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Skip Navigation LinksJRI > Archive > January-March 2007, Volume 7, Issue 4 > Studying the effects of TGF-β2 and BMP-2 growth factors on differentiation of murine embryonic stem cells into cardiomyocytes



Volume 7, Issue 4, Number 29 / January-March
(pages 338-348)


Studying the effects of TGF-β2 and BMP-2 growth factors on differentiation of murine embryonic stem cells into cardiomyocytes




Department of Biology, Faculty of Sciences, Islamic Azad University, Science & Research Campus, Tehran, Iran

Department of Biology, Faculty of Sciences, Islamic Azad University, Science & Research Campus, Tehran, Iran

Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

Monoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran

1- Monoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran
2- Immunology Department, Faculty of Medical Science, Tehran University of Medical Science, Tehran, Iran

1- Nanobiotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran
2- Department of Immunology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran

Faculty of Medical Tehran University of Medical Sciences, Tehran, Iran

Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran

 Corresponding Author
Monoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran


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Abstract
Introduction: Stem cell therapy seems promising for the treatment of incurable diseases such as injured myocardium. Directed in vitro differentiation of embryonic stem cells (ES) to cardiomyo-cytes is invaluable. TGFβ-2 and BMP-2, two different growth factors from the transforming growth factor-β superfamily (TGF-β), exhibit their effects on growth, immigration and different-tiation and other cellular functions during embryonic development. In this study, the effects of these factors on differentiation of ES cells into cardiomyocytes were evaluated. Materials & Methods: Murine embryonic stem cells (CCB derived from mouse strain 129) were used for cardiomyocyte differentiation. To increase the effects of the factors, ES cells were grown on inactivated fibroblasts for 24 hours in differentiating culture medium containing 8 ng/ml of TGF-β2 and 5 ng/ml of BMP-2 while decreasing serum concentration from 20% to 7.5%. Later, embryoid bodies (EB) were formed in 800-cells hanging drops and suspension from ES cells and eventually, EBs were cultured on gelatin-coated plates. After continuation of these steps, ES cells differentiated into beating cells. To determine the effects of the growth factors on ES cell differen-tiation into cardiomyocytes, expression of primary transcribing factors, MEF2, Nkx2.5, and specific cardiac genes including MHC, ANF, MLC-2v were assessed by semiquantitative RT-PCR and expression of desmin and α-actinin in differentiated cardiomyocytes was studied by western blotting and immunostaining. Results: After completion of the differentiation process, the EBs in the experimental groups exhibited beating compared with the control group. Considering the quantitative comparison of the dectrophoretic bands of RT-PCR and PCR products of the plated EBs in different media, it was seen that TGF-2 from BMP-2 and both factors together, compared to each one alone, is more effective on differentiation of embryonic stem cells into cardiomyocytes. In addition, in plated EBs, in a medium containing both factors, the expression of desmin and -actinin were determined by western blotting and the presence of cardiomyocytes, by marking desmin, by immunohistochemical staining. Conclusion: The results showed that the synchronized effects of both factors, while decreasing serum concentration during ES and EB cell cultures, increases differentiation of ES cells into cardiomyocytes.

Keywords: Embryonic stem cell, Differentiation, Cardiomyocyte, TGF-B2, BMP-2


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