Cell and Tissue Research

, Volume 361, Issue 3, pp 745–753 | Cite as

Three-dimensional differentiation of adipose-derived mesenchymal stem cells into insulin-producing cells

  • Layasadat KhorsandiEmail author
  • Ali Khodadadi
  • Fereshteh Nejad-Dehbashi
  • Sadegh Saremy
Regular Article


The aim of this study is to evaluate the collagen/hyaluronic acid (Col/HA) scaffold effect on the differentiation of insulin-producing cells (IPCs) from adipose-derived mesenchymal stem cells (ASCs). In this experimental study, ASCs were cultured and seeded in a Col/HA scaffold (3D culture) and then treated with induction media. After induction, the presence of IPCs was evaluated using gene expression (PDX-1, GLUT-2 and insulin) analysis and immunocytochemistry, while functional maturity was determined by measuring insulin release in response to low- and high-glucose media. The induced IPCs were morphologically similar to pancreatic islet-like cells. Expression of the islet-associated genes PDX-1, GLUT-2 and insulin genes in 3D-cultured cells was markedly higher than the 2D-cultured cells exposure differentiation media. Compared to the 2D culture of ASCs-derived IPCs, the insulin release from 3D ASCs-derived IPCs showed a nearly 4-fold (p < 0.05) increase when exposed to a high glucose (25 mmol) medium. The percentage of insulin-positive cells in the 3D experimental group showed an approximately 4-fold increase compared to the 2D experimental culture cells. The results of this study demonstrated that the COL/HA scaffold can enhance the differentiation of IPCs from rat ASCs.


Collagen/hyaluronic acid scaffold Adult stem cells PDX-1 GLUT-2 Insulin 



The financial cost of this project has been provided by the research council of the Ahvaz Jundishapur University of Medical Sciences (Grant number: CMRC-48).

Supplementary material

441_2015_2140_MOESM1_ESM.xml (9 kb)
ESM 1 (XML 9 kb)


  1. Aloysious N, Nair PD (2014) Enhanced survival and function of islet-like clusters differentiated from adipose stem cells on a three-dimensional natural polymeric scaffold: an in vitro study. Tissue Eng Part A 20:1508–22CrossRefPubMedGoogle Scholar
  2. Bayati V, Hashemitabar M, Gazor R, Nejatbakhshi R, Bijannejad D (2013) Expression of surface markers and muogenic potential of rat bone marrow-and adipose derived stem cell a comparative study. Anat Cell Biol 46:113–121PubMedCentralCrossRefPubMedGoogle Scholar
  3. Bonner-Weir S, Taneja M, Weir GC, Tatarkiewicz K, Song KH, Sharma A, O’Neil JJ (2000) In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci U S A 97:7999–8004PubMedCentralCrossRefPubMedGoogle Scholar
  4. Bose B, Shenoy SP, Konda S, Wangikar P (2012) Human embryonic stem cell differentiation into insulin secreting β-cells for diabetes. Cell Biol Int 36:1013–20CrossRefPubMedGoogle Scholar
  5. Brun T, Franklin I, St-Onge L, Biason-Lauber A, Schoenle EJ, Wollheim CB, Gauthier BR (2004) The diabetes-linked transcription factor PAX4 promotes b-cell proliferation and survival in rat and human islets. J Cell Biol 167:1123–1135PubMedCentralCrossRefPubMedGoogle Scholar
  6. Calderon L, Collin E, Velasco-Bayon D, Murphy M, O’Halloran D, Pandit A (2010) Type II collagen-hyaluronan hydrogel-a step towards a scaffold for intervertebral disc tissue engineering. Eur Cell Mater 20:134–48PubMedGoogle Scholar
  7. Chen PY, Huang LL, Hsieh HJ (2007) Hyaluronan preserves the proliferation and differentiation potentials of long-term cultured murine adipose-derived stromal cells. Biochem Biophys Res Commun 360:1–6CrossRefPubMedGoogle Scholar
  8. Chun SY, Mack DL, Moorefield E, Oh SH, Kwon TG, Pettenati MJ, Yoo JJ, Coppi PD, Atala A, Soker S (2012) Pdx1 and controlled culture conditions induced differentiation of human amniotic fluid-derived stem cells to insulin-producing clusters. J Tissue Eng Regen Med. doi: 10.1002/term.1631 PubMedCentralGoogle Scholar
  9. Cras-Méneur C, Elghazi L, Czernichow P, Scharfmann R (2001) Epidermal growth factor increases undifferentiated pancreatic embryonic cells in vitro: a balance between proliferation and differentiation. Diabetes 50:1571–9CrossRefPubMedGoogle Scholar
  10. Davidenko N, Campbell JJ, Thian ES, Watson CJ, Cameron RE (2010) Collagen-hyaluronic acid scaffolds for adipose tissue engineering. Acta Biomater 6:3957–68CrossRefPubMedGoogle Scholar
  11. Dunbar AJ, Goddard C (2000) Structure-function and biological role of betacellulin. Int J Biochem Cell Biol 32:805–15CrossRefPubMedGoogle Scholar
  12. Ebrahimie M, Esmaeili F, Cheraghi C, Houshmand F, Shabani L, Ebrahimie E (2014) Efficient and simple production of insulin-producing cells from embryonal carcinoma stem cells using mouse neonate pancreas extract as a natural inducer. PLoS ONE 9:e90885PubMedCentralCrossRefPubMedGoogle Scholar
  13. Edlund H (1998) Transcribing pancreas. Diabetes 47:1817–1823CrossRefPubMedGoogle Scholar
  14. Eyre DR, Matsui Y, Wu JJ (2002) Collagen polymorphisms of the intervertebral disc. Biochem Soc Trans 30:844–848CrossRefPubMedGoogle Scholar
  15. Florio P, Luisi S, Marchetti P, Lupi R, Cobellis L, Falaschi C, Sugino H, Navalesi R, Genazzani AR, Petraglia F (2000) Activin A stimulates insulin secretion in cultured human pancreatic islets. J Endocrinol Investig 23:231–234CrossRefGoogle Scholar
  16. Gabr MM, El-Far MA, Ibrahim RY, El-Halawani SM, Ismail AM, Zakaria MM, Sherry MK, Ghoneim MA (2012) Biochemical induction of adult bone marrow hematopeitic stem cells into insulin producing cells. Curr Top Biochem Res 14:45–56Google Scholar
  17. Grayson WL, Ma T, Bunnell B (2004) Human mesenchymal stem cells tissue development in 3D PET matrices. Biotechnol Prog 20:905–12CrossRefPubMedGoogle Scholar
  18. Guo W, Miao C, Liu S, Qiu Z, Li J, Duan E (2009) Efficient differentiation of insulin-producing cells from skin-derived stem cells. Cell Prolif 42:49–62CrossRefPubMedGoogle Scholar
  19. Hammar EB, Irminger JC, Rickenbach K, Parnaud G, Ribaux P, Bosco D, Rouiller DG, Halban PA (2005) Activation of NF-kB by extracellular matrix is involved in spreading and glucose-stimulated insulin secretion of pancreatic b cells. J Biol Chem 280:30630–30637CrossRefPubMedGoogle Scholar
  20. Hardikar AA, Marcus-Samuels B, Geras-Raaka E, Raaka BM, Gershengorn MC (2003) Human pancreatic precursor cells secrete FGF2 to stimulate clustering into hormone expressing islet-like cell aggregates. Proc Natl Acad Sci USA 100:7117–7122Google Scholar
  21. Herbst RS (2004) Review of epidermal growth factor receptor biology. Int J Radiat Oncol Biol Phys 59:21–6CrossRefPubMedGoogle Scholar
  22. Huotari MA, Palgi J, Otonkoski T (1998) Growth factor-mediated proliferation and di€erentiation of insulin-pro- ducing INS-1 and RINm5F cells; identification of betacellulin as a novel b-cell mitogen. Endocrinology 139:1494–1499PubMedGoogle Scholar
  23. Kayoko TO, Yoshinori O, Maki M, Akio H, Akira A, Akira T, Abe K, Tamaki T (2010) Micro 3D culture system using hyaluronan-collagen capsule for skeletal muscle-derived stem cells. Open Tissue Eng Regen Med J 3:18–27CrossRefGoogle Scholar
  24. Khorsandi L, Mirhoseini M, Mohamadpour M, Orazizadeh M, Khaghani S (2013) Effect of curcumin on dexamethasone-induced testicular toxicity in mice. Pharm Biol 51:206–12CrossRefPubMedGoogle Scholar
  25. Knight KR, Uda Y, Findlay MW, Brown DL, Cronin KJ, Jamieson E, Tai T, Keramidaris E, Penington AJ, Rophael J, Harrison LC, Morrison WA (2006) Vascularized tissue-engineered chambers promote survival and function of transplanted islets and improve glycemic control. FASEB J 20:565PubMedGoogle Scholar
  26. Liu SH, Lee LT (2012) Efficient differentiation of mouse embryonic stem cells into insulin-producing cells. Exp Diabetes Res 2012:201295PubMedCentralPubMedGoogle Scholar
  27. Liu J, Liu Y, Wang H, Hao H, Han Q, Shen J, Shi J, Li C, Mu Y, Han W (2013) Direct differentiation of hepatic stem-like WB cells into insulin-producing cells using small molecules. Sci Rep 3:1–8Google Scholar
  28. Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R (2001) Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 292:1389–1394CrossRefPubMedGoogle Scholar
  29. Maleki A, Kjoniksen AL, Nystrom B (2007) Characterization of the chemical degradation of hyaluronic acid during chemical gelation in the presence of different cross-linker agents. Carbohydr Res 342:2776–2792CrossRefPubMedGoogle Scholar
  30. Mashima H, Shibata H, Mine T, Kojima I (1996) Formation of insulin-producing cells from pancreatic acinar AR42J cells by hepatocyte growth factor. Endocrinology 137:3969–76PubMedGoogle Scholar
  31. Mason MN, Arnold CA, Mahoney MJ (2009) Entrapped collagen type 1 promotes differentiation of embryonic pancreatic precursor cells into glucose-responsive beta-cells when cultured in three-dimensional PEG hydrogels. Tissue Eng Part A 15:3799–808PubMedCentralCrossRefPubMedGoogle Scholar
  32. Mohr JC, de Pablo JJ, Palecek SP (2006) 3-D microwell culture of human embryonic stem cells. Biomaterials 27:6032–42CrossRefPubMedGoogle Scholar
  33. Mukherjee A, Sidis Y, Mahan A, Raher MJ, Xia Y, Rosen ED, Bloch KD, Thomas MK, Schneyer AL (2007) FSTL3 deletion reveals roles for TGFbeta family ligands in glucose and fat homeostasis in adults. Proc Natl Acad Sci U S A 104:1348–1353PubMedCentralCrossRefPubMedGoogle Scholar
  34. Olson AL, Pessin JE (1996) Structure function, and regulation of the mammalian facilitative glucose transporter gene family. Annu Rev Nutr 16:235–56CrossRefPubMedGoogle Scholar
  35. Pandey S (2010) Stem cell transplantation: a future for diabetic paitient. Int J Pharm Sci Rev Res 1:68–71Google Scholar
  36. Park MK, Han C, Lee KH, Hong SH, Kim HS, Lee YJ, Jeong IK, Noh JH, Yang TY, Lee MS, Kim KW, Lee MK (2007) Effects of activin A on pancreatic ductal cells in streptozotocin-induced diabetic rats. Transplantation 83:925–930CrossRefPubMedGoogle Scholar
  37. Parnaud G, Hammar E, Ribaux P, Donath MY, Berney T, Halban PA (2009) Signaling pathways implicated in the stimulation of b-cell proliferation by extracellular matrix. Mol Endocrinol 23:1264–1271CrossRefPubMedGoogle Scholar
  38. Peshavaria M, Cissell MA, Henderson E, Petersen HV, Stein R (2000) The PDX-1 activation domain provides specific functions necessary for transcriptional stimulation in pancreatic beta-cells. Mol Endocrinol 14:1907–1917PubMedGoogle Scholar
  39. Puissant B, Barreau C, Bourin P, Clavel C, Corre J, Bousquet C, Taureau C, Cousin B, Abbal M, Laharrague P, Penicaud L, Casteilla L, Blancher A (2005) Immunomodulatory effect of human adipose tissue-derived adult stem cells: comparison with bone marrow mesenchymal stem cells. Br J Haematol 129:118–29CrossRefPubMedGoogle Scholar
  40. Richardson SM, Walker RV, Parker S, Rhodes NP, Hunt JA, Freemont AJ, Hoyland JA (2006) Intervertebral disc cell-mediated mesenchymal stem cell differentiation. Stem Cells 24:707–716CrossRefPubMedGoogle Scholar
  41. Risbud MV, Bhonde RR (2001) Suitability of cellulose molecular dialysis membrane for bioartificial pancreas: in vitro biocompatibility studies. J Biomed Mater Res 54:436–444CrossRefPubMedGoogle Scholar
  42. Rooney P, Kumar S (1993) Inverse relationship between hyaluronan and collagens in development and angiogenesis. Differentiation 54:1–9PubMedGoogle Scholar
  43. Schmeichel KL, Bissell MJ (2003) Modeling tissue-specific signaling and organ function in three dimensions. J Cell Sci 116:2377–88PubMedCentralCrossRefPubMedGoogle Scholar
  44. Skoudy A, Rovira M, Savatier P, Martin F, León-Quinto T, Soria B, Real FX (2004) Transforming growth factor (TGF) beta, fibroblast growth factor (FGF) and retinoid signalling pathways promote pancreatic exocrine gene expression in mouse embryonic stem cells. Biochem J 379:749–56PubMedCentralCrossRefPubMedGoogle Scholar
  45. Speier S, Gjinovci A, Charollais A, Meda P, Rupnik M (2007) Cx36-mediated coupling reduces b-cell heterogeneity, confines the stimulating glucose concentration range, and affects insulin release kinetics. Diabetes 56:1078–1086CrossRefPubMedGoogle Scholar
  46. Sun Y, Chen L, Hou XG, Hou WK, Dong JJ, Sun L, Tang KX, Wang B, Song J, Li H, Wang KX (2007) Differentiation of bone marrow-derived mesenchymal stem cells from diabetic patients into insulin-producing cells in vitro. Chin Med J (Engl) 120:771–6Google Scholar
  47. Takeuchi H, Nakatsuji N, Suemori H (2014) Endodermal differentiation of human pluripotent stem cells to insulin-producing cells in 3D culture. Sci Rep 4:4488PubMedCentralPubMedGoogle Scholar
  48. Tang S, Vickers SM, Hsu HP, Spector M (2007) Fabrication and characterization of porous hyaluronic acid-collagen composite scaffolds. J Biomed Mater Res A 82:323–335CrossRefPubMedGoogle Scholar
  49. Tholpady SS, Katz AJ, Ogle RC (2003) Mesenchymal stem cells from rat visceral fat exhibit multipotential differentiation in vitro. Anat Rec A 272:398–402CrossRefGoogle Scholar
  50. Wang X, Ye K (2009) Three-dimensional differentiation of embryonic stem cells into islet-like insulin-producing clusters. Tissue Eng Part A 15:1941–52CrossRefPubMedGoogle Scholar
  51. Wang LX, Zhao H, Jiang B, Ding Y (2009) Adhesion and growth of human periodontal ligament cells on hyaluronic acid/collagen scaffold. Hua Xi Kou Qiang Yi Xue Za Zhi 27:220–3PubMedGoogle Scholar
  52. Watada H, Kajimoto Y, Miyagawa J, Hanafusa T, Hamaguchi K, Matsuoka T, Yamamoto K, Matsuzawa Y, Kawamori R, Yamasaki Y (1996) PDX-1 induces insulin and glucokinase gene expressions in alphaTC1 clone 6 cells in the presence of betacellulin. Diabetes 45:1826–31CrossRefPubMedGoogle Scholar
  53. Watt FM, Hogan BL (2000) Out of Eden: stem cells and their niches. Science 287:1427–30CrossRefPubMedGoogle Scholar
  54. Weber LM, Hayda KN, Anseth KS (2008) Cell-matrix interactions improve b-cell survival and insulin secretion in three-dimensional culture. Tissue Eng Part A 14:1959–1968PubMedCentralCrossRefPubMedGoogle Scholar
  55. Wong RS (2011) Extrinsic factors involved in the differentiation of stem cells into insulin-producing cells: an overview. Exp Diabetes Res. doi: 10.1155/2011/406182 PubMedCentralPubMedGoogle Scholar
  56. Woods EJ, Walsh CM, Sidner RA, Zieger MA, Lakey JR, Ricordi C, Critser JK (2004) Improved in vitro function of islets using small intestinal submucosa. Transplant Proc 36:1175CrossRefPubMedGoogle Scholar
  57. Xu W, Hu R, Fan E, Han D (2011) Adipose-derived mesenchymal stem cells in collagen-hyaluronic acid gel composite scaffolds for vocal fold regeneration. Ann Otol Rhinol Laryngol 120:123–30CrossRefPubMedGoogle Scholar
  58. Yang L, Li S, Hatch H, Ahrens K, Cornelius JG, Petersen BE (2002) In vitro transdifferentiation of adult hepatic stem cells into pancreatic endocrine hormoneproducing cells. Proc Natl Acad Sci U S A 99:8078–8083PubMedCentralCrossRefPubMedGoogle Scholar
  59. Yang Z, Chen Z, Liu K, Bai Y, Jiang T, Feng D, Feng G (2013) Experimental study on tissue engineered cartilage complex three-dimensional nano-scaffold with collagen type II and hyaluronic acid in vitro. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 27:1240–5PubMedGoogle Scholar
  60. Zahran F, Salama M, Lotfy A, El-Deen IM (2012) Isolation and characterization of adipose tissue-derived stem cells: an in vitro study. Basic Res J Med Clin Sci 1:88–94Google Scholar
  61. Zalzman M, Anker-Kitai L, Efrat S (2005) Differentiation of human liver-derived, insulin-producing cells toward the cell phenotype. Diabetes 54:2568–75CrossRefPubMedGoogle Scholar
  62. Zavan B, Cortivo R, Tonello C, Abatangelo G (2003) Gland cell cultures into 3D hyaluronan-based scaffolds. J Mater Sci Mater Med 14:727–729CrossRefPubMedGoogle Scholar
  63. Zhang H, Wei YT, Tsang KS, Sun CR, Li J, Huang H, Cui FZ, An YH (2008) Implantation of neural stem cells embedded in hyaluronic acid and collagen composite conduit promotes regeneration in a rabbit facial nerve injury model. J Transl Med 6:67PubMedCentralCrossRefPubMedGoogle Scholar
  64. Zhao FQ, Keating AF (2007) Functional properties and genomics of glucose transporters. Curr Genomics 8:113–128PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Layasadat Khorsandi
    • 1
    • 2
    Email author
  • Ali Khodadadi
    • 1
    • 3
  • Fereshteh Nejad-Dehbashi
    • 1
  • Sadegh Saremy
    • 1
  1. 1.Cell & Molecular Research Center, Faculty of MedicineAhvaz Jundishapur University of Medical SciencesAhvazIran
  2. 2.Department of Anatomical Sciences, Faculty of MedicineAhvaz Jundishapur University of Medical SciencesAhvazIran
  3. 3.Department of Immunology and Petroleum and Environmental Pollutants Research Center, Faculty of MedicineJundishapur University of Medical SciencesAhvazIran

Personalised recommendations