Sequencing of CRISPR engineered GFP-Nkx1.2 expressing hES cells (H9 line). Sequencing GFP-Nkx1.2 expressing hES cells

Robust protocols for directed differentiation of human pluripotent cells are required to determine whether mechanisms operating in model organisms are relevant to our own development. Recent work in vertebrate embryos has identified neuromesodermal progenitors as a bipotent cell population that contributes to paraxial mesoderm and spinal cord. However, precise protocols for in vitro differentiation of human spinal cord progenitors are lacking. Informed by signalling activities during spinal cord generation in amniote embryos, we show here that transient dual-SMAD inhibition, together with retinoic acid (dSMADi-RA), provides rapid and reproducible induction of human spinal cord progenitors from neuromesodermal progenitor-like cells. We additionally demonstrate extension of this protocol to generate motorneurons. Using CRISPR-Cas9 to engineer a GFP-reporter for a neuromesodermal progenitor-associated transcription factor Nkx1.2 in human embryonic stem cells, to facilitate selection of this cell population. RNA-sequencing was used to identify human and conserved neuromesodermal progenitor transcriptional signatures, validate this differentiation protocol and implicate new pathways and processes in human neural differentiation. This optimised protocol, novel reporter line and transcriptomic data are useful resources with which to dissect molecular mechanisms regulating generation of human spinal cord and allow scale-up of distinct cell populations for global analyses, including proteomic, biochemical and chromatin interrogation.