Stemmacs chondrodiff media

For differentiation into adipocytes and osteoblasts, WJ-MSCs cultured in FBS and XF conditions were plated in 6-well plates at a concentration of 1 × 10⁵ cells. After the cells reached 70–80% confluency, they were treated with StemMACS™ Adipodiff Media and OsteoDiff Media (Miltenyi Biotec, Bergisch Gladbach, Germany), following the manufacturer’s instructions. The medium was changed every 3 days. After 3 weeks of induction, Oil Red O (Abcam, Cambridge, UK) staining or Alizarin Red S (Sigma-Aldrich, St. Louis, MO) staining was performed to evaluate lipid-laden fat cells or calcium deposition, respectively. For chondrogenic differentiation, 2.5 × 10⁵ cells were placed in a 15-mL polypropylene tube and maintained with 1 mL of StemMACS™ Chondrodiff Media (Miltenyi Biotec), following the manufacturer’s instructions. The cells were allowed to undergo differentiation for 3 weeks, with media change every 3 days. After differentiation, the round pellets were embedded in paraffin and cut into 3-µm sections that were stained by Alcian Blue (Merk Millipore, Darmstadt, Germany) to detect glycosaminoglycans.

The O9-1 mouse cranial neural crest cell line was purchased from MilliporeSigma (SCC049) and cultured in complete ES cell medium containing 15% FBS and LIF (MilliporeSigma, ES-101-B) with 100 units/ml penicillin-streptomycin (Invitrogen) at 37°C in a humidified atmosphere containing 5% CO₂ according to the manufacturer’s instructions. For osteogenic or chondrogenic differentiation, O9-1 cells were cultured with StemMACS OsteoDiff Media or StemMACS ChondroDiff Media (Milteny Biotec), respectively. Osteogenic differentiation and chondrogenic differentiation were evaluated by alizarin red or alcian blue staining as previously described [70 (link),73 (link)]. To knockdown Tmem2 expression in O9-1 cells, we used lentivirus-mediated shRNA transduction. Lentivirus particles expressing an shRNA that is validated to deplete mouse Tmem2 (Mission shRNA, TRC Clone ID: TRCN0000295501, MilliporeSigma) and control lentivirus particles expressing an shRNA that does not target any known genes (Mission shRNA, MilliporeSigma SHC005) were purchased from MilliporeSigma. Lentivirus particles were added to O9-1 cells cultured in growth media supplemented with 5 μg/ml polybrene and cultured for 2 days. Cells transduced with lentiviral shRNAs were selected and maintained in the presence of 10 μg/mL puromycin.

A total of 150,000 cells were cultured in the tip of a 15 mL conical tube (Greiner, Kremsmünster, Austria) to enable cell culture in micromass with chondrogenic medium (Stem MACS Chondro Diff Media, Miltenyi Biotec). Cells were resuspended carefully and cultured at 37 °C in a 5% CO₂ humidified atmosphere with the cap slightly screwed. Half of the chondrogenic medium was replaced weekly. On Day 21, aggregates were stained with Alcian blue (Sigma) to highlight cartilage proteoglycans. In some cases, cryosectioned pellets were stained with Alcian blue to confirm chondrogenic differentiation.

For scaffold seeding, hCh and mnCPC were thawed and cultured in monolayer until 80–90 % confluence. Scaffolds were seeded either with 1.0 × 10⁶ hCh or mnCPC (passage 2) per scaffold as published previously [11 (link)]. Seeded scaffolds were cultured in StemMACS ChondroDiff Media (Miltenyi Biotec) supplemented with 0.5 % gentamycin (Biochrom) for up to 42 days in a humidified atmosphere with 5 % CO₂ at 37 °C. Medium was changed twice a week. Scaffolds were analyzed on days 14, 28 and 42.

The MSCs were plated at a density of 5 × 10⁴ cells/well in 12-well plates and induced to differentiate into adipocytes with culture medium supplemented with 100 µM indomethacin (Sigma-Aldrich Co. LLC, United States), 1 µM dexamethasone (FUJIFILM Wako Pure Chemical Corporation, Japan), 0.5 µM IBMX (Sigma-Aldrich), and 10 μg/mL insulin (Sigma-Aldrich) for 2 weeks. Then, the cells were stained with Oil Red O (Sigma-Aldrich) (He et al., 2014 (link)). UC-MSCs were cultured for 4 weeks using a StemPro osteogenesis differentiation kit (Thermo Fisher Scientific Inc., United States) in accordance with the manufacturer’s instructions, and their osteogenic differentiation was evaluated. The cells were stained with alizarin red (Sigma-Aldrich). In the chondrogenic differentiation assay, we used a pellet culture system using Stem MACS™ Chondro Diff Media (Miltenyi Biotec GmbH; Germany) at 2.5 × 10⁵ in 15 mL conical tubes for 3 weeks. The cells were fixed with 4% formaldehyde and stained with toluidine blue (Sigma-Aldrich).

The differentiation of equine and human MSCs into adipocytes, osteocytes, and chondrocytes was performed using the mesenchymal stem cell assays developed by Miltenyi Biotec. For adipogenic differentiation, StemMACS AdipoDiff Media (Miltenyi, Auburn, CA, USA 130-091-677) was used, and cells were stained with Oil Red O (Millipore Sigma, ST Louis, MO, USA cat# 0975525G). For osteogenic differentiation, StemMACS OsteoDiff Media (Miltenyi, Auburn, CA, USA, 130-091-678) was used, followed by the detection of alkaline phosphatase activity using SIGMAFAST™ BCIP^®/NBT (Sigma-Aldrich, ST Louis, MO, USA, cat# B5655-25TAB). For chondrogenic differentiation, StemMACS™ ChondroDiff Media (Miltenyi, Auburn, CA, USA, 130-091-679) was used, followed by Alcian blue (Millipore Sigma, ST Louis, MO, USA, cat# TMS010C) staining of chondrocyte “nodules”. MSCs from three separate horses and MSCs from two diabetic and two control human subjects were used in duplicate.