Stromal Lineage Differentiation Assay

The ability of BMSCs and ASCs to differentiate into multiple stromal lineages was tested as previously described [17 (link)]. Briefly, for adipogenic differentiation, cells in HPL and FBS were cultured in StemPro® adipogenic differentiation medium (Invitrogen) or standard growth medium (control). After 14 days, intracellular lipid formation was assessed via Oil red O (Sigma-Aldrich) staining. For quantification, the stain was extracted using 99% isopropanol (Sigma-Aldrich) and absorbance was measured at 540 nm using a microplate reader. For osteogenic differentiation, cells in HPL and FBS were cultured in osteogenic differentiation medium prepared by adding final concentrations of 0.05 mM L-ascorbic acid 2-phosphate, 10 nM dexamethasone, and 10 mM β glycerophosphate (all from Sigma-Aldrich) to the respective growth media. Cells in standard growth medium served as controls. After 21 days, extracellular calcium deposition was evaluated via Alizarin red S staining (Sigma-Aldrich). For quantification, the stain was dissolved in cetylpyridinium chloride (Sigma-Aldrich) and absorbance was measured at 540 nm using the microplate reader.