Alizarin red

Umbilical cords (UCs) were harvested from 19-day-pregnant Wistar rats. The isolation of UC-derived mesenchymal stem cells (UC-MSCs) was performed following our previously established protocol11. The isolated UC-MSCs were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Gibco, Invitrogen, NY, USA), 1.5% penicillin/streptomycin (Gibco), and 0.25% amphotericin B (Gibco). The cultures were maintained at 37°C in a humidified incubator with 5% CO₂ and the medium was refreshed every three days. Once the cells reached 80% confluency, they were passage into new culture flasks. UC-MSCs at passage 5 were utilized for subsequent experiments.
To characterize UC-MSCs, flow cytometry analysis was conducted at passage 3 following the manufacturer’s protocol. Briefly, cells were stained with rat anti-CD90-FITC, CD29-PE, CD31-PerCP, and CD45-APC antibodies (BD Biosciences, San Jose, CA, USA) and incubated for 20 minutes in the dark at room temperature. Following incubation, the stained cells were analysed using a BD FACS Lyric, and data interpretation was performed using BD FACS Suite (BD Biosciences).
The multilineage differentiation potential of UC-MSCs at passage 5 was further evaluated by inducing adipogenic and osteogenic differentiation. Cells were cultured in standard growth media until they reached 95% confluency, after which the medium was replaced with Rat MesenCult™ adipogenic and osteogenic differentiation basal media (Stem Cell Technologies, Singapore) supplemented with their respective differentiation supplements, 1% L-glutamine, 1% penicillin, and 0.25% amphotericin B (Gibco). Media changes were performed every three days, and after 21 days of induction, adipogenic and osteogenic differentiation was confirmed by staining lipid and calcium deposits with Oil Red O and Alizarin Red, respectively (Sigma-Aldrich).