DIFFERENTIATION OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS INTO HEPATOCYTES INDUCED BY RAT FIBROTIC LIVER TISSUE EXTRACTS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YANCheng ^1,2 , XUEGai ² , WULiying ² , LIUJianfang ² ,  HOUYanning ²

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

Corresponding author：

HOUYanning, Email: biph2014@163.com

Keywords：

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

DOI：

10.7507/1002-1892.20150189

Video：

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Objective To investigate the differentiation potential of human umbilical cord mesenchymal stem cells (HUCMSCs) into hepatocytes induced by rat fibrotic liver tissue extracts. Methods Liver fibrosis was induced in the Sprague Dawley rats (weighting, 180-220 g) by repeated intraperitoneal injections of 3% thioacetamide-saline at a dose of 200 mg/kg twice a week for 4 weeks;fibrotic liver tissues were used to prepare liver homogenate supernatants. The HUCMSCs at passage 3 were cultured in DMEM/F12 with 10% fetal bovine serum (FBS) (control group) and in DMEM/F12 with 10%FBS and 50 g/L liver homogenate supernatants (experimental group) for 7 days. The morphological changes of the cells were recorded;the protein levels of cytokeratin 18 (CK18), alpha fetoprotein (AFP), and CYP3A4 were measured using Western blot. The glycogen storing ability of the cells was detected by periodic acid-schiff (PAS) staining. Furthermore, the synthesis of albumin (ALB) and blood urea nitrogen (BUN) was measured. Results In experimental group, after 1 day of induction, the stem cells of fusiform shape began to lose sharp edges and progressively shrunk, and then they changed into hepatocyte-like cells with round and irregular shape at 7 days. Positive expressions of AFP, CK18, and CYP3A4 were observed in the experimental group, but negative expression in the control group. The concentrations of BUN and ALB were (0.43±0.07) mmol/L and (8.08±0.41) μg/mL in the control group and were (2.52±0.20) mmol/L and (41.48±4.11) μg/mL in the experimental group, showing significant differences (t=24.160, P=0.000;t=19.810, P=0.000). PAS staining results showed navy blue nucleus and lavender cytoplasm in the control group, but dark purple cell body and visible nucleus in the experimental group. Conclusion HUCMSCs could differentiate into hepatocyte-like cells induced by rat fibrotic liver tissue extracts, which have hepatocyte biomarkers (AFP, CK18, and CYP3A4) and hepatocyte-specific functions of glycogen storage, urea production and ALB secretion, so they could partially replace the function of hepatocytes, that may be one of the therapeutic mechanisms of stem cell transplantation.

Citation： YANCheng, XUEGai, WULiying, LIUJianfang, HOUYanning. DIFFERENTIATION OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS INTO HEPATOCYTES INDUCED BY RAT FIBROTIC LIVER TISSUE EXTRACTS. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(7): 878-883. doi: 10.7507/1002-1892.20150189 Copy

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2.	Wei R,Yang J,Hou W,et al.Insulin-producing cells derived from human embryonic stem cells:comparison of definitive endoderm- and nestin-positive progenitor-based differentiation strategies.PLoS One,2013,8(8):e72513.
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4.	Srivastava D,Ivey KN.Potential of stem-cell-based therapies for heart disease.Nature,2006,441(7097):1097-1099.
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11.	郑盛,殷芳,肖琼怡,等.脐带间充质干细胞移植对急性肝功能衰竭的修复及移植途径的优化.肝脏,2014,19(12):918-923.
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13.	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.
14.	Wang H,Wu G,Park HJ,et al.Protective effect of Phellinus linteus polysaccharide extracts against thioacetamide-induced liver fibrosis in rats:a proteomics analysis.Chin Med,2012,7(1):23.
15.	中华医学会感染病学分会肝衰竭与人工肝学组,中华医学会肝病学分会重型肝病与人工肝学组.肝衰竭诊治指南(2012年版).中华肝脏病杂志,2013,21(3):177-183.
16.	Dianat N,Steichen C,Vallier L,et al.Human pluripotent stem cells for modelling human liver diseases and cell therapy.Curr Gene Ther,2013,13(2):120-132.
17.	Zhou R,Li Z,He C,et al.Human umbilical cord mesenchymal stem cells and derived hepatocyte-like cells exhibit similar therapeutic effects on an acute liver failure mouse model.PLoS One,2014,9(8):e104392.
18.	Li L,Xie T.Stem cell niche:structure and function.Annu Rev Cell Dev Biol,2005,21(3):605-631.
19.	Lee CC,Kim KD.Stem cell microenvironment as a potential therapeutic target.Regen Med,2012,7(1):3-5.
20.	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.
21.	Tan Y,Xiao EH,Xiao LZ,et al.VEGF₁₆₅ expressing bone marrow mesenchymal stem cells differentiate into hepatocytes under HGF and EGF induction in vitro.Cytotechnology,2012,64:635-647.
22.	Zhang S,Chen L,Liu T,et al.Human umbilical cord matrix stem cells efficiently rescue acute liver failure through paracrine effects rather than hepatic differentiation.Tissue Eng Part A,2012,18(13-14):1352-1364.
23.	Hosny S,Abdel-Rahman NZ,Eman M,et al.Peripheral vein infusion of autologous mesenchymal stem cells in Egyptian HCV-positive patients with end-stage liver disease.Stem Cell Res Ther,2014,5(3):70.
24.	Liang L,Ma T,Chen W,et al.Therapeutic potential and related signal pathway of adipose-derived stem cell transplantation for rat liver injury.Hepatology Res,2009,39(8):822-832.
25.	何宏亮,高志良.间充质干细胞的肝细胞分化及其治疗终末期肝病的临床研究进展.中华肝脏病杂志,2014,22(5):397-400.

1. van der Kooy D,Weiss S.Why stem cells? Science,2000,287(5457):1439-1441.
2. Wei R,Yang J,Hou W,et al.Insulin-producing cells derived from human embryonic stem cells:comparison of definitive endoderm- and nestin-positive progenitor-based differentiation strategies.PLoS One,2013,8(8):e72513.
3. Cantz T,Manns MP,Ott M.Stem cells in liver regeneration and therapy.Cell Tissue Res,2008,331(1):271-282.
4. Srivastava D,Ivey KN.Potential of stem-cell-based therapies for heart disease.Nature,2006,441(7097):1097-1099.
5. Morigi M,Introna M,Imberti B,et al.Human bone marrow mesenchymal stem cells accelerate recovery of acute renal injury and prolong survival in mice.Stem Cells,2008,26(8):2075-2082.
6. Tsai PC,Fu TW,Chen YM,et al.The therapeutic potential of human umbilical mesenchymal stem cells from Wharton's jelly in the treatment of rat liver fibrosis.Liver Transpl,2009,15(5):484-495.
7. Jung KH,Shin HP,Lee S,et al.Effect of human umbilical cord blood-derived mesenchymal stem cells in a cirrhotic rat model.Liver Int,2009,29(6):898-909.
8. Zheng LH,Chu JD,Shi YQ,et al.Bone marrow-derived stem cells ameliorate hepatic fibrosis by down-regulating interleukin-17.Cell Biosci,2013,3(2):46.
9. Peng L,Xie DY,Lin BL,et al.Autologous bone marrow mesenchymal stem cell transplantation in liver failure patients caused by hepatitis B:short-term and long-term outcomes.Hepatology,2011,54(3):820-828.
10. 薛改,李建立,刘建芳,等.PKH26标记示踪人脐带间充质干细胞在肝硬化大鼠体内的迁移.中华肝脏病杂志,2014,22(12):910-914.
11. 郑盛,殷芳,肖琼怡,等.脐带间充质干细胞移植对急性肝功能衰竭的修复及移植途径的优化.肝脏,2014,19(12):918-923.
12. Rubio D,Garcia S,Paz MF,et al.Molecular characterization of spontaneous mesenchymal stem cell transformation.PLoS One,2008,3(1):e1398.
13. 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.
14. Wang H,Wu G,Park HJ,et al.Protective effect of Phellinus linteus polysaccharide extracts against thioacetamide-induced liver fibrosis in rats:a proteomics analysis.Chin Med,2012,7(1):23.
15. 中华医学会感染病学分会肝衰竭与人工肝学组,中华医学会肝病学分会重型肝病与人工肝学组.肝衰竭诊治指南(2012年版).中华肝脏病杂志,2013,21(3):177-183.
16. Dianat N,Steichen C,Vallier L,et al.Human pluripotent stem cells for modelling human liver diseases and cell therapy.Curr Gene Ther,2013,13(2):120-132.
17. Zhou R,Li Z,He C,et al.Human umbilical cord mesenchymal stem cells and derived hepatocyte-like cells exhibit similar therapeutic effects on an acute liver failure mouse model.PLoS One,2014,9(8):e104392.
18. Li L,Xie T.Stem cell niche:structure and function.Annu Rev Cell Dev Biol,2005,21(3):605-631.
19. Lee CC,Kim KD.Stem cell microenvironment as a potential therapeutic target.Regen Med,2012,7(1):3-5.
20. 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.
21. Tan Y,Xiao EH,Xiao LZ,et al.VEGF₁₆₅ expressing bone marrow mesenchymal stem cells differentiate into hepatocytes under HGF and EGF induction in vitro.Cytotechnology,2012,64:635-647.
22. Zhang S,Chen L,Liu T,et al.Human umbilical cord matrix stem cells efficiently rescue acute liver failure through paracrine effects rather than hepatic differentiation.Tissue Eng Part A,2012,18(13-14):1352-1364.
23. Hosny S,Abdel-Rahman NZ,Eman M,et al.Peripheral vein infusion of autologous mesenchymal stem cells in Egyptian HCV-positive patients with end-stage liver disease.Stem Cell Res Ther,2014,5(3):70.
24. Liang L,Ma T,Chen W,et al.Therapeutic potential and related signal pathway of adipose-derived stem cell transplantation for rat liver injury.Hepatology Res,2009,39(8):822-832.
25. 何宏亮,高志良.间充质干细胞的肝细胞分化及其治疗终末期肝病的临床研究进展.中华肝脏病杂志,2014,22(5):397-400.

Chinese Journal of Reparative and Reconstructive Surgery

DIFFERENTIATION OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS INTO HEPATOCYTES INDUCED BY RAT FIBROTIC LIVER TISSUE EXTRACTS

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