Molecular Biology Reports

, Volume 39, Issue 3, pp 2585–2595 | Cite as

Over-expression of Nkx2.5 and/or cardiac α-actin inhibit the contraction ability of ADSCs-derived cardiomyocytes

  • Lili Zhao
  • Dapeng Ju
  • Qian Gao
  • Xueli Zheng
  • Gongshe Yang


Adipose tissue-derived stromal cells (ADSCs) can differentiate into cardiomyocytes, which provide a source of new cardiomyocyte progenitors for tissue engineering. Here, we showed that ADSCs isolated from subcutaneous adipose tissues of mouse were largely negative for CD31, CD34, but positive for CD105. About 1.62% cells in these cells can spontaneously differentiate into cardiac-like cells (cells expressing cardiac marker proteins) when cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented only with penicillin, streptomycin, and 20% newborn bovine serum (NBS), expressed cardiac markers such as MF20, Connexin45, cMHC, cTnT, a-actin, Nkx2.5, and GATA4, and part of these cells (account for about 0.47% of inoculated cells) showed spontaneous contractions accompanied by transient Ca2+ activity in culture. In vitro, although over-expression of Nkx2.5 and/or cardiac α-actin increased the number of cardiac-like cells expressing cardiac-specific proteins, but while inhibited the contraction function of ADSCs-derived cardiomyocytes.


Adipose Stromal cells Cardiomyogenic differentiation Cardiomyocytes Nkx2.5 Cardiac α-actin 



We thank Prof. Zhiying Zhang (College of Animal Science and Technology, Northwest A&F University, Shaanxi, China) for his suggestions on experimental design. Contract Grant Sponsor: Key and Specific National Project for Creating New Biological Species Transgenically; Contract Grant Number: 2009ZX08009-157B.

Supplementary material

Supplementary material 1 (MPG 10996 kb)

Supplementary material 2 (MPG 15348 kb)


  1. 1.
    Vidarsson H, Hyllner J, Sartipy P (2010) Differentiation of human embryonic stem cells to cardiomyocytes for in vitro and in vivo applications. Stem Cell Rev Rep 6:108–120CrossRefGoogle Scholar
  2. 2.
    Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, Kasahara H, Rota M, Musso E, Urbanek K, Leri A, Kajstura J, Nadal-Ginard B, Anversa P (2003) Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 114:763–776PubMedCrossRefGoogle Scholar
  3. 3.
    Pallante BA, Duignan I, Okin D, Chin A, Bressan MC, Mikawa T, Edelberg JM (2007) Bone marrow Oct3/4+ cells differentiate into cardiac myocytes via age-dependent paracrine mechanisms. Circ Res 100:1–11CrossRefGoogle Scholar
  4. 4.
    Oyama T, Nagai T, Wada H, Naito AT, Matsuura K, Iwanaga K, Takahashi T, Goto M, Mikami Y, Yasuda N, Akazawa H, Uezumi A, Takeda S, Komuro I (2007) Cardiac side population cells have a potential to migrate and differentiate into cardiomyocytes in vitro and in vivo. J Cell Biol 176:329–341PubMedCrossRefGoogle Scholar
  5. 5.
    Zhang J, Wilson GF, Soerens AG, Koonce CH, Yu J, Palecek SP, Thomson JA, Kamp TJ (2009) Functional cardiomyocytes derived from human induced pluripotent stem cells. Circ Res 104:30–41CrossRefGoogle Scholar
  6. 6.
    Madonna R, Geng YJ, De Caterina R (2009) Adipose tissue-derived stem cells: characterization and potential for cardiovascular repair. Arterioscler Thromb Vasc Biol 29:1723–1729PubMedCrossRefGoogle Scholar
  7. 7.
    Léobon B, Roncalli J, Joffre C, Mazo M, Boisson M, Barreau C, Calise D, Arnaud E, André M, Pucéat M, Pénicaud L, Prosper F, Planat-Bénard V, Casteilla L (2009) Adipose-derived cardiomyogenic cells: in vitro expansion and functional improvement in a mouse model of myocardial infarction. Cardiovasc Res 83:757–767PubMedCrossRefGoogle Scholar
  8. 8.
    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
  9. 9.
    Bacou F, el Andalousi RB, Daussin PA, Micallef JP, Levin JM, Chammas M, Casteilla L, Reyne Y, Nouguès J (2004) Transplantation of adipose tissue-derived stromal cells increases mass and functional capacity of damaged skeletal muscle. Cell Transplant 13:103–111PubMedGoogle Scholar
  10. 10.
    Planat-Bénard V, Menard C, André M, Puceat M, Perez A, Garcia-Verdugo JM, Pénicaud L, Casteilla L (2004) Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells. Circ Res 94:223–229PubMedCrossRefGoogle Scholar
  11. 11.
    Kim MR, Jeon ES, Kim YM, Lee JS, Kim JH (2009) Thromboxane A2 induces differentiation of human mesenchymal stem cells to smooth muscle-like cells. Stem Cells 27:191–199PubMedCrossRefGoogle Scholar
  12. 12.
    Erickson GR, Gimble JM, Franklin DM, Rice HE, Awad H, Guilak F (2002) Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo. Biochem Biophys Res Commun 290:763–769PubMedCrossRefGoogle Scholar
  13. 13.
    Gimble J, Guilak F (2003) Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy 5:362–369PubMedCrossRefGoogle Scholar
  14. 14.
    Rangappa S, Fen C, Lee EH, Bongso A, Sim EK (2003) Transformation of adult mesenchymal stem cells isolated from the fatty tissue into cardiomyocytes. Ann Thorac Surg 75:775–779PubMedCrossRefGoogle Scholar
  15. 15.
    Choi YS, Dusting GJ, Stubbs S, Arunothayaraj S, Han XL, Collas P, Morrison WA, Dilley RJ (2010) Differentiation of human adipose-derived stem cells into beating cardiomyocytes. J Cell Mol Med 14:878–889PubMedCrossRefGoogle Scholar
  16. 16.
    Jumabay M, Zhang R, Yao Y, Goldhaber JI, Boström KI (2010) Spontaneously beating cardiomyocytes derived from white mature adipocytes. Cardiovasc Res 85:17–27PubMedCrossRefGoogle Scholar
  17. 17.
    Matsumoto T, Kano K, Kondo D, Fukuda N, Iribe Y, Tanaka N, Matsubara Y, Sakuma T, Satomi A, Otaki M, Ryu J, Mugishima H (2007) Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential. J Cell Physiol 215:210–222CrossRefGoogle Scholar
  18. 18.
    Kazama T, Fujie M, Endo T, Kano K (2008) Mature adipocyte-derived dedifferentiated fat cells can transdifferentiate into skeletal myocytes in vitro. Biochem Biophys Res Commun 377:780–785PubMedCrossRefGoogle Scholar
  19. 19.
    Briggs LE, Takeda M, Cuadra AE, Wakimoto H, Marks MH, Walker AJ, Seki T, Oh SP, Lu JT, Sumners C, Raizada MK, Horikoshi N, Weinberg EO, Yasui K, Ikeda Y, Chien KR, Kasahara H (2008) Perinatal loss of Nkx2-5 results in rapid conduction and contraction defects. Circ Res 103:580–590PubMedCrossRefGoogle Scholar
  20. 20.
    Yamada Y, Sakurada K, Takeda Y, Gojo S, Umezawa A (2007) Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 undergo the stochastic cardiomyogenic fate and behave like transient amplifying cells. Exp Cell Res 313:698–706PubMedCrossRefGoogle Scholar
  21. 21.
    Sassoon DA, Garner I, Buckingham M (1988) Transcripts of α-cardiac and α-skeletal actins are early markers for myogenesis in the mouse embryo. Development 104:155–164PubMedGoogle Scholar
  22. 22.
    Chen CY, Schwartz RJ (1996) Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac a-actin gene transcription. Mol Cell Biol 16:6372–6384PubMedGoogle Scholar
  23. 23.
    Tholpady SS, Katz AJ, Ogle RC (2003) Mesenchymal stem cells from rat visceral fat exhibit multipotential differentiation in vitro. Anat Rec A Discov Mol Cell Evol Biol 272:398–402PubMedCrossRefGoogle Scholar
  24. 24.
    Madonna R, De Caterina R (2008) In vitro neovasculogenic potential of resident adipose tissue precursors. Am J Physiol Cell Physiol 295:1271–1280CrossRefGoogle Scholar
  25. 25.
    He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B (1998) A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 95:2509–2514PubMedCrossRefGoogle Scholar
  26. 26.
    Dubois SG, Floyd EZ, Zvonic S, Kilroy G, Wu X, Carling S, Halvorsen YD, Ravussin E, Gimble JM (2008) Isolation of human adipose-derived stem cells from biopsies and liposuction specimens. Methods Mol Biol 449:69–79PubMedCrossRefGoogle Scholar
  27. 27.
    Kodama H, Hirotani T, Suzuki Y, Ogawa S, Yamazaki K (2002) Cardiomyogenic differentiation in cardiac myxoma expressing lineage-specific transcription factors. Am J Pathol 161:381–389PubMedCrossRefGoogle Scholar
  28. 28.
    Armiñán A, Gandía C, Bartual M, García-Verdugo JM, Lledó E, Mirabet V, Llop M, Barea J, Montero JA, Sepúlveda P (2009) Cardiac differentiation is driven by NKX2.5 and GATA4 nuclear translocation in tissue-specific mesenchymal stem cells. Stem Cells Dev 18:907–918PubMedCrossRefGoogle Scholar
  29. 29.
    Skerjanc IS, Petropoulos H, Ridgeway AG, Wilton S (1998) Myocyte enhancer factor 2C and Nkx2-5 up-regulate each other’s expression and initiate cardiomyogenesis in P19 cells. J Biol Chem 273:34904–34910PubMedCrossRefGoogle Scholar
  30. 30.
    Wei L, Roberts W, Wang L, Yamada M, Zhang S, Zhao Z, Rivkees SA, Schwartz RJ, Imanaka-Yoshida K (2001) Rho kinases play an obligatory role in vertebrate embryonic organogenesis. Development 128:2953–2962PubMedGoogle Scholar
  31. 31.
    Yamada Y, Yokoyama S, Wang XD, Fukuda N, Takakura N (2007) Cardiac stem cells in brown adipose tissue express CD133 and induce bone marrow nonhematopoietic cells to differentiate into cardiomyocytes. Stem Cells 25:1326–1333PubMedCrossRefGoogle Scholar
  32. 32.
    Monzen K, Zhu W, Kasai H, Hiroi Y, Hosoda T, Akazawa H, Zou Y, Hayashi D, Yamazaki T, Nagai R, Komuro I (2002) Dual effects of the homeobox transcription factor Csx/Nkx2-5 on cardiomyocytes. Biochem Biophys Res Commun 298:493–500PubMedCrossRefGoogle Scholar
  33. 33.
    Riazi AM, Lee H, Hsu C, Van Arsdell G (2005) CSX/Nkx2.5 modulates differentiation of skeletal myoblasts and promotes differentiation into neuronal cells in vitro. J Biol Chem 280:10716–10720PubMedCrossRefGoogle Scholar
  34. 34.
    Tanaka M, Chen Z, Bartunkova S, Yamasaki N, Izumo S (1999) The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development. Development 126:1269–1280PubMedGoogle Scholar
  35. 35.
    Chen CY, Schwartz RJ (1997) Competition between negative acting YY1 versus positive acting serum response factor and tinman homologue Nkx-2.5 regulates cardiac alpha-actin promoter activity. Mol Endocrinol 11:812–822PubMedCrossRefGoogle Scholar
  36. 36.
    Morkin E (2000) Control of cardiac myosin heavy chain gene expression. Microsc Res Tech 50:522–531PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Lili Zhao
    • 1
  • Dapeng Ju
    • 1
  • Qian Gao
    • 1
  • Xueli Zheng
    • 1
  • Gongshe Yang
    • 1
  1. 1.Laboratory of Animal Fat Deposition and Muscle Development, College of Animal Science and TechnologyNorthwest A&F UniversityYanglingPeople’s Republic of China

Personalised recommendations