At the level of immunolabeling, expression of stem cell markers Venetoclax research buy was abolished in iN cells,

consistent with a conversion of H1 ESCs into iN cells (Figure 2A). Quantitative RT-PCR analyses revealed that iN cells expressed increased levels of endogenous Ngn2 as well as of two neuronal markers, NeuN and MAP2, whose levels were elevated ∼100-fold (Figure 2B). In addition, we observed an even larger induction of the expression of the transcription factors Brn2 and FoxG1, which are markers for excitatory cortical neurons (Figure 2B). Immunoblotting experiments showed that the neuronal precursor cell (NPC) markers nestin and Sox2 were only detectable in the ESCs and iPSCs, whereas a series of well-established synaptic genes were only expressed in 3-week-old Ngn2 iN cells (Figures Panobinostat price 2C and S2A). Quantitative RT-PCR measurements of the expression of the NPC markers Sox2 and nestin in the first 2 weeks after Ngn2 induction revealed a transient brief increase in these markers immediately after induction, with a rapid decline in expression (Figure S2B). Furthermore, upon coculture with mouse astrocytes, H1-cell-derived iN cells formed synapses with each other and with cocultured COS cells expressing neuroligin-1 (Figures 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. Measurements of the yield of iN cell conversion in three stem cell lines, H1 ESCs and two different iPSC

lines, showed that nearly 100% of surviving lentivirally infected ESCs and iPSCs were converted into neurons, revealing an unprecedented efficiency of conversion (Figure 2D). When we calculated the number of iN cells generated as a function of starting ESCs or iPSCs, we observed an apparent increase with H1 ESC-derived iN cells but not with the two iPSC-line-derived iN cells (Figure 2D). The increase in cell numbers TCL in H1 ESC-derived iN cells is due to the continuing division of H1 cells after plating; iPS-cell derived iN cells do not show such increased cell numbers because they exhibit some cell death in response to culture splitting and lentiviral infection, resulting in a partial loss of the iPSCs as iN cells are being generated. Overall, these data demonstrate that forced expression of a single transcription factor—Ngn2—induces neuronal differentiation with high yield. We next aimed to gain insight into the nature of the neurons generated and, more importantly, to assess the reproducibility of Ngn2-induced production of iN cells from different ESC and iPSC lines. Toward this end, we quantitatively analyzed expression of 73 genes at the single-cell level using fluidigm-dependent mRNA measurements (Pang et al., 2011; Table S1). All fluidigm-mediated quantitative RT-PCR assays were validated using standard curves (Table S1).