Cell Biochemistry and Biophysics

, Volume 66, Issue 2, pp 309–318 | Cite as

Differentiation of Reprogrammed Mouse Cardiac Fibroblasts into Functional Cardiomyocytes

  • Bo Jiang
  • Hongyan Dong
  • Qingpeng Li
  • Yong Yu
  • Zhifeng Zhang
  • Yazhou Zhang
  • Gang WangEmail author
  • Zhongming ZhangEmail author
Original Paper


Fibroblasts can be reprogrammed by ectopic expression of reprogramming factors to yield induced pluripotent stem (iPS) cells that are capable of transdifferentiating into diverse types of somatic cell lines. In this study, we examined if functional cardiomyocytes (CMs) can be produced from mouse cardiac fibroblasts (CFs), using iPS cell factor-based reprogramming. CFs were isolated from Oct4-GFP-C57 mice and infected with a retrovirus expressing the Yamanaka reprogramming factors, Oct4, Sox2, Klf4, and c-Myc to reprogram the CFs into a CF-iPS cell line. Primary mouse embryonic fibroblast cells (MEFs) were used as a control. We found that the dedifferentiated CF-iPS cells showed similar biological characteristics (morphology, pluripotent factor expression, and methylation level) as embryonic stem cells (ESs) and MEF-iPS cells. We used the classical embryoid bodies (EBs)-based method and a transwell CM co-culture system to simulate the myocardial paracrine microenvironment for performing CF-iPS cell cardiogenic differentiation. Under this simulated myocardial microenvironment, CF-iPS cells formed spontaneously beating EBs. The transdifferentiated self-beating cells expressed cardiac-specific transcription and structural factors and also displayed typical myocardial morphology and electrophysiological characteristics. CFs can be dedifferentiated into iPS cells and further transdifferentiated into CMs. CFs hold great promise for CM regeneration as an autologous cell source for functional CM in situ without the need for exogenous cell transplantation in ischemic heart disease.


Cardiac fibroblasts Induced pluripotent stem cells Directed differentiation Cardiomyocyte Myocardial regeneration 



This study was supported by the National Natural Science Foundation of China (31071307) to Zhongming Zhang, Ministry of Science and Technology (2009CB941100) and Chinese Academy of Sciences (XDA01010401) to Gang Wang. We thank Dr. Jingwen Yin, Xiaona Chen, Zhen Liu and Yu Fu for their excellent technical assistance and all the members in Prof. Gang Wang’s lab for their helpful discussion in this study. We are grateful to Prof. Anning Lin for his kind help.

Conflict of interest

The authors confirm that there are no conflicts of interest.

Supplementary material

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Supplementary material 1 (DOC 34 kb)
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Supplementary material 2 (DOC 27 kb)
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Supplementary material 3 (DOC 27 kb)
12013_2012_9487_MOESM4_ESM.tif (914 kb)
Supplementary material 4 (TIF 914 kb)

Supplementary material 5 (AVI 611 kb)


  1. 1.
    Segers, V. F., & Lee, R. T. (2008). Stem-cell therapy for cardiac disease. Nature, 451, 937–942.PubMedCrossRefGoogle Scholar
  2. 2.
    Reinecke, H., Minami, E., Zhu, W. Z., & Laflamme, M. A. (2008). Cardiogenic differentiation and transdifferentiation of progenitor cells. Circulation Research, 103, 1058–1071.PubMedCrossRefGoogle Scholar
  3. 3.
    Dong, H. Y., Xu, Z. W., Zhang, Z. M., et al. (2009). Mobilization of bone marrow-derived Nk2-5+ cardiac progenitor cells under acute myocardial ischemia. Acta Physiologica Sinica, 61, 189–193.Google Scholar
  4. 4.
    Nussbaum, J., Minami, E., Laflamme, M. A., Virag, J. A., Ware, C. B., Masino, A., et al. (2007). Transplantation of undifferentiated murine embryonic stem cells in the heart: Teratoma formation and immune response. FASEB Journal, 21, 1345–1357.PubMedCrossRefGoogle Scholar
  5. 5.
    Kuzmenkin, A., Liang, H., Xu, G., et al. (2009). Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro. FASEB Journal, 23, 4168–4180.PubMedCrossRefGoogle Scholar
  6. 6.
    Zhang, J., Wilson, G. F., Soerens, A. G., Koonce, C. H., Yu, J., Palecek, S. P., et al. (2009). Functional cardiomyocytes derived from human induced pluripotent stem cells. Circulation Research, 104, e30–e41.PubMedCrossRefGoogle Scholar
  7. 7.
    Nelson, T. J., Martinez-Fernandez, A., Yamada, S., et al. (2009). Repair of acute myocardial infarction by human stemness factors induced pluripotent stem cells. Circulation, 120, 408–416.PubMedCrossRefGoogle Scholar
  8. 8.
    Camelliti, P., Borg, T. K., & Kohl, Peter. (2005). Structural and functional characterisation of cardiac fibroblasts. Cardiovascular Research, 65, 40–51.PubMedCrossRefGoogle Scholar
  9. 9.
    Anversa, P., & Nadal-Ginard, B. (2002). Myocyte renewal and ventricular remodeling. Nature, 415, 240–243.PubMedCrossRefGoogle Scholar
  10. 10.
    Etzion, S., Barbash, I. M., Feinberg, M. S., et al. (2002). Cellular cardiomyoplasty of cardiac fibroblasts by adenoviral delivery of MyoD ex vivo: An unlimited source of cells for myocardial repair. Circulation, 106, 125–130.CrossRefGoogle Scholar
  11. 11.
    Ieda, M., Fu, J. D., Delgado-Olguin, P., Vedantham, V., Hayashi, Y., Bruneau, B. G., et al. (2010). Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell, 142, 375–386.PubMedCrossRefGoogle Scholar
  12. 12.
    Hochedlinger, K., & Jaenisch, R. (2006). Nuclear reprogramming and pluripotency. Nature, 441, 1061–1067.PubMedCrossRefGoogle Scholar
  13. 13.
    Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126, 663–676.PubMedCrossRefGoogle Scholar
  14. 14.
    Okita, K., Ichisaka, T., & Yamanaka, S. (2007). Generation of germline-competent induced pluripotent stem cells. Nature, 448, 313–317.PubMedCrossRefGoogle Scholar
  15. 15.
    Ieda, M., Tsuchihashi, T., Ive, K. N., et al. (2009). Cardiac fibroblasts regulate myocardial proliferation through beta1 integrin signaling. Developmental Cell, 16, 233–244.PubMedCrossRefGoogle Scholar
  16. 16.
    Jiang, B., Dong, H., Zhang, Z., et al. (2007). Hypoxic response elements control expression of human vascular endothelial growth factor165 genes transferred to ischemia myocardium in vivo and in vitro. Journal of Gene Medicine, 9, 788–796.PubMedCrossRefGoogle Scholar
  17. 17.
    Pucéat, M. (2008). Protocols for cardiac differentiation of embryonic stem cells. Methods, 45, 168–171.PubMedCrossRefGoogle Scholar
  18. 18.
    Aasen, T., Raya, A., Barrero, M. J., Garreta, E., Consiglio, A., Gonzalez, F., et al. (2008). Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nature Biotechnology, 26, 1276–1284.PubMedCrossRefGoogle Scholar
  19. 19.
    Nakagawa, M., Koyanagi, M., Tanabe, K., et al. (2007). Generation of high quality iPS cells. Neuroscience Research, 58(Suppl 1), S19.Google Scholar
  20. 20.
    Carey, B. W., Markoulaki, S., Hanna, J., et al. (2009). Reprogramming of murine and human somatic cells using a single polycistronic vector. Proceedings of the National Academy of Sciences, 106(1), 157–162.CrossRefGoogle Scholar
  21. 21.
    Hanna, J., Wernig, M., Markoulaki, S., et al. (2007). Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science, 318, 1920–1923.PubMedCrossRefGoogle Scholar
  22. 22.
    Park, I. H., Arora, N., Huo, H., et al. (2008). Disease-specific induced pluripotent stem cells. Cell, 134, 877–886.PubMedCrossRefGoogle Scholar
  23. 23.
    Chen, H. S., Kim, C., & Mercola, M. (2009). Electrophysiological challenges of cell-based myocardial repair. Circulation, 120, 2496–2508.PubMedCrossRefGoogle Scholar
  24. 24.
    Mummery, C., Ward-van Oostwaard, D., Doevendans, P., et al. (2003). Differentiation of human embryonic stem cells to cardiomyocytes: Role of coculture with visceral endoderm-like cells. Circulation, 107, 2733–2740.PubMedCrossRefGoogle Scholar
  25. 25.
    Zhang, Y. M., Hartzell, C., Narlow, M., & Dudley, S. C., Jr. (2002). Stem cell-derived cardiomyocytes demonstrate arrhythmic potential. Circulation, 106, 1294–1299.PubMedCrossRefGoogle Scholar
  26. 26.
    Sun, Y., Kiani, M. F., Postlethwaite, A. E., & Weber, K. T. (2002). Infarct scar as living tissue. Basic Research in Cardiology, 97, 343–347.PubMedCrossRefGoogle Scholar
  27. 27.
    Zannad, F., Dousset, B., & Alla, F. (2001). Treatment of congestive heart failure: interfering the aldosterone-cardiac extracellular matrix relationship. Hypertension, 38, 1227–1232.PubMedCrossRefGoogle Scholar
  28. 28.
    Cho, H. J., Lee, C. S., Kwon, Y. W., et al. (2010). Induction of pluripotent stem cells from adult somatic cells by protein-based reprogramming without genetic manipulation. Blood, 116, 386–395.PubMedCrossRefGoogle Scholar
  29. 29.
    Maherali, N., Sridharan, R., Xie, W., et al. (2007). Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell, 1, 55–70.PubMedCrossRefGoogle Scholar
  30. 30.
    Maherali, N., Sridharan, R., Xie, W., et al. (2002). Stem cell differentiation requires a paracrine pathway in the heart. FASEB Journal, 16, 1558–1566.CrossRefGoogle Scholar
  31. 31.
    Kofidis, T., de Bruin, J. L., Yamane, T., et al. (2005). Stimulation of paracrine pathways with growth factors enhances embryonic stem cell engraftment and host-specific differentiation in the heart after ischemic myocardial injury. Circulation, 111, 2486–2493.PubMedCrossRefGoogle Scholar
  32. 32.
    Sun, Y. (2009). Myocardial repair/remodelling following infarction: Roles of local factors. Cardiovascular Research, 81, 482–490.PubMedCrossRefGoogle Scholar
  33. 33.
    Snider, P., Standley, K. N., Wang, J., et al. (2009). Origin of cardiac fibroblasts and the role of periostin. Circulation Research, 105, 934–947.PubMedCrossRefGoogle Scholar
  34. 34.
    Yoshida, Y., Takahashi, K., Okita, K., et al. (2009). Hypoxia enhances the generation of induced pluripotent stem cells. Cell Stem Cell, 5, 237–241.PubMedCrossRefGoogle Scholar
  35. 35.
    Polo, J. M., Liu, S., Figueroa, M. E., Kulalert, W., Eminli, S., Tan, K. Y., et al. (2010). Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells. Nature Biotechnology, 28, 848–855.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Bo Jiang
    • 1
    • 4
  • Hongyan Dong
    • 2
  • Qingpeng Li
    • 4
  • Yong Yu
    • 3
  • Zhifeng Zhang
    • 2
  • Yazhou Zhang
    • 2
  • Gang Wang
    • 3
    Email author
  • Zhongming Zhang
    • 1
    • 4
    Email author
  1. 1.Department of Thoracic and Cardiovascular SurgeryThe First Affiliated Hospital of Nanjing Medical UniversityNanjingChina
  2. 2.Department of BiologyXuzhou Medical CollegeXuzhouChina
  3. 3.State Key Laboratory of Molecular BiologyInstitute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of SciencesShanghaiChina
  4. 4.Institute of Cardiovascular DiseaseAffiliated Hospital of Xuzhou Medical CollegeXuzhouChina

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