Astrocyte Differentiation from Human Neural Progenitor Cells

Human astrocyte differentiation was performed as described by^{79 (link)} with minor adaptations. hNPCs were passaged with Accutase at 80% confluency and counted using a Neubauer improved cell counting chamber (Carl Roth, PK361). Cells were resuspended in astrocyte differentiation medium (50 % DMEM/F12-GlutaMAX, 50% Neurobasal supplemented with 1% B27 (17504044, Life Technologies), 0.5% N2, 0.5x GlutaMaxx (Thermo Fisher Scientific, 35050061), 0.5x non-essential amino acids, 0.02% beta-mercaptoethanol, 2.5 µg/ml human Insulin (Sigma, I3536-100MG), 5 ng/ml CNTF (Miltenyi Biotec, 130-096-337), 10 ng/ml BMP4 (Miltenyi Biotec, 130-111-165), 10 ng/ml EGF (Miltenyi Biotec, 130-093-825), 8 ng/ml FGF2 (Miltenyi Biotec, 130-093-839), 10 ng/ml Heregulin1/Neuregulin1b (Biomol, 97642.1) containing 10 µM ROCK inhibitor Y27632 (Enzo Life Sciences, ALX-270333-M005) and were seeded on 15 µg/mL poly-L-ornithine and 10 µg/mL laminin coated 6-well plates at a density of 500.000 cells/well. Astrocytes were differentiated in astrocyte differentiation medium at 37 °C, 7% CO₂, 21% O₂ for at least 35 days with daily medium changes. At 80 % confluency, astrocytes were passaged with Accutase and seeded on 15 µg/mL poly-L-ornithine and 10 µg/mL laminin coated plates.