Neuronal differentiation was initiated by growth factor withdrawal from NPs. Successful neuronal commitment was demonstrated in E neurons by an increase in the expression of pan-neuronal markers, tubulin β-III (TUB3) and microtubule associated protein 2 (MAP2) [39,40] (Fig 3A and 3B). Expression of CTIP2, a marker for deep cortical neurons [41], was significantly increased by four weeks of neuronal differentiation (L) (Fig 3C). Expression levels of the synaptic markers, synapsin 1 (SYN1) and synaptophysin (SYP), was positively correlated with the time of neuronal differentiation. (Fig 3D and 3E). Gene expression of several neuronal ion channel proteins also tended to increase as NPs matured into neurons (S3 Fig), although some of the markers did not reach statistical significance. Collectively, the gene expression analyses indicate that E neurons already exhibit key neuronal identifiersbut markers for neuronal function continue to increase their expression level with further differentiation to L neurons.
