TRANSPLANTATION OF HUMAN AMNIOTIC EPITHELIAL CELLS IN TREATMENT OF HEPATIC FIBROSIS IN IMMUNE RATS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

XUNQuan ¹ ,  LUOHongwu ² , HUANGXiangjun ² , HUANGFeizhou ²

1. First Department of General Surgery, the People's Hospital of Liuyang, Liuyang Hunan, 410300, P. R. China;
2. First Department of General Surgery, Xiangya Third Hospital, Central South University;

Corresponding?author：

LUOHongwu, Email: xun155067@163.com

Keywords：

Human amniotic epithelial cells; Cell transplantation; Hepatic fibrosis; Human Alu gene repeat sequence; Rat

DOI：

10.7507/1002-1892.20150075

Video：

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Objective To observe the survival, migration, and effect of human amniotic epithelial cells (hAECs) on hepatic fibrosis in immune rats so as to provide the experimental theory for the clinical treatment with hAECs. Methods Sixty-four 10-week-old male Sprague Dawley rats (weighing, 220-280 g) were randomly divided into 4 groups, sixteen rats in each group. Rat hepatic fibrosis model was induced in groups A, B, and C; hepatic fibrosis rats were injected with 4×106 hAECs in group A, and with normal saline in group B, and no treatment was given in group C; group D served as control group. After 2 weeks of transplantation, the expression of human Alu gene repeat sequence was detected by DNA-PCR method and human leucocyte antigen G (HLA-G) by immunohistochemical staining in heart, liver, spleen, kidney, lung, and brain in group A, and then the percentage of positive expression was compared between organs except spleen. Semi-quantitative analysis was done for liver fibrosis with HE staining according to Chevallier semi-quantitative histological liver fibrosis scoring system, and immunohistochemical staining for TGF-β1 was used to record immunohistochemical score (ISH), the concentrations of aspartate transaminase (AST), alanine aminotransferase (ALT), and albumin (ALB) were determined to analyze hepatic fibrosis. Results Alu gene repeat sequence and HLA-G could be detected in liver, heart, brain, lung, and kidney in group A, the percentage of positive expression in the liver was significantly higher than that in the other organs (P<0.05). The histological semi-quantitative score of group A (10.47±3.20) was significantly lower than that of groups B and C[(13.84±3.46) and (13.85±3.16)](P<0.05), but no significant difference was found between groups B and C (P>0.05). The ISH scores in groups A, B, C, and D were 3.60±1.50, 5.38±2.60, 5.50±2.40, and 1.87±1.36, respectively; groups A, B, and C were significantly higher than group D, and group A was significantly lower than groups B and C (P<0.05), but there was no significant difference between groups B and C (P>0.05). The concentrations of ALT and AST in groups A, B, and C were significantly higher than those in group D, and group A was significantly lower than groups B and C (P<0.05), but there was no significant difference between groups B and C (P>0.05). The concentration of ALB in groups A, B, and C was significantly lower than that in group D, and group A was significantly higher than groups B and C (P<0.05), but there was no significant difference between groups B and C (P>0.05). Conclusion hAECs can survive in immune rats by intrasplenic transplantation and migrate to liver, heart, brain, lung, and kidney, and the liver shows the largest migration. The transplantation of hAECs in immune rat with cirrhosis can alleviate hepatic fibrosis and improve the serum indexes of liver function.

Citation： XUNQuan, LUOHongwu, HUANGXiangjun, HUANGFeizhou. TRANSPLANTATION OF HUMAN AMNIOTIC EPITHELIAL CELLS IN TREATMENT OF HEPATIC FIBROSIS IN IMMUNE RATS. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(3): 356-363. doi: 10.7507/1002-1892.20150075 Copy

1.	Jiao J, Friedman SL, Aloman C. Hepatic fibrosis. Curr Opin Gastroenterol, 2009, 25(3):223-229.
2.	Bilir BM, Guinette D, Karrer DA, et al. Hepatocyte transplantation in acute liver failure. Liver Transpl, 2000, 6(1):32-40.
3.	Sancho-Bru P, Najimi M, Caruso M, et al. Stem and progenitor cells for liver repopulation:can we standardise the process from bench to bedside? Gut, 2009, 58(4):594-603.
4.	Nussler A, Konig S, Ott M, et al. Present status and perspectives of cell-based therapies for liver diseases. J Hepatol, 2006, 45(1):144-159.
5.	Fang CH, Jin J, Joe JH, et al. In vivo differentiation of human amniotic epithelial cells into cardiomyocyte-like cells and cell transplantation effect on myocardial infarction in rats:comparison with cord blood and adipose tissue-derived mesenchymal stem cells. Cell Transplant, 2012, 21(8):1687-1696.
6.	Liu YH, Vaghjiani V, Tee JY, et al. Amniotic epithelial cells from the human placenta potently suppress a mouse model of multiple sclerosis. PLoS One, 2012, 7(4):e35758.
7.	Moodley Y, Ilancheran S, Samuel C, et al. Human amnion epithelial cell transplantation abrogates lung fibrosis and augments repair. Am J Respir Crit Care Med, 2010, 182(5):643-651.
8.	Sankar V, Muthusamy R. Role of human amniotic epithelial cell transplantation in spinal cord injury repair research. Neuroscience, 2003, 118(1):11-17.
9.	Yawno T, Schuilwerve J, Moss TJ, et al. Human amnion epithelial cells reduce fetal brain injury in response to intrauterine inflammation. Dev Neurosci, 2013, 35(2-3):272-282.
10.	Kim TH, Park YW, Ahn JS, et al. Effects of conditioned media from human amniotic epithelial cells on corneal alkali injuries in rabbits. J Vet Sci, 2013, 14(1):61-67.
11.	Manuelpillai U, Tchonque J, Lourensz D, et al. Transplantation of human amnion epithelial cells reduces hepatic fibrosis in immunocompetent CCl(4)-treated mice. Cell Transplant, 2010, 19(9):1157-1168.
12.	Manuelpillai U, Lourensz D, Vaghjiani V, et al. Human amniotic epithelial cell transplantation induces markers of alternative macrophage activation and reduces established hepatic fibrosis. PLoS One, 2012, 7(6):e38631.
13.	Lanthier N, Rubbia-Brandt L, Spahr L. Liver progenitor cells and therapeutic potential of stem cells in human chronic liver diseases. Acta Gastroenterol Belg, 2013, 76(1):3-9.
14.	羅宏武, 黃湘俊, 黃飛舟, 等. 人羊膜上皮細胞橫向分化及脾內移植的初步研究. 中國修復重建外科雜志, 2011, 25(2):144-148.
15.	Marongiu F, Gramignoli R, Dorko K, et al. Hepatic differentiation of amniotic epithelial cells. Hepatology, 2011, 53(5):1719-1729.
16.	劉紅梅, 張玉玲, 康金玲, 等. 復合因素誘導大鼠肝硬化模型的建立. 寧夏醫科大學學報, 2012, 34(3):310-312.
17.	羅宏武, 尋權, 黃湘俊, 等. 人羊膜上皮細胞移植急性肝損傷小鼠的定量效果分析. 中國組織工程研究, 2013, 17(32):5834-5839.
18.	Chevallier M, Guerret S, Chossegros P, et al. A histological semiquantitative scoring system for evaluation of hepatic fibrosis in needle liver biopsy specimens:comparison with morphometric studies. Hepatology, 1994, 20(2):349-355.
19.	Soslow RA, Dannenberg AJ, Rush D, et al. COX-2 is expressed in human pulmonary, colonic, and mammary tumors. Cancer, 2000, 89(12):2637-2645.
20.	Fernandes M, Sridhar MS, Sangwan VS, et al. Amniotic membrane transplantation for ocular surface reconstruction. Cornea, 2005, 24(6):643-653.
21.	Kakishita K, Nakao N, Sakuragawa N, et al. Implantation of human amniotic epithelial cells prevents the degeneration of nigral dopamine neurons in rats with 6-hydroxydopamine lesions. Brain Res, 2003, 980(1):48-56.
22.	Wei JP, Zhang TS, Kawa S, et al. Human amnion-isolated cells normalize blood glucose in streptozotocin-induced diabetic mice. Cell Transplant, 2003, 12(5):545-552.
23.	Hou Y, Huang Q, Liu T, et al. Human amnion epithelial cells can be induced to differentiate into functional insulin-producing cells. Acta Biochim Biophys Sin (Shanghai), 2008, 40(9):830-839.
24.	Sant'Anna LB, Cargnoni A, Ressel L, et al. Amniotic membrane application reduces liver fibrosis in a bile duct ligation rat model. Cell Transplant, 2011, 20(3):441-453.
25.	Lysy PA, Campard D, Smets F, et al. Stem cells for liver tissue repair:current knowledge and perspectives. World J Gastroenterol, 2008, 14(6):864-875.
26.	Evangelista M, Soncini M, Parolini O. Placenta-derived stem cells:new hope for cell therapy? Cytotechnology, 2008, 58(1):33-42.
27.	Murphy S, Rosli S, Acarya R, et al. Amnion epithelial cell isolation and characterization for clinical use. Curr Protoc Stem Cell Biol, 2010, Chapter 1:Unit 1E. 6.
28.	Zhou K, Koike C, Yoshida T, et al. Establishment and characterization of immortalized human amniotic epithelial cells. Cell Reprogram, 2013, 15(1):55-67.
29.	Bossard P, Zaret KS. GATA transcription factors as potentiators of gut endoderm differentiation. Development, 1998, 125(24):4909-4917.
30.	Takashima S, Ise H, Zhao P, et al. Human amniotic epithelial cells possess hepatocyte-like characteristics and functions. Cell Struct Funct, 2004, 29(3):73-84.
31.	Miki T, Lehmann T, Cai H, et al. Stem cell characteristics of amniotic epithelial cells. Stem Cells, 2005, 23(10):1549-1559.
32.	Miki T, Marongiu F, Ellis EC, et al. Production of hepatocyte-like cells from human amnion. Methods Mol Biol, 2009, 481:155-168.
33.	羅宏武, 黃湘俊, 黃飛舟, 等. 人羊膜上皮細胞具有向肝細胞樣細胞分化的特性. 中華肝臟病雜志, 2011, 19(8):623-624.
34.	Huang X, Luo H, Huang FZ, et al. Labeling and tracking human amniotic epithelial cells with green fluorescent protein in an adeno-associated virus vector. Cell Mol Biol Lett, 2011, 16(2):201-213.
35.	Bailo M, Soncini M, Vertua E, et al. Engraftment potential of human amnion and chorion cells derived from term placenta. Transplantation, 2004, 78(10):1439-1448.
36.	Cargnoni A, Gibelli L, Tosini A, et al. Transplantation of allogeneic and xenogeneic placenta-derived cells reduces bleomycin-induced lung fibrosis. Cell Transplant, 2009, 18(4):405-422.
37.	Carosella ED, Favier B, Rouas-Freiss N, et al. Beyond the increasing complexity of the immunomodulatory HLA-G molecule. Blood, 2008, 111(10):4862-4870.
38.	King A, Burrows TD, Hiby SE, et al. Surface expression of HLA-C antigen by human extravillous trophoblast. Placenta, 2000, 21(4):376-387.
39.	Bahri R, Hirsch F, Josse A, et al. Soluble HLA-G inhibits cell cycle progression in human alloreactive T lymphocytes. J Immunol, 2006, 176(3):1331-1339.
40.	Parolini O, Alviano F, Bagnara GP, et al. Concise review:isolation and characterization of cells from human term placenta:outcome of the first international Workshop on Placenta Derived Stem Cells. Stem Cells, 2008, 26(2):300-311.
41.	Kamimura R, Ishii T, Sasaki N, et al. Comparative study of transplantation of hepatocytes at various differentiation stages into mice with lethal liver damage. Cell Transplant, 2012, 21(11):2351-2362.
42.	Li C, Kong Y, Wang H, et al. Homing of bone marrow mesenchymal stem cells mediated by sphingosine 1-phosphate contributes to liver fibrosis. J Hepatol, 2009, 50(6):1174-1183.
43.	Yang X, Song L, Wu N, et al. An experimental study on intracerebroventricular transplantation of human amniotic epithelial cells in a rat model of Parkinson's disease. Neurol Res, 2010, 32(10):1054-1059.
44.	Liu T, Wu J, Huang Q, et al. Human amniotic epithelial cells ameliorate behavioral dysfunction and reduce infarct size in the rat middle cerebral artery occlusion model. Shock, 2008, 29(5):603-611.
45.	Parekkadan B, van Poll D, Megeed Z, et al. Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells. Biochem Biophys Res Commun, 2007, 363(2):247-252.
46.	Ueta M, Kweon MN, Sano Y, et al. Immunosuppressive properties of human amniotic membrane for mixed lymphocyte reaction. Clin Exp Immunol, 2002, 129(3):464-470.

1. Jiao J, Friedman SL, Aloman C. Hepatic fibrosis. Curr Opin Gastroenterol, 2009, 25(3):223-229.
2. Bilir BM, Guinette D, Karrer DA, et al. Hepatocyte transplantation in acute liver failure. Liver Transpl, 2000, 6(1):32-40.
3. Sancho-Bru P, Najimi M, Caruso M, et al. Stem and progenitor cells for liver repopulation:can we standardise the process from bench to bedside? Gut, 2009, 58(4):594-603.
4. Nussler A, Konig S, Ott M, et al. Present status and perspectives of cell-based therapies for liver diseases. J Hepatol, 2006, 45(1):144-159.
5. Fang CH, Jin J, Joe JH, et al. In vivo differentiation of human amniotic epithelial cells into cardiomyocyte-like cells and cell transplantation effect on myocardial infarction in rats:comparison with cord blood and adipose tissue-derived mesenchymal stem cells. Cell Transplant, 2012, 21(8):1687-1696.
6. Liu YH, Vaghjiani V, Tee JY, et al. Amniotic epithelial cells from the human placenta potently suppress a mouse model of multiple sclerosis. PLoS One, 2012, 7(4):e35758.
7. Moodley Y, Ilancheran S, Samuel C, et al. Human amnion epithelial cell transplantation abrogates lung fibrosis and augments repair. Am J Respir Crit Care Med, 2010, 182(5):643-651.
8. Sankar V, Muthusamy R. Role of human amniotic epithelial cell transplantation in spinal cord injury repair research. Neuroscience, 2003, 118(1):11-17.
9. Yawno T, Schuilwerve J, Moss TJ, et al. Human amnion epithelial cells reduce fetal brain injury in response to intrauterine inflammation. Dev Neurosci, 2013, 35(2-3):272-282.
10. Kim TH, Park YW, Ahn JS, et al. Effects of conditioned media from human amniotic epithelial cells on corneal alkali injuries in rabbits. J Vet Sci, 2013, 14(1):61-67.
11. Manuelpillai U, Tchonque J, Lourensz D, et al. Transplantation of human amnion epithelial cells reduces hepatic fibrosis in immunocompetent CCl(4)-treated mice. Cell Transplant, 2010, 19(9):1157-1168.
12. Manuelpillai U, Lourensz D, Vaghjiani V, et al. Human amniotic epithelial cell transplantation induces markers of alternative macrophage activation and reduces established hepatic fibrosis. PLoS One, 2012, 7(6):e38631.
13. Lanthier N, Rubbia-Brandt L, Spahr L. Liver progenitor cells and therapeutic potential of stem cells in human chronic liver diseases. Acta Gastroenterol Belg, 2013, 76(1):3-9.
14. 羅宏武, 黃湘俊, 黃飛舟, 等. 人羊膜上皮細胞橫向分化及脾內移植的初步研究. 中國修復重建外科雜志, 2011, 25(2):144-148.
15. Marongiu F, Gramignoli R, Dorko K, et al. Hepatic differentiation of amniotic epithelial cells. Hepatology, 2011, 53(5):1719-1729.
16. 劉紅梅, 張玉玲, 康金玲, 等. 復合因素誘導大鼠肝硬化模型的建立. 寧夏醫科大學學報, 2012, 34(3):310-312.
17. 羅宏武, 尋權, 黃湘俊, 等. 人羊膜上皮細胞移植急性肝損傷小鼠的定量效果分析. 中國組織工程研究, 2013, 17(32):5834-5839.
18. Chevallier M, Guerret S, Chossegros P, et al. A histological semiquantitative scoring system for evaluation of hepatic fibrosis in needle liver biopsy specimens:comparison with morphometric studies. Hepatology, 1994, 20(2):349-355.
19. Soslow RA, Dannenberg AJ, Rush D, et al. COX-2 is expressed in human pulmonary, colonic, and mammary tumors. Cancer, 2000, 89(12):2637-2645.
20. Fernandes M, Sridhar MS, Sangwan VS, et al. Amniotic membrane transplantation for ocular surface reconstruction. Cornea, 2005, 24(6):643-653.
21. Kakishita K, Nakao N, Sakuragawa N, et al. Implantation of human amniotic epithelial cells prevents the degeneration of nigral dopamine neurons in rats with 6-hydroxydopamine lesions. Brain Res, 2003, 980(1):48-56.
22. Wei JP, Zhang TS, Kawa S, et al. Human amnion-isolated cells normalize blood glucose in streptozotocin-induced diabetic mice. Cell Transplant, 2003, 12(5):545-552.
23. Hou Y, Huang Q, Liu T, et al. Human amnion epithelial cells can be induced to differentiate into functional insulin-producing cells. Acta Biochim Biophys Sin (Shanghai), 2008, 40(9):830-839.
24. Sant'Anna LB, Cargnoni A, Ressel L, et al. Amniotic membrane application reduces liver fibrosis in a bile duct ligation rat model. Cell Transplant, 2011, 20(3):441-453.
25. Lysy PA, Campard D, Smets F, et al. Stem cells for liver tissue repair:current knowledge and perspectives. World J Gastroenterol, 2008, 14(6):864-875.
26. Evangelista M, Soncini M, Parolini O. Placenta-derived stem cells:new hope for cell therapy? Cytotechnology, 2008, 58(1):33-42.
27. Murphy S, Rosli S, Acarya R, et al. Amnion epithelial cell isolation and characterization for clinical use. Curr Protoc Stem Cell Biol, 2010, Chapter 1:Unit 1E. 6.
28. Zhou K, Koike C, Yoshida T, et al. Establishment and characterization of immortalized human amniotic epithelial cells. Cell Reprogram, 2013, 15(1):55-67.
29. Bossard P, Zaret KS. GATA transcription factors as potentiators of gut endoderm differentiation. Development, 1998, 125(24):4909-4917.
30. Takashima S, Ise H, Zhao P, et al. Human amniotic epithelial cells possess hepatocyte-like characteristics and functions. Cell Struct Funct, 2004, 29(3):73-84.
31. Miki T, Lehmann T, Cai H, et al. Stem cell characteristics of amniotic epithelial cells. Stem Cells, 2005, 23(10):1549-1559.
32. Miki T, Marongiu F, Ellis EC, et al. Production of hepatocyte-like cells from human amnion. Methods Mol Biol, 2009, 481:155-168.
33. 羅宏武, 黃湘俊, 黃飛舟, 等. 人羊膜上皮細胞具有向肝細胞樣細胞分化的特性. 中華肝臟病雜志, 2011, 19(8):623-624.
34. Huang X, Luo H, Huang FZ, et al. Labeling and tracking human amniotic epithelial cells with green fluorescent protein in an adeno-associated virus vector. Cell Mol Biol Lett, 2011, 16(2):201-213.
35. Bailo M, Soncini M, Vertua E, et al. Engraftment potential of human amnion and chorion cells derived from term placenta. Transplantation, 2004, 78(10):1439-1448.
36. Cargnoni A, Gibelli L, Tosini A, et al. Transplantation of allogeneic and xenogeneic placenta-derived cells reduces bleomycin-induced lung fibrosis. Cell Transplant, 2009, 18(4):405-422.
37. Carosella ED, Favier B, Rouas-Freiss N, et al. Beyond the increasing complexity of the immunomodulatory HLA-G molecule. Blood, 2008, 111(10):4862-4870.
38. King A, Burrows TD, Hiby SE, et al. Surface expression of HLA-C antigen by human extravillous trophoblast. Placenta, 2000, 21(4):376-387.
39. Bahri R, Hirsch F, Josse A, et al. Soluble HLA-G inhibits cell cycle progression in human alloreactive T lymphocytes. J Immunol, 2006, 176(3):1331-1339.
40. Parolini O, Alviano F, Bagnara GP, et al. Concise review:isolation and characterization of cells from human term placenta:outcome of the first international Workshop on Placenta Derived Stem Cells. Stem Cells, 2008, 26(2):300-311.
41. Kamimura R, Ishii T, Sasaki N, et al. Comparative study of transplantation of hepatocytes at various differentiation stages into mice with lethal liver damage. Cell Transplant, 2012, 21(11):2351-2362.
42. Li C, Kong Y, Wang H, et al. Homing of bone marrow mesenchymal stem cells mediated by sphingosine 1-phosphate contributes to liver fibrosis. J Hepatol, 2009, 50(6):1174-1183.
43. Yang X, Song L, Wu N, et al. An experimental study on intracerebroventricular transplantation of human amniotic epithelial cells in a rat model of Parkinson's disease. Neurol Res, 2010, 32(10):1054-1059.
44. Liu T, Wu J, Huang Q, et al. Human amniotic epithelial cells ameliorate behavioral dysfunction and reduce infarct size in the rat middle cerebral artery occlusion model. Shock, 2008, 29(5):603-611.
45. Parekkadan B, van Poll D, Megeed Z, et al. Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells. Biochem Biophys Res Commun, 2007, 363(2):247-252.
46. Ueta M, Kweon MN, Sano Y, et al. Immunosuppressive properties of human amniotic membrane for mixed lymphocyte reaction. Clin Exp Immunol, 2002, 129(3):464-470.

Chinese Journal of Reparative and Reconstructive Surgery

TRANSPLANTATION OF HUMAN AMNIOTIC EPITHELIAL CELLS IN TREATMENT OF HEPATIC FIBROSIS IN IMMUNE RATS

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