100 18b 1mg

Skin fibroblasts from healthy subjects (CTRL M, Z and B) and patients PT1, PT2 and PT3 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with high glucose (Euro Clone, ECB7501L), supplemented with 10% fetal bovine serum (FBS, Gibco, 100-18B-1MG), 1% penicillin/streptomycin (Gibco, ECB3001D), 1% L-glutamine (Gibco, ECB3000D), 10 μg/mL of Fibroblast grow factor (FGF, Peprotech, 100-18B-1 MG) and kept at 37°C in a 5% CO₂ incubator. Pioglitazone (PGZ, Sigma-Aldrich, CDS021593-50MG) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, D2650-100ML) to a stock solution of 10 mM and added to the cell growth medium at the final concentration of 5 µM for 48 h. As untreated condition, cells were cultured with an equal volume of DMSO in the growth medium. The medium was exchanged every day with fresh medium. In some experiments, cells were treated with Insulin at the final concentration of 1 µM for 5 days and H₂O₂ at the final concentration of 800 µM for 48 h.

ReNcell VM neural progenitor cells (Merck Millipore SCC008) were used in this study as an in vitro model of hNSC. They were grown either as neurospheres or as adherent monolayer on laminin coated plasticware in the following culture medium: 50% DMEM:F12 (GIBCO 11554546), 50% Neurobasal (GIBCO 21103049), 0.5% Penicillin-Streptomycin (Sigma-Aldrich P4333-100ML), 1% N2 supplement (GIBCO 17502048), 2% B27 NeuroMix (GIBCO 17504044), heparin (5μg/ml, Sigma-Aldrich 9041-08-1). Human Epidermal Growth Factor (hEGF, GIBCO AF-100-15-1MG) and human Fibroblast Growth Factor (hFGF, GIBCO 100-18B-1MG) were added to the cell culture medium just before use, both at a concentration of 20 ng/ml. Growth medium was replaced with fresh medium every 2 days.
Transfection of hNSC was performed by electroporation using a Nucleofector^TM 2b Device (Lonza) with Nucleofector^TM Kits for Mouse Neural Stem Cells as per manufacturer instructions (Lonza VPG-1004).