Immunoblotting experiments showed that the neuronal precursor cell (NPC) markers nestin and Sox2 were only expressed in the ES and iPS cells, whereas a series of well-established synaptic genes were only expressed in 3-week old Ngn2 iN cells (Figs. 2C and S2A). Quantitative RT-PCR measurements of the expression of the NPC markers Sox2 and nestin in the first two weeks after Ngn2 induction revealed a transient brief increase in these markers immediately after induction, with a rapid decline in expression (Fig. S2B). Furthermore, upon co-culture with mouse astrocytes H1-cell derived iN cells formed synapses with each other and with co-cultured COS cells expressing neuroligin-1 (Figs. S2C–S2E). Thus, iN cell generation involves a switch from a stem cell to a neuronal gene expression phenotype with stimulation of endogenous Ngn2 expression.
