Here, we characterize a population of cells emigrating from hESC-derived Lomitapide Mesylate adherent neurospheres plated on fibronectin and provide evidence that they mimic the dorsal neural tube containing premigratory NCSCs and resembling their site of origin in vivo. The adherent neurospheres are mainly composed of neuroepithelial rosettes, which express Pax3 and Sox9, markers of dorsal neuroepithelium and premigratory NC, but are negative for the ventral neural tube marker Nkx2.2. Cells that leave neuroepithelial clusters and start migrating on fibronectin uniformly upregulate Sox10, a critical transcription factor for NC development. The clusters remain negative for Sox10 and can be mechanically isolated. After replating on fibronectin, they give rise to a second wave of emigrating cells, which also upregulate Sox10. Most of the cells within replated clusters remain negative for Sox10 3 days after replating. It is likely that virtually all cells within the original adherent neurospheres are competent to emigrate and upregulate Sox10. Indeed, over 80% of cells within adherent neurospheres are positive for the markers of dorsal neuroepithelium and lack the ventral markers, consistent with the cells being premigratory NC. Recent evidence in avian embryos suggests that the onset of NC specification occurs during gastrulation, where epiblast cells that express the transcription factor Pax7, are specified to form NC cells in the absence of exogenous factors but in a manner that requires Pax7 itself. In the mouse NC, the Pax3 protein may be functionally equivalent to the Pax7. We observed early and robust upregulation of Pax3 during hESC neutralization, such that by day 5 of neuralization,80% of cells were positive for Pax3. Remarkably nearly 60% of the cells were positive for Sox9, a marker of premigratory NCSCs. These findings suggest that under current neuralization conditions most neural progenitors express markers associated with NC competence and specification. Despite their early specification at gastrula stages, NC cells do not delaminate from the neural tube until late neurulation and do not initiate differentiation until late migratory and post-migratory stages. Furthermore, execution of NC cell fates in vivo requires continuous Wnt signaling while various growth factors appear to direct differentiation along distinct pathways. Wnts and BMPs mediate NC induction in several model species. In a previous study using human cells, melanocytes were generated from hESCs using Wnt3a-conditioned media; however the presence of multipotent NCSCs was not demonstrated. Indeed, microarray analysis of neurosphere Dexrazoxane hydrochloride cultures confirmed the upregulation of Wnt1, Wnt3a, BMP4 and BMP5, as well as the expression of transcription factors downstream of Wnt and BMP signaling. EmNCSCs expressed several transcription factors characteristic of early NC specification in chick, mouse, and Xenopus. For instance, MSX1 and ZIC1, two of the earliest NC genes, were upregulated 48 and 72 hours, respectively, after the start of neuralization. These two genes have been demonstrated to be pivotal in the development of the NC and the expression of later NC-specific genes. The later NC-specifying genes include Sox9, Sox10 and Snail, which proceed to regulate the EMT and migration of NC cells from the neural tube. Consistent with these data, expression of Sox9, Sox10 and SNAIL was upregulated during neuralization of hESCs. The chronological order of upregulation of some NC genes in the hESC cultures may follow a different timetable compared to known in vivo models; whether this is due to an in vitro artifact or reflects species differences remains to be determined. In vivo, the NC competency zone is mediated by BMP and Wnt signaling with additional signals, including Wnts mediating the induction of the NC and delamination of NC from the neural tube. Different from our approach, most protocols for human NC derivation from ES cells use exogenous BMP to facilitate the differentiation. To test if BMP pathway is also important for the generation of emNCSCs, we treated neurosphere cultures with Noggin.