class="head no_bottom_margin" id="sec1title">IntroductionThe neural crest is a uniquely vertebrate cell population characterized by its remarkable ability to form numerous differentiated derivatives, as diverse as facial skeleton and peripheral nervous system. From a stem cell biology perspective, the neural crest is an ideal embryonic source of multipotent stem cells for the purposes of regenerative medicine. Numerous studies have successfully isolated cell lines that differentiate into neural crest derivatives either in vitro or after transplantation into the embryo (, , ). However, it remains controversial whether neural crest cells are indeed true stem cells or simply a transient multipotent progenitor population.A cardinal feature of stem cells is their ability to give rise to multiple lineages and to self-renew. In contrast to true stem cells, primary cultures of embryonic neural crest cells examined to date have only limited capacity to self-renew in vitro (, ). Likewise, neural crest cells derived from peripheral nerve or facial skin have self-renewal capacity but tend to form a limited set of cell types reflecting their tissue of origin (, , , , ). In general, adult neural-crest-derived “stem cells” have limited ability for long-term self-renewal or differentiation into a large repertoire of derivatives (, ). This raises the important question of whether a true neural crest stem cell that is multipotent and capable of self-renewal can be identified in either the embryo or adult.Embryonic neural crest cells arise from the dorsal portion of the developing CNS around the time of neural tube closure. At this premigratory phase, neural crest precursors are characterized by combined expression of transcription factors such as FOXD3, SOXE, SNAIL, and AP2α (). They subsequently undergo an epithelial to mesenchymal transition (EMT), enabling them to leave the neural tube, and migrate extensively throughout the embryo. Single cell lineage analyses in vivo (, ) and clonal analyses in vitro (href="#bib9" rid="bib9" class=" bibr popnode">Calloni et al., 2009) have shown that the majority of embryonic neural crest cells are multipotent. Indeed, premigratory precursors contribute not only to neural crest, but also to dorsal neural tube lineages (href="#bib2" rid="bib2" class=" bibr popnode">Baggiolini et al., 2015, href="#bib8" rid="bib8" class=" bibr popnode">Bronner-Fraser and Fraser, 1989). After emigration, however, their developmental potential is thought to become restricted by cues present in the environment and final site of localization. Finally, neural crest cells differentiate into many cell types that far exceed the repertoire traditionally considered as “ectodermal,” including sensory and autonomic neurons and glia, bone and cartilage of the face, smooth muscle cells, adipocytes, melanocytes, and various endocrine cells (href="#bib12" rid="bib12" class=" bibr popnode">Dupin and Coelho-Aguiar, 2013).Development of new methods for generating neural crest cells with self-renewal capacity is complicated by the transience of the embryonic premigratory crest stage. Our goal was to maintain, for extended time periods in vitro, primary neural crest stem cells derived from the embryo or human embryonic stem cells (hESCs) in a self-renewing state that reflects their premigratory character; under appropriate conditions, these can then be differentiated into multiple derivatives. Such long-term maintenance of neural crest stem cells is useful not only for regenerative medicine, but also for understanding neural-crest-related birth defects.
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