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

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

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

95 Citations (Scopus)

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 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+CD45eCSCs 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.

Original languageEnglish
Pages (from-to)1662-1681
Number of pages20
JournalNature Protocols
Volume9
Issue number7
Early online date19 Jun 2014
DOIs
Publication statusPublished - Jul 2014

Keywords

  • SIDE POPULATION CELLS
  • PROGENITOR CELLS
  • IN-VITRO
  • CARDIOMYOCYTES
  • REGENERATION
  • MULTIPOTENT
  • THERAPY
  • REPAIR
  • DIFFERENTIATION
  • INTRACORONARY

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