Human ES cells can reproducibly differentiate in vitro into EBs with three embryonic germ layers. Given the fact that this process recapitulates early embryogenesis, we hypothesize that neural crest-like cell population are present in EBs. In order to isolate neural crest-like cells from EBs, we identified two cell surface markers, frizzled-3 and cadherin-11 that are expressed in neural crest cells during embryogenesis. We then detected robust expression of frizzled-3 and cadherin-11 in a subpopulation within EBs (). This enabled us to tightly gate the FACS sorting to obtain a homogenous frizzled-3+
population. As a result, contamination of cells with alternative lineages was significantly limited (). With appropriate inducing signals, FACS-sorted frizzled-3+
cells were able to differentiate to various neural crest lineages, such as neurons, glial cells, smooth muscle cells, chondrocytes, and osteoblasts ( and ). Moreover, these FACS-sorted frizzled-3+
cells were capable of self-renewing and maintaining their differentiation potential in short-term cultures (). A recent study reported the derivation of neural crest stem cells from human ES cells at the neural rosette stage. It is surprising that large number of cells with a neural crest profile is present in rosette cultures considering that similar conditions are routinely used for the generation of hES cell–derived central nervous system progeny [11
Neural crest cells are multipotent stem cells that contribute to a diverse array of tissues throughout the embryo. During craniofacial development, cranial neural crest contributes extensively to the formation of mesenchymal structures in the head and neck. The major difference between the cranial and trunk neural crest is the ability of cranial neural crest to generate skeletal derivatives. Fate maps of the cranial neural crest suggest that the majority of the head skeleton is neural crest derived [32
], with the exception of parietal bones having a mesoderm origin [34
Given its osteogenic differentiation potential, frizzled-3+/cadherin-11+ cells behave more like cranial neural crest than trunk neural crest. Therefore, bone tissues generated from frizzled-3+/cadherin-11+ CNCLMP cells share similar developmental origin with craniofacial bones, thereby representing a superior therapeutic tissue source for craniofacial bone repair.
A common theme in vertebrate organogenesis is the reciprocal interaction between epithelium and mesenchyme. During dental development a specialized region of the oral epithelium known as the dental placode signals to an underlying cranial neural crest derived population of cells to alter their cell fate and to differentiate as dental mesenchyme including a terminal phenotype of odontoblasts. It will be interesting to utilize the unique properties of reciprocal tissue interactions occurring during odontogenesis to test the competency of the ES cell-derived CNCLMP cells to respond to signals from a dental epithelium. Previous studies has demonstrated that premigratory neural crest cells and other non-odontogenic CNC cells, when recombined with a competent epithelium, can receive signals causing them to differentiate to odontogenic lineages [28
]. The emergence of induced genes consistent with an odontogenic phenotype would argue that the CNCLMP cells are as plastic in their ability to respond to inductive signals as are authentic populations of cranial neural crest cells that reside in the first brachial arch.
At least some neural crest cells are multipotent, as shown by clonal analysis in vitro [31
] and dye labeling in vivo [37
]. It is possible that FACS-sorted CNCLMP cells are a mixed population in which a given cell does not have the potential to differentiate to all the cranial neural crest lineages. To address this question, further investigation is necessary to establish single-colony derived strains from CNCLMP cells and subsequently to carry out fate-mapping analyses. It has been shown that bone morphogenetic protein (BMP) and Wnt synergistically suppress differentiation and maintain multipotency of neural crest stem cells (NCSCs) [40
]. Neural crest stem cells generated from hES cell-derived neural rosette are capable of forming proliferating neurosphere-like structures in response to FGF2/EGF exposure [11
]. We observed limited self-renewal of FACS-sorted CNCLMP cells (). It will be interesting to examine whether CNCLMP cells respond in a similar way to these signaling molecules, and consequently maintain long-term self-renewal activity.