Here, we characterize a population of cells emigrating from hESC-derived 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.
Scheme of early emNCSCs derivation from hESCs and differentiation into NC lineages.
Analysis of global gene expression profile of cells within the clusters and emNCSCs revealed selective upregulation of the NC-related genes in the migratory cells. For example, homeobox gene PhOX2b
is essential for the development of both autonomic and sympathetic neurons 
and regulates the dynamic expression of RGS4 
. The upregulation of DLK1
in emNCSCs is consistent with the role of Notch signaling in NC differentiation and maintenance 
. We detected selective upregulation of gonadotropin-releasing hormone 2 (GnRH2) in emNCSCs. Indeed, some cells releasing GnRH arise from cranial NC 
and their differentiation is modulated by the NC transcription factors SOX10
and FOXD3 
. It is tempting to speculate that these differences between the migratory cells and adherent clusters will provide insight into the genes and pathways involved in delamination of human NC.
In contrast to previous reports 
, a much larger fraction (~100-fold increase) of emNCSCs is positive for the mesenchymal cell marker CD73, which is a unique characteristic of cephalic NC. Cephalic NC cells are known to emigrate earlier than trunk NC in all species studied, including recently derived human NC cells 
. Previously described NCSCs lack the expression of connexin 43 (
supplementary microarray data), which could be due to the extensive passaging. In contrast, the emNCSCs described in this study uniformly express connexin 43, previously found to be characteristic of early migrating NC 
. Finally, previous studies in chick demonstrated that BMP inhibition (e.g.,
with Noggin) can block NC specification during early, but not late stages of NC development 
. Treatment with Noggin quantitatively abolishes the appearance of emNCSCs. Taken together these data strongly argue that we have identified and characterized early migratory NCSCs, likely resembling cephalic NCSCs, derived from human ES cells.
EmNCSCs express the classical marker of NCSC, p75; however, we found that in our setting, dorsal neuroepithelium-like cells also express p75. Similarly, both cell types are positive for HNK-1, a well-known NC marker in avian but not murine embryos 
. Indeed, at the time of NC emigration in mice the most dorsal part of the neural tube, including some premigratory NC cells residing in that area, are positive for p75 
. Similarly, human neural tube cells express p75 
. Previously, when hESCs were neuralized using stromal feeder cells 
, only a proportion of cells in culture (e.g.,
the most outer part of neuroepithelieal rosettes) were found positive for both p75 and HNK-1 
. The difference in the neuralization protocols is a likely explanation for the observed discrepancies. The heterogeneous p75 reactivity within neuroepithelial clusters was observed using three different antibodies; rabbit polyclonal serum and two mouse monoclonal antibodies (including one directly conjugated, previously used for isolation of human NCSCs 
.) For HNK-1 staining, we used the same monoclonal antibody staining conditions as previously reported 
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 cultures confirmed the upregulation of Wnt1
, 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
, 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
, which proceed to regulate the EMT and migration of NC cells from the neural tube 
. Consistent with these data, expression of Sox9
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.
the NC competency zone (area around the neuroectoderm border) 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. After treatment with Noggin, the expression of NC markers Sox10, p75 and HNK-1 was nearly ablated compared to controls. Treatment with the Wnt/β-catenin antagonist Dkk1 resulted in the ablation of Sox10 and HNK1, but not p75. These results are consistent with the distinct mechanisms of action of Noggin and Dkk1. While Noggin (produced by the notochord in vivo
) re-specifies the dorsal neuroepithelium into ventral fates, Dkk1 inhibits the early steps of NC induction, without altering the axial cell fates or general patterning 
. In our hands, the dorsal neuroepithelium-like clusters (adherent neurspheres) are positive for p75. The loss of Sox10 and p75 after the addition of Noggin is consistent with re-specification of dorsal neuroepithelium into ventral neuroepithelium, which typically does not express NC markers. Following the same logic, the addition of Dkk1 may inhibit the NC specification, i.e.
appearance of Sox10- and HNK-1-positive emNCSCs, but does not re-specify the dorsal neuroepithelium-like cells, which remain positive for p75. Indeed, the human neural tube cells were found positive for the p75 antigen at the time when the NC is generated 
. It remains to be determined if this expression is localized to the premigratory neural crest in the dorsal part of human neural tube.
In addition to the patterning growth factors discussed above, matrix elasticity may affect hESC differentiation towards NC lineages, similar to that seen for muscle differentiation 
. It will be important to investigate hESC differentiation into emNCSC using various elasticity matrixes.
Finally, we assessed the ability of emNCSCs to migrate in vivo,
incorporate into NC derivatives and differentiate appropriately. Grafted emNCSCs efficiently contributed to a variety of NC-derived tissues and differentiated appropriately. This finding demonstrates that emNCSCs are competent to contributing to NC derivatives such as the trigeminal ganglia, as cells that do not normally incorporate into the trigeminal ganglia are excluded from the developing ganglia even when they are immediately adjacent to it 
. Furthermore, emNCSCs contributed to connective tissues including smooth muscle. Therefore, emNCSCs are capable of extensive migration to appropriate NC cell destinations and appear to have the ability to interact with adjacent host NC cells and differentiate efficiently compared to late emNCSCs, in vivo 
. Critically, transplanted human emNCSCs do not contribute to the non-NC cell types, such as CNS tissue (e.g.,
Given the fact that these cells are derived in an antigen free environment (no co-culture with murine tissue) the protocol for the derivation of emNCSCs is ideal for generating emNCSC-derived tissues in the culture dish that can subsequently be used for patient treatment (e.g., neurocristopahties). To determine the therapeutic potential of these cells for treating a disease model, we investigated the ability of emNCSCs to colonize aganglionic embryonic guts in organotypic cultures. EmNCSCs were found to be capable of colonizing aganglionic guts and differentiating into neurofilament-positive cells, presumably enteric neurons, in ex vivo gut cultures. This suggests that emNCSCs might be useful in cell replacement therapies to treat neurocristopathies.