Differentiation of Dopaminergic and Astrocytic Cells

Primary human astrocytes (#1800, ScienCell Research Laboratories) were cultured as previously described (Pajarillo, Digman, et al., 2021 (link)) with a slight modification. Lund human mesencephalic (LUHMES, CRL-2927, ATCC) dopaminergic-like cells were maintained in DMEM/F-12 with 1% N2 supplement (Gibco) and 40 ng/ml basic fibroblast growth factor (PeproTech) and subcultured on culture flasks precoated with 50 μg/ml poly-l-ornithine and 1 μg/ml fibronectin. After plated in 24-well or 6-well tissue culture plates, LUHMES cells were differentiated into morphologically and biochemically mature dopamine-like neurons with a differentiation media containing tetracycline (MilliporeSigma), glial cell–derived neurotrophic factor, and dibutyryl-cAMP (PeproTech), followed by treatment with designated compounds and downstream analysis. Human H4 astrocytes (HTB-148, ATCC) were maintained in DMEM supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin and maintained at 37°C in a 95% air, 5% CO₂ incubator. Mouse cath.a-differentiated (CAD) catecholaminergic-like cells (MilliporeSigma) were differentiated into morphologically and biochemically mature neurons by replacing them with serum-free media.