Abstract
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.
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Acknowledgements
We acknowledge the technical assistance of R. Williams and S. Broadfoot of Liverpool John Moores University. This work was carried out with funding support from the British Heart Foundation (PG 08/085), from CARE-MI FP7 (Health F5-2010-242038), Endostem FP7 (Health F5-2010-241440) large-scale collaborative projects, from a Marie Curie International Reintegration FP7 grant (PIRG02-GA-2007-224853), from FIRB-Futuro-in-Ricerca (RBFR081CCS, RBFR1213KA) and from the Italian Ministry of Health (GR-2008-1142673, GR-2010-2318945).
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G.M.E., B.N.-G. and D.T. designed the protocols; A.J.S., F.C.L., I.A. and C.D.W. performed cell isolations and refined the protocols; A.J.S., F.C.L., I.A., A.N. and V.A. performed flow cytometry on freshly isolated and clonal eCSCs; A.J.S. and G.M.E. performed cell differentiation and immunocharacterization; A.J.S., F.C.L., I.A., V.A., D.T. and G.M.E. analyzed data; and A.J.S., G.M.E., B.N.-G. and D.T. wrote and edited the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Flow cytometric analysis: Forward scatter/side scatter gating.
(a) Unsorted mouse cardiac small cells. (b) Mouse eCSCs. (c) Unsorted rat cardiac small cells. (d) Rat eCSCs.
Supplementary information
Supplementary Figure 1
Flow cytometric analysis: Forward scatter/side scatter gating. (PDF 172 kb)
Cardiomyocyte cells exhibit functional synchronized rhythmic beating.
The cardiomyocyte cells derived from eCSCs exhibit functional synchronized rhythmic beating which is stable and maintained for the duration of the culture. (AVI 4210 kb)
Individual CardioStem Sphere–derived cells maintain rhythmic beating phenotype.
Individually plated cardiomyocyte cells derived from CardioStem Spheres following sphere disaggregation maintain a rhythmic beating phenotype in culture. (AVI 2709 kb)
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Smith, A., Lewis, F., Aquila, I. et al. Isolation and characterization of resident endogenous c-Kit+ cardiac stem cells from the adult mouse and rat heart. Nat Protoc 9, 1662–1681 (2014). https://doi.org/10.1038/nprot.2014.113
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DOI: https://doi.org/10.1038/nprot.2014.113
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