. Cell Prolif, 2015, 48(2): 157-165.
24. Patcharee P, Wilairat L. Developing a new two-step protocol to generate functional hepatocytes from Wharton’s jelly-derived mesenchymal stem cells under hypoxic condition. Stem Cells Int, 2013, (2013): 762196.
25. Tan Y, Xiao EH, Xiao LZ, et al. VEGF(165) expressing bone marrow mesenchymal stem cells differentiate into hepatocytes under HGF and EGF induction in vitro. Cytotechnology, 2012, 64(6): 635-647.
26. Sherif SA, Moharib MN, El-Lakkany NM, et al. The use of microencapsulated hepatocytes transplantation reduces mortality and liver alterations in Schistosoma mansoni infected hamsters. J Egypt Soc Parasitol, 2014, 44(1): 229-241.
27. Shi XL, Gao Y, Yan Y, et al. Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes. Cell Res, 2016, 26(2): 206-216.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF BLOOD MICROENVIRONMENT OF RATS WITH HEPATIC FIBROSIS ON DIFFERENTIATION OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS INTO HEPATOCYTES AND ITS MECHANISMS

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