This article will provide a method for isolating and culturing quail or chicken HH14– valve endocardial cells and HH25 valve cushion mesenchymal cells.
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.
1. Preparation
2. Pre-EMT Endocardial Cell Isolation and Culture
3. Post-EMT AV Cushion Mesenchymal Cell Isolation and Culture
4. Representative Results
Figure 1: Examples of embryos. (A) HH14- quail embryo, and (B) HH25 chicken embryo are shown here.
Figure 2: HH14- valve endocardial explants after 2 hours of culture. (A) with the myocardium present, or (B) with the myocardium removed.
Figure 3: HH14- valve endocardial explants. (A) With the myocardium present, many of the cells undergo endocardial to mesenchymal transformation and have a spindle-shaped, mesenchymal phenotype after 48 hours of culture. (B) When the myocardium is removed all of the cells maintain a cobblestone-shaped, endocardial phenotype after 48 hours in culture.
Figure 4: HH25 valve mesenchymal cells in a collagen gel. Cells are rounded immediately after seeding.
Figure 5: HH25 mesenchymal cells in culture. (A) After 48 hours of culture in a constrained collagen gel the HH25 mesenchymal cells are beginning to spread. (B) HH25 cells in a stress-free collagen gel 7 days after seeding and 6 days after liberation have compacted the gel to approximately 50% of the original area.
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 factors may regulate this process. Isolating embryonic progenitor mesenchymal cells from HH25 AV valve cushions using the method described by Butcher et al. provides an opportunity to determine what signals drive differentiation toward mature valvular interstitial cells3. Culturing the cells in mechanically stressed or stress-free conditions allows the researcher to probe biomechanical stimuli, and factors added to the culture medium can provide biochemical signals. In vitro models that help to decipher mechanisms affecting EMT, cell differentiation, and structural remodeling can be used to better understand the causes of congenital heart defects and could help determine ways to drive stem cells toward mature valve cell phenotypes for tissue engineering applications.
The authors have nothing to disclose.
This research is supported by The Hartwell Foundation, the American Heart Association (Scientist Development Grant #0830384N) and the Foundation Leducq: Mitral 07CVD04.
Material Name | Typ | Company | Catalogue Number | Comment |
---|---|---|---|---|
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 | 2041S | ||
4-well culture plates | Nunc | 176740 | ||
Sterile disposable filter units, PES, 0.2 μm pore size membrane | Nalgene | 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 |