Dissecting Neural Crest Ontogenesis and Axial Specification in Vitro Using Human Pluripotent Stem Cells

Neural crest (NC) is a multipotent embryonic population integral to vertebrate development owing
to its broad contribution to an array of derivatives including peripheral ganglia and the craniofacial
skeleton. The NC cells originate from the dorsal tip of the neural tube along the anteroposterior
axis and based on their positional identity, which is coupled with their ability to generate distinct
cell types, are subdivided into cranial, cardiac and vagal, trunk and sacral NC in an order that
corresponds to the adjacent regions of the spinal cord. The importance of NC integrity is
demonstrated by the wide spectrum of NC-related pathologies including tumours, termed
neurocristopathies, that predominantly arise from defects in the development of NC and their
derivatives in multiple tissues. Our knowledge of NC ontogenesis and pathophysiology primarily
comes from studies with animal models, however, the model organisms don’t always recapitulate
human development. Thus, human pluripotent stem cells (hPSCs) are an attractive platform to
study NC ontogenesis and pathogenesis in human context in vitro. Several studies using directed
differentiation have decoded the signalling requirements for cranial NC specification from hPSCs,
but the signals and transcription factors that drive the acquisition of a posterior axial identity that
corresponds to vagal and trunk levels in in vitro-derived NC have been less studied. Recently, it
was demonstrated that vagal NC cells are derived from cranial crest through the action of the
caudalising factor retinoic acid (RA), whereas their trunk counterparts are generated downstream
of a neuromesodermal progenitor (NMP) population. The work presented here defines Wnt along
with cell density as critical factors for vagal NC specification in vitro and demonstrates the ability
of RA-induced vagal NC to give rise to their enteric nervous system (ENS) derivatives upon
further differentiation using two different hPSC lines. It also shows that Notch pathway inhibition
as part of the culture regimen enhances neural and glial differentiation in ENS cultures.
Additionally, it reveals a previously unknown role of the pro-mesodermal factor TBXT together
with Wnt signalling effectors in regulating, through chromatin remodelling, the adoption of
posterior axial identity in NMP-derived trunk NC. We believe that these insights contribute to our
knowledge of posterior patterning in post-otic NC and they can be applied in the future to optimise
NC differentiation strategies with practical implications in biomedicine.