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

Nature Protocols volume 9, pages 1662–1681 (2014)Cite this article

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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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Figure 1: Perfusion apparatus and system.
Figure 2: Mouse aorta cannulation procedure and hanging of the heart on the perfusion system.
Figure 3: Flow cytometric analysis of unsorted mouse cardiac small cells.
Figure 4: Flow cytometric analysis of CD45+ and CD45− sorted fractions shows that mouse cardiac small cells are completely depleted of CD45+ cells by magnetic sorting.
Figure 5: Flow cytometric analysis shows magnetic-activated cell sorting of the CD45− selection for c-Kit yields a high purity of c-Kit+ eCSCs from the mouse heart.
Figure 6: Rat aorta cannulation procedure and hanging of the heart on the perfusion system.
Figure 7: Analysis of unsorted rat cardiac small cells.
Figure 8: Flow cytometric analysis of CD45+ and CD45− sorted fractions shows that rat cardiac small cells are completely depleted of CD45+ cells by magnetic sorting.
Figure 9: Analysis shows magnetic activated cell sorting of the CD45− selection for c-Kit yields a high purity of c-Kit+ eCSCs from the rat heart.
Figure 10: Flow cytometric characterization of freshly isolated c-Kit+CD45− mouse eCSCs.
Figure 11: Isolated c-Kit+CD45− eCSCs from both mouse and rat are clonogenic, self-renewing and form CardioStem spheres.
Figure 12: Immunocytochemistry of c-Kit+CD45− eCSCs and their multipotency.
Figure 13: Differentiation of c-Kit+CD45− eCSCs into functional beating cardiomyocytes in vitro.

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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).

Author information

Author notes

  1. Daniele Torella and Georgina M Ellison: These authors jointly directed this work.

Authors and Affiliations

  1. Centre of Human & Aerospace Physiological Sciences, School of Biomedical Sciences, King's College London, London, UK

    Andrew J Smith, Fiona C Lewis, Bernardo Nadal-Ginard & Georgina M Ellison

  2. Centre for Stem Cells and Regenerative Medicine, King's College London, London, UK

    Andrew J Smith, Fiona C Lewis, Bernardo Nadal-Ginard & Georgina M Ellison

  3. Department of Medical and Surgical Sciences, Laboratory of Molecular and Cellular Cardiology, Magna Graecia University, Catanzaro, Italy

    Iolanda Aquila, Daniele Torella & Georgina M Ellison

  4. Stem Cell and Regenerative Biology Unit (Biostem), Liverpool John Moores University, Liverpool, UK

    Cheryl D Waring

  5. Department of Experimental and Clinical Medicine, Laboratory of Molecular Oncology, Magna Graecia University, Catanzaro, Italy

    Aurora Nocera & Valter Agosti

  6. CIS for Genomics and Molecular Pathology, Magna Graecia University, Catanzaro, Italy

    Aurora Nocera & Valter Agosti

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Contributions

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.

Corresponding authors

Correspondence to Daniele Torella or Georgina M Ellison.

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Competing interests

The authors declare no competing financial interests.

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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