Human neural crest induction by temporal modulation of WNT activation.

The developmental biology of neural crest cells in humans remains unexplored due to technical and ethical challenges. The use of pluripotent stem cells to model human neural crest development has gained momentum. We recently introduced a rapid chemically defined approach to induce robust neural crest by WNT/β-CATENIN activation. Here we investigate the temporal requirements of ectopic WNT activation needed to induce neural crest cells. By altering the temporal activation of canonical WNT/β-CATENIN with a GSK3 inhibitor we find that a 2 Day pulse of WNT/β-CATENIN activation via GSK3 inhibition is optimal to generate bona fide neural crest cells, as shown by their capacity to differentiate to neural crest specific fates including peripheral neurons, glia, melanoblasts and ectomesenchymal osteocytes, chondrocytes and adipocytes. Although a 2 Day pulse can impart neural crest character when GSK3 is inhibited days after seeding, optimal results are obtained when WNT is activated from the beginning, and we find that the window of competence to induce NCs from non-neural ectodermal/placodal precursors closes by day 3 of culture. The reduced requirement for exogenous WNT activation offers an approach that is cost-effective, and we show that this adherent 2-dimensional approach is efficient in a broad range of culture platforms ranging from 96-well vessels to 10 cm dishes.