Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells into Insulin Producing Cells Using Minimal Differentiation Factors

Authors

1 Physiology Research Center, Kerman University of Medical Sciences, Kerman, Iran

2 Assistant Professor of Pathology, Stem Cell Research Center, Kerman University of Medical Sciences, Kerman, Iran

3 Assistant Professor, Department of Physiology, College of Sciences, Shiraz Branch, Islamic Azad University, Shiraz, Iran

Abstract

Background & Aims: Type 1 diabetes, or insulin-dependent diabetes, is an autoimmune disease in which pancreatic beta cells are destroyed by the immune system. Hitherto, no definite treatment has been found for this condition. Mesenchymal stem cells (MSCs) are multipotent, self-renewing cells that have the ability to differentiate into mesodermal tissues. This ability has attracted the attention of researchers toward MSCs as therapeutic agents. The aim of this study was to inspect the in vitro differentiation of human adipose-derived tissue stem cells (hADSCs) into insulin producing cells (IPCs) using minimal differentiation factors to provide a source of cells for the purpose of diabetic cell therapy. Methods: The hADSCs were obtained from liposuction aspirates and induced to differentiate into IPCs under a two-stage protocol. In the pre-induction stage, a combination of low-glucose DMEM medium, 20% (FBS), β-mercaptoethanol, and nicotinamide, and in the induction stage, high-glucose DMEM, β- mercaptoethanol, and nicotinamide without FBS was used. Differentiation was evaluated through morphological analysis, dithizone (DTZ) staining, and reverse transcription polymerase chain reaction (RTPCR). In order to evaluate the performance of differentiated cells, insulin production level was measured. Results: Morphological changes were observed using an inverted microscope at the end of the differentiation stage. Based on dithizone staining, differentiated cells were positive. Furthermore, RT-PCR confirmed the expression of insulin, pancreatic duodenal homeobox (PDX-1), paired box gene 4 (PAX-4), and glucose transporter type 2 (GLUT2) in differentiated cells. Moreover, insulin production by the IPCs was confirmed using enzyme-linked immunosorbent assay (ELISA). Conclusion: It can be concluded that hADSCs can differentiate into IPCs using minimal differentiation factors.

Keywords


  1. Raslova K. An update on the treatment of type 1 and type 2 diabetes mellitus: focus on insulin detemir, a long-acting human insulin analog. Vasc Health Risk Manag 2010; 6: 399-410.
  2. Noguchi H. Pancreatic islet transplantation. World J Gastrointest Surg 2009; 1(1): 16-20.
  3. Vija L, Farge D, Gautier JF, Vexiau P, Dumitrache C, Bourgarit A, et al. Mesenchymal stem cells: Stem cell therapy perspectives for type 1 diabetes. Diabetes Metab 2009; 35(2): 85-93.
  4. Yoshimura K, Suga H, Eto H. Adipose-derived stem/progenitor cells: roles in adipose tissue remodeling and potential use for soft tissue augmentation. Regen Med 2009; 4(2): 265-73.
  5. The American Society forAesthetic Plastic Surgery. ASAPS/ASPS Position Statement on Stem Cells and Fat Grafting. Aesthetic Surgery Journal 2011; 31(6): 716-7.
  6. Gaustad KG, Boquest AC, Anderson BE, Gerdes AM, Collas P. Differentiation of human adipose tissue stem cells using extracts of rat cardiomyocytes. Biochem Biophys Res Commun 2004; 314(2): 420-7.
  7. Brown SA, Levi B, Lequeux C, Wong VW, Mojallal A, Longaker MT. Basic science review on adipose tissue for clinicians. Plast Reconstr Surg 2010; 126(6): 1936-46.
  8. Gir P, Oni G, Brown SA, Mojallal A, Rohrich RJ. Human adipose stem cells: current clinical applications. Plast Reconstr Surg 2012; 129(6): 1277-90.
  9. Sordi V, Malosio ML, Marchesi F, Mercalli A, Melzi R, Giordano T, et al. Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets. Blood 2005; 106(2): 419-27.
  10. Abdi R, Fiorina P, Adra CN, Atkinson M, Sayegh MH. Immunomodulation by mesenchymal stem cells: a potential therapeutic strategy for type 1 diabetes. Diabetes 2008; 57(7): 1759-67.
  11. Krampera M, Pasini A, Pizzolo G, Cosmi L, Romagnani S, Annunziato F. Regenerative and immunomodulatory potential of mesenchymal stem cells. Curr Opin Pharmacol 2006; 6(4): 435-41.
  12. Tyndall A, Walker UA, Cope A, Dazzi F, De BC, Fibbe W, et al. Immunomodulatory properties of mesenchymal stem cells: a review based on an interdisciplinary meeting held at the Kennedy Institute of Rheumatology Division, London, UK, 31 October 2005. Arthritis Res Ther 2007; 9(1): 301.
  13. Patel SA, Sherman L, Munoz J, Rameshwar P. Immunological properties of mesenchymal stem cells and clinical implications. Arch Immunol Ther Exp (Warsz ) 2008; 56(1): 1-8.
  14. Moshtagh PR, Emami SH, Sharifi AM. Differentiation of human adipose-derived mesenchymal stem cell into insulin-producing cells: an in vitro study. J Physiol Biochem 2013; 69(3): 451-8.
  15. Limbert C, Seufert J. In vitro (re)programming of human bone marrow stromal cells toward insulin-producing phenotypes. Pediatr Diabetes 2009; 10(6): 413-9.
  16. Marappagounder D, Somasundaram I, Dorairaj S, Sankaran RJ. Differentiation of mesenchymal stem cells derived from human bone marrow and subcutaneous adipose tissue into pancreatic islet-like clusters in vitro. Cell Mol Biol Lett 2013; 18(1): 75-88.
  17. Taha MF, Hedayati V. Isolation, identification and multipotential differentiation of mouse adipose tissue-derived stem cells. Tissue Cell 2010; 42(4): 211-6.
  18. Talens-Visconti R, Bonora A, Jover R, Mirabet V, Carbonell F, Castell JV, et al. Human mesenchymal stem cells from adipose tissue: Differentiation into hepatic lineage. Toxicol In Vitro 2007; 21(2): 324-9.
  19. Li X, Zhang Y, Qi G. Evaluation of isolation methods and culture conditions for rat bone marrow mesenchymal stem cells. Cytotechnology 2013; 65(3): 323-34.
  20. Li L, Li F, Qi H, Feng G, Yuan K, Deng H, et al. Coexpression of Pdx1 and betacellulin in mesenchymal stem cells could promote the differentiation of nestin-positive epithelium-like progenitors and pancreatic islet-like spheroids. Stem Cells Dev 2008; 17(4): 815-23.
  21. Wang N, Adams G, Buttery L, Falcone FH, Stolnik S. Alginate encapsulation technology supports embryonic stem cells differentiation into insulin-producing cells. J Biotechnol 2009; 144(4): 304-12.
  22. Sun B, Roh KH, Lee SR, Lee YS, Kang KS. Induction of human umbilical cord blood-derived stem cells with embryonic stem cell phenotypes into insulin producing islet-like structure. Biochem Biophys Res Commun 2007; 354(4): 919-23.
  23. Tsai PJ, Wang HS, Shyr YM, Weng ZC, Tai LC, Shyu JF, et al. Transplantation of insulin-producing cells from umbilical cord mesenchymal stem cells for the treatment of streptozotocin-induced diabetic rats. J Biomed Sci 2012; 19: 47.
  24. Hayashi KY, Tamaki H, Handa K, Takahashi T, Kakita A, Yamashina S. Differentiation and proliferation of endocrine cells in the regenerating rat pancreas after 90% pancreatectomy. Arch Histol Cytol 2003; 66(2): 163-74.
  25. Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 2001; 292(5520): 1389-94.
  26. Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M. Insulin production by human embryonic stem cells. Diabetes 2001; 50(8): 1691-7.
  27. Zanini C, Bruno S, Mandili G, Baci D, Cerutti F, Cenacchi G, et al. Differentiation of Mesenchymal Stem Cells Derived from Pancreatic Islets and Bone Marrow into Islet-Like Cell Phenotype. PLoS ONE 2011; 6(12): e28175.
  28. Chao KC, Chao KF, Fu YS, Liu SH. Islet-Like Clusters Derived from Mesenchymal Stem Cells in Wharton's Jelly of the Human Umbilical Cord for Transplantation to Control Type 1 Diabetes. PLoS ONE 2008; 3(1): e1451.
  29. Chandra V, Swetha G, Muthyala S, Jaiswal A, Bellare J, Nair PD, et al. Islet-Like Cell Aggregates Generated from Human Adipose Tissue Derived Stem Cells Ameliorate Experimental Diabetes in Mice. PLoS ONE 2011; 6(6): e20615.
  30. Lee J, Han DJ, Kim SC. In vitro differentiation of human adipose tissue-derived stem cells into cells with pancreatic phenotype by regenerating pancreas extract. Biochem Biophys Res Commun 2008; 375(4): 547-51.
  31. Otto TC, Lane MD. Adipose development: from stem cell to adipocyte. Crit Rev Biochem Mol Biol 2005; 40(4): 229-42.
  32. Wu XH, Liu CP, Xu KF, Mao XD, Zhu J, Jiang JJ, et al. Reversal of hyperglycemia in diabetic rats by portal vein transplantation of islet-like cells generated from bone marrow mesenchymal stem cells. World J Gastroenterol 2007; 13(24): 3342-9.