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Isolation and characterization of resident endogenous c-Kit+ cardiac stem cells from the adult mouse and rat heart

2014; Nature Portfolio; Volume: 9; Issue: 7 Linguagem: Inglês

10.1038/nprot.2014.113

1754-2189

Andrew J. Smith, Fiona Lewis, Iolanda Aquila, Cheryl D. Waring, Aurora Nocera, Valter Agosti, Bernardo Nadal‐Ginard, Daniele Torella, Georgina M. Ellison,

Pluripotent Stem Cells Research

A protocol on how to isolate c-kit-positive cardiac stem cells via magnetic activated cell sorting and how to then differentiate the cells into the three main cardiac lineages: functional, beating cardiomyocytes, smooth muscle and endothelial cells. 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 ∼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.