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J Vis Exp. 2016 Mar 18;(109). doi: 10.3791/53750.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration.

Author information

1
Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry; Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry.
2
Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry.
3
Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry; Department of Medicine, University of Rochester School of Medicine and Dentistry; Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry; Eric_Small@urmc.rochester.edu.

Abstract

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

PMID:
27023710
PMCID:
PMC4829037
DOI:
10.3791/53750
[Indexed for MEDLINE]
Free PMC Article

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