Molecular Biology Reports

, Volume 40, Issue 4, pp 3023–3031

Mesenchymal stem cells as an appropriate feeder layer for prolonged in vitro culture of human induced pluripotent stem cells



Feeder layers have been applied extensively to support the growth and stemness potential of stem cells for in vitro cultures. Mouse embryonic fibroblast and mouse fibroblast cell line (SNL) are common feeder cells for human induced pluripotent stem cells (hiPSCs) culture. Because of some problems in the use of these animal feeders and in order to simplify the therapeutic application of hiPSCs, we tested human adult bone marrow mesenchymal stem cells (hMSCs) as a potent feeder system. This method benefits from prevention of possible contamination of animal origin feeder systems. hiPSCs transferred onto mitotically inactivated hMSCs and passaged every 5 days. Prior to this culture, MSCs were characterized by flow cytometry of their surface markers and evaluation of their osteogenic and adipogenic differentiation potentials. The morphology, expressions of some specific pluripotency markers such as SSEA-3, NANOG and TRA-1-60, alkaline phosphates activity, formation embryoid bodies and their differentiation potentials of iPSCs on SNL and MSC feeder layers were evaluated. To investigate the prolonged maintenance of pluripotency, the quantitative transcriptions of some pluripotency markers including OCT4, SOX2, NANOG and REX1 were compared in the iPS clones on SNL or MSC feeders. Human iPSCs cultured on human MSCs feeder were slightly thinner and flatter than ones on the other feeder system. Interestingly MSCs supported the prolonged in vitro proliferation of hiPSCs along with maintenance of their pluripotency. Altogether our results suggest human mesenchymal stem cells as an appropriate feeder layer for human iPSCs culture for clinical applications and cell therapy.


Embryoid body Feeder layer Human induced pluripotent stem cell Mesenchymal stem cell Mouse fibroblastic feeder 

Supplementary material

11033_2012_2376_MOESM1_ESM.doc (24 kb)
Supplementary material 1 (DOC 23 kb)


  1. 1.
    Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872PubMedCrossRefGoogle Scholar
  2. 2.
    Park IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA, Lerou PH, Lensch MW, Daley GQ (2008) Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451(7175):141–146PubMedCrossRefGoogle Scholar
  3. 3.
    Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318(5858):1917–1920PubMedCrossRefGoogle Scholar
  4. 4.
    Moretti A, Bellin M, Welling A, Jung CB, Lam JT, Bott-Flugel L, Dorn T, Goedel A, Hohnke C, Hofmann F, Seyfarth M, Sinnecker D, Schomig A, Laugwitz KL (2010) Patient-specific induced pluripotent stem-cell models for long-QT syndrome. N Engl J Med 363(15):1397–1409PubMedCrossRefGoogle Scholar
  5. 5.
    Hu BY, Weick JP, Yu J, Ma LX, Zhang XQ, Thomson JA, Zhang SC (2010) Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency. Proc Natl Acad Sci USA 107(9):4335–4340PubMedCrossRefGoogle Scholar
  6. 6.
    Narsinh KH, Sun N, Sanchez-Freire V, Lee AS, Almeida P, Hu S, Jan T, Wilson KD, Leong D, Rosenberg J, Yao M, Robbins RC, Wu JC (2011) Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells. J Clin Invest 121(3):1217–1221PubMedCrossRefGoogle Scholar
  7. 7.
    Feng Q, Lu SJ, Klimanskaya I, Gomes I, Kim D, Chung Y, Honig GR, Kim KS, Lanza R (2010) Hemangioblastic derivatives from human induced pluripotent stem cells exhibit limited expansion and early senescence. Stem Cells 28(4):704–712PubMedCrossRefGoogle Scholar
  8. 8.
    Ben-Nun IF, Montague SC, Houck ML, Tran HT, Garitaonandia I, Leonardo TR, Wang YC, Charter SJ, Laurent LC, Ryder OA, Loring JF (2011) Induced pluripotent stem cells from highly endangered species. Nat Methods 8(10):829–831PubMedCrossRefGoogle Scholar
  9. 9.
    Bendall SC, Stewart MH, Menendez P, George D, Vijayaragavan K, Werbowetski-Ogilvie T, Ramos-Mejia V, Rouleau A, Yang J, Bosse M, Lajoie G, Bhatia M (2007) IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature 448(7157):1015–1021PubMedCrossRefGoogle Scholar
  10. 10.
    James D, Levine AJ, Besser D, Hemmati-Brivanlou A (2005) TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 132(6):1273–1282PubMedCrossRefGoogle Scholar
  11. 11.
    Wang L, Li L, Menendez P, Cerdan C, Bhatia M (2005) Human embryonic stem cells maintained in the absence of mouse embryonic fibroblasts or conditioned media are capable of hematopoietic development. Blood 105(12):4598–4603PubMedCrossRefGoogle Scholar
  12. 12.
    Pekkanen-Mattila M, Ojala M, Kerkela E, Rajala K, Skottman H, Aalto-Setala K (2012) The effect of human and mouse fibroblast feeder cells on cardiac differentiation of human pluripotent stem cells. Stem Cells Int 2012:875059PubMedGoogle Scholar
  13. 13.
    Takahashi K, Narita M, Yokura M, Ichisaka T, Yamanaka S (2009) Human induced pluripotent stem cells on autologous feeders. PLoS One 4(12):e8067PubMedCrossRefGoogle Scholar
  14. 14.
    Amit M, Margulets V, Segev H, Shariki K, Laevsky I, Coleman R, Itskovitz-Eldor J (2003) Human feeder layers for human embryonic stem cells. Biol Reprod 68(6):2150–2156PubMedCrossRefGoogle Scholar
  15. 15.
    Richards M, Fong CY, Chan WK, Wong PC, Bongso A (2002) Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 20(9):933–936PubMedCrossRefGoogle Scholar
  16. 16.
    Ma T (2010) Mesenchymal stem cells: from bench to bedside. World J Stem Cells 2(2):13–17PubMedCrossRefGoogle Scholar
  17. 17.
    Aggarwal S, Pittenger MF (2005) Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105(4):1815–1822PubMedCrossRefGoogle Scholar
  18. 18.
    Le Blanc K, Tammik L, Sundberg B, Haynesworth SE, Ringden O (2003) Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex. Scand J Immunol 57(1):11–20PubMedCrossRefGoogle Scholar
  19. 19.
    Bakhshandeh B, Soleimani M, Hafizi M, Ghaemi N (2012) A comparative study on nonviral genetic modifications in cord blood and bone marrow mesenchymal stem cells. Cytotechnology 64(5):523–540PubMedCrossRefGoogle Scholar
  20. 20.
    Sakata N, Chan NK, Chrisler J, Obenaus A, Hathout E (2010) Bone marrow cell cotransplantation with islets improves their vascularization and function. Transplantation 89(6):686–693PubMedCrossRefGoogle Scholar
  21. 21.
    Montes R, Ligero G, Sanchez L, Catalina P, de la Cueva T, Nieto A, Melen GJ, Rubio R, Garcia-Castro J, Bueno C, Menendez P (2009) Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media: distinct requirements for TGF-beta and IGF-II. Cell Res 19(6):698–709PubMedCrossRefGoogle Scholar
  22. 22.
    Bakhshandeh B, Soleimani M, Hafizi M, Paylakhi SH, Ghaemi N (2012) MicroRNA signature associated with osteogenic lineage commitment. Mol Biol Rep 39(7):7569–7581PubMedCrossRefGoogle Scholar
  23. 23.
    Volarevic V, Ljujic B, Stojkovic P, Lukic A, Arsenijevic N, Stojkovic M (2011) Human stem cell research and regenerative medicine: present and future. Br Med Bull 99:155–168PubMedCrossRefGoogle Scholar
  24. 24.
    Xu C, Jiang J, Sottile V, McWhir J, Lebkowski J, Carpenter MK (2004) Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth. Stem Cells 22(6):972–980PubMedCrossRefGoogle Scholar
  25. 25.
    Hovatta O, Mikkola M, Gertow K, Stromberg AM, Inzunza J, Hreinsson J, Rozell B, Blennow E, Andang M, Ahrlund-Richter L (2003) A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 18(7):1404–1409PubMedCrossRefGoogle Scholar
  26. 26.
    Cheng L, Hammond H, Ye Z, Zhan X, Dravid G (2003) Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. Stem Cells 21(2):131–142PubMedCrossRefGoogle Scholar
  27. 27.
    Eiselleova L, Peterkova I, Neradil J, Slaninova I, Hampl A, Dvorak P (2008) Comparative study of mouse and human feeder cells for human embryonic stem cells. Int J Dev Biol 52(4):353–363PubMedCrossRefGoogle Scholar
  28. 28.
    Zhang K, Cai Z, Li Y, Shu J, Pan L, Wan F, Li H, Huang X, He C, Liu Y, Cui X, Xu Y, Gao Y, Wu L, Cao S, Li L (2011) Utilization of human amniotic mesenchymal cells as feeder layers to sustain propagation of human embryonic stem cells in the undifferentiated state. Cell Reprogram 13(4):281–288PubMedCrossRefGoogle Scholar
  29. 29.
    Park JH, Kim SJ, Oh EJ, Moon SY, Roh SI, Kim CG, Yoon HS (2003) Establishment and maintenance of human embryonic stem cells on STO, a permanently growing cell line. Biol Reprod 69(6):2007–2014PubMedCrossRefGoogle Scholar
  30. 30.
    Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK (2001) Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 19(10):971–974PubMedCrossRefGoogle Scholar
  31. 31.
    Ying QL, Stavridis M, Griffiths D, Li M, Smith A (2003) Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat Biotechnol 21(2):183–186PubMedCrossRefGoogle Scholar
  32. 32.
    Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH (2004) Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 10(1):55–63PubMedCrossRefGoogle Scholar
  33. 33.
    Klingemann H, Matzilevich D, Marchand J (2008) Mesenchymal stem cells: sources and clinical applications. Transfus Med Hemother 35(4):272–277PubMedCrossRefGoogle Scholar
  34. 34.
    Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8(4):315–317PubMedCrossRefGoogle Scholar
  35. 35.
    Fathke C, Wilson L, Hutter J, Kapoor V, Smith A, Hocking A, Isik F (2004) Contribution of bone marrow-derived cells to skin: collagen deposition and wound repair. Stem Cells 22(5):812–822PubMedCrossRefGoogle Scholar
  36. 36.
    Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Scholer H, Smith A (1998) Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95(3):379–391PubMedCrossRefGoogle Scholar
  37. 37.
    Niwa H, Miyazaki J, Smith AG (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24(4):372–376PubMedCrossRefGoogle Scholar
  38. 38.
    Pesce M, Gross MK, Scholer HR (1998) In line with our ancestors: oct-4 and the mammalian germ. Bioessays 20(9):722–732PubMedCrossRefGoogle Scholar
  39. 39.
    Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R (2003) Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev 17(1):126–140PubMedCrossRefGoogle Scholar
  40. 40.
    Kalmar T, Lim C, Hayward P, Munoz-Descalzo S, Nichols J, Garcia-Ojalvo J, Martinez Arias A (2009) Regulated fluctuations in nanog expression mediate cell fate decisions in embryonic stem cells. PLoS Biol 7(7):e1000149PubMedCrossRefGoogle Scholar
  41. 41.
    Pan G, Li J, Zhou Y, Zheng H, Pei D (2006) A negative feedback loop of transcription factors that controls stem cell pluripotency and self-renewal. FASEB J 20(10):1730–1732PubMedCrossRefGoogle Scholar
  42. 42.
    Chambers I (2004) The molecular basis of pluripotency in mouse embryonic stem cells. Cloning Stem Cells 6(4):386–391PubMedCrossRefGoogle Scholar
  43. 43.
    Xu RH, Sampsell-Barron TL, Gu F, Root S, Peck RM, Pan G, Yu J, Antosiewicz-Bourget J, Tian S, Stewart R, Thomson JA (2008) NANOG is a direct target of TGFbeta/activin-mediated SMAD signaling in human ESCs. Cell Stem Cell 3(2):196–206PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  1. 1.Department of Developmental BiologyFaculty of Biological Science, Kharazmi UniversityTehranIran
  2. 2.Stem Cell Biology DepartmentStem Cell Technology Research CenterTehranIran
  3. 3.Department of Hematology, Faculty of Medical ScienceTarbiat Modares UniversityTehranIran
  4. 4.Department of Biotechnology, College of ScienceUniversity of TehranTehranIran
  5. 5.Department of Biology, Science and Research BranchIslamic Azad UniversityTehranIran

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