Astrocyte Co-culture Increases Neuronal Maturation and Endogenous Astrocytes Arise at Later Time-Points in Differentiation.

<p>A) Schematic of differentiation up to 100 days. For astrocyte co-culture, astrocytes were added to neuronal cultures at ∼day 24 of differentiation. Endogenous astrocytes gradually emerged over the course of 100 days, after day 40. B) Day 42 and day 100 neuronal cultures were immunostained and imaged for GFAP. Scale bars = 50 µm. C) After 40–50 (D40) or 100 days (D100), cells were lysed, RNA extracted, and expression of 150 genes analyzed using the NanoString platform. A subset of neuronal markers (C) and synaptic markers (D) are shown. Data are from at least 6 independent differentiations (3 lines). For day 40–50 n = 29–38, for day 100 n = 15–19. E) Neuron cultures with or without astrocytes were immunostained and imaged using confocal microscopy at day 40. Insets in right column show VGLUT1 staining along the length of a neuronal process. Representative images are shown. Scale bars = 50 µm. F) qPCR was performed using RNA harvested from day 40 cultures. Data normalized to <i>GAPDH</i> expression. For neurons alone n = 20 for MAP2, TBR1, CUX1, GAD1, n = 37 for SYP, n = 38 for VGLUT1; for astrocyte co-culture n = 17 for GAD1, n = 18 for MAP2, TBR1, CUX1, n = 27 for SYP, n = 25 for VGLUT1. For C–F, data are represented as mean ± SEM. Significance determined by student’s t-test: **, p<0.01; ***, p<0.001; ****, p<0.0001.