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

Summary

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

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.

Introduction

The epicardium is a single layer of mesothelial cells that lines the heart and impacts cardiac development, maturation and repair. Through a highly coordinated exchange of paracrine signals, the intimate dialogue between the myocardium and epicardium is indispensable for cardiac growth and the formation of non-myocyte cardiac lineages¹. Epicardial-derived cells (EPDC) emerge from a subset of epicardial cells through an epithelial-to-mesenchymal transition (EMT)², invade the subepicardial space and underlying myocardium, and differentiate largely into coronary vascular mural cells and cardiac fibroblasts, and to a lesser extent, endothelial cells ....

Protocol

NOTE: All experimentation with mice were approved by the University Committee on Animal Resources at the University of Rochester.

1. Epicardial Outgrowth Culture Assay (Figure 1A)

1. Preparations
   1. Prepare 5 ml medium A for primary epicardial cell isolation by supplementing Medium 199 (M199) with 5% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (Pen/Strep).
   2. Pre-warm medium A and 100 ml Hanks' Balanced Salt Solution (HBSS) in a 37 °C water bath.
   3. Allow collagen-coated chamber slides to warm to room temperature for 30 min.
   4. Add 100 µl medium A to each well of the chamber slide and gently ro....

Results

The epicardium can be efficiently isolated using an outgrowth culture assay by taking advantage of its exterior location and exploiting the developmental plasticity and intrinsic migratory behavior of EPDC. Murine embryonic hearts are isolated at E11.5 prior to epicardial EMT and cultured dorsal side down on collagen-coated chamber slides²⁶ (Figure 1A, B). Explanted hearts will continue to contract; however, the epicardium will adhere to the collagen matrix and.......

Discussion

Here we outline detailed methods to isolate primary epicardial cell by outgrowth and track EPDC migration in ex vivo heart cultures. For outgrowth cultures, the appropriate time to remove the epicardium-depleted heart varies slightly between experiments. Periodic monitoring of the explants after an overnight incubation is recommended to gauge the extent of epicardial outgrowth and to extract the heart before fibroblasts appear. To ensure purity, hearts should be removed within 24 hr of explanting to prevent accu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium	Hyclone	SH3002201	DMEM
Hanks' Balanced Salt Solution	Hyclone	SH3003102	HBSS
Medium 199	Hyclone	SH3025301	M199
Dulbecco’s Phosphate-Buffered Saline	Hyclone	SH3002802	DPBS
Fetal Bovine Serum	Gemini	100-106	FBS
Penicillin/Streptomycin Solution	Hyclone	SV30010
Corning Biocoat Collagen I, 4-Well Culture Slides	Corning	354557
Multiwell 24-well plates	Falcon	08-772-1
Dissecting microscope	Leica	M50	Equipped with Leica KL300 LED might source
Confocal miscroscope	Olympus	1X81	Equipped with muli-argon laser 405/488/515, FV10-MCPSU laser 405/440/635, 559 laser, and mercury lamp
Bioruptor	Diagenode	UCD-200
Transforming growth factor beta 1	R&D Systems	100-B-001	TGF-β1
Platelet-derived growth factor BB	R&D Systems	220-BB-010	PDGF-BB
Trizol Reagent	Applied Biosystems	15596-026	Caution, hazardous material
TURBO DNA-free Kit	Life Technologies	AM1907	DNaseI
iScript cDNA Synthesis Kit	BioRad	1708891
UltraPure Glycogen	Life Technologies	10814-010
SYBR Green	BioRad	170-8880
Protease inhibtor cocktail tablets	Roche	11836170001
Alexa Goat-anti-rabbit 488	Life Technologies	A11008	Secondary antibody
Alexa Goat anti-rabbit 594	Life Technologies	A-11012	Secondary antibody
Alexa Goat anti-mouse 594	Life Technologies	A-11005	Secondary antibody
Mouse anti-smooth muscle alpha actin, Cy3-conjugated	Sigma-Aldrich	C6198	monoclonal antibody, clone 1A4
Mouse anti-Wilms tumor 1 (WT1)	Life Technologies	MA1-46028	monoclonal antibody, clone 6F-H2
Rabbit anti-ZO1	Life Technologies	40-2200	polyclonal antibody
Rabbit anti-Collagen Type 4	Millipore	AB756P	polyclonal antibody
4',6-Diamidino-2-Phenylindole, Dihydrochloride	Life Technologies	D1306	DAPI
Fluorescent mounting medium	DAKO	S3023
16% Paraformaldehyde solution	Electron Microscopy Sciences	15710	PFA diluted to 4% in DPBS. Caution, hazardous material
Sucrose	Sigma-Aldrich	S0389
Tissue Freezing Medium	Triangle Biomedical Sciences	TFM-B
Pap pen	DAKO	S2002
Disposable mictrotome blades	Sakura Finetek	4689
Microscope slides	Globe Scientific	1358W	positively charged, 25 mm x 75 mm x 1 mm
Cryostat	Leica	CM1950
Forceps	Fine Science Tools	11295-00
Surgical Scissors	Fine Science Tools	91460-11
Disposable base molds	Fisher Scientific	22-363-553
Ketamine	Hospira	NDC 0409-2051-05
Xylazine	Akorn	NADA 139-236