Generation and characterisation of pre-neural progenitors and neural crest from human embryonic stem cells.

The spinal cord emerges from a niche of neuromesodermal progenitors (NMPs) formed and maintained by Wnt/FGF signals in the posterior end of the embryo. NMP-like cells can be generated from human pluripotent stem cells providing a promising source for spinal cord replacement therapies. However, these progenitors are often transient and unable to produce the full range of rostrocaudal spinal cord identities in vitro. Here we report the generation of NMP-derived pre-neural progenitors (PNPs). These PNPs maintain pre-spinal cord identity by co-expressing the transcription factors SOX2 and CDX2, while losing the mesodermal potential of NMPs by downregulating TBXT. They gradually adopt more posterior identity by activating collinear HOX gene expression, and in parallel undergo an epithelial-to-mesenchymal transition to give rise to neural crest (NC) cells with corresponding rostrocaudal identity. Overall design: Bulk mRNA sequencing was completed using 47 RNA samples extracted from differentiated WA09 hESCs. Each experiment (n=3) consisted of 12 time-points: undifferentiated WA09 human embryonic stem cells (t0), NMPs-likes cells (36h) and PNPs/NC cells (P1-P10). P9 samples contain 2 experimental repeats. Four additional samples were collected during each experiment: At passage 5 and passage 8 mesenchymal (MES) or epithelial (EPI) cells were enriched by selective detachment.