Differentiated neurons showed increased expression of several markers of cortical layer glutamatergic neurons. Throughout the differentiation, pallium marker PAX6 showed a constant expression, cortical progenitor marker TBR2 [39] was slightly decreased and telencephalic neuron maturation marker BF1 [40] increased towards day 49, indicative of maturing neurons (Fig 2E). The cortical upper layer (2–4) markers CUX1, SATB2 [41] showed increasing levels at day 49, whereas BRN2 progenitor expression [42] showed a decreased level (Fig 2F). High expression of the late-onset deep layer markers TBR1 and CTIP2 [43] was observed at day 18 and day 49 (Fig 2G). CTIP2 had a significant increase at day 49 compared to day 5 and day 8, indicating neural stem cells differentiation towards deep layer cortical neurons. Rosette and neural stem cell markers HES5, DACH1, PLZF and SOX2 expressed at the hNES cell stage (Fig 1A–1G) showed decrease in expression during differentiation indicating neuronal maturation (Fig 2H). Specifically, neural stem cell markers HES5 and DACH1 showed a significant reduction from day 8 to day 49 indicating loss of neural stem cell identity. Finally, we performed immunocytochemical
