Abstract
Cardiac resident stem/progenitor cells are critical to the cellular and functional integrity of the heart by maintaining myocardial cell homeostasis. Given their central role in myocardial biology, resident cardiac progenitor cells have become a major focus in cardiovascular research. Identification of putative cardiac progenitor cells within the myocardium is largely based on the presence or absence of specific cell surface markers. Additional purification strategies take advantage of the ability of stem cells to efficiently efflux vital dyes such as Hoechst 33342. During fluoresence activated cell sorting (FACS) such Hoechst-extruding cells appear to the side of Hoechst-dye retaining cells and have thus been termed side population (SP) cells. We have shown that cardiac SP cells that express stem cell antigen 1 (Sca-1) but not CD31 are cardiomyogenic, and thus represent a putative cardiac progenitor cell population. This chapter describes the methodology for the isolation of resident cardiac progenitor cells utilizing the SP phenotype combined with stem cell surface markers.
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References
Laugwitz, K. L., Moretti, A., Lam, J., Gruber, P., Chen, Y., Woodard, S., Lin, L. Z., Cai, C. L., Lu, M. M., Reth, M., Platoshyn, O., Yuan, J. X., Evans, S., and Chien, K. R. (2005) Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages, Nature 433, 647–653.
Martin, C. M., Meeson, A. P., Robertson, S. M., Hawke, T. J., Richardson, J. A., Bates, S., Goetsch, S. C., Gallardo, T. D., and Garry, D. J. (2004) Persistent expression of the ATP-binding cassette transporter, Abcg2, identifies cardiac SP cells in the developing and adult heart, Dev Biol 265, 262–275.
Messina, E., De Angelis, L., Frati, G., Morrone, S., Chimenti, S., Fiordaliso, F., Salio, M., Battaglia, M., Latronico, M. V., Coletta, M., Vivarelli, E., Frati, L., Cossu, G., and Giacomello, A. (2004) Isolation and expansion of adult cardiac stem cells from human and murine heart, Circ Res 95, 911–921.
Oh, H., Bradfute, S. B., Gallardo, T. D., Nakamura, T., Gaussin, V., Mishina, Y., Pocius, J., Michael, L. H., Behringer, R. R., Garry, D. J., Entman, M. L., and Schneider, M. D. (2003) Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction, Proc Natl Acad Sci U S A 100, 12313–12318.
Pfister, O., Mouquet, F., Jain, M., Summer, R., Helmes, M., Fine, A., Colucci, W. S., and Liao, R. (2005) CD31− but Not CD31+ cardiac side population cells exhibit functional cardiomyogenic differentiation, Circ Res 97, 52–61.
Beltrami, A. P., 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., and Anversa, P. (2003) Adult cardiac stem cells are multipotent and support myocardial regeneration, Cell 114, 763–776.
Hierlihy, A. M., Seale, P., Lobe, C. G., Rudnicki, M. A., and Megeney, L. A. (2002) The post-natal heart contains a myocardial stem cell population, FEBS Lett 530, 239–243.
Goodell, M. A., Brose, K., Paradis, G., Conner, A. S., and Mulligan, R. C. (1996) Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo, J Exp Med 183, 1797–1806.
Zhou, S., Schuetz, J. D., Bunting, K. D., Colapietro, A. M., Sampath, J., Morris, J. J., Lagutina, I., Grosveld, G. C., Osawa, M., Nakauchi, H., and Sorrentino, B. P. (2001) The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype, Nat Med 7, 1028–1034.
Challen, G. A., and Little, M. H. (2006) A side order of stem cells: the SP phenotype, Stem Cells 24, 3–12.
Pfister, O., Oikonomopoulos, A., Sereti, K. I., Sohn, R. L., Cullen, D., Fine, G. C., Mouquet, F., Westerman, K., and Liao, R. (2008) Role of the ATP-binding cassette transporter Abcg2 in the phenotype and function of cardiac side population cells, Circ Res 103, 825–835.
Acknowledgments
This work was supported by NIH grants HL71775, HL86967, HL73756, HL 88533, and HL93148. The authors would like to thank Mr. Darragh Cullen for technical assistance. Mr. Grigoriy Losyev at BWH Cardiovascular FACS Core is acknowledged for assistance with cell sorting.
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Pfister, O., Oikonomopoulos, A., Sereti, KI., Liao, R. (2010). Isolation of Resident Cardiac Progenitor Cells by Hoechst 33342 Staining. In: Lee, R. (eds) Stem Cells for Myocardial Regeneration. Methods in Molecular Biology, vol 660. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-705-1_4
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DOI: https://doi.org/10.1007/978-1-60761-705-1_4
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