Stempro adipogenesis and osteogenesis differentiation kit

Assessment of adipogenic and osteogenic differentiation potential for MSCs isolated from randomly selected patient samples were performed in accordance with the ISCT recommendation. StemPro® Adipogenesis and Osteogenesis Differentiation Kit (Gibco, USA) was used according to the manufacturer instructions. Cells at passages 3–5 were used in differentiation experiments. To detect adipogenic differentiation, oil red O stain was used. To detect osteogenic differentiation, alizarin red S stain was used.

To analyze the potential of PD-MSCs^PRL-1 to differentiate into mesodermal lineages, PD-MSCs^PRL-1 (passage = 5) were plated at a density of 5 × 10³ cells/cm² in various differentiation induction media using the StemPro adipogenesis and osteogenesis differentiation kit (Gibco) according to the manufacturer’s instructions. After approximately 21 days, PD-MSCs^PRL-1 were fixed in 4% paraformaldehyde and incubated for 1 h with Oil Red O (Sigma-Aldrich) to stain lipids to visualize lipid vesicles and von Kossa with 5% silver nitrate (Sigma-Aldrich) under the light to evaluate the accumulation of calcium deposits.
To induce adipogenic differentiation, normal and GO-derived OFs (5 × 10³ cells/cm²) were seeded and incubated in serum-free DMEM/F12 supplemented with 33 μM biotin, 17 μM pantothenic acid, 10 μg/mL transferrin, 0.2 nM triiodothyronine (T₃), 1 μM insulin (all from Sigma-Aldrich), 0.2 μM carbaprostacyclin (cPGI₂; Cayman Chemical, Ann Arbor, MI, USA), 1 μM dexamethasone, and 0.1 mM isobutylmethylxanthine (IBMX; all from Sigma-Aldrich) for the first 4 days. To induce the maturation of adipocytes, the medium was supplemented except 1 μM dexamethasone and 0.1 mM IBMX (all from Sigma-Aldrich) for 6 days and was replaced every other day. Lipid accumulation and adipocyte morphology were visualized by Oil Red O staining.

Cells were differentiated into adipogenic and osteogenic lineages using Stempro® Adipogenesis and Osteogenesis differentiation kit (Gibco). Briefly, 6×10⁴ cells/well were cultured onto 24-well plate for 48-72 hours until they reached 100% confluency (for osteogenic differentiation) and 80% confluency (for adipogenic differentiation). The growth medium was then replaced with osteogenic or adipogenic induction medium, whereas for control (no induction), a complete RPMI growth medium was used instead. Cells were incubated for 21 days for osteogenic differentiation, and 14 days for adipogenic differentiation. The medium was replaced every three days. The capability of cells to differentiate into osteogenic was determined by deposition of calcium visualised by staining with 2% Alizarin Red S (Sigma-Aldrich), while the formation of adipocytes determined the capability of cells to differentiate into adipogenic visualised by staining with 0.3% Oil Red O (Sigma-Aldrich).