title: Isolation and characterization of resident endogenous c-Kit+ cardiac stem cells from the adult mouse and rat heart.
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.
authors.affiliation: 1] Centre of Human & Aerospace Physiological Sciences, School of Biomedical Sciences, King's College London, London, UK. [2] Centre for Stem Cells and Regenerative Medicine, King's College London, London, UK.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Smith
authors.affiliation: 1] Centre of Human & Aerospace Physiological Sciences, School of Biomedical Sciences, King's College London, London, UK. [2] Centre for Stem Cells and Regenerative Medicine, King's College London, London, UK.
authors.forename: Fiona C
authors.initials: FC
authors.lastname: Lewis
authors.affiliation: Laboratory of Molecular and Cellular Cardiology, Department of Medical and Surgical Sciences, Magna Graecia University, Catanzaro, Italy.
authors.forename: Iolanda
authors.initials: I
authors.lastname: Aquila
authors.affiliation: Stem Cell and Regenerative Biology Unit (Biostem), Liverpool John Moores University, Liverpool, UK.
authors.forename: Cheryl D
authors.initials: CD
authors.lastname: Waring
authors.affiliation: 1] Laboratory of Molecular Oncology, Department of Experimental and Clinical Medicine, Magna Graecia University, Catanzaro, Italy. [2] CIS for Genomics and Molecular Pathology, Magna Graecia University, Catanzaro, Italy.
authors.forename: Aurora
authors.initials: A
authors.lastname: Nocera
authors.affiliation: 1] Laboratory of Molecular Oncology, Department of Experimental and Clinical Medicine, Magna Graecia University, Catanzaro, Italy. [2] CIS for Genomics and Molecular Pathology, Magna Graecia University, Catanzaro, Italy.
authors.forename: Valter
authors.initials: V
authors.lastname: Agosti
authors.affiliation: 1] Centre of Human & Aerospace Physiological Sciences, School of Biomedical Sciences, King's College London, London, UK. [2] Centre for Stem Cells and Regenerative Medicine, King's College London, London, UK.
authors.forename: Bernardo
authors.initials: B
authors.lastname: Nadal-Ginard
authors.affiliation: 1] Laboratory of Molecular and Cellular Cardiology, Department of Medical and Surgical Sciences, Magna Graecia University, Catanzaro, Italy. [2].
authors.forename: Daniele
authors.initials: D
authors.lastname: Torella
authors.affiliation: 1] Centre of Human & Aerospace Physiological Sciences, School of Biomedical Sciences, King's College London, London, UK. [2] Centre for Stem Cells and Regenerative Medicine, King's College London, London, UK. [3] Laboratory of Molecular and Cellular Cardiology, Department of Medical and Surgical Sciences, Magna Graecia University, Catanzaro, Italy. [4].
authors.forename: Georgina M
authors.initials: GM
authors.lastname: Ellison
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D019070:Cell Lineage; D002478:Cells, Cultured; D051379:Mice; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism*; D051381:Rats; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D019009:Proto-Oncogene Proteins c-kit