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Human dental tissues are sources of neural crest origin multipotent stem cells whose regenerative potential is a focus of extensive studies. Rational programming of clinical applications requires a more detailed knowledge of the characters inherited from neural crest. Investigation of neural crest cells generated from human pluripotent stem cells provided opportunity for their comparison with the postnatal dental cells. The purpose of this study was to investigate the role of the culture conditions in the expression by dental cells of neural crest characters. The results of the study demonstrate that specific neural crest cells requirements, serum-free, active WNT signaling and inactive SMAD 2/3, are needed for the activity of the neural crest characters in dental cells. Specifically, the decreasing concentration of fetal bovine serum (FBS) from regularly used for dental cells 10% to 2% and below, or using serum-free medium, led to emergence of a subset of epithelial-like cells expressing the two key neural crest markers, p75 and HNK-1. Further, the serum-free medium supplemented with neural crest signaling requirements (WNT inducer BIO and TGF-β inhibitor REPSOX), induced epithelial-like phenotype, upregulated the p75, Sox10 and E-Cadherin and downregulated the mesenchymal genes (SNAIL1, ZEB1, TWIST). An expansion medium containing 2% FBS allowed to obtain an epithelial/mesenchymal SHED population showing high proliferation, clonogenic, multi-lineage differentiation capacities. Future experiments will be required to determine the effects of these features on regenerative potential of this novel SHED population., Competing Interests: The authors have declared that no competing interests exist.