期刊
NATURE PROTOCOLS
卷 9, 期 7, 页码 1662-1681出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2014.113
关键词
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资金
- British Heart Foundation [PG 08/085]
- CARE-MI FP7 project [Health F5-2010-242038]
- Endostem FP7 project [Health F5-2010-241440]
- Marie Curie International Reintegration FP7 grant [PIRG02-GA-2007-224853]
- FIRB-Futuro-in-Ricerca [RBFR081CCS, RBFR1213KA]
- Italian Ministry of Health [GR-2008-1142673, GR-2010-2318945]
This protocol describes the isolation of endogenous c-Kit (also known as CD117)-positive (c-Kit(+)), CD45-negative (CD45(-)) cardiac stem cells (eCSCs) from whole adult mouse and rat hearts. The heart is enzymatically digested via retrograde perfusion of the coronary circulation, resulting in rapid and extensive breakdown of the whole heart. Next, the tissue is mechanically dissociated further and cell fractions are separated by centrifugation. The c-Kit(+)CD45(-) eCSC population is isolated by magnetic-activated cell sorting technology and purity and cell numbers are assessed by flow cytometry. This process takes similar to 4 h for mouse eCSCs or 4.5 h for rat eCSCs. We also describe how to characterize c-Kit(+)CD45(-) eCSCs. The c-Kit(+)CD45(-) eCSCs exhibit the defining characteristics of stem cells: they are self-renewing, clonogenic and multipotent. This protocol also describes how to differentiate eCSCs into three main cardiac lineages: functional, beating cardiomyocytes, smooth muscle, and endothelial cells. These processes take 17-20 d.
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