Stemdiff neural progenitor supplement a

To analyze the differentiation of human cells in direct contact with biomaterials, neural progenitor cells derived from human induced pluripotent stem cells (hiPSC-NPC) were used; these were cultured as previously described from the hiPSC line, 1-DL-01, from WiCell [104 (link)]. Experiments using hiPSC-NPC were conducted with the approval of the University of Victoria’s Human Ethics Committee under protocol number #12-187. Briefly, the cells were cultured in STEMdiff™ Neural progenitor medium (NPM) (05834, Stemcell Technologies, Vancouver, BC, Canada) containing 1X STEMdiff™ Neural Progenitor Supplement A (05836, Stemcell Technologies, Vancouver, BC, Canada), 1X STEMdiff™ Neural Progenitor Supplement B (05837, Stemcell Technologies, Vancouver, BC, Canada), and 1% penicillin-streptomycin (P4333, Sigma Aldrich, St. Louis, MO, USA). These medium supplements were used to promote the efficient conversion of hiPSC to CNS-type NPCs and inhibit the unwanted differentiation of non-CNS-type cells. The cultured hiPSC-NPC were maintained at 37 °C with 5% CO₂, and media changes were performed daily.

The HDF51i-509iPSCs were differentiated into NPCs as described previously (Murphy et al. 2017 (link)). NPCs were then maintained in STEMdiff™ Neural Progenitor Medium (STEMCELL Technologies) supplemented with 1 × STEMdiff neural progenitor supplement A (STEMCELL Technologies) and 1 × STEMdiff neural progenitor supplement B (STEMCELL Technologies) at 37 °C and 5% CO₂. Neural differentiation was initiated by replacing 50% v/v of the culture medium every 2–3 days with BrainPhysTM neural medium (STEMCELL Technologies) supplemented with 2% v/v Neurocult™ SM1 Neuronal Supplement (STEMCELL Technologies), 1% v/v N2 Supplement-A (STEMCELL Technologies), 20 ng/mL human recombinant brain-derived neurotrophic factor, 20 ng/mL human recombinant glial cell line–derived neurotrophic factor (Peprotech), 1 mM dibutyryl cyclic-AMP sodium salt (Sigma-Aldrich), and 200 nM ascorbic acid (STEMCELL Technologies), making complete BrainPhys™ neural medium (Sundaramoorthy et al. 2020a ).