Extended Data Fig. 3 ∣. PCCOs exhibit astrocytic maturation.

a, PCCOs at div74 were harvested and fixed for immunostaining against anti-EAAT1 to reveal the astrocytes in PCCOs. Bar: 100 μm. Zoom-in bar: 40 μm. GFP in green labels PLCs. EAAT1 in red marks astrocytes. DAPI in blue represents nucleus. Arrow points the star-shape astrocyte. Double arrow: astrocytic end-feet (picture represents the results from three individual PCCOs generated from two unrelated iPSC lines and H1 cells). n = 12 includes four biological replicates (PCCOs generated from H1, iPSC-Line1, iPSC-Line2, iPSC-Line3) and three technical replicates (three PCCOs for each of the biological replicates). b, mRNA levels of astrocyte marker genes increased in PCCOs compared to COs. COs and PCCOs at div74 were harvested for RNA extraction followed by qPCR using reported mature and immature astrocytes markers. GLUL-1, HIST1H2BC, NUSAP1, CHI3L, and TPX2 represent mature astrocytes. ALDH1L1, DTYMK and TLR4 represent immature astrocytes. CD49f indicates functional astrocytes. GAPDH used as reference. n = 12 includes 3 independent biological replicates (three individual PCCOs generated from two unrelated iPSC lines and H1 cells, see color code shown in b) and 4 technical replicates for each biological replicate, error bar represents mean±1 SD.