Mesencult osteogenic differentiation kit

For peripheral neuron differentiation, NCCs were cultured in neuron differentiation media containing DMEM/F12, N2 supplement (Gibco), BDNF (10 ng/mL, R&D Systems), GDNF (10 ng/mL, R&D Systems), NGF (10 ng/mL, Peprotech), neurotrophin-3 (10 ng/mL, Peprotech), sodium l-ascorbic acid salt (200 μM) and dbcAMP (0.5 mM, Sigma) for 12–14 days. The medium was changed every 2 days. For mesenchymal differentiation, hiPSC-NCCs were cultured in media containing DMEM/F12, 10% FBS (Gibco), 1% penicillin-streptomycin (Corning), 1% l-alanyl-l-glutamine (Gibco), and 2-mercaptoethanol (0.1 mM, Sigma). The differentiated cells were passaged every 4 to 5 days and the media were changed every 2 days. Adipoblast, osteoblast, and chondroblast differentiation were performed according to the manufacturer’s directions using MesenCult Osteogenic Differentiation Kit (catalog 05465), MesenCult Adipogenic Differentiation Kit (catalog 05412), and MesenCult-ACF Chondrogenic Differentiation Kit (catalog 05455) (STEMCELL Technologies), respectively. For corneal keratocyte differentiation, NCCs were cultured in matrigel-coated plates and keratocyte differentiation media containing DMEM/F12, FGF2 (10 ng/mL), ascorbic acid-2-phosphate (1 mM, Sigma), 1% ITS, and 1% NEAA.

SCAP-Ss and DPSCs at indicated passages were seeded at a density of 5 × 10⁴/cm² in the aforementioned culture medium as reported [2 (link), 8 (link)]. When cells reached 80% confluency, the medium was changed into adipogenic differentiation medium (MesenCult Adipogenic Differentiation Kit, Stem Cell Technologies) or osteogenic differentiation medium (MesenCult Osteogenic Differentiation Kit, Stem Cell Technologies). The differentiation medium was changed every 3.5 days as we described previously [2 (link), 3 (link), 23 (link)]. 21 days later, the SCAP-S and DPSC-derived cells were stained with Oil Red O or Alizarin Red staining buffer, respectively. Then, the cells were photographed with a Nikon Eclipse Ti-U microscope (Nikon, Tokyo, Japan).

Twenty-four hours after seeding, half samples were maintained in control medium–CM (DMEM + 10% FBS) and half samples were moved to osteogenic medium-OM (MesenCult™ Osteogenic Differentiation Kit, Stemcell Technologies, Cambridge, MA, USA). hMSCs were maintained for 21 days at 37 °C, 5% CO₂ and 95% humidity, and medium replaced twice a week. At time 1 (24 h after seeding, Time 1 CM), 10 and 21 days (Times 10 CM/OM, control or osteogenic, and 21 CM/OM) hMSCs and scaffolds were analyzed.