Rencell nsc maintenance media

Induced neural stem cells (iNSCs) were generated from normal donor skin fibroblasts (GM05659; Coriell Institute for Medical Research, Camden, NJ, USA) and NPC patient-derived human Dermal Fibroblasts (hDFs, GM03123 (NPC1P237S/I1061T), GM18453 (NPC1I1061T/I1061T); Coriell Institute for Medical Research). Viral production and transduction were performed as described previously. Briefly, retroviral pMX-SOX2 and pMX-HMGA2 were transfected into 293 FT cells along with VSV-G and gag/pol plasmids using Fugene 6 transfection reagent (Roche, Indianapolis, IN, USA). The viral supernatants were collected at 48- and 72-h post-transfection and used to infect hDFs with 5 μg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA). For neural stem cell induction, the medium was changed to NSC maintenance medium (ReNcell NSC maintenance media; Millipore, Billerica, MA, USA) with basic fibroblast growth factor (bFGF; Sigma-Aldrich, St. Louis, MO, USA) and epidermal growth factor (EGF; Sigma-Aldrich, St. Louis, MO, USA) after expansion of the infected cells. NSC-like colonies were picked and cultured in neurosphere culture conditions. To generate a homogenous population of iNSCs, cells were maintained as neurospheres and cultured as attached cells on PLO/FN-coated dishes, repeatedly. NPC-iNSC lines from NPC1 mutant human fibroblast were generated up to 10 independent clones.

Embryonic bodies were produced after treating iPSC colonies with dispase and placing them in human neural stem cell differentiate medium (50 % DMEM/F12 plus 50 % Neural basal medium (Gibco) containing 1 % N2 supplement (Gibco), 2 % B27 supplement (Gibco), 1 μM Dorsomorphin (Sigma-Aldrich), 10 μM SB431542 (Sigma-Aldrich)) in a 100 mm petri dish. Medium was changed every two days. After 2 weeks, spheres were seeded on polyornithine (Sigma-Aldrich) and laminin (Sigma-Aldrich) coated dishes and kept culturing in the same medium for another 7-14 days for neural rosette formation. Neural rosettes were collected with Stem Diff neural rosette selection reagent and re-plated onto polyornithine/laminin coated dishes as a single cell suspension in neural stem cell culture medium (ReNcell NSC maintenance media (Millipore) containing, 1 % N2 supplement, 1 % NEAA, 20 ng/ml bFGF and 20 ng/ml EGF). For three germ layers differentiation, iPSC colonies were digested with dispase and culture in DMEM with 10 % FBS for 4 days in 10 cm petri dish. After 4 days in suspension culture, floating embryonic bodies were re-seeded onto gelatin-coated dishes in the same culture medium for 10 days. The medium was changed every other day.