Single-cell RNA sequencing reveals differentiation trajectories directing human neural crest formation and differentiation

Abstract

Neural crest (NC) is a transient population of multipotent progenitor cells in vertebrates, contributing to the formation of various organ systems. NC induction occurs at the neural plate border and involves the orchestration of multiple events. After NC cells emerge from the closing neural tube, they delaminate from the neural tube through an epithelium-mesenchymal transition and then migrate extensively to their target sites, depending on the axial level of their origin. NC cells are of ectodermal origin but exhibit a remarkably broad differentiation potential. The different phases of NC development are mediated by unique sets of specifiers and that confer to the cells the ability to migrate and differentiate. A stepwise differentiation protocol has been established for generation of NC from human pluripotent stem cells (hPSC). However, how the in vitro differentiation relates to embryonic development is still unclear. Here, we applied single-cell RNA sequencing to establish the differentiation trajectories directing human NC formation and differentiation in vitro. Due to asynchrony in differentiation, five distinct cell populations were identified from hPSC-derived NC cells. They represent cells at the neural plate border, specified NC and the post-migratory NC, resembling the key intermediate states during embryonic development. Intriguingly, after NC specification, NC differentiation diverges into three transitional states in which the NC intermediates possess distinctive differentiation plasticity towards the neurogenic, skeletogenic and myogenic lineages and with different proliferation capacity. In summary, NC formation and its stereotypical sequence of intermediate states can be nicely recapitulated in vitro using hPSC.