Isolation and identification of human adipose tissue-derived mesenchymal stem cells and differentiation into hepatocyte-like cells in vitro

doi:10.3969/j.issn.1006-7795.2014.05.018

Abstract

Abstract:

Objective To explore the method of isolation, culture and identification of human adipose-derived mesenchymal stem cells (hADSCs) and induce the hADSCs into hepatocyte-like cells in vitro.Methods hADSCs was obtained by collagenase I digestion centrifugation for primary culture and the stem cell surface antigens including CD13, CD73, CD34, CD45 and HLA-DR were determined by flow cytometry (FCM). The cells were induced into adipocytes and osteocytes by using induction medium. The hADSCs were induced by using the two-stage cytokine induction method and then the morphological changes were observed. The liver cell-related genes such as AFP, CK18, CK19 and so on were tested in the hepatocyte-like cells by RT-PCR. The expression of the above-mentioned molecules of the hepatocyte-like cells were tested by cell immunohisto- chemical method and glycogen synthesis function of the hepatocyte-like cells was tested by PAS staining. Results hADSCs obtained in the digestion centrifugation are of the uniform size and are fusiform fibroblast-like cells, their proliferation was good. Cells could survive and kept undifferentiated for a long time in vitro. Flow cytometry showed that cells highly expressed CD13, CD73, but the expression of CD34, CD45 and HLA-DR were low; hADSCs could be induced into adipocytes and osteocytes. After being induced into hepatocyte-like cells, the human adipose-derived cells, cellular morphology gradually became polygonal or round, showed high expression of liver cell-related gene, including alpha-fetal protein (AFP), albumin (ALB), and cytochrome CYP3A4. Besides, the obtained hepatocyte-like cells also expressed liver cell-related proteins incuding HepPar-1, AFP, CK18, CK19. PAS staining showed that the cytoplasm of hepatocyte-like cells were in red. Conclusion hADSCs could be isolated and obtained using digestion centrifugation, the obtained hADSCs could be repeatedly passaged and could survive for long time, and could be induced into functional hepatocyte-like cells in vitro, which provide experimental basis for ADSCs as seed cells of cell therapy, bioartificial liver and liver tissue engineering.

Key words: human adipose tissue-derived mesenchymal stem cells (hADSCs), isolation, identification, differentiation, hepatocyte-like cells

CLC Number:

R329

Zhang Yunwei, Xu Lijuan, Wang Shufang, Yan Li. Isolation and identification of human adipose tissue-derived mesenchymal stem cells and differentiation into hepatocyte-like cells in vitro[J]. Journal of Capital Medical University, 2014, 35(5): 608-616.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://journal03.magtech.org.cn/Jweb_sdykdxxb/EN/10.3969/j.issn.1006-7795.2014.05.018

https://journal03.magtech.org.cn/Jweb_sdykdxxb/EN/Y2014/V35/I5/608

References

[1] Liu J, Fan D. Hepatitis B in China[J]. Lancet, 2007, 369(9573):1582-1583.
[2] Dhawan A, Puppi J, Hughes R D, et al. Human hepatocyte transplantation: current experience and future[J]. Nat Rev Gastroenterol Hepatol,2010,7(5):288-298.
[3] Okura H, Komoda H, Saga A, et al. Properties of hepatocyte-like cell clusters from human adipose tissue-derived mesenchymal stem cells[J]. Tissue Eng Part C Methods, 2010,16(4):761-770.
[4] Zaret KS, Grompe M. Generation and regeneration of cells of the liver and pancreas[J]. Science,2008,322(5907):1490-1494.
[5] Terry C, Hughes R D, Mitry R R, et al. Cryopreservation-induced nonattachment of human hepatocytes: role of adhesion molecules[J].Cell Transplant,2007,16(6):639-647.
[6] Thomson J A, Itskovitz-Eldor J, Shapiro S S, et al. Embryonic stem cell lines derived from human blastocysts[J].Science,1998,282(5391):1145-1147.
[7] Gallicano G I, Mishra L. Hepatocytes from induced pluripotent stem cells:a giant leap forward for hepatology[J]. Hepatology,2010,51(1):20-22.
[8] Lee K D, Kuo T K, WhangPeng J, et al. In vitro hepatic differentiation of human mesench-ymal stem cells[J]. Hepatology,2004,40(6):1275-1284.
[9] Snykers S, Vanhaecke T, Papeleu P, et al. Sequential exposure to cytokines reflecting-embryogenesis: the key for in vitro differentiation of adult bone marrow stem cells into functional hepatocyte-like cells[J]. Toxicol Sci,2006,94(2):330-341,discussion:235-339.
[10] Lee M J, Jung J, Na K H, et al. Anti-fibrotic effect of chorionic plate-derived mesenchymal stem cells isolated from human placenta in a rat model of CCl(4)-injured liver: potential application to the treatment of hepatic diseases[J].J Cell Biochem,2010,111(6):1453-1463.
[11] Shin K S, Lee H J, Jung J, et al. Culture and in vitro hepatogenic differentiation of placenta-derived stem cells, using placental extract as an alternative to serum[J]. Cell Prolif,2010,43(5):435-444.
[12] Lagasse E, Connors H, Al-Dhalimy M,et al. Purified hematopoietic stem cellscandifferentiate into hepatocytes in vivo[J]. Nat Med,2000,6(11):1229-1234.
[13] Zuk P A, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies[J].Tissue Eng,2001,7(2):211-228.
[14] Zuk P A, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells[J]. Mol Biol Cell,2002,13(12):4279-4295.
[15] di Bonzo L V, Ferrero I, Cravanzola C, et al. Human mesenchymal stem cells as a twoedged sword in hepatic regenerative medicine: engraftment and hepatocyte differentiation versus profibrogenic potential[J]. Gut,2008,57(2):223-231.
[16] Chen Y, Dong X J, Zhang G R, et al. In vitro differentiation of mouse bone marrow stromal stem cells into hepatocytes induced by conditioned culture medium of hepatocytes[J]. J Cell Biochem,2007, 102(1): 52-63.
[17] Matsunaga T, Sakamaki S, Kohgo Y, et al. Recombinant human granulocyte Colony-stimulating factor can mobilize sufficient amounts of peripheral blood stem cells in healthy volunteers for allogeneic transplantation[J]. Bone Marrow Transplant,1993,11(2):103-108.
[18] Kasai M, Kiyama Y, Kawamura A. Application of peripheral blood stem cells(PBSC) mobilized by recombinant human granulocyte colony stimulating factor for allogeneic PBSC transplantation and the comparison of allogeneic PBSC transplantation and bone marrow transplantation[J]. Transfus Apher Sci,2002,26(2):121-127.
[19] Conget P A, Min guell J J. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells[J].J Cell Physiol,1999,181(1):67-73.
[20] Seo M J, Suh S Y, Bae Y C, et al. Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo. Biochem[J]. Biophys Res Commun,2005,328: 258-264.
[21] Safford K M, Hicok K C, Safford S D, et al. Neurogenic differentiation of murine and human adipose-derived stromal cells[J]. BiochemBiophys Res Commun,2002,294:371-379.
[22] Lee R H, Kim B, Choi Iet al. Characterization and expression analysis of mesenchymal stem cells from human bone mar-row and adipose tissue[J]. Cell Physiol Biochem,2004,14(4-6):311-324.
[23] Katz A J, Tholpady A, Tholpady S S, et al.Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells[J]. Stem Cells,2005,23(3):412-423.
[24] Gronthos S, Franklin D M, Leddy H A, et al. Surface protein characterization of human adipose tissue-derived stro-mal cells[J]. J Cell Physiol,2001,189(1):54-63.
[25] Kern S, Eichler H, Stoeve J, et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood or adi-pose tissue[J]. Stem Cells,2006,24(5):1294-1301.
[26] Strem B M, Hicok K C, Zhu M, et al. Multipotential differentiation of adipose tissue-derived stem cells[J]. Keio J Med,2005,54(3):132-141.
[27] Dicker A, Le Blanc K, Astrom G,et al. Functional studies of mesenchymal stem cells derived from adult human adipose tissue[J]. Exp Cell Res,2005,308(2):283-290.
[28] Im G I, Shin Y W, Lee K B. Do adipose tissue-derived mesenchymal stem cells have the same osteogenic and chondrogenic potential as bone marrow-derived cells?[J]. Osteoarthritis Cartilage,2005,13(10):845-853.
[29] Safford K M, Hicok K C, Safford S D, et al. Neurogenic differentiation of murine and human adipose-derived stromal cells[J]. BiochemBiophys Res Commun,2002,294(2):371-379.
[30] Secco M, Moreira Y B, Zucconi E, et al. Gene expression profile of mesenchymal stem cells from paired umbilical cord units: cord is different from blood[J]. Stem Cell Rev, 2009,5(4):387-401.
[31] 王晓辉,惠玲,杨霄鹏,等.人脐带间充质干细胞体外培养、鉴定及其诱导分化的研究[J]. 解放军医药杂志,2013,25(10):11-15,30.
[32] Ruhnke M, Ungefroren H, Nussler A, et al. Differentiation of in vitro modified human peripheral blood monocytes into hepatocytelike and pancreatic isletlike cells[J]. Gastroenterology, 2005,128(7):1774-1786.
[33] Izadpanah R, Trygg C, Patel B, et al. Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue[J]. J Cell Biochem,2005,99(5): 1285-1297.
[34] He Z, Feng M. Activation, isolation, identification and culture of hepatic stem cells from porcine liver tissues[J]. Cell Prolif,2011,44(6):558-566.
[35] Yan L, Han Y, Wang J, et al. Peripheral blood monocytes from patients with HBV related decompensated liver cirrhosis can differentiate into functional hepatocytes[J]. Am J Hematol,2007,82(11):949-954.
[36] 尹鹏,梁庆威.外源性骨形态发生蛋白7联合血管内皮生长因子诱导大鼠脂肪干细胞向成骨细胞分化的实验研究[J]. 中国医科大学学报,2011,(10):897-901.
[37] Banas A, Teratani T, Yamamoto Y, et al. Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes[J]. Hepatology, 2007, 46(1): 219-228.
[38] Aurich H, Sgodda M, Kaltwasser P,et al. Hepatocyte differentiation of mesenchymal stem cells from human adipose tissue in vitro promotes hepatic integration in vivo[J]. Gut,2009,58(4):570-581.
[39] Fiegel H C, Lioznov M V, Cortes-Dericks L, et al. Liver-Specific gene expression in cultured human hematopoietic stem cells[J]. Stem Cells,2003, 21(1):98-104.
[40] Takeda M, Yamamoto M, Isoda K, et al.Availability of bone marrow stromal cells in three-dimensional coculture with hepatocytes and transplantation into liver damaged mice[J]. J Biosci Bioeng,2005,100(1):77-81.
[41] Wang P P, Wang J H, Yan Z P, et al. Expression of hepatocyte-like phenotypes in bone marrow stromal cells after HGF induction[J].Biochem Biophys Res Commun,2004,320(3):712-716.
[42] 张刚庆,方驰华,池达智.肝细胞生长因子诱导骨髓间充质干细胞向肝细胞分化的实验研究[J].中华外科杂志,2005,43(11):716-720.
[43] Schwartz R E, Reyes M, Koodie L, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells[J]. J Clin Invest, 2002, 109(10): 1291-1302.