Osteogenic differentiation medium

The cells were cultured up to 80%–90% confluence and changed to the osteogenic differentiation medium or the adipogenic differentiation medium (STEMCELL Technologies, Vancouver, BC, Canada). Osteoblastic differentiation was analyzed by alizarin red staining. Adipocytic differentiation was identified by Oil Red-O staining.

Murine mesenchymal stem cells (MSCs) (1.0 × 10⁴ per well) from STEMCELL (Seattle, WA, United States) were plated in 48-well plates and cultured in MesenCult^TM basal expansion medium with 10% 10x Supplement (Stemcell) for 72 h. Next, the cells were rinsed with PBS and the medium was replaced with osteogenic differentiation medium (Stemcell). MSCs cultured in expansion medium were served as the negative control. Half of the medium was changed every 3 days, and cells were harvested at 0, 5, and 7 days after induction.

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.