Differentiation and gene expression of region-specific astrocytes. (A) Schematic showing the process of subtype astrocyte differentiation. Human PSCs were induced to neuroepithelia (NE) in the presence of DMH1 and SB431542 (SB) in the first week, followed by patterning to region-specific progenitors in the second week in the presence or absence of morphogens. The neural progenitors (NP) were then expanded in suspension for 5.5 months before differentiating to astrocytes (Astro) in an adherent culture in the presence of BMP4 and CNTF for 1 week. AP, astrocyte progenitor. (B) ESC-derived regional neural progenitors (60 days) were stained for forebrain (OTX2), ventral forebrain (NKX2-1), cortical (FOXG1), ventral spinal cord (NKX2-2) and cervical spinal cord (HOXA3) markers. (C) Representative images of ESC-derived regional astrocytes that were stained for S100β, SOX9 and GFAP or HEPACAM. Cell nuclei were stained for Hoechst (blue). Scale bars: 75 μm.