Gm05659

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.

The human embryo kidney cell line HEK293T, human cervical cancer-derived cell line HeLa, and human T-lymphocyte-derived cell line Jurkat were obtained from the American Type Culture Collection (ATCC). The TZM-bl cell line is a HeLa-derived cell line expressing CD4, CXCR4, CCR5, and the HIV LTR-luciferase, and this cell line was obtained from the NIH AIDS Reagent Program (NIH, Bethesda, MD, USA). HEK293T, HeLa, and TZM-bl cells were maintained in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin at 37 °C in a 5% CO₂ humidified incubator. Jurkat cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin at 37 °C in a 5% CO₂ humidified incubator. Primary fibroblasts from an Niemann–Pick disease type C1 patient (GM03123) and healthy donor (GM05659) were obtained from Coriell Institute (Camden, NJ, USA) and maintained in DMEM medium supplemented with 15% fetal bovine serum (not inactivated), 100 U/mL of penicillin, and 100 μg/mL of streptomycin at 37 °C in a 5% CO₂ humidified incubator.