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Engineering

In Vitro Culture of Epicardial Cells From Mouse Embryonic Heart

Published: April 27th, 2016

DOI:

10.3791/53993

1Program in Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, 2National Heart Research Institute Singapore, National Heart Centre Singapore, 3The Hatter Cardiovascular Insititute, University College London
* These authors contributed equally

The epicardium is an essential source of multipotent cardiovascular progenitor cells and paracrine factors that are required for cardiovascular development and regeneration. We describe here a method to culture mouse embryonic epicardial cells.

During embryogenesis, the epicardial contribution to coronary vasculature development has been very well established. Cells derived from the epicardium differentiate into smooth muscle cells, fibroblasts and endothelial cells that contribute to the formation of coronary vessels. Here we have established an in vitro culture method for embryonic epicardial cells. Using genetic labelling, we have demonstrated that the majority of the migrating cells in our explant culture are of epicardial origin. Epicardial explant cells also retain the expression of epicardial markers (Wt1 and Tbx18). Furthermore, we provide evidence that epicardial explant cells undergo epithelial to mesenchymal transition (EMT), migrate and differentiate into smooth muscle cells after Transforming growth factor beta 1 (TGF-β1) treatment in a manner indistinguishable from that of epicardial cells in vivo. In conclusion, we provide a novel method for the culture of embryonic epicardial cells, which will help to explore the role of specific genes in epicardial cell biology.

A wealth of experimental data have illustrated that the epicardium influences critical steps in cardiac development. During development, the septum transversum gives rise to a clump of mesothelial cells known as the proepicardium1-4. Cells from the proepicardium then migrate and envelope the myocardium forming the epicardium. Following this, a subset of epicardial cells undergo EMT giving rise to a migratory population of epicardium-derived cells (EPDCs) that subsequently invade the myocardium. Genetic as well as retroviral lineage tracing experiments have demonstrated that EPDCs differentiate into various lineages including smooth muscle cells, fibroblasts....

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All experiments were approved by the Duke-NUS Graduate Medical School Institutional Animal Care and Use Committee.

1. Retrieving the Embryonic Ventricles

  1. Sacrifice a timed pregnant mouse at the desired embryonic stage (E11.5 or E12.5) using a euthanasia chamber with carbon dioxide gas supply or another approved euthanasia method.
  2. Place the mouse on its back on a dissecting table. Disinfect the abdomen of the female with 70% ethanol. Lift the skin over the belly and make a.......

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Using this culture protocol, primary epicardial cells can be isolated with high purity for downstream applications. The cultured cells are able to undergo EMT, migrate and differentiate just as epicardial cells do in vivo.

To determine the purity of our primary epicardial cell culture, we analyzed the epicardial explants generated from Sema3dGFPCre/+;R26Tom/+ emb.......

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It is pivotal to develop techniques that facilitate the study of the epicardium to cater to the increasing significance of the epicardium in cardiac development and regeneration. The epicardial culture system poses significant advantages for epicardial research.

An alternative way to isolate epicardial cells is to use fluorescence-activated cell sorting (FACS). This method relies on the use of epicardial markers (or epicardium-cell-specific transgenic expression of a fluorescent protein) to se.......

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This work was supported by funds from DUKE-NUS Graduate Medical School Singapore, Goh foundation and Singapore NRF fellowship (NRF-NRFF2016-01) to Manvendra K. Singh.

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Name Company Catalog Number Comments
Dulbecco’s modified Eagle’s medium (DMEM) Life tech invitrogen  11995065
Penicillin/streptomycin solution Life tech invitrogen  15140122
Fetal bovine serum (FBS)  Life tech invitrogen  10500064
Paraformaldehyde Sigma P6148-5KG
Recombinant fibroblast growth factor 2 (FGF2) PeproTech 450-33
Recombinant transforming growth factor beta 1 (TGF-β1) PeproTech 100-21
ZO-1 antibody Life tech invitrogen  40-2200
α-Tubulin antibody Sigma T 6074
α-smooth muscle actin (SMA) antibody Sigma A 2547
Phalloidin antibody Life tech invitrogen  A12379
3D Collagen Culture kit  Millipore  ECM 675
8-well chamber slide Fisher Scientific NNU 154534-PK
Trizol reagent Life Technologies 15596-018
ViiA 7 Real-Time PCR System Life Technologies 4453536
Superscript First Strand Synthesis kit Life Technologies 11904-018

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