Cell and Tissue Research

, Volume 347, Issue 2, pp 383–395 | Cite as

Enhancement of adipogenic and osteogenic differentiation of human bone-marrow-derived mesenchymal stem cells by supplementation with umbilical cord blood serum

  • Chandana Tekkatte
  • Prasanna Vidyasekar
  • Nand Kishore Kapadia
  • Rama S Verma
Regular Article


Umbilical cord blood serum (UCBS) is a promising replacement for animal sera for the culture of human mesenchymal stem cells (hMSC), the unique serum composition of UCBS appearing to have variable effects on their proliferation and differentiation. Conditioning UCBS with methods such as charcoal stripping assists specific processes such as adipogenesis and osteogenesis in hMSCs. The charcoal stripping of serum removes lipophilic materials such as oestrogens, which are known inhibitors of adipogenesis. hMSC cultures supplemented with charcoal-stripped UCBS (CS-UCBS) show enhanced adipogenesis in adipogenic induction medium (AIM) containing indomethacin, 3-isobutyl-1-methylxanthine and dexamethasone. To obtain efficient adipogenesis without CS-UCBS, we have developed a modified protocol in which cells cultured separately with UCBS and CS-UCBS are constantly treated with minimal doses of insulin (1.1 μg/ml) for 10 days prior to the addition of AIM. hMSC cultures differentiated by using the modified protocol show improved adipogenesis under fetal bovine serum (FBS), UCBS and CS-UCBS conditions, with levels of adipogenesis being highest in UCBS, thereby eliminating the need for charcoal stripping. Furthermore, in each of the three sera, the insulin-pre-treated hMSCs accumulate lipid droplets faster and exhibit improved adipogenesis overall when compared with normal AIM-induced adipogenesis. We have also compared the levels of osteogenesis in hMSCs by using an induction medium devoid of dexamethasone. Maximum calcium deposition has been observed in hMSCs cultured with UCBS, as compared with those cultured with FBS or CS-UCBS. Our newly developed methods with a humanized serum supplement thus enhance the differentiation of cultured hMSCs.


Mesenchymal stem cells Umbilical cord blood serum Adipogenesis Charcoal stripping Insulin 


  1. Bost F, Aouadi M, Caron L, Binetruy B (2005) The role of MAPKs in adipocyte differentiation and obesity. Biochimie 87:51–56PubMedCrossRefGoogle Scholar
  2. Cheng S-L, Yang JW, Rifas L, Zhang S-F, Avioli LV (1994) Differentiation of human bone marrow osteogenic stromal cells in vitro: induction of the osteoblast phenotype by dexamethasone. Endocrinology 134:277–286PubMedCrossRefGoogle Scholar
  3. Chuang CC, Yang RS, Tsai KS, Ho FM, Liu SH (2007) Hyperglycemia enhances adipogenic induction of lipid accumulation: involvement of extracellular signal-regulated protein kinase 1/2, phosphoinositide 3-kinase/Akt, and peroxisome proliferator-activated receptor gamma signalling. Endocrinology 148:4267–4275PubMedCrossRefGoogle Scholar
  4. Cifrian E, Guidry A, Marquardt WW (1996) Role of milk fractions, serum, and divalent cations in protection of mammary epithelial cells of cows against damage by Staphylococcus aureus toxins. Am J Vet Res 57:308–312PubMedGoogle Scholar
  5. Dang ZC, Lowik CW (2005) Removal of serum factors by charcoal treatment promotes adipogenesis via a MAPK-dependent pathway. Mol Cell Biochem 268:159–167PubMedCrossRefGoogle Scholar
  6. Dao MA, Creer MH, Nolta JA, Verfaillie CM (2007) Biology of umbilical cord blood progenitors in bone marrow niches. Blood 110:74–81PubMedCrossRefGoogle Scholar
  7. Davis RJ (1993) The mitogen-activated protein kinase signal transduction pathway. J Biol Chem 268:14553–14556PubMedGoogle Scholar
  8. Delalat B, Pourfathollah AA, Soleimani M, Mozdarani H, Ghaemi SR, Movassaghpour AA, Kaviani S (2009) Isolation and ex vivo expansion of human umbilical cord blood-derived CD34+ stem cells and their cotransplantation with or without mesenchymal stem cells. Hematology 14:125–132PubMedCrossRefGoogle Scholar
  9. Erices A, Conget P, Minguell JJ (2000) Mesenchymal progenitor cells in human umbilical cord blood. Br J Haematol 109:235–242PubMedCrossRefGoogle Scholar
  10. Girard J, Perdereau D, Foufelle F, Prip-Buus C, Ferré P (1994) Regulation of lipogenic enzyme gene expression by nutrients and hormones. FASEB J 8:36–42PubMedGoogle Scholar
  11. Gutkind JS (1998) The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades. J Biol Chem 273:1839–1842PubMedCrossRefGoogle Scholar
  12. Huang GP, Pan ZJ, Jia BB, Zheng Q, Xie CG, Gu JH, McNiece IK, Wang JF (2007) Ex vivo expansion and transplantation of hematopoietic stem/progenitor cells supported by mesenchymal stem cells from human umbilical cord blood. Cell Transplant 16:579–585PubMedCrossRefGoogle Scholar
  13. Jeong S, Yoon M (2011) 17β-Estradiol inhibition of PPARγ-induced adipogenesis and adipocyte-specific gene expression. Acta Pharmacol Sin 32:230–238PubMedCrossRefGoogle Scholar
  14. Jung J, Moon N, Ahn JY, Oh EJ, Kim M, Cho CS, Shin JC, Oh IH (2009) Mesenchymal stromal cells expanded in human allogenic cord blood serum display higher self-renewal and enhanced osteogenic potential. Stem Cells Dev 18:559–571PubMedCrossRefGoogle Scholar
  15. Pagès G, Lenormand P, L'Allemain G, Chambard JC, Meloche S, Pouysségur J (1993) Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci USA 90:8319–8323PubMedCrossRefGoogle Scholar
  16. Phadnis SM, Joglekar MV, Venkateshan V, Ghaskadbi SM, Hardikar AA, Bhonde RR (2006) Human umbilical cord blood serum promotes growth, proliferation, as well as differentiation of human bone marrow-derived progenitor cells. In Vitro Cell Dev Biol Anim 42:283–286PubMedGoogle Scholar
  17. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147PubMedCrossRefGoogle Scholar
  18. Porter RM, Huckle WR, Goldstein AS (2003) Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells. J Cell Biochem 90:13–22PubMedCrossRefGoogle Scholar
  19. Qu Q, Perälä-Heape M, Kapanen A, Dahllund J, Salo J, Väänänen HK, Härkönen P (1998) Estrogen enhances differentiation of osteoblasts in mouse bone marrow culture. Bone 22:201–209PubMedCrossRefGoogle Scholar
  20. Ramírez-Zacarías JL, Castro-Muñozledo F, Kuri-Harcuch W (1992) Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O. Histochemistry 97:493–497PubMedCrossRefGoogle Scholar
  21. Rosen ED, MacDougald OA (2006) Adipocyte differentiation from the inside out. Nat Rev Mol Cell Biol 7:885–896PubMedCrossRefGoogle Scholar
  22. Rosen ED, Spiegelman BM (2000) Molecular regulation of adipogenesis. Annu Rev Cell Biol 16:145–171CrossRefGoogle Scholar
  23. Sankaranarayanan K, Tekkatte C, Gunasingh GP, V Malini, U Prithika, Renny CM, Raghavan U, R Sai Babu, Guhathakurta S, Cherian KM (2011) Humanised substitutes for animal sera in human mesenchymal stem cell culture and differentiation. Cell Biol Int (in press)Google Scholar
  24. Schlessinger J (1993) How receptor tyrosine kinases activate ras. Trends Biochem Sci 18:273–275PubMedCrossRefGoogle Scholar
  25. Shetty P, Bharucha K, Tanavde V (2007) Human umbilical cord blood serum can replace fetal bovine serum in the culture of mesenchymal stem cells. Cell Biol Int 31:293–298PubMedCrossRefGoogle Scholar
  26. Skolnik EY, Batzer A, Li N, Lee CH, Lowenstein E, Mohammadi M, Margolis B, Schlessinger J (1993) The function of GRB2 in linking the insulin receptor to Ras signaling pathways. Science 260:1953–1955PubMedCrossRefGoogle Scholar
  27. Spees JL, Gregory CA, Singh H, Tucker HA, Peister A, Lynch PJ, Hsu SC, Smith J, Prockop DJ (2004) Internalized antigens must be removed to prepare hypoimmunogenic mesenchymal stem cells for cell and gene therapy. Mol Ther 9:747–756PubMedCrossRefGoogle Scholar
  28. Tekkatte C, Gunasingh GP, Cherian KM, Sankaranarayanan K (2011) "Humanized" stem cell culture techniques: the animal serum controversy. Stem Cells Int 2011:504723PubMedGoogle Scholar
  29. Wyrsch A, dalle Carbonare V, Jansen W, Chklovskaia E, Nissen C, Surbek D, Holzgreve W, Tichelli A, Wodnar-Filipowicz A (1999) Umbilical cord blood from preterm human fetuses is rich in committed and primitive hematopoietic progenitors with high proliferative and self-renewal capacity. Exp Hematol 27:1338–1345PubMedCrossRefGoogle Scholar
  30. Yohay DA, Zhang J, Thrailkill KM, Arthur JM, Quarles LD (1994) Role of serum in the developmental expression of alkaline phosphatase in MC3T3-E1 osteoblasts. J Cell Physiol 158:467–475PubMedCrossRefGoogle Scholar
  31. Zhang HH, Huang J, Düvel K, Boback B, Wu S, Squillace RM, Wu CL, Manning BD (2009) Insulin stimulates adipogenesis through the Akt-TSC2-mTORC1 pathway. PLoS One 4:e6189PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Chandana Tekkatte
    • 1
  • Prasanna Vidyasekar
    • 1
  • Nand Kishore Kapadia
    • 2
  • Rama S Verma
    • 1
  1. 1.Stem cell and Molecular Biology laboratory, Department of BiotechnologyIndian Institute of Technology MadrasChennaiIndia
  2. 2.Department of Cardio-thoracic SurgeryFortis Malar HospitalChennaiIndia

Personalised recommendations