Tissue Engineering and Regenerative Medicine

, Volume 9, Issue 6, pp 311–319 | Cite as

Direct comparison of distinct cardiomyogenic induction methodologies in human cardiac-derived c-kit positive progenitor cells

Original Article


Cardiac stem/progenitor cells can be differentiated into cardiomyocytes in vitro using several differentiation methodologies. However, the methodology of cardiomyogenic induction in human c-kit positive progenitor cells (hCPCsc-kit+) was not fully demonstrated. Thus, the purpose of our study was to directly evaluate each cardiomyocyte induction system using hCPCsc-kit+. In this study, cardiomyocyte induction methodologies were divided into the following three groups; treatment with dexamethasone, 5-azacytidine, and co-treatment with 5-azacytidine and Transforming Growth Factor Beta 1 (TGF-β1), using different serum concentrations [2% or 10% fetal bovine serum (FBS)]. GATA4 and Nkx2-5, cardiac-specific transcription factors, were expressed in our hCPCsckit+. However, the GATA4 and Nkx2-5 expressions were significantly decreased in 10% FBS/cardiomyogenic induction system (p < 0.01), whereas the GATA4 and Nkx2-5 expressions were preserved in 2% FBS/cardiomyogenic induction system (p > 0.05). GATA4 and Nkx2-5 is crucial roles in cardiac development, thus we considered the low serum conditions more affected in our cardiomyogenic induction system. In addition, c-kit expression decreased significantly during cardiomyogenic differentiation. Importantly, we demonstrated that co-treated with 5-azacytidine and TGF-β1 led to an earlier expression pattern of alpha-sarcomeric actin (α-SA), implying that this cardiomyocyte induction system facilitates early cardiomyocyte differentiation of hCPCsc-kit+. Thus, the present study provides a pivotal cardiomyogenic differentiation methodology using hCPCsc-kit+for basic or clinical research.

Key words

5-azacytidine cardiomyocyte differentiation dexamethasone hCPCsc-kit+ TGF-β


  1. 1.
    A Leri, J Kajstura and P Anversa, Cardiac stem cells and mechanisms of myocardial regeneration, Physi Rev, 85, 1373 (2005).CrossRefGoogle Scholar
  2. 2.
    E Messina, LDe Angelis, G Frati, et al., Isolation and expansion of adult cardiac stem cells from human and murine heart, Circulation Research, 95, 911 (2004).PubMedCrossRefGoogle Scholar
  3. 3.
    C Bearzi, M Rota, T Hosoda, et al., Human cardiac stem cells, Proc Nat Aca Sci Unit Stat America, 104, 14068 (2007).CrossRefGoogle Scholar
  4. 4.
    TS Li, T Komota, M Ohshima, et al., TGF-beta induces the differentiation of bone marrow stem cells into immature cardiomyocytes, Biochem Bioph Res Comm, 366, 1074 (2008).CrossRefGoogle Scholar
  5. 5.
    Y Zhu, T Liu, K Song, et al., ADSCs differentiated into cardiomyocytes in cardiac microenvironment, Molecular and Cell Biochem, 324, 117 (2009).CrossRefGoogle Scholar
  6. 6.
    S Makino, K Fukuda, S Miyoshi, et al., Cardiomyocytes can be generated from marrow stromal cells in vitro, J Clin Invest, 103, 697 (1999).PubMedCrossRefGoogle Scholar
  7. 7.
    D Durocher, F Charron, R Warren, et al., The cardiac transcription factors Nkx2-5 and GATA-4 are mutual cofactors, EMBO J, 16, 5687 (1997).PubMedCrossRefGoogle Scholar
  8. 8.
    SH Orkin, GATA-binding transcription factors in hematopoietic cells, Blood, 80, 575 (1992).PubMedGoogle Scholar
  9. 9.
    WY Huang, E Cukerman, CC Liew, Identification of a GATA motif in the cardiac alpha-myosin heavy-chain-encoding gene and isolation of a human GATA-4 cDNA, Gene, 155, 219 (1995).PubMedCrossRefGoogle Scholar
  10. 10.
    RJ Arceci, AA King, MC Simon, et al., Mouse GATA-4: a retinoic acid-inducible GATA-binding transcription factor expressed in endodermally derived tissues and heart, Mol and Cell Biol, 13, 2235 (1993).Google Scholar
  11. 11.
    S Tamura, XH Wang, M Maeda, et al., Gastric DNA-binding proteins recognize upstream sequence motifs of parietal cellspecific genes, Proc Nati Aca Sci Unit Stat Am, 90, 10876 (1993).CrossRefGoogle Scholar
  12. 12.
    F Charron, M Nemer, GATA transcription factors and cardiac development, Seminars in Cell & Develo Biology, 10, 85 (1999).CrossRefGoogle Scholar
  13. 13.
    PB Burton, MC Raff, P Kerr, et al., An intrinsic timer that controls cell-cycle withdrawal in cultured cardiac myocytes, Develo Biol, 216, 659 (1999).CrossRefGoogle Scholar
  14. 14.
    I Lyons, LM Parsons, L Hartley, et al., Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5, Genes & Develo, 9, 1654 (1995).CrossRefGoogle Scholar
  15. 15.
    M Farokhpour, K Karbalaie, S Tanhaei, et al., Embryonic stem cellderived cardiomyocytes as a model system to study cardioprotective effects of dexamethasone in doxorubicin cardiotoxicity, Toxicology in vitro: an int J publ asso with BIBRA, 23, 1422 (2009).CrossRefGoogle Scholar
  16. 16.
    WS Shim, S Jiang, P Wong, et al., Ex vivo differentiation of human adult bone marrow stem cells into cardiomyocyte-like cells, Biochem Bioph Res Comm, 324, 481 (2004).CrossRefGoogle Scholar
  17. 17.
    AM Smits, P van Vliet, CH Metz, et al., Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes: an in vitro model for studying human cardiac physiology and pathophysiology, Nature Prot, 4, 232 (2009).CrossRefGoogle Scholar
  18. 18.
    MJ Goumans, TP de Boer, AM Smits, et al., TGF-beta1 induces efficient differentiation of human cardiomyocyte progenitor cells into functional cardiomyocytes in vitro, Stem Cell Res, 1, 138 (2007).PubMedCrossRefGoogle Scholar
  19. 19.
    BC Heng, H Haider, EK Sim, et al., Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro, Cardiovas Res, 62, 34 (2004).CrossRefGoogle Scholar
  20. 20.
    H Oh, SB Bradfute, TD Gallardo, et al., Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction, Proc Nat Aca Sci Unit Stat of Am, 100, 12313 (2003).CrossRefGoogle Scholar
  21. 21.
    F Limana, A Zacheo, D Mocini, et al., Identification of myocardial and vascular precursor cells in human and mouse epicardium, Cir Res, 101, 1255 (2007).CrossRefGoogle Scholar
  22. 22.
    KL Laugwitz, A Moretti, J Lam, et al., Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages, Nature, 433, 647 (2005).PubMedCrossRefGoogle Scholar
  23. 23.
    Y He, WY Zhang, M Gong, et al., Low serum concentration facilitates the differentiation of hepatic progenitor cells, Saudi Med J, 32, 128 (2011).PubMedGoogle Scholar
  24. 24.
    SC Choi, J Yoon, WJ Shim, et al., 5-azacytidine induces cardiac differentiation of P19 embryonic stem cells, Exper Mol Med, 36, 515 (2004).Google Scholar
  25. 25.
    P Antonitsis, E Ioannidou-Papagiannaki, A Kaidoglou, et al., In vitro cardiomyogenic differentiation of adult human bone marrow mesenchymal stem cells. the role of 5-azacytidine, Int Cardiovas Thora Surg, 6, 593 (2007).CrossRefGoogle Scholar
  26. 26.
    Q Qian, H Qian, X Zhang, et al., 5-Azacytidine induces cardiac differentiation of human umbilical cord-derived mesenchymal stem cells by activating extracellular regulated kinase, Stem Cells Devel, 21, 67 (2012).CrossRefGoogle Scholar
  27. 27.
    K Fukuda, Regeneration of cardiomyocytes from bone marrow: use of mesenchymal stem cell for cardiovascular tissue engineering, Cytotech, 41, 165 (2003).CrossRefGoogle Scholar
  28. 28.
    HS Kim, JW Cho, K Hidaka, et al., Activation of MEK-ERK by heregulin-beta1 promotes the development of cardiomyocytes derived from ES cells, Biochem Bioph Res Comm, 361, 732 (2007).CrossRefGoogle Scholar
  29. 29.
    A Bel, E Messas, O Agbulut, et al., Transplantation of autologous fresh bone marrow into infarcted myocardium: a word of caution, Circul, 108Suppl 1, II247 (2003).Google Scholar
  30. 30.
    SM Taylor, PA Jones, Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine, Cell, 17, 771 (1979).PubMedCrossRefGoogle Scholar
  31. 31.
    SA Chamuleau, E van Belle, PA Doevendans, Enhancing cardiac stem cell differentiation into cardiomyocytes, Cardiovas Res, 82, 385 (2009).CrossRefGoogle Scholar
  32. 32.
    AP Beltrami, L Barlucchi, D Torella, et al., Adult cardiac stem cells are multipotent and support myocardial regeneration, Cell, 114, 763 (2003).PubMedCrossRefGoogle Scholar
  33. 33.
    K Matsuura, T Nagai, N Nishigaki, et al., Adult cardiac Sca-1-positive cells differentiate into beating cardiomyocytes, J Biolo Chem, 279, 11384 (2004).CrossRefGoogle Scholar
  34. 34.
    O Pfister, F Mouquet, M Jain, et al., CD31− but Not CD31+ cardiac side population cells exhibit functional cardiomyogenic differentiation, Cir Res, 97, 52 (2005).CrossRefGoogle Scholar

Copyright information

© The Korean Tissue Engineering and Regenerative Medicine Society and Springer Netherlands 2012

Authors and Affiliations

  1. 1.Laboratory of Cardiovascular Regeneration, Division of Cardiovascular Medicine, Seoul St. Mary’s HospitalThe Catholic University of Korea School of MedicineSeoulKorea
  2. 2.Laboratory of Vascular Medicine and Stem Cell Biology, Department of PhysiologyPusan National University School of MedicineYangsanKorea
  3. 3.Stem Cell Translational ResearchInstitute of Biomedical Research and Innovation/RIKKEN Center of Developmental BiologyKobeJapan
  4. 4.Department of Regenerative Medicine ScienceTokai University School of MedicineIseharaJapan
  5. 5.College of PharmacyAjou UniversitySuwonKorea

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