Chondrogenic differentiation medium

Multilineage differentiation potential of AML-MSCs and HD-MSCs was identified as we reported [12 (link), 14 (link), 17 (link)]. In brief, 5×10⁴ cells were seeded into 12-well plate wells in MSC culture medium for 2–3 days. After reaching 60–80% confluence, the indicated AML-MSCs and HD-MSCs were cultured in adipogenic-, osteogenic-, and chondrogenic-differentiation medium (Stem Cell Technologies) according to the manufacturer’s instructions, respectively. After 3 weeks’ induction, the MSC-derived adipocytes, osteoblasts, and chondrocytes were identified by Oil Red O staining, Alizarin Red S staining, and Alcian Blue staining, respectively.

hUMSCs were extracted using the tissue block culture attachment method, as previously reported [19 (link),20 (link)]. The preparation of P5 hUMSCs was conducted in a GMP facility following good clinical practice (GCP) guidelines. The identification of hUMSCs was detected by flow cytometry (BD Biosciences, FACSAria2, USA). The antibodies used in this experiment included CD73-BV421 (cat.562430, BD Biosciences, USA), CD105-APC (cat. 562408, BD Biosciences, USA), CD90-FITC (cat.555595, BD Biosciences, USA), HLA-DR-PE (cat. 555812, BD Biosciences, USA), CD34-PerCP-Cy™5.5 (cat. 347203; BD Biosciences, USA), and CD45-FITC (cat.555482; BD Biosciences, USA). Osteogenic differentiation medium (catalog#05465, STEMCELL Technologies, China), adipogenic differentiation medium (catalog#05412, STEMCELL Technologies, China), and chondrogenic differentiation medium (catalog#05455, STEMCELL Technologies, China) were used to assess the osteogenic, adipogenic, and chondrogenic differentiation potential of hUMSCs, following the manufacturer’s instructions. Oil Red O solution (O1391, Sigma-Aldrich, USA), Alizarin Red S (A5533, Sigma-Aldrich, USA), and Alcian Blue staining solution (TMS-010-C, Sigma-Aldrich, USA) were used for adipogenic, osteogenic, and chondrogenic staining, respectively, according to the manufacturer’s instructions.

For adipogenic differentiation, passage 2 cADSC were seeded on 12-well plates at a concentration 4 × 10⁴ cells/well and cultured in DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic solution until 80% confluency. The medium was then replaced with 1 mL of adipogenesis differentiation medium (Thermo Fisher Scientific) and changed every 3 days. Adipogenesis was analyzed by Oil Red O staining after 21 days.
For osteogenic differentiation, passage 2 cADSC were seeded on 12-well plates at a concentration 2 × 10⁵ cells/well and incubated in DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic solution for 24 h. The medium was then replaced with 1 mL of osteogenesis differentiation medium (Thermo Fisher Scientific) and changed every 3 days. For osteogenic analysis, mineral deposits were analyzed quantitatively by Alizarin Red staining after 21 days.
For chondrogenic differentiation, 2 × 10⁵ passage 2 cADSC were suspended in 1 mL of chondrogenic differentiation medium (STEMCELL Technologies, Vancouver, BC, Canada) and 0.5 mL was aliquoted into a 15 mL conical tube and then centrifuged at 300× g for 5 min. The lid was then loosened and the cells were incubated for 3 days, after which 0.5 mL of medium was added and replaced every 3 days. Chondrogenic differentiation was evaluated by Alcian Blue staining after 21 days.