Chicken serum

Eggs were purchased from Charles River Labs (#10100326) and incubated in a rotating egg incubator at 37°C with 55% humidity until Hamburger Hamilton Stage 30–31 (HH30–31), a stage of active valve remodeling. Embryos were then removed from the shell, and the mitral valves were dissected from the heart and stored in sterile PBS until all eggs were dissected. Mitral valves were pooled, and cells were dissociated with Trypsin containing EDTA (Corning, #25–053-CL). Cells were cultured in 100 mm dishes from Sigma (#93100), and maintained in M199 medium (Hyclone, #SH30253.01) supplemented with 5% heat-inactivated chicken serum (Bioworld, #30611183–1), 1% Pen-Strep (Life Technologies, 15070–063), and 0.1% Insulin-Transferrin-Selenium (ITS) (Gibco, #41400–045). Treatment groups for cell experiments included: Untreated, DHH 1μg/ml of recombinant mouse DHH C23II N-Terminus ligand (Novus Biologicals, #NBP2–35175), Antibodies (0.5μg/ml) against SHH (Santa Cruz, #sc-365112), IHH (Santa Cruz, #sc-166685), and DHH (Santa Cruz, #sc-271168), Cyclopamine 25μM (Tocris, #1623), and Cytochalasin D 1μM (Sigma, #C8273). All treatments, unless otherwise specified, were performed in low serum containing media: M199, 0.1% chicken serum, 0.1% ITS, 1% Pen-Strep.

Primary adult human dermal fibroblasts (HDF) (ATCC, Manassas, VA) and
human cardiac fibroblasts (HCF) (Cell Applications, San Diego, CA) cells were
purchased at passage 2 from LGC Standards, France and Tebu-bio, France
respectively, and cultured in 5% CO2 in a 37°C incubator. HDF were
maintained in fibroblast basal medium (ATCC, Manassas, VA) with fibroblast
growth kit-low serum (ATCC, Manassas, VA), and HCF were maintained in HCF growth
medium (Cell Applications, San Diego, CA) according to the manufacturers’
instructions. BJ Fibroblasts were purchased from ATCC (Manassas, VA) and
cultured in DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin
(Gibco, Grand Island, NW). Chicken valvular interstitial cells (cVICs) were
isolated from the anterior mitral leaflet of chicken embryos at HH40 as
described before^{18 (link)}. cVICs were
maintained in Medium 199 (Invitrogen, Waltham,MA) containing 5% of chicken serum
(Bioworld, Philadelphia, PA), 0.1% ITS (Gibco, Grand Island, NW) and 1%
Penicillin-Streptomycin (Gibco, Grand Island, NW).