Tgfb3

The palate was dissected and explanted from the E15.0 embryo and cultured on track-etched polycarbonate membrane filter (Nuclepore) in Trowell type organ culture with serumless, chemically defined BGJb medium (Gibco). In our dissection, the primary palate and the nasal septum were not excluded from our culture system. Affi-Gel beads (Bio-Rad) were incubated in TGFB3 (100 ng/μl, R&D Systems). Bovine serum albumin (BSA; Sigma-Aldrich) was used instead of recombinant protein for the control beads. The beads were immersed in recombinant protein or BSA at 37 °C for 60 min and placed on the primary palate of the explants using a pipette tube. After culture, the in vitro explants were fixed at each stage in 4% paraformaldehyde overnight and then processed for histological examination and qPCR analyses.

The dissected palate of the E15.0 mutants was cultured on a Nuclepore filter (Whatman) in Trowell-type organ culture in chemically defined medium (BGJb, Gibco/Life Technologies). Affi-Gel beads (Bio-Rad) were incubated in TGFB3 (100 ng/μl, R&D Systems) and placed on the primary palate of the Cbfb mutant explants, as described previously (Sarper et al., 2018 (link)). BSA was used for the control beads. Fusion of the palatal process was evaluated histologically. The anterior portion of the palates was also dissected under the microscope and total RNA was extracted from these samples for analysis by qPCR.
To evaluate the possible rescue of cleft palate in Cbfb mutants by folic acid application, the palatal explants were cultured for 48 h in BGJb (Gibco) culture medium containing folic acid (N⁵-formyl-5,6,7,8-tetrahydropteroyl-L-glutamic acid, Sigma-Aldrich) at a final concentration of 100 μg/ml. After culture, the in vitro explants were fixed at each stage in 4% paraformaldehyde overnight and then processed for histological observation.

For chondrogenesis in hMSCs, 250,000 MSCs were centrifuged at 500 g, 7 min in 15 ml tubes to form pellets at high-density culture. Chondrogenesis was induced for 18 days with MEM medium supplemented with 50 μg/ml L-ascorbic acid-2-phosphate (Sigma-Aldrich.), 1% ITS+1 (BD Bioscience), 10⁻⁷ M dexamethasone (Sigma-Aldrich), and 10 ng/ml TGFb3 (R&D Systems, Wiesbaden, Germany). The aggregated cells were cultured in tubes with 0.5-1 ml medium/pellet at 37°C in a humidified atmosphere containing 95% air and 5% CO₂. The medium was replaced every other day for 18 days [25 (link)].

The chondrogenic potential of SMSCs was evaluated by established methods using samples from 12 donors [10 (link)]. A total of 250,000 passage 1 cells were placed in a 15-mL polypropylene tube (Falcon®; Corning Inc., Corning, NY, USA) and centrifuged at 1500 rpm for 5 min. Subsequently, the cells were cultured in chondrogenic medium: high-glucose Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 5000 ng/mL BMP-2 (Sigma-Aldrich), 10 ng/mL TGF-b3 (R&D Systems, Minneapolis, MN, USA), 10⁻⁷ M dexamethasone (Sigma-Aldrich), 50 mg/mL ascorbate2-phosphate (Sigma-Aldrich), 40 mg/mL proline (Sigma-Aldrich), 100 mg/mL pyruvate (Sigma-Aldrich), and 5 mg/mL ITS 1 Premix (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), which was replaced every 3–4 days for 21 days. The chondrogenic pellets were fixed with 4% PFA and photographed, and the Feret diameters of the pellets were measured using the NIH ImageJ software to compare their sizes.