In vitro differentiation of rat mesenchymal stem cells to hepatocyte lineage

Document Type: Original Article

Authors

1 Department of Biology, Faculty of Biological Sciences, North Branch of Islamic Azad University, Tehran, Iran

2 Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran

3 Department of Biology, Faculty of Basic Science, Sciences and Researches branch of Islamic Azad University, Tehran, Iran

4 Department of Anatomy, School of medicine, Iran University of Medical Sciences, Tehran, Iran

5 Department of Biology, Faculty of Science, University of Zabol, Zabol, Iran. Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran

Abstract

Objective(s): Mesenchyme is a type of undifferentiated loose connective tissue that is derived mostly from mesoderm. Recently, mesenchymal stem cells (MSCs), as adult stem cells (ASCs) able to divide into a variety of different cells, are of utmost importance for stem cell research. In this research, ability of the liver extract to induce differentiation of rat derived omentum tissue mesenchymal stem cells (rOT-MSCs) into hepatocyte cells (HCs) was investigated.
Materials and Methods: After isolation and confirmation of rOT-MSCs they were co-cultured with liver extract and hepatogenic differentiation was monitored. Expressions of mesenchymal stem cell markers were also analyzed via flow cytometry. Moreover, expressions of octamer-binding transcription factor-4 (Oct-4), Wilm's tumor suppressor gene-1 (WT-1), albumin (ALB), alpha fetoprotein (AFP), cytokeratin-18 (CK-18), and mRNAs were analyzed using RT-PCR on days 16, 18 and 21. ALB production was analyzed by immunocytochemistry and western blot. Furthermore, glycogen and urea production were determined via periodic acid-Schiff (PAS) staining and colorimetric assays respectively.
Results:The phenotypic characterization revealed the positive expressions of CD90, CD44 and negative expression of CD45 inrOT-MSCs. These cells also expressed mRNA of Oct-4 and WT-1 as markers of omentum tissue. Differentiated rOT-MSCs in presence of 6 µg/ml liver extract expressed ALB, AFP, CK-18, glycogen and urea as specific markers of HCs.
Conclusion: These observations suggest that liver extract is potentially able to induce differentiation of MSCs into hepatocyte lineage and can be considered an available source for imposing tissue healing on the damaged liver.

Keywords


1.Bellentani S, Tiribelli C. The spectrum of liver disease in the general population: Lessons from the Dionysos study. J Hepatol 2001; 35:531-537.

2.Alpdogan O. Advances in immune regulation in transplantation. Discov Med 2013; 15:150-159.

3.Ekser B, Ezzelarab M, Hara H, Van der Windt DJ, Wijkstrom M, Bottino R, et al. Clinical xenotransplantation: the next medical revolution? Lancet 2012; 18: 672-683.

4.Lacaille F. Liver transplantation and liver cell transplantation. Clin Res Hepatol Gastroenterol 2012; 36:304-307.

5.Yang XY, Wang W, Li X. In vitro hepatic differentiation of human endometrial stromal stem cells. In vitro Cell Dev Biol-Anim 2013; 50:162-170.

6.Jameson E. Cellular transplantation for liver diseases. Gastroen Res 2008; 1:8-13.

7.Puglisi MA, Saulnier N, Piscaglia AC, Tondi P, Agnes S, Gasbarrini A. Adipose tissue-derived mesenchymal stem cells and hepatic differentiation: old concepts and future perspectives. Eur Rev Med Pharm Sci 2011; 15:355-364.

8.Khoshdel A, Lotfi AS, Soleimani M, Daliri M, Mota A. Evaluation of biochemical markers in hepatocyte like cells differentiated from adipose derivation stem cells. World Appl Sci J 2012; 16:693-698.

9.Theise ND, Badve S, Saxena R, Henegariu O, Sell S, Crawford JM, et al. Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 2000; 31:235-240.

10.Shah S, Lowery E, Braun RK, Martin A, Huang N, Medina M, et al. Cellular basis of tissue regeneration by omentum. PLoS One 2012; 7:e38368.

11.Dhanasekaran M, Indumathi S, Harikrishnan R, Mishra R, Lissa RP, Rajkumar JS, et al. Human omentum fat-derived mesenchymal stem cells trans-differentiates into pancreatic islet-like cluster. Cell Biochem Funct 2013; 31:612-619.

12.Klopp AH, Zhang Y, Solley T, Amaya-Manzanares F, Marini F, Andreeff M, et al. Omental adipose tissue-derived stromal cells promote vascularization and growth of endometrial tumors. Clin Cancer Res 2012; 18:771-782.

13.Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000; 24:372-376.

14.Ochiya T. Advances of stem cell-based liver regeneration: science and the future. Nihon Rinsho 2011; 69:2156-2160.

15.Herrick SE, Mutsaers SE. The potential of mesothelial cells in tissue engineering and regenerative medicine applications. Int J Artif Organs 2007; 30:27-40.

16.Pancholi N,  Patel J, Gudehithlu K, Kraus M, Dunea G, Arruda J, et al. Culture of omentum-induced regenerating liver yielded hepatocyte-committed stem cells. Transl Res 2010; 156: 358-368.

17.Liu YX, Yue W, Ji L, Nan X. Pei XT. Production of erythroid cells from human embryonic stem cells by fetal liver cell extract treatment. BMC Dev Biol 2010; 10:85.

18.Margl W, Prehn C, Bogumil R, Rohring C, Suhre K, Adamski J. Procedure for tissue sample preparation and metabolite extraction for high-throughput targeted metabolomics. Metabolomics 2012; 8:133-142.

19.Liu XN, Yin QI, Zhang HAO, Zhang H, Zhu SJ, Wei YJ et al. Tissue extracts from infarcted myocardium of rats in promoting the differentiation of bone marrow stromal cells into cardiomyocyte-like cells. Biomed Environ Sci 2008; 21:110-117.

20.Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, et al. Horwitz Minimal criteria for defining multipotent mesenchymal stromal cells. Cytotherapy 2006; 8:315-317.

21.Kolf CM, Cho E, Tuan RS. Biology of adult mesenchymal stem cells: regulation of niche, self-renewal and differentiation. Arthritis Res Ther 2007; 9:204.

22.Chen KS, Chen WS, Chen HY, Lee CC, Kao HS, Chen YC, et al. Experience in primary culture of human peritoneal mesothelial cell. Chin J Physiol 2012; 55:274-283.

23.Bruce A, Flaat M, Gagliardi C, Patel B. Adipose-derived stem cells: Isolation, expansion and differentiation. Methods 2008; 45:115-120.

24.Miryounesi M, Nayernia K, Dianatpour M, Mansouri F, Modarressi MH. Co culture of mouse embryonic stem cells with sertoli cells promote                  in vitro generation of germ cells. Iran J Basic Med Sci 2013; 16:779-783.

25.Ramroodi N, Niazi AA, Sanadgol N, Ganjali Z, Sarabandi V. Evaluation of reactive epstein–barr virus (EBV) in Iranian patient with different subtypes of multiple sclerosis (MS). Brazil J Infect Dis 2013; 7:156-163.

26.Sanadgol N, Mostafaie A, Bahrami GH, Mansouri K, Ghanbari F, Bidmeshkipour A. Elaidic acid sustains LPS and TNF-α induced ICAM-1 and VCAM-I expression onhuman bone marrow endothelial cells. Clin Biochem 2010; 43:968-972.

27.Ogasawara K, Terada T, Asaka J, Katsura T, Inui K. Hepatocyte nuclear factor-4a regulates the human organic anion transporter 1 gene in the kidney. Am J Physiol Renal Physiol 2007; 292:1819-1826.

28.Gu N, Adachi T, Matsunaga T, Tsujimoto G, Ishihara A, Yasuda K, et al. HNF-1a participates in glucose regulation of sucrase-isomaltase gene expression in epithelial intestinal cells. Biochem Biophys Res Commun 2007; 353:617-622.

29.Kania G, Blyszczuk P, Jochbeim A, Ott M, Wobus AM. Generation of glycogen and albumin producing hepatocyte-like cells from embryonic stem cells. Biol Chen 2004; 385:943-953.

30.Kang XQ, Zang WJ, Song TS, Xu XL, Yu XJ, Li DL,              et al. Rat bone marrow mesenchymal stem cells differentiate into hepatocytes in vitro. World J Gastroenterol 2005; 11:3479-3484.

31.Lee KD. Applications of mesenchymal stem cells: an updated review. Chang Gung Med J 2008; 31:228-236.

32.Togel F, Hu Z, Weiss K, Isaac J, Lange C, Westenfelder C. Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms. Am J Renal Physiol 2005; 28931-42.

33.Stieger B, Peters R, Sidler MA, Meier PJ. Hepatocyte transplantation: potential of hepatocyte progenitor cells and bone marrow derived stem cells. Swiss Med Wkly 2006; 136:552-556.

34.Karaoz E, Aksoy A, Ayhan S, Sarıboyaci AE, Kaymaz F, Kasap M. Characterization of mesenchymal stem cells from rat bone marrow: ultrastructural properties, differentiation potential and mmunopheno- typic markers. Histochem Cell Biol 2013; DOI 10.1007/s00418-009-0629-6.

35.Prasajak P, Leeanansaksiri W. 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.

36.Chen KS, Chen WS, Chen HY, Lee CC, Kao HS, Chen YC, et al. Experience in primary culture of hman peritoneal mesothelial cell. Chin J Physiol 2012; 55:274-283.

37.Peng L, Zhu HP, Zheng YB, Xie C, Chong YT, Gao ZL. Mesenchymal stem cells differentiate into hepatocyte-like cells under different induction systems in hepatitis B patients with liver failure. Afr J Biotechnol 2011; 10:840-847.

38.          Snykers S, Kock JD, Rogiers V, Vanhaecke T. In Vitro differentiation of embryonic and adult stem cells into hepatocytes: state of the art. Stem Cells 2009; 27:577-605.

39.Mardani M, Kabiri A, Esfandiari E, Esmaeili A, Pourazar AA, Ansar M, et al.  The effect of platelet rich plasma on chondrogenic differentiation of human adipose derived stem cells in transwell culture. Iran J Basic Med Sci 2013; 16: 1163-1169.

40.Cho CH, Parashurama N, Park EY, Suganuma K,  Nahmias Y,  Tilles J, et al. Homogeneous differentiation of hepatocyte-like cells from embryonic stem cells: applications for the treatment of liver failure. FASEB J 2008; 22:898-909.

41.Nikoozad Z, Ghorbanian MT, Rezaei A. Comparison of the liver function and hepatic specific genes expression in cultured mesenchymal stem cells and hepatocytes. Iran J Basic Med Sci 2014; 17:27-33.

42.Wagers AJ, Weissman IL. Plasticity of adult stem cells. Cell 2004; 116:639-648.

43.Dhanasekaran M,  Indumathi S, Kanmani A, Poojitha R, Revathy KM, Rajkumar JS, et al. Surface antigenic profiling of stem cells from human omentum fat in comparison with subcutaneous fat and bone marrow. Cytotechnology 2012; 64:497-509.

44.Dimitroff CJ, Lee JY, Rafii S, Fuhlbrigge RC, Sackstein R. CD44 is a major E-selectin ligand on human hematopoietic progenitor cells. J Cell Biol 2001; 153:1277-1286.

45.Dominici M, Le Blanc K, Mueller I, Cortenbach IS, Marini FC, Krausc DS, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. Cytotherapy 2006; 8:315-317.