Recombinant human transforming growth factor β1

In vitro differentiation ability of culture expanded BAL-MSCs into adipocytes, chondrocytes and osteocytes was examined as previously described (Khatri et al., 2009 (link)). For adipogenesis, BAL-MSCs were cultured in adipogenic induction medium consisting of C-DMEMS, 1 μM dexamethasone, 10 μg/ml insulin, and 0.2 mM indomethacin for 2 weeks. The cells were fed with fresh medium every 3-4 days. Generation of lipid droplets indicative of adipogenesis was detected by 0.3% Sudan III staining.
To induce the differentiation of BAL-MSCs into osteocytes, these cells were cultured in C-DMEM, 100 nM dexamethasone, 10 mM β-glycerophosphate and 0.05 mM L-ascorbic acid-2-phosphate for 3 weeks. Calcium deposition in differentiated osteocytes was detected by Alizarin S staining (Khatri et al., 2009 (link)).
For chondrogenic differentiation, BAL-MSCs were incubated for 3 weeks in C-DMEM medium supplemented with 50 mM ascorbic acid, 0.5 mg/ml insulin (Sigma) and 10 ng/ml recombinant human transforming growth factor-β1 (Life Technologies). Cultures were fed with fresh medium every 3-4 days. Sulfated glycosaminoglycans in differentiated cultures were detected by 1% Alcian blue staining.

All experiments employed the immortalized human vocal fold fibroblast cell line created in our laboratory, referred to as HVOX.¹⁴ This line has been shown to stable through multiple population doublings; the current experiments employed cells in passages 20–30. All cells were maintained in DMEM supplemented with 10% FBS and antibiotic/antimycotic. Recombinant human transforming growth factor-β1 (Life Technologies, Grand Island, NY) was employed in all experiments.