Transdifferentiation of human fibroblasts

NHF-1-hTERT human fibroblasts were transdifferentiated, as described previously (17 (link)). Briefly, 1 × 10⁵ NHF-1-hTERT cells were seeded in six-well plate supplemented with 10% FBS DMEM media overnight. The next day (day 0), cells were transduced with LV-GFP-TRAIL, LV-mCherry-FLuc, or LV-GFP-NLuc for 24 hours in 2% FBS DMEM media supplemented with polybrene (8 μg/ml) (Thermo Fisher Scientific). The cells were transduced again (day 1) with a cocktail of LV-SOX2 and LV-rtTA in 2% FBS DMEM media supplemented with polybrene (8 μg/ml) for 24 hours. Before use, 1 × 10⁶ cells were seeded in T-175 flask with regular 10% FBS DMEM media overnight. Starting from day 2, cells were cultured in STEMdiff neural induction media (STEMCELL Technologies) with doxycycline (2 μg/ml; Sigma-Aldrich), and media was replenished every other day. Cells were detached with Accutase cell detachment solution (STEMCELL Technologies) for use on day 6.