A1007201

Differentiation experiments were carried out in technical triplicates and maintained in culture for three weeks. The media were exchanged every three days. The controls were cultured in DMEM supplemented with 10% FCS, 1% Pen/Strep and 1% amphotericin B. For adipogenic and osteogenic differentiation, 4000 MSCs were seeded per well of a 12-well plate in DMEM supplemented with 10% FCS, 1% Pen/Strep and 1% amphotericin B for 48 h. After washing with PBS +/+, adipogenesis (ThermoFisher, Waltham, MA, USA, A1007001) or osteogenesis (ThermoFisher, A1007201) differentiation media were added. For chondrogenic differentiation, 350,000 MSCs were pelleted per 15 mL Falcon tube and resuspended in chondrogenesis differentiation media (ThermoFisher, A1007101).

Experimental differentiation procedures were conducted in triplicates (passage 2), and the cultures were maintained for a duration of three weeks. The cultural media were refreshed every three days. The control samples were cultured in DMEM supplemented with 10% FCS, 1% Pen/Strep, and 1% amphotericin B. Adipogenic and osteogenic differentiations were performed by seeding 4000 MSCs per well of a 12-well plate in DMEM supplemented with 10% FCS, 1% Pen/Strep, and 1% amphotericin B for 48 h. Subsequently, the cells were washed with PBS and treated with either adipogenesis ((ThermoFisher, Waltham, MA, USA, A1007001)) or osteogenesis (ThermoFisher, A1007201) differentiation media. For chondrogenic differentiation, 350,000 MSCs were collected in 15 mL Falcon tubes and pelleted, followed by resuspension in chondrogenesis (ThermoFisher, A1007101) differentiation media.

For flow cytometry, P3 rat ADSCs (passage 3) were treated with 0.05% trypsin (Gibco BRL) and resuspended in PBS containing 1% bovine serum albumin (BSA). They were then treated with antibodies for 30 min in a dark room at room temperature. Fluorescein isothiocyanate (FITC)-conjugated antibodies were used for the analysis of CD31 (Abcam, Cambridge, UK), CD34, CD105, CD73 (Bioss Antibodies, Woburn, USA), CD45, and CD90 (BD Biosciences, San Jose, USA). Cells were analyzed using a flow cytometer (BD Accuri C6 Plus Flow Cytometer; BD Biosciences) and the data were analyzed using BD Accuri C6 Software (BD Biosciences).
In vitro differentiation of rat ADSCs was performed in the same manner as previously reported^{64 (link)}. ADSCs were cultured 3 to 7 times and recovered by treatment with 0.05% trypsin (Gibco BRL). The cells were counted using 0.4% trypan blue (Gibco BRL) staining and dispensed into 12-well plates at 1 × 10⁴ cells/cm². Differentiation was induced using adipogenic differentiation medium (A1007001; Gibco BRL), chondrogenic differentiation medium (A1007101; Gibco BRL), and osteogenic differentiation medium (A1007201; Gibco BRL). The differentiation medium was changed once every 3 to 4 days. After differentiation into adipose, bone, and cartilage, the cells were stained with Oil Red O (Sigma), Alizarin Red S (Sigma), and Alcian Blue (Sigma), respectively.

The cells were subjected to induced differentiation using commercial kits (StemPro A10070-01, A10071-01, and A10072-01; Gibco) according to the manufacturer's instructions. Briefly, for the chondrogenic cell differentiation, the eBMmsc, eADmsc, and eUCmsc were plated in 5 μl droplets at a concentration of 1.6 × 10⁷ cells/ml and cultured under standard conditions for 2 h; then, the differentiation medium was added. The medium was replaced every 4 days for 2 weeks. For the osteogenic differentiation, 5 × 10³ cells/cm² were plated and incubated for 24 h; then, the IMDM medium was replaced by a specific differentiation medium, which was replaced every 4 days for 2 weeks. For the adipogenic differentiation, 1 × 10⁴ cells/cm² were plated and incubated for 24 h; then, the IMDM medium was replaced by a specific differentiation medium, which was replaced every four days for two weeks. The cells in the control group were plated under the same conditions but were maintained in IMDM medium. At the end of the two-week period, the cells were fixed and stained using Sudan black dye (Sigma-Aldrich) for the adipogenesis differentiation detection, Alizarin red (Sigma-Aldrich) for the osteogenesis detection, and Alcian blue (Sigma-Aldrich) for the chondrogenesis differentiation detection.

Palatal tissue was dissected from control and Wnt1^Cre;Kdm6b^fl/fl mice at E13.5 and cultured as previously described. Then, the differentiation assay was conducted according to the manufacturer’s protocol (Gibco, A1007201) (Chen et al., 2020 (link)). Briefly, mesenchymal cells were seeded into cell culture plates at the desired concentration followed by incubation at 36°C in a humidified atmosphere of 5% CO₂ for the required time (a minimum of 2 hr and up to 4 days). Then, the growth medium was replaced by complete differentiation medium and cells were continuously incubated for 3 weeks under osteogenic conditions. After specific periods of cultivation, cells were stained using 2% Alizarin red S solution (PH 4.2) solution. Images were acquired using EPSON Scan and Keyence BZ-X710/810 microscopes. Quantification of the Alizarin red S staining was conducted according to the manufacturer’s protocol (ScienCell, 8678).