At day 7 of induction, the medium was switched to 2% horse serum/DMEM media until day 14.
The next day, C2C12 cell medium was switched to differentiation medium, including 10 µL/mL of pooled fractions enriched in Collagen XXV-GFP or the same amount of the corresponding fractions from GFP-alone conditioned media, or 0.5 ng/mL of recombinant GFP (Novus biologicals).
24 h later, the medium was switched to CDF12 medium with medium changes every day.
When the medium was switched to adipogenic and osteogenic differentiation medium, the MSC concentrations were approximately 30, 60, 80, and 100%, respectively.
After 48 h, the medium was switched to α-MEM with 2% (v/v) fetal bovine serum (FBS) in order to initiate differentiation.
Then the medium was switched to the RPMI1640 medium supplemented with Albumin, Ascorbic acid, transferrin, selenite, 5 ng/mL BMP4 (R&D Systems), and CHIR99021 to induce cardiac mesoderm formation.
Once a stable current was observed, the synthetic medium was switched to the desired PDWW concentration buffered with 50 mM KH2PO4, pH 7.0 (Jiang et al. 2009), and MFCs were operated in continuous mode for 60 days.
The medium was switched to serum-free medium (DMEM/F12 with L-glutamine, penicillin/streptomycin, gentamicin and 550 nM hydrocortisone) after the cells reached confluence.
Then the medium was switched to 2 ml of medium containing 100 nM of α-amanitin and time –lapse movie acquisition was continued.
After 16 h culture medium was switched to regular growth medium and cells were allowed to incubate for additional 48 hours.
Another 24 h later, the medium was switched to ESC medium [31] for a total period of 10 d, with replacement on alternate days.
