Differentiation of GBA1-/- hESCs into Neurons

A GBA1−/− human embryonic stem cell (hESCs) line was previously generated and characterized [9 (link)]. Neural precursor cells (NPCs) were generated from the hESCs using a modified dual SMAD inhibition protocol [28 (link)], as previously described [29 (link),30 ]. For neuronal differentiation, NPC were dissociated with 0.05% trypsin-EDTA (ThermoFisher, Cat. 25300054) and plated on poly-L-ornithine/laminin (PLO)-coated dishes at a density of 10,000–15,000 cells/cm2 in N2B27 medium supplemented with 100 ng/ml FGF-8 (PeproTech, Cat.100–25), 200 ng/ml sonic hedgehog (PeproTech, Cat. 100–45), and 100 μM ascorbic acid 2-phosphate (Sigma, Cat. A8960–5G) for seven days. Finally, the NPCs were plated on PLO-coated plates at a density of 50,000 cells/cm2 in BGAA medium, which was composed of N2B27 medium supplemented with 20 ng/ml BDNF (R&D System, Cat. 248-BDB-01M/CF), 10 ng/ml GDNF (PeproTech, Cat. 450–10), 500 μM Dibutyryl-cAMP (STEMCELL Technologies, Cat. 100–0244), and 100 μM ascorbic acid 2-phosphate (Sigma, Cat. A8960–5G). Neuronal cultures were maintained in BGAA medium for 6weeks, and the cell culture medium was changed every 4 days.