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

Abstract

Article:Smith, AJ orcid.org/0000-0001-7283-5611, Lewis, FC, Aquila, I et al. (6 more authors) (2014) Isolation and characterization of resident endogenous c-Kit cardiac stem cells ⁺ from the adult mouse and rat heart. Nature Protocols, 9 (7). pp. 1662 -1681 . ISSN 1754 -2189 https://doi.org/10.1038/nprot.2014.113 © 2014, Rights Managed by Nature Publishing Group. This is an author produced version of a paper published in Nature Protocols. Uploaded in accordance with the publisher's self-archiving policy.eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ Reuse Unless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. The publisher or other rights-holder may allow further reproduction and re-use of this version -refer to the White Rose Research Online record for this item. Where records identify the publisher as the copyright holder, users can verify any specific terms of use on the publisher's website. TakedownIf you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. ABSTRACTThis protocol describes the isolation of endogenous c-kit-positive (c-kit pos ), CD45-negative (CD45 neg ) 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 separated by centrifugation. The c-kit pos CD45 neg eCSC population is isolated by magnetic activated cell sorting technology and purity and cell number assessed by flow cytometry. This process takes approximately 4 hours for mouse eCSCs or 4.5 hours for rat eCSCs. We also describe how to characterise c-kit pos CD45 neg eCSCs. The c-kit pos CD45 neg eCSCs exhibit the defining characteristics of stem cells; being self-renewing, clonogenic and multipotent. This protocol also describes how to differentiate eCSCs into the three main cardiac lineages: functional, beating cardiomyocytes, smooth muscle and endothelial cells. These processes take between 17 and 20 days.

Response to Molkentin’s Letter to The Editor Regarding Article, “The Absence of Evidence Is Not Evidence of Absence: The Pitfalls of Cre Knock-Ins in the c-kit Locus”

“…, which are several-fold higher in number than the CPCs. [4][5][6] These data have been nicely corroborated by van Berlo. 3 van Berlo 3 and Molkentin's letter 2 also disregard that among the c-kit + cells of wild-type animals, the CPCs and hematopoietic stem cells express c-kit at a significantly lower level than the c-kit + mast cells and endothelial progenitor cells.…”

“…3 van Berlo 3 and Molkentin's letter 2 also disregard that among the c-kit + cells of wild-type animals, the CPCs and hematopoietic stem cells express c-kit at a significantly lower level than the c-kit + mast cells and endothelial progenitor cells. [4][5][6][7] They did notice 3 that in ckit-mutated animals (constitutive and regulated), Cre expression depends on only 1 of the alleles. Therefore, it is to be expected that c-kit (and therefore Cre) expression would be only 50% in the already low expressing c-kit…”

How to make a cardiomyocyte

“…The observed discrepancies could be due to different experimental methodologies (for further discussion see Molkentin and Houser, 2013;Molkentin and Houser, 2014;Nadal-Ginard et al, 2014;Torella et al, 2014). As c-Kit is a relatively ubiquitous marker (including endothelial, hematopoietic, tissue monocytes), its expression alone is insufficient to define a specific c-Kit + cell population (Smith et al, 2014). Therefore, cell surface marker signatures will be needed to define such c-Kit + subtypes.…”

Dnmt3a-mediated inhibition of Wnt in cardiac progenitor cells improves differentiation and remote remodeling after infarction

“…Both processes involve activation of endogenous cardiac progenitor cells (CPCs) in cardiac niches that, upon specific stimuli, undergo proliferation and differentiation toward vascular or muscle lineages and release paracrine factors acting on neighboring cells to modulate tissue repair (1). A number of progenitor cells have been identified in the adult mouse and/or human heart based on their expression of specific surface markers, e.g., c-kit (2) and Sca-1 (3), or transcription factors, such as homeobox gene islet1 (Isl1) (4), or their ability to efflux Hoechst dye 33342 (identified as side population cells, ref. 5).…”