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
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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 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. |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Biomedical Sciences (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 29 Jul 2016 13:19 |
Last Modified: | 18 Jan 2018 17:48 |
Published Version: | http://dx.doi.org/10.1038/nprot.2014.113 |
Status: | Published |
Publisher: | Nature Publishing Group |
Identification Number: | 10.1038/nprot.2014.113 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:102048 |