, Volume 18, Issue 5, pp 578–588 | Cite as

Decrease of apoptosis markers during adipogenic differentiation of mesenchymal stem cells from human adipose tissue

  • Debora Lo Furno
  • Adriana C. E. Graziano
  • Silvia Caggia
  • Rosario E. Perrotta
  • Maria Stella Tarico
  • Rosario Giuffrida
  • Venera CardileEmail author
Original Paper


Although the proliferation and differentiation of mesenchymal stem cells (MSCs) from adipose tissue (AT) have been widely studied, relatively little information is available on the underlying mechanism of apoptosis during the adipogenic differentiation. Thus, the aim of this study was to analyze how the expression of some apoptotic markers is affected by in vitro expansion during adipogenic differentiation of AT-MSCs. The cultures incubated or not with adipogenic medium were investigated by Western blot at 7, 14, 21, and 28 days for the production of p53, AKT, pAKT, Bax, PDCD4 and PTEN. MSCs were recognized for their immunoreactivity to MSC-specific cell types markers by immunocytochemical procedure. The effectiveness of adipogenic differentiation was assessed by staining with Sudan III and examination of adipogenic markers expression, such as PPAR-γ and FABP, at different time points by Western blot. The adipogenic differentiation medium led to the appearance, after 7 days, of larger rounded cells presenting numerous vacuoles containing lipids in which it was evident a red–orange staining, that increased in size in a time-dependent manner, parallel to an increase of the levels of expression of PPAR-γ and FABP. More than 50 % of human MSCs were fully differentiated into adipocytes within the four-week induction period. The results showed that during adipogenic differentiation of AT-MSCs the PI3K/AKT signaling pathway is activated and that p53, PTEN, PDCD4, and Bax proteins are down-regulated in time-dependent manner. Our data provide new information on the behavior of some apoptotic markers during adipogenic differentiation of AT-MSCs to apply for tissues repair and regeneration.


Adipogenesis Apoptosis Cell death Differentiation Human cultures MSC 


  1. 1.
    Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7:211–228PubMedCrossRefGoogle Scholar
  2. 2.
    Walker MR, Patel KK, Stappenbeck TS (2009) The stem cell niche. J Pathol 217:169–180PubMedCrossRefGoogle Scholar
  3. 3.
    Kern S, Eichler H, Stoeve J, Klüter H, Bieback K (2006) Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 24:1294–1301PubMedCrossRefGoogle Scholar
  4. 4.
    Tuan RS, Boland G, Tuli R (2003) Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Res Ther 5:32–45PubMedCrossRefGoogle Scholar
  5. 5.
    Friedenstein AJ, Deriglasova UF, Kulagina NN, Panasuk AF, Rudakowa SF, Luriá EA, Ruadkow IA (1974) Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol 2:83–92PubMedGoogle Scholar
  6. 6.
    Lin G, Garcia M, Ning H, Banie L, Guo YL, Lue TF, Lin CS (2008) Defining stem and progenitor cells within adipose tissue. Stem cells dev 17:1053–1064PubMedCrossRefGoogle Scholar
  7. 7.
    Magun R, Gagnon AM, Yaraghi Z, Sorisky A (1998) Expression and regulation of neuronal apoptosis inhibitory protein during adipocyte differentiation. Diabetes 47:1948–1952PubMedCrossRefGoogle Scholar
  8. 8.
    Planat-Benard V, Silvestre JS, Cousin B, André M, Nibbelink M, Tamarat R, Clergue M, Manneville C, Saillan-Barreau C, Duriez M, Tedgui A, Levy B, Pénicaud L, Casteilla L (2004) Plasticity of human adipose lineage cells toward endothelial cells. physiological and therapeutic perspectives. Circulation 109:656–663PubMedCrossRefGoogle Scholar
  9. 9.
    Barry FP, Murphy JM (2004) Mesencymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 36:568–584PubMedCrossRefGoogle Scholar
  10. 10.
    Dicker A, Le Blanc K, Aström G, van Harmelen V, Götherström C, Blomqvis L, Arner P, Rydén M (2005) Functional studies of mesenchymal stem cells derived from adult human adipose tissue. Exp Cell Res 308:283–290PubMedCrossRefGoogle Scholar
  11. 11.
    Jeon ES, Kang YJ, Song HY, Woo JS, Jung JS, Kim YK, Kim JH (2005) Role of MEK-ERK pathway in sphingosylphosphorylcholine-induced cell death in human adipose tissue-derived mesenchymal stem cells. Biochim Biophys Acta 1734:25–33PubMedCrossRefGoogle Scholar
  12. 12.
    Kim YJ, Bae YC, Suh KT, Jung JS (2006) Quercetin, a flavonoid, inhibits proliferation and increase osteogenic differentiation in human adipose stromal cells. Biochem Pharmacol 72:1268–1278PubMedCrossRefGoogle Scholar
  13. 13.
    Strissel KJ, Stancheva Z, Miyoshi H, Perfield JW II, DeFuria J, Jick Z, Greenberg AS, Obin MS (2007) Adipocyte death, adipose tissue remodeling, and obesity complications. Diabetes 56:2910–2918PubMedCrossRefGoogle Scholar
  14. 14.
    Magun R, Boone DL, Tsang BK, Sorisky A (1998) The effect of adipocyte differentiation on the capacity of 3T3-L1 cells to undergo apoptosis in response to growth factor deprivation. Int J Obes Relat Metab Disord 22:567–571PubMedCrossRefGoogle Scholar
  15. 15.
    Niesler CU, Urso B, Prins JB, Siddle K (2000) IGF-1 inhibits apoptosis induced by serum withdrawal, but potentiates TNF-α-induced apoptosis, in 3T3-L1 preadipocytes. J Endocrinol 167:165–174PubMedCrossRefGoogle Scholar
  16. 16.
    Gagnon A, Artemenko Y, Crapper T, Sorisky A (2003) Regulation of endogenous SH2 domain-containing inositol 5-phosphatase (SHIP2) in 3T3-L1 and human preadipocytes. J Cell Physiol 197:243–250PubMedCrossRefGoogle Scholar
  17. 17.
    Fischer-Posovszky P, Rews D, Horenburg S, Debatin KM, Wabitsch M (2012) Differential function of AKt1 and AKt2 in human adipocytes. Mol Cell Endocrinol 358:135–143PubMedCrossRefGoogle Scholar
  18. 18.
    Zebisch K, Voigt V, Wabitsch M, Brandsch M (2012) Protocol for effective differentiation of 3T3-L1 cells to adipocytes. Anal Biochem 425:88–90PubMedCrossRefGoogle Scholar
  19. 19.
    Staiger H, Loffler G (1998) The role of PDGF-dependent suppression of apoptosis in differentiating 3T3-L1 preadipocytes. Eur J Cell Biol 77:220–227PubMedCrossRefGoogle Scholar
  20. 20.
    Li H, Fong C, Chen Y, Caia G, Yang M (2010) Beta-adrenergic signals regulate adipogenesis of mouse mesenchymal stem cells via cAMP/PKA pathway. Mol Cell Endocrinol 323:201–207PubMedCrossRefGoogle Scholar
  21. 21.
    Yu W, Chen Z, Zhang J, Zhang L, Ke H, Huang L, Peng Y, Zhang X, Li S, Lahn BT, Xiang AP (2008) Critical role of phosphoinositide 3-kinase cascade in adipogenesis of human mesenchymal stem cells. Mol Cell Biochem 310:11–18PubMedCrossRefGoogle Scholar
  22. 22.
    Aubin D, Gagnon A, Sorisky A (2005) Phosphoinositide 3-kinase is required for human adipocyte differentiation in culture. Int J Obes (Lond) 29:1006–1009CrossRefGoogle Scholar
  23. 23.
    Kohn AD, Summers SA, Birnbaum MJ, Roth RA (1996) Expression of a constitutively active Akt Ser/Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem 271:31372–31378PubMedCrossRefGoogle Scholar
  24. 24.
    Magun R, Burgering BM, Coffer PJ, Pardasani D, Lin Y, Chabot J, Sorisky A (1996) Expression of a constitutively activated form of protein kinase B (c-Akt) in 3T3-L1 preadipose cells causes spontaneous differentiation. Endocrinology 137:3590–3593PubMedCrossRefGoogle Scholar
  25. 25.
    Armstrong JF, Kaufman MH, Harrison DJ, Clarke AR (1995) High-frequency developmental abnormalities in p53-deficient mice. Curr Biol 5:931–936PubMedCrossRefGoogle Scholar
  26. 26.
    Choi J, Donehower LA (1999) p53 in embryonic development: maintaining a fine balance. Cell Mol Life Sci 55:38–47PubMedCrossRefGoogle Scholar
  27. 27.
    Hall PA, Lane DP (1997) Tumor suppressors: a developing role for p53? Curr Biol 7:R144–R147PubMedCrossRefGoogle Scholar
  28. 28.
    Almog N, Rotter V (1997) Involvement of p53 in cell differentiation and development. Biochem Biophys Acta 1333:F1–F27PubMedGoogle Scholar
  29. 29.
    Zambetti GP, Horwitz EM, Schipani E (2006) Skeletons in the p53 tumor suppressor closet: genetic evidence that p53 blocks bone differentiation and development. J Cell Biol 172:795–797PubMedCrossRefGoogle Scholar
  30. 30.
    Constance CM, Morgan JI, Umek RM (1996) C/EBPalpha regulation of the growth-arrest associated gene gadd45. Mol Cell Biol 16:3878–3883PubMedGoogle Scholar
  31. 31.
    Berberich SJ, Litteral V, Mayo LD, Tabesh D, Morris D (1999) mdm-2 gene amplification in 3T3-L1 preadipocytes. Differentiation 64:205–212PubMedCrossRefGoogle Scholar
  32. 32.
    Inoue N, Yamamoto T, Ishikawa M, Watanabe K, Matsuzaka T, Nakagawa Y, Takeuchi Y, Kobayashi K, Takahashi A, Suzuki H, Hasty AH, Toyoshima H, Yamada N, Shimano H (2008) Cyclin-dependent kinase inhibitor, p21WAF1/CIP1, is involved in adipocyte differentiation and hypertrophy, linking to obesity, and insulin resistance. J Biol Chem 283:21220–21229PubMedCrossRefGoogle Scholar
  33. 33.
    Hong Y, Cervantes RB, Tichy E, Tischfield JA, Stambrook PJ (2007) Protecting genomic integrity in somatic cells and embryonic stem cells. Mutat Res 614:48–55PubMedCrossRefGoogle Scholar
  34. 34.
    Sabbatini P, McCormick F (1999) Phosphoinositide 3-OH kinase (PI3K) and PKB/Akt delay the onset of p53-mediated, transcriptionally dependent apoptosis. J Biol Chem 274:24263–24269PubMedCrossRefGoogle Scholar
  35. 35.
    Vogelstein B, Lane D, Levine AJ (2000) Surfing the p53 network. Nature 408:307–310PubMedCrossRefGoogle Scholar
  36. 36.
    Gross A, McDonnell JM, Korsmeyer SJ (1999) BCL-2 family members and the mitochondria in apoptosis. Genes Dev 13:1899–1911PubMedCrossRefGoogle Scholar
  37. 37.
    Castedo M, Ferri KF, Blanco J, Roumier T, Larochette N, Barretina J, Amendola A, Nardacci R, Métivier D, Este JA, Piacentini M, Kroemer G (2001) Human immunodeficiency virus 1 envelope glycoprotein complex-induced apoptosis involves mammalian target of rapamycin/FKBP12-rapamycin-associated protein-mediated p53 phosphorylation. J Exp Med 194:1097–1110PubMedCrossRefGoogle Scholar
  38. 38.
    Castedo M, Perfettini JL, Roumier T, Kroemer G (2002) Cyclin dependent kinase-1: linking apoptosis to cell cycle and mitotic catastrophe. Cell Death Differ 9:1287–1293PubMedCrossRefGoogle Scholar
  39. 39.
    Marzo I, Brenner C, Zamzami N, Jürgensmeier JM, Susin SA, Vieira HL, Prévost MC, Xie Z, Matsuyama S, Reed JC, Kroemer G (1998) Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281:2027–2031PubMedCrossRefGoogle Scholar
  40. 40.
    Oltvai ZN, Milliman CL, Korsmeyer SJ (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74:609–619PubMedCrossRefGoogle Scholar
  41. 41.
    Myers MP, Pass I, Batty IH, Van der Kaay J, Stolarov JP, Hemmings BA, Wigler MH, Downes CP, Tonks NK (1998) The lipid phosphatase activity of PTEN is critical for its tumor suppressor function. Proc Natl Acad Sci USA 95:13513–13518PubMedCrossRefGoogle Scholar
  42. 42.
    Wu X, Senechal K, Neshat MS, Whang YE, Sawyers CL (1998) The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway. Proc Natl Acad Sci USA 95:15587–15591PubMedCrossRefGoogle Scholar
  43. 43.
    Franke TF, Kaplan DR, Cantley LC, Toker A (1997) Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4- bisphosphate. Science 275:665–668PubMedCrossRefGoogle Scholar
  44. 44.
    Sun H, Lesche R, Li DM, Liliental J, Zhang H, Gao J, Gavrilova N, Mueller B, Liu X, Wu H (1999) PTEN modulates cell cycle progression and cell survival by regulating phosphatidylinositol 3,4,5,-trisphosphate and Akt/protein kinase B signaling pathway. Proc Natl Acad Sci USA 96:6199–6204PubMedCrossRefGoogle Scholar
  45. 45.
    Blanco-Aparicio C, Renner O, Leal JFM, Carnero A (2007) PTEN, more than the AKT pathway. Carcinogenesis 28:1379–1386PubMedCrossRefGoogle Scholar
  46. 46.
    Cardone MH, Roy N, Stennicke HR, Salvesen GS, Franke TF, Stanbridge E, Frisch S, Reed JC (1998) Regulation of cell death protease caspase-9 by phosphorylation. Science 282:1318–1321PubMedCrossRefGoogle Scholar
  47. 47.
    Kops GJ, de Ruiter ND, De Vries-Smits AM, Powell DR, Bos JL, Burgering BM (1999) Direct control of the Forkhead transcription factor AFX by protein kinase B. Nature 398:630–634PubMedCrossRefGoogle Scholar
  48. 48.
    Yilmaz OH, Valdez R, Theisen BK, Guo W, Ferguson DO, Wu H, Morrison SJ (2006) Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature 441:475–482PubMedCrossRefGoogle Scholar
  49. 49.
    Zhang J, Grindley JC, Yin T, Jayasinghe S, He XC, Ross JT, Haug JS, Rupp D, Porter-Westpfahl KS, Wiedemann LM, Wu H, Li L (2006) PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention. Nature 441:518–522PubMedCrossRefGoogle Scholar
  50. 50.
    Groszer M, Erickson R, Scripture-Adams DD, Dougherty JD, Le Belle J, Zack JA, Geschwind DH, Liu X, Kornblum HI, Wu H (2006) PTEN negatively regulates neural stem cell self-renewal by modulating G0–G1 cell cycle entry. Proc Natl Acad Sci USA 103:111–116PubMedCrossRefGoogle Scholar
  51. 51.
    Lankat-Buttgereit B, Göke R (2009) The tumour suppressor Pdcd4: recent advances in the elucidation of function and regulation. Biol Cell 101:309–317PubMedCrossRefGoogle Scholar
  52. 52.
    Mudduluru G, Medved F, Grobholz R, Jost C, Gruber A, Leupold JH, Post S, Jansen A, Colburn NH, Allgayer H (2007) Loss of programmed cell death 4 expression marks adenoma-carcinoma transition, correlates inversely with phosphorylated protein kinase B, and is an independent prognostic factor in resected colorectal cancer. Cancer 110:1697–1707PubMedCrossRefGoogle Scholar
  53. 53.
    Dorrello NV, Peschiaroli A, Guardavaccaro D, Colburn NH, Sherman NE, Pagano M (2006) S6K1- and betaTRCP-mediated degradation of PDCD4 promotes protein translation and cell growth. Science 314:467–471PubMedCrossRefGoogle Scholar
  54. 54.
    Caggia S, Libra M, Malaponte G, Cardile V (2011) Modulation of YY1 and p53 expression by transforming growth factor-β3 in prostate cell lines. Cytokine 56:403–410PubMedCrossRefGoogle Scholar
  55. 55.
    Sonenberg N, Pause A (2006) Signal transduction. Protein synthesis and oncogenesis meet again. Science 314:428–429PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Debora Lo Furno
    • 1
  • Adriana C. E. Graziano
    • 1
  • Silvia Caggia
    • 1
  • Rosario E. Perrotta
    • 2
  • Maria Stella Tarico
    • 2
  • Rosario Giuffrida
    • 1
  • Venera Cardile
    • 1
    Email author
  1. 1.Department of Bio-medical Sciences, Section of PhysiologyUniversity of CataniaCataniaItaly
  2. 2.Dipartimento di Specialità Medico-ChirurgicheCannizzaro HospitalCataniaItaly

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