We also closely examined the morphology of GFAP+ cells after dissociation. When maintained in monolayer in defined serum-free culture conditions28, astrocytes extended abundant thin projections (Fig. 3e). To investigate whether these cells could respond to reactive cues in vitro, we added serum, which is a potent activator of reactive astrogliosis29, to the culture medium. Within several days, the cells adopted a reactive phenotype with polygonal morphologies and upregulated expression of genes associated with in vivo astrogliosis, including GFAP, VIM and LCN2 (ref. 30) (Supplementary Fig. 6). Finally, using electron microscopy, we confirmed that the thin GFAP+ processes dispersed throughout the hCSs contained numerous glycogen granules (Fig. 3f), which are predominantly localized in astrocytes in the mammalian brain31.
