This suggests that the same ventral fate seen in NT2 cells is present within the NT2N population. Up-regulation of the genes responsible for the dorsal telencephalon progenitors is seen following ATRA differentiation. However, the XAV939 expression of NGN1 is undetectable. NGN2 is a downstream target for PAX6 and its up-regulation is most likely a direct result of PAX6 expression. In NT2 cells RA has been reported to up-regulate NGN1 throughout the 21 days of the ATRA differentiation. It appears that the loss of NGN1 expression may be due to the removal of ATRA or that the use of the mitotic inhibitors may have removed the population of NGN1 expressing progenitors. Up-regulation of NGN2, EMX2, and PAX6 define the dorsal telencephalon by inhibiting expression of ventral markers. This leads to the development of glutamatergic projection neurons. Pyramidal neurons preferentially express SLC17A7. The development of pyramidal neurons is consistent with the up-regulation of SLC17A7 and no change in the expression of SLC17A6 seen in the NT2N population. NT2 cells express all of the spinal cord dorsoventral axis transcription factors studied here with high expression of ASCL1, PAX3, PAX7, NKX6.1, and OLIG2 indicating the present of both dorsal and ventral neural progenitors. Given the expression of transcription factors expressed in NT2 cells the intermediate progenitor domains of ventral progenitor 0, and vp1 and the most ventral domain vp3 are poorly expressed or lacking. Following exposure of NT2 cells to ATRA the NT2N population expresses nearly the same progenitor populations as expressed in NT2 cells. NKX2.2 expression is totally absent following ATRA and would suggest the absence of vp3 progenitors. The expression of OLIG2 would suggest the possible development of motor neurons which requires the association of OLIG2 with NGN2. However, the development of oligodendrocytes requires the interaction of OLIG2 with NKX2-2. Since NKX2-2 is undetectable in the NT2N population the lack of oligodendrocyte development seen in this population is consistent with the loss of NKX2.2 expression. An increase in the progenitor population from vp0, and vp1 is seen with the NT2N population due to the up-regulation of DBX2 which is consistent with the reported dependence of these progenitor regions on RA for their development. NT2N described here appear to be immature due to the lack of restriction of TAU to axons and their lack of formation of functional synapses. The punctate staining seen with Synapsin 1 has been previously reported to occur in NT2N cultured with or without astrocytes. Here we report that the NT2N possess intrinsic electrical SCH727965 excitability characteristic of neurons but fail to establish functional synaptic contacts despite their extensive network of neural processes. This result appears to be in agreement with previous reports that the NT2N as well as other neurons generated from hES cell require cell contact with mature astrocytes in order to induce synaptogenesis. The presence of different neurotransmitter phenotypes has been previously reported for both monolayer and aggregate differentiation methods. In agreement with previous reports, the population of NT2N characterized here displayed upregulation of genes directly linked to the GABAergic, glutamatergic, catecholaminergic, and cholinergic phenotypes. We also report the presence of astrocytes in the ATRAdifferentiated cell population before isolation of NT2N and within the cell clusters present in the purified NT2N population. The generation of oligodendrocytes, was not seen. This may be due to the inability of NT2 cells to differentiate into oligodendrocytes, as supported by the lack of NKX2-2 expression, and is agreement with other studies using human neurospheres or hES cell lines.