SCD  Vol.3 No.1 , January 2013
Towards the development of a reliable protocol for mesenchymal stem cell cardiomyogenesis
Author(s) Faizal Z. Asumda*

The specific transformative steps that occur from the multipotent through the mature cardiomyocyte state are determined in large part by changes in gene expression. The exact differentiation and developmental programs are tightly regulated in a step-wise systematic fashion based on the influence of specific instigating and signaling factors. Crucial to the observed cell behavior and tissue specific phenotypic differences are alterations in the organization, translocation and expression of nuclear proteins during differentiation. The cardiomyogenic differentiation of Mesenchymal Stem Cells (MSCs) remains a precarious process. Transplanted MSCs must respond to endogenous signaling molecules involved in early embryonic cardiomyogenesis by activating the specific gene regulatory network required for successful cardiomyogenesis and transdifferentiation. To do that, transplanted MSCs would have to be genuinely reprogrammed genetically to become functional cardiac myocytes. A consideration of recent experimental findings suggests that Bone morphogenic protein (BMP-2/4), insulin-like growth factor (IGF-1) and fibroblast growth factor (FGF-2) in combination is a potent inducer of MSC cardiomyogenesis. The development of an optimum and reliable in vitro culture milieu for directing their cardiomyogenesis will provide an indispensable model system for molecular studies and genetic manipulation.

Cite this paper
Z. Asumda, F. (2013) Towards the development of a reliable protocol for mesenchymal stem cell cardiomyogenesis. Stem Cell Discovery, 3, 13-21. doi: 10.4236/scd.2013.31003.
[1]   Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S. and Marshak, D.R. (1999) Multilineage potential of adult human mesenchymal stem cells. Science, 284, 143-147. doi:10.1126/science.284.5411.143

[2]   Deans, R.J. and Moseley, A.B. (2000) Mesenchymal stem cells: Biology and potential clinical uses. Experimental Hematology, 28, 875-884. doi:10.1016/S0301-472X(00)00482-3

[3]   Kolf, C.M., Cho, E. and Tuan, R.S. (2007) Mesenchymal stromal cells. Biology of adult mesenchymal stem cells: Regulation of niche, self-renewal and differentiation. Arthritis Research & Therapy, 9, 204-215. doi:10.1186/ar2116

[4]   Williams, A.R. and Hare, J.M. (2011) Mesenchymal stem cells: Biology, pathophysiology, translational findings, and therapeutic implications for cardiac disease. Circulation Research, 109, 923-940. doi:10.1161/CIRCRESAHA.111.243147

[5]   Keating, A. (2012) Mesenchymal stromal cells: New directions. Cell Stem Cell, 10, 709-716. doi:10.1016/j.stem.2012.05.015

[6]   Heldman, A.W., Zambrano, J.P. and Hare, J.M. (2011) Cell therapy for heart disease: Where are we in 2011? Journal of the American College of Cardiology, 57, 466-468. doi:10.1016/j.jacc.2010.09.028

[7]   Asumda, F. (2012) Bone marrow mesenchymal stem aging: Implications for cellular cardiomyoplasty: A theoretical exposition. Lambert Academic Publishing, Saarbrücken.

[8]   Asumda, F.Z. and Chase, P.B. (2012) Age-related changes in rat bone-marrow mesenchymal stem cell plasticity. BMC Cell Biology, 12, 44-55. doi:10.1186/1471-2121-12-44

[9]   Greco, S.J., Liu, K. and Rameshwar, P. (2007) Functional similarities among genes regulated by Oct4 in human mesenchymal and embryonic stem cells. Stem Cells, 25, 3143-3154. doi:10.1634/stemcells.2007-0351

[10]   Gonzalez, R., Maki, C.B., Pacchiarotti, J., Csontos, S., Pham, J.K., Slepko, N., Patel, A. and Silva, F. (2007) Pluripotent marker expression and differentiation of human second trimester mesenchymal stem cells. Biochemical and Biophysical Research Communications, 362, 491-497. doi:10.1016/j.bbrc.2007.08.033

[11]   Asumda, F.Z. and Chase, P.B. (2012) Nuclear cardiac troponin and tropomyosin are expressed early in cardiac differentiation of rat mesenchymal stem cells. Differentiation, 83, 106-115. doi:10.1016/j.diff.2011.10.002

[12]   Young, D.A., DeQuach, J.A. and Christman, K.L. (2011) Human cardiomyogenesis and the need for systems boilogy analysis. Wiley Interdisciplinary Reviews Systems Biology and Medicine, 3, 666-680. doi:10.1002/wsbm.141

[13]   Heng, B.C., Haider, H.K., Sim, E.K., Cao, T. and Ng, S.C. (2004) Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro. Cardiovascular Research, 62, 34-42. doi:10.1016/j.cardiores.2003.12.022

[14]   Chien, K.R. (2004) Stem cells: Lost in translation. Nature, 428, 607-608. doi:10.1038/nature02500

[15]   Chien, K.R. (2008) Regenerative medicine and human models of human disease. Nature, 453, 302-305. doi:10.1038/nature07037

[16]   Armi?án, A., Gandía, C., García-Verdugo, J.M., Lledó, E., Mullor, J.L., Montero, J.A. and Sepúlveda, P. (2010) Cardiac transcription factors driven lineage-specification of adult stem cells. Journal of Cardiovascular Translational Research, 3, 61-65. doi:10.1007/s12265-009-9144-3

[17]   Nishiyama, N., Miyoshi, S., Hida, N., Uyama, T., Okamoto, K., Ikegami, Y., Miyado, K., Segawa, K., Terai, M., Sakamoto, M., et al. (2007) The significant cardiomyogenic potential of human umbilical cord blood-derived mesenchymal stem cells in vitro. Stem Cells, 25, 2017-2024. doi:10.1634/stemcells.2006-0662

[18]   Spees, J.L., Olson, S.D., Ylostalo, J., Lynch, P.J., Smith, J., Perry, A., Peister, A., Wang, M.Y. and Prockop, D.J. (2003) Differentiation, cell fusion, and nuclear fusion during ex vivo repair of epithelium by human adult stem cells from bone marrow stroma. Proceedings of the National Academy of Sciences of the United States of America, 100, 2397-2402. doi:10.1073/pnas.0437997100

[19]   Labovsky, V., Hofer, E.L., Feldman, L., Fernández, V.V., García, R.H., Bayes-Genis, A., Hernando, I.A., Levin, M.J. and Chasseing, N.A. (2010) Cardiomyogenic differentiation of human bone marrow mesenchymal cells: Role of cardiac extract from neonatal rat cardiomyocytes. Differentiation, 79, 93-101. doi:10.1016/j.diff.2009.10.001

[20]   Valiunas, V., Doronin, S., Valiuniene, L., Potapova, I., Zuckerman, J., Walcott, B., Robinson, R.B., Rosen MR, Brink, P.R. and Cohen, I.S. (2004) Human mesenchymal stem cells make cardiac connexins and form functional gap junctions. The Journal of Physiology, 555, 617-626. doi:10.1113/jphysiol.2003.058719

[21]   Ramkisoensing, A., Pijnappels, D., Schalij, M., de Vries, A. and Atsma, D. (2012) Connexin43 expression is essential for functional cardiomyogenic differentiation of human fetal mesenchymal stem cells. Journal of the American College of Cardiology, 59, E855. doi:10.1016/S0735-1097(12)60856-0

[22]   Garbade, J., Schubert, R., Lenz, D., Walther, T., Gummert, J., Dhein, S. and Mohr, F.W. (2005) Fusion of bone marrow-derived stem cells with cardiomyocytes in a heterologous in vitro model. European Journal of Cardio-Thoracic Surgery, 28, 685-691. doi:10.1016/j.ejcts.2005.06.047

[23]   Liu, Y., Song, J., Liu, W., Wan, Y., Chen, X. and Hu, C. (2003) Growth and differentiation of rat bone marrow stromal cells: Does 5-azacytidine trigger their cardiomyogenic differentiation? Cardiovascular Research, 58, 460-468. doi:10.1016/S0008-6363(03)00265-7

[24]   Rangappa, S., Fen, C., Lee, E.H., Bongso, A. and Sim, E.K. (2000) Transformation of adult mesenchymal stem cells isolated from the fatty tissue into cardiomyocytes. Annals of Thoracic Surgery, 75, 775-779. doi:10.1016/S0003-4975(02)04568-X

[25]   Antonitsis, P., Papagiannaki, E.I., Kaidoglou, A. and Papakonstantinou, C. (2007) In vitro cardiomyogenic differentiation of adult human bone marrow mesenchymal stem cells: The role of 5-azacytidine. Interactive Cardiovascular and Thoracic Surgery, 6, 593-597. doi:10.1510/icvts.2007.157875

[26]   Zhang, Y., Chu, Y., Shen, W. and Dou, Z. (2009) Effect of 5-azacytidine induction duration on differentiation of human first-trimester fetal mesenchymal stem cells towards cardiomyocyte-like cells. Interactive Cardiovascular and Thoracic Surgery, 9, 943-946. doi:10.1510/icvts.2009.211490

[27]   Makino, S., Fukuda, K., Miyoshi, S., Konishi, F., Kodama, H., Pan, J., Sano, M., Takahashi, T, Hori, S. and Abe, H., et al. (1999) Cardiomyocytes can be generated from marrow stromal cells in vitro. The Journal of Clinical Investigation, 103, 697-705. doi:10.1172/JCI5298

[28]   Qian, Q., Qian, H., Zhang, X., Zhu, W., Yan, Y., Ye, S., Peng, X., Li, W., Xu, Z., Sun, L. and Xu, W. (2012) 5-Azacytidine induces cardiac differentiation of human umbilical cord-derived mesenchymal stem cells by activating extracellular regulated kinase. Stem Cells Development, 21, 67-75. doi:10.1089/scd.2010.0519

[29]   Balana, B., Nicoletti, C., Zahanich, I., Graf, E. M., Christ, T., Boxberger, S. and Ravens, U. (2006) 5-Azacytidine induces changes in electrophysiological properties of human mesenchymal stem cells. Cell Research, 16, 949-960. doi:10.1038/

[30]   Yao, Y., Li, W., Wu, J., Germann, U.A., Su, M.S., Kuida, K. and Boucher, D.M. (2003) Extracellular signal-regulated kinase 2 is necessary for mesoderm differentiation. Proceedings of the National Academy of Sciences of the United States of America, 28, 12759-12764. doi:10.1073/pnas.2134254100

[31]   Jaiswal, R.K., Jaiswal, N., Bruder, S.P., Mbalaviele, G., Marshak, D.R. and Pittenger, M.F. (2000) Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. Journal of Biological Chemistry, 275, 9645-9652. doi:10.1074/jbc.275.13.9645

[32]   Kim, H.S., Cho, J.W., Hidaka, K. and Morisaki, T. (2007) Activation of MEK-ERK by heregulin-beta1 promotes the development of cardiomyocytes derived from ES cells. Biochemical and Biophysical Research Communications, 28, 732-738. doi:10.1016/j.bbrc.2007.07.045

[33]   Tortorella, L.L., Milasincic, D.J. and Pilch, P.F. (2001) Critical proliferation-independent window for basic fibroblast growth factor repression of myogenesis via the p42/ p44 MAPK signaling pathway. Journal of Biological Chemistry, 276, 13709-13717. doi:10.1002/jgm.583

[34]   Takeda, Y., Mori, T., Imabayashi, H., Kiyono, T., Gojo, S., Miyoshi, S., Hida, N., Ita, M., Segawa, K. and Ogawa, S. (2004) Can the life span of human marrow stromal cells be prolonged by bmi-1, E6, E7, and/or telomerase without affecting cardiomyogenic differentiation? Journal of Gene Medicine, 6, 833-845.

[35]   Taylor, S.M. and Jones, P.A. (1982) Mechanism of action of eukaryotic DNA methyltransferase. Use of 5-azacytosine-containing DNA. Journal of Molecular Biology, 162, 679-692. doi:10.1016/0022-2836(82)90395-3

[36]   Creusot, F., Acs, G. and Christman, J.K. (1982) Inhibition of DNA methyltransferase and induction of Friend erythroleukemia cell differentiation by 5-azacytidine and 5-aza-29-deoxycytidine. Journal of Biological Chemistry, 257, 2041-2048.

[37]   Taylor, S.M. and Jones, P.A. (1979) Multiple new phenoltypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine. Cell, 17, 771-779. doi:10.1016/0092-8674(79)90317-9

[38]   Cifarelli, R.A., Conconi, M.T., Marmo, R., Liddo, R., Dininno, C., Ferraro, S., Cellini, F. and Parnigotto, P. (2012) 5-Azacytidine makes human preadipocytes able to differentiate into mesoderm-derived cell lineages. Stem Cells Development, 21, 76-85. doi:10.1089/scd.2010.0464

[39]   Fishman, M.C. and Chien, K.R. (1997) Fashioning the vertebrate heart: Earliest embryonic decisions. Development, 124, 2099-2117.

[40]   Roura, S., Farré, J., Hove-Madsen, L., Prat-Vidal, C., Soler-Botija, C., Gálvez-Montón, C., Vilalta, M. and Bayes-Genis, A. (2010) Exposure to cardiomyogenic stimuli fails to transdifferentiate human umbilical cord blood-derived mesenchymal stem cells. Basic Research in Cardiology, 105, 419-430. doi:10.1007/s00395-009-0081-8

[41]   van Wijk, B., Moorman, A.F. and van den Hoff, M.J. (2007) Role of bone morphogenetic proteins in cardiac differentiation. Cardiovascular Research, 74, 244-255. doi:10.1016/j.cardiores.2006.11.022

[42]   Kawai, T., Takahashi, T., Esaki, M., Ushikoshi, H., Nagano, S., Fujiwara, H. and Kosai, K. (2004) Efficient cardiomyogenic differentiation of embryonic stem cells by fibroblast growth factor 2 and bone morphogenetic protein. Circulation, 68, 691-702. doi:10.1253/circj.68.691

[43]   Bartunek, J., Croissant, J.D., Wijns, W., Gofflot, S., de Lavareille, A., Vanderheyden, M., Kaluzhny, Y., Mazouz, N., Willemsen, P. and Penicka, M. (2007) Pretreatment of adult bone marrow mesenchymal stem cells with cardiomyogenic growth factors and repair of the chronically infarcted myocardium. American Journal of Physiology. Heart and Circulatory Physiology, 292, H1095-H1104. doi:10.1152/ajpheart.01009.2005

[44]   Laflamme, M.A., Chen, K.Y., Naumova, A.V., Muskheli, V., Fugate, J.A., Dupras, S.K., Reinecke, H., Xu, C., Hassanipou, M. and Police, S. (2007) Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nature Biotechnology, 25, 1015-1024. doi:10.1038/nbt1327

[45]   Behfar, A., Yamada, S., Crespo-Diaz, R., Nesbitt, J.J., Rowe, L.A., Perez-Terzic, C., Gaussin, V., Homsy, C., Bartunek, J. and Terzic, A. (2010) Guided cardiopoiesis enhances therapeutic benefit of bone marrow human mesenchymal stem cells in chronic myocardial infarction. Journal of American College of Cardiology, 56, 722-734. doi:10.1016/j.jacc.2010.03.066

[46]   Hahn, J.Y., Cho, H.J., Kang, H.J., Kim, T.S., Kim, M.H., Chung, J.H., Bae, J.W., Oh, B.H., Park, Y.B. and Kim, H.S. (2008) Pre-treatment of mesenchymal stem cells with a combination of growth factors enhances gap junction formation, cytoprotective effect on cardiomyocytes, and therapeutic efficacy for myocardial infarction. Journal of American College of Cardiology, 51, 933-943. doi:10.1016/j.jacc.2007.11.040

[47]   Burridge, P. W., Thompson, S., Millrod, M., Weinberg, S., Yuan, X., Peters, A., Mahairaki, V., Koliatsos, V.E., Tung, L. and Zambidis, E.T., (2011) A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PloS One, 6, e18293. doi:10.1371/journal.pone.0018293

[48]   Redig, J.K. and Adler, E. (2011) Doing the dirty work: Progress in the search for a reliable protocol for cardiomyogenesis. Stem Cell Research & Therapy, 2, 35. doi:10.1186/scrt76

[49]   Schlange, T., Andree, B., Arnold, H.H. and Brand, T. (2000) BMP2 is required for early heart development during a distinct time period. Mechanisms of Development, 91, 259-270. doi:10.1016/S0925-4773(99)00311-1

[50]   Schultheiss, T.M., Burch, J.B. and Lassar, A.B. (1997) A role for bone morphogenetic proteins in the induction of cardiac myogenesis. Genes & Development, 11, 451-462. doi:10.1101/gad.11.4.451

[51]   Monzen, K., Nagai, R. and Komuro, I. (2002) A role for bone morphogenetic protein signaling in cardiomyocyte differentiation. Trends in Cardiovascular Medicine, 12, 263-269. doi:10.1016/S1050-1738(02)00172-X

[52]   Sugi, Y., Yamamura, H., Okagawa, H. and Markwald, R.R. (2004) Bone morphogenetic protein-2 can mediate myocardial regulation of atrioventricular cushion mesenchymal cell formation in mice. Developmental Biology, 269, 505-518. doi:10.1016/j.ydbio.2004.01.045

[53]   Qi, X., Li, T.G., Hao, J., Hu, J., Wang, J. and Simmons, H. (2004) BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways. Proceedings of the National Academy of Sciences of the United States of America, 101, 6027-6032. doi:10.1073/pnas.0401367101

[54]   Rosenblatt-velin, N., Lepore, M.G., Cartoni, C., Beermann, F. and Pedrazzini, T. (2005) FGF-2 controls the differentiation of resident cardiac precursors into functional cardiomyocytes. The Journal of Clinical Investigation, 115, 1724-1733. doi:10.1172/JCI23418

[55]   Barron, M., Gao, M. and Lough, J. (2000) Requirement for BMP and FGF signaling during cardiogenic induction in non-precardiac mesoderm specific, transient, and cooperative. Developmental Dynamics, 218, 383-393. doi:10.1002/(SICI)1097-0177(200006)218:2<383::AID-DVDY11>3.0.CO;2-P

[56]   Lough, J., Barron, M., Brogley, M., Sugi, Y., Bolender, D.L. and Zhu, X. (1996) Combined BMP-2 and FGF-4, but neither factor alone, induces cardiogenesis in non-precardiac embryonic mesoderm. Developmental Biology, 178, 198-202. doi:10.1006/dbio.1996.0211

[57]   Siegel, G., Krause, P., W?hrle, S., Nowak, P., Ayturan, M., Kluba, T., Brehm, B.R., Neumeister, B., K?hler, D. and Rosenberger, P. (2012) Bone marrow-derived human mesenchymal stem cells express cardiomyogenic proteins but do not exhibit functional cardiomyogenic differentiation potential. Stem Cells Development. Epub Ahead of Print. doi:10.1089/scd.2011.0626

[58]   Sadat, S., Gehmert, S., Song, Y.H., Yen, Y., Bai, X., Gaiser, S., Klein, H. and Alt, E. (2007) The cardioprotective effect of mesenchymal stem cells is mediated by IGF-I and VEGF. Biochemical and Biophysical Research Communications, 363, 674-679. doi:10.1016/j.bbrc.2007.09.058

[59]   Lee, W.L., Chen, J.W., Ting, C.T., Ishiwata, T., Lin, S.J., Korc, M. and Wang, P.H. (1999) Insulin-like growth factor I improves cardiovascular function and suppresses apoptosis of cardiomyocytes in dilated cardiomyopathy. Endocrinology, 140, 4831-4840. doi:10.1210/en.140.10.4831

[60]   Solem, M.L. and Thomas, A.P. (1998) Modulation of cardiac Ca2+ channels by IGF1. Biochemical and Biophysical Research Communications, 252, 151-155. doi:10.1006/bbrc.1998.9626

[61]   Aberg, N.D., Blomstrand, F., Aberg, M.A., Bj?rklund, U., Carlsson, B., Carlsson-Skwirut, C., Bang, P., R?nnb?ck, L. and Eriksson, P.S. (2003) Insulin-like growth factor-I increases astrocyte intercellular gap junctional communication and connexin43 expression in vitro. Journal of Neuroscience Research, 74, 12-22. doi:10.1002/jnr.10734

[62]   Urbanek, K., Rota, M., Cascapera, S., Bearzi, C., Nascimbene, A., De Angelis, A., Hosoda, T., Chimenti, S., Baker, M. and Limana, F. (2005) Cardiac stem cells possess growth factor-receptor systems that after activation regenerate the infarcted myocardium, improving ventricular function and long-term survival. Circulation Research, 97, 663-673. doi:10.1161/01.RES.0000183733.53101.11

[63]   Kofidis, T., de Bruin, J.L., Yamane, T., Balsam, L.B., Lebl, D.R. and Swijnenburg, R.J. (2004) Insulin-like growth factor promotes engraftment, differentiation, and functional improvement after transfer of embryonic stem cells for myocardial restoration. Stem Cells, 22, 1239-1245. doi:10.1634/stemcells.2004-0127

[64]   Perez-Terzic, C., Faustino, R. S., Boorsma, B. J., Arrell, D. K., Niederlander, N. J., Behfar, A. and Terzic, A. (2007) Stem cells transform into a cardiac phenotype with remodeling of the nuclear transport machinery. Nature Clinical Practice Cardiovascular Medicine, 1, S68-S76. doi:10.1038/ncpcardio0763

[65]   Arminan, A., Gandia, C., Bartual, M., Garcia-Verdugo, J.M., Lledo, E., Mirabet, V., Llop, M., Barea, J., Montero, J.A. and Sepúlveda, P. (2009) Cardiac differentiation is driven by NKX2.5 and GATA4 nuclear translocation in tissue-specific mesenchymal stem cells. Stem Cells and Development, 18, 907-918. doi:10.1089/scd.2008.0292

[66]   Naito, A.T., Tominaga, A., Oyamada, M., Oyamada, Y., Shiraishi, I., Monzen, K., Komuro, I. and Takamatsu, T. (2003) Early stage-specific inhibitions of cardiomyocyte differentiation and expression of Csx/Nkx-2.5 and GATA-4 by phosphatidylinositol 3-kinase inhibitor LY294002. Experimental Cell Research, 291, 56-69. doi:10.1016/S0014-4827(03)00378-1

[67]   Durocher, D., Charron, F., Warren, R., Schwartz, R.J. and Nemer, M. (1997) The cardiac transcription factors Nkx2-5 and GATA-4 are mutual cofactors. The EMBO Journal, 16, 5687-5696. doi:10.1093/emboj/16.18.5687

[68]   Bayes-Genis, A., Roura, S., Soler-Botija, C., Farré, J., Hove-Madsen, L., Llach, A and Cinca, J. (2005) Identification of cardiomyogenic lineage markers in untreated human bone marrow-derived mesenchymal stem cells. Transplantation Proceedings, 37, 4077-4079. doi:10.1016/j.transproceed.2005.09.103

[69]   Dulce, R., Balkan, W., Hare, J.M. and Schulman, I.H. (2011) Wnt signalling: a mediator of the heart-bone marrow axis after myocardial injury? European Heart Journal, 2, 2-4.