Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Summary

This article will provide a method for isolating and culturing quail or chicken HH14^- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Abstract

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

Protocol

1. Preparation

1. Incubate fertile quail or chicken eggs to stage 14^- (about 2 days) or 25 (about 4.5 days) at 60% humidity and 37°C.
2. Prepare sterile Earl's Balanced Salt Solution (EBSS)
   1. Add 100 mL 10X EBSS to 600 mL 18 MΩ water
   2. Add 2.2 g sodium bicarbonate
   3. Adjust the pH of the solution to 7.2
   4. Bring the solution up to 1000 mL
   5. Sterilize the solution by passing through a 0.2 μm filter
3. Prepare sterile M199 culture medium.
   1. Add 1 package of M199 powder to 700 mL 18 MΩ water.
   2. Add 2.2 g sodium bicarbonate

Discussion

The method for isolating endocardial cells from stage HH14^- hearts originally developed by Runyan and Markwald provides a controlled, in vitro environment to study the factors that initiate and modulate embryonic EMT². HH14^- endocardial explants cultured without myocardium will not undergo EMT without biomechanical or biochemical intervention. If the myocardium is left on the explant it will signal a subset of endocardial cells to undergo mesenchymal transformation, but external .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Extra fine Bonn scissors, curved	Fine Science Tools	14085-08
Dumont tweezers #55	World Precision Instruments, Inc.	14099
Dumont tweezers #5	World Precision Instruments, Inc.	14098
Sterile transfer pipettes	Samco Scientific, Thermo Fisher Scientific	2041S
4-well culture plates	Nalge Nunc international	176740
Sterile disposable filter units, PES, 0.2 μm pore size membrane	Nalge Nunc international	566-0020
Sterile 15 mL centrifuge tubes	Corning	430828
Sterile petri dishes, 100 mm x 15 mm	VWR international	25384-342
Laboratory tape	VWR international	89097-920
Rat-tail collagen I	BD Biosciences	354236
10X Earl’s Balanced Salt Solution	Quality Biological, Inc.	119-064-131
M199 powder	Invitrogen	31100-035
Penicillin-Streptomycin	Invitrogen	15140-122
Insulin-Transferrin-Selenium-G supplement (100X)	Invitrogen	41400-045
Chicken serum	Invitrogen	16110-082
0.25% Trypsin-EDTA	Invitrogen	25200-056
Sodium bicarbonate	Sigma-Aldrich	S5761
Sodium hydroxide solution, 1.0 N	Sigma-Aldrich	S2770
Fertile quail eggs (Coturnix coturnix)	Lake Cumberland Game Bird Farm
Fertile chicken eggs (Gallus gallus)	Cornell University Poultry Farm