Abstract
Epicardium is the outer cell layer of the heart. Its integrity and function are essential for normal heart development. To study the role of epicardium in both fetal and adult hearts, it is desirable to isolate and culture pure populations of these cells. Here we describe methods with Cre-loxP technology to lineage tag epicardial cells (EpiCs) and epicardium-derived cells (EPDCs), dissociate and isolate them by flow-activated cytometry sorting (FACS), and characterize them by quantitative PCR and immunostaining. This platform allows further characterization and manipulation of EpiCs and EPDCs for expression studies and functional assays.
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References
Zhou, B., and Pu W.T. (2008) More than a cover: epicardium as a novel source of cardiac progenitor cells, Regen Med 3 633–635.
Manner, J., Perez-Pomares J.M., Macias, D., and Munoz-Chapuli, R. (2001) The origin, formation and developmental significance of the epicardium: a review. Cells Tissues, Organs 169 89–103.
Perez-Pomares, J.M., Carmona, R., Gonzalez-Iriarte, M., Atencia, G., Wessels, A., and Munoz-Chapuli, R. (2002) Origin of coronary endothelial cells from epicardial mesothelium in avian embryos, Int J Dev Biol 46 1005–1013.
Gittenberger-de Groot, A. C., Vrancken Peeters, M. P., Mentink, M. M., Gourdie, R. G., and Poelmann, R. E. (1998) Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions, Circ Res 82, 1043–1052.
Yang, J.T., Rayburn, H., and Hynes, R.O. (1995) Cell adhesion events mediated by alpha 4 integrins are essential in placental and cardiac development, Development 121 549–560.
Moore, A. W., McInnes, L., Kreidberg, J., Hastie, N. D., and Schedl, A. (1999) YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland and throughout nephrogenesis, Development 126, 1845–1857.
Zamora, M., Manner, J., and Ruiz-Lozano, P. (2007) Epicardium-derived progenitor cells require beta-catenin for coronary artery formation, Proc Natl Acad Sci U S A 104 18109–18114.
Wu, H., Lee, S.H., Gao, J., Liu, X., and Iruela-Arispe, M.L. (1999) Inactivation of erythropoietin leads to defects in cardiac morphogenesis, Development 126 3597–3605.
Merki, E., Zamora, M., Raya, A., Kawakami, Y., Wang, J., Zhang, X., Burch, J., Kubalak, S.W., Kaliman, P., Belmonte, J.C., Chien, K.R., and Ruiz-Lozano, P. (2005) Epicardial retinoid X receptor alpha is required for myocardial growth and coronary artery formation, Proc Natl Acad Sci U S A 102 18455–18460.
Wilm, B., Ipenberg, A., Hastie, N.D., Burch, J.B., and Bader, D.M. (2005) The serosal mesothelium is a major source of smooth muscle cells of the gut vasculature, Development 132 5317–5328.
Dettman, R.W., Denetclaw, W. Jr., Ordahl, C.P., and Bristow, J. (1998) Common epicardial origin of coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts in the avian heart, Dev Biol 193 169–181.
Cai, C.L., Martin, J.C., Sun, Y., Cui, L., Wang, L., Ouyang, K., Yang, L., Bu, L., Liang, X., Zhang, X., Stallcup, W.B., Denton, C.P., McCulloch, A., Chen, J., and Evans, S.M. (2008) A myocardial lineage derives from Tbx18 epicardial cells, Nature 454 104–108.
Zhou, B., Ma, Q., Rajagopal, S., Wu, S. M., Domian, I., Rivera-Feliciano, J., Jiang, D., von Gise, A., Ikeda, S., Chien, K. R., and Pu, W. T. (2008) Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart, Nature 454, 109–113.
Limana, F., Zacheo, A., Mocini, D., Mangoni, A., Borsellino, G., Diamantini, A., De Mori, R., Battistini, L., Vigna, E., Santini, M., Loiaconi, V., Pompilio, G., Germani, A., and Capogrossi, M. C. (2007) Identification of myocardial and vascular precursor cells in human and mouse epicardium, Circ Res 101, 1255–1265.
Kreidberg, J. A., Sariola, H., Loring, J. M., Maeda, M., Pelletier, J., Housman, D., and Jaenisch, R. (1993) WT-1 is required for early kidney development, Cell 74, 679–691.
Perez-Pomares, J. M., Phelps, A., Sedmerova, M., Carmona, R., Gonzalez-Iriarte, M., Munoz-Chapuli, R., and Wessels, A. (2002) Experimental studies on the spatiotemporal expression of WT1 and RALDH2 in the embryonic avian heart: a model for the regulation of myocardial and valvuloseptal development by epicardially derived cells (EPDCs), Dev Biol 247, 307–326.
Zhou, B, A von Gise, Q Ma, YW Hu, and WT Pu. (2010). Genetic fate mapping demonstrates contribution of epicardium-derived cells to the annulus fibrosis of the mammalian heart. Dev Biol 338, 251–261.
Feil, R., Wagner, J., Metzger, D., and Chambon, P. (1997) Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains, Biochem Biophys Res Commun 237, 752–757.
Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L., and Luo, L. (2007) A global double-fluorescent Cre reporter mouse, Genesis 45, 593–605.
Mao, X., Fujiwara, Y., and Orkin, S. H. (1999) Improved reporter strain for monitoring Cre recombinase-mediated DNA excisions in mice, Proc Natl Acad Sci U S A 96, 5037–5042.
Vintersten, K., Monetti, C., Gertsenstein, M., Zhang, P., Laszlo, L., Biechele, S., and Nagy, A. (2004) Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals, Genesis 40 241–246.
Lepilina, A., Coon, A. N., Kikuchi, K., Holdway, J. E., Roberts, R. W., Burns, C. G., and Poss, K. D. (2006) A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration, Cell 127 607–619.
Tarnavski, O., McMullen, J. R., Schinke, M., Nie, Q., Kong, S., and Izumo, S. (2004) Mouse cardiac surgery: comprehensive techniques for the generation of mouse models of human diseases and their application for genomic studies, Physiol Genomics 16 349–360.
Acknowledgments
The authors would like to thank Elizabeth Boush for aiding FACS isolation. This work was supported by funding from NIH RO1 HL094683 (WTP), an American Heart Association Postdoctoral Fellowship (BZ), and charitable support from James Smith and Gail Federici-Smith (WTP), and the Simeon Burt Wolbach Research Fund (BZ).
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Zhou, B., Pu, W.T. (2012). Isolation and Characterization of Embryonic and Adult Epicardium and Epicardium-Derived Cells. In: Peng, X., Antonyak, M. (eds) Cardiovascular Development. Methods in Molecular Biology, vol 843. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-523-7_15
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DOI: https://doi.org/10.1007/978-1-61779-523-7_15
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