In order to confirm preservation of cortical fate of the neuronal co-cultures, immunocytochemical stainings for cortical markers TBR1, CTIP2, and SATB2 were performed (Fig. 2c). Three weeks after final plating the cultures contain–next to the added astrocytes and undifferentiated NPCs–about 60% neurons (HuCHuD positive nuclei 62% ± 11%), of which 38% (±6%) were TBR1 positive, 41% (±3%) CTIP2 positive, and 12% (±4%) SATB2 positive, suggesting that early generated deep layer neurons were predominantly present.
