Differentiation of T-MSCs into Schwann Cells

To differentiate T-MSCs into SCs, we used the technique of Razavi et al. [41 (link)] in which human T-MSCs were induced to form neurospheres. We harvested human T-MSCs (80%–90% confluence) and then plated them in plastic dishes at (1.5–2) × 10⁵ cells/cm² in DMEM/F-12 (Welgene Inc., Daegu, Korea) supplemented with 20 ng/mL basic fibroblast growth factor (bFGF, PeproTech, London, UK), 20 ng/mL epidermal growth factor (EGF, PeproTech), and 2% B27 supplement (1:50, Gibco, Life Technologies, Burlington, ON, Canada) at 37 °C under 5% CO₂ in humidified air. We replenished the cultures with fresh medium every 3–4 days. After 7 days, neurospheres were triturated using a 25-gauge needle and replated in laminin-coated cell culture plates containing DMEM/F12 supplemented with 10% FBS, 14 µM forskolin (Sigma-Aldrich), 5 ng/mL platelet-derived growth factor-AA (PDGF, PeproTech), 10 ng/mL bFGF (PeproTech) and 200 ng/mL recombinant human heregulin-β1 (PeproTech) for terminal differentiation. The cells were incubated for 9 days under these conditions, and then harvested for further investigations.