Immortalized Human Neural Progenitor Cell Culture and Induced Pluripotent Stem Cell Differentiation

Immortalized human NPCs (ReNcell VM Human Neural Progenitor Cell Line; Millipore, Billerica, MA, USA) were cultured on CTS CELLstart Substrate (Thermo Fisher Scientific, Waltham, MA, USA)-coated dishes in StemPro NSC SFM (Thermo Fisher Scientific) supplemented with 200 mM GlutaMAX (Thermo Fisher Scientific), 100 U/mL penicillin, and 100 µg/mL streptomycin (Thermo Fisher Scientific). The hiPSC cell line 253G1 was obtained from the RIKEN cell bank and maintained on laminin-521 (BioLamina, Sundbyberg, Sweden) or hESC-qualified matrigel (Corning, Corning, NY, USA)-coated dishes in mTeSR1 or mTeSR Plus media (Veritas, Tokyo, Japan). For differentiation of hiPSCs into NPCs, hiPSCs were seeded onto hESC-qualified matrigel-coated 6-well plates or 60-mm dishes and cultured in STEMdiff SMADi Neural Induction medium (Veritas) for at least 16 days according to the manufacturer’s instruction.
The constructed Ad serotype 5 vector and AAV1, 2, 5, and 6 vectors were used in this study^{16 (link)}. These vectors drive the expression of a full-length iCasp9 with a hemagglutinin (HA) tag (YPYDVPDYAA) at its C-terminus under the control of the CMV promoter.