Adipogenic differentiation kit

Osteogenic and adipogenic differentiation were carried out according to the method described by Gao et al. (18 (link)). hUCMs (1×10⁴cell/cm²) were detached from the substratum and cultured on glass slides. For osteogenic differentiation, the cells were fed with osteogenic medium that consisted of low glucose DMEM (L-DMEM) supplemented with 10% FBS, 10 nM dexamethasone, 50 μmol/L ascorbic acid and 10 mM L β-glycerol phosphate for 21 days. The induced cells were stained by alizarin red and observed under an inverted microscope (Olympus, Japan). For adipogenic differentiation, the cells were treated for 14 days with an adipogenic differentiation kit as recommended by the manufacturer (Invitrogen, USA, A1007001). Differentiated cells were stained by oil red O and analyzed by a light microscope. As controls, hUCM cells were maintained in the complete medium without inducing agents.

To assess the osteogenic ability of BMMSCs, BMMSCs were cultured in osteoinductive medium containing 10 mM β-glycerophosphate (Sigma, USA), 100 μM L-ascorbic acid 2-phosphate (Sigma), and 10 nM dexamethasone (Sigma, USA). After 7 days of induction, the cells were stained with an alkaline phosphatase (ALP) staining kit (Sigma, USA) or collected for ALP activity testing using alkaline phosphatase yellow (pNPP) liquid ELISA substrate (Sigma, USA). For the adipo-induction, an adipogenic differentiation kit (Invitrogen, USA) was used. After 4 weeks of induction, the cells were stained with Oil Red O (Sigma, USA). After photographing, lipid droplets were then dissolved with 100% isopropanol, and OD value was measured at 492 nm. For real-time RT-PCR assays, total mRNA was isolated from BMMSCs after one week of induction. All assays were done in duplicate from at least three independent experiments.