Modeling Metabolic Stress in NPCs

AGS NPCs (Neuronascent, Gaithersburg, MD, USA) and mouse NPCs (gift of Song lab, Baltimore, MD) have been previously described (Drew et al., 2016 (link); Ma et al., 2009 (link)). They were grown under standard conditions at 37°C and 5% CO₂ with NeuroCult basal media (STEMCELL, Vancouver, BC, CA) with EGF (50 ng/ml, PeproTech, Inc, Rocky Hill, NJ, USA), FGF (100 ng/ml, PeproTech, Inc), heparin (0.002%), and proliferation supplements (STEMCELL). Early passage cultures (P2) were expanded and frozen and thawed in batches for use in experiments. These cultures contain cells ubiquitously expressing the NPC marker, Nestin, and the proliferation marker, Ki-67 (Figure 1—figure supplement 1). For in vitro modeling of metabolic stress, cells were exposed to either: (i) 1% hypoxia in a specialized incubator (Nuaire, Plymouth, MN, USA) saturated with Nitrogen/5% CO₂; (ii) hypothermia in standard incubators maintained at lower temperatures; and (iii) complex I inhibition with the addition of rotenone to cell media. For cell proliferation determination, wells were seeded in triplicate with 50,000 cells. On subsequent consecutive days, cells were detached with Accutase (STEMCELL) and counted by automated cytometry (Nanoentek, Waltham, MA, USA).