, 2007). Critically, however, the human SFEBq cultures were not reported to produce any late neurons with markers of upper cortical layers, despite some being cultured for as long as 106 days (Eiraku et al., 2008). More recently, similar results with hESCs and hiPSCs were obtained through a simpler embryoid body (EB)-based method, with a high efficiency of dorsal telencephalic specification (Li et al., 2009 and Zeng et al., 2010). EBs were cultured without growth factors for 2 weeks until

neural CB-839 rosettes formed. Gene expression analysis showed that certain Wnt morphogens (dorsalizing signals) were strongly induced during the second week, and nearly all the neural rosette cells were Foxg1+/Pax6+ by the third week. The cells exhibited the same responsiveness to dorsoventral patterning cues (Wnt versus Sonic hedgehog [SHH]) that Sasai’s group originally described (Watanabe et al., 2005). The progenitor cells generated Tbr1+ and Ctip2+ glutamatergic neurons but again, the production of late cortical neurons with markers typical of upper layers was not reported. A remarkably simple protocol for producing cortical neurons from mESCs was reported

by Vanderhaeghen’s group (Gaspard et al., 2008). In this method, mESCs were plated at low density in default differentiation medium. The cells naturally adopted a telencephalic identity, but in contrast to aggregate cultures, a majority of telencephalic cells expressed ventral progenitor cell markers within 2 weeks and differentiated

into GABAergic neurons. Noting that SHH expression was induced during the period of neural conversion, the authors treated the cells with Gefitinib mw a SHH antagonist, resulting in nearly complete suppression of ventral markers and yielding glutamatergic neurons with pyramidal morphology, indicating a dorsal fate shift. These cells also exhibited the known sequence of neuronal subtype production, with Reelin+ and Tbr1+ neurogenesis Dichloromethane dehalogenase peaking first, followed by production of Ctip2+ and then Cux1+ and Satb2+ neurons. However, the authors also noted a large underrepresentation of Cux1+ and Satb2+ neurons when they analyzed the expected proportions of each subtype, suggesting that in vivo cues are important for the full generation of late neurons destined for upper cortical layers. Surprisingly, the cortical cells derived by Gaspard et al. (2008) displayed specific areal identity upon transplantation into the frontal cortex of neonatal mice, extending axonal projections to a repertoire of subcortical targets that would be expected from neurons in the visual/occipital cortex. Prior to grafting, most of the mESC-derived neurons expressed Coup-TF1, which is expressed in the caudal but not rostral cortex. This suggested that the cells have an innate differentiation program that requires neither intracortical (e.g., FGF, Wnt, BMP gradients) nor extracortical (e.g., thalamocortical afferents) patterning cues to acquire area-specific neuronal properties.