PT  - JOURNAL ARTICLE
AU  - Laure Verrier
AU  - Lindsay Davidson
AU  - Marek Gierliński
AU  - Kate G. Storey
TI  - Generation, selection and transcriptomic profiling of human neuromesodermal and spinal cord progenitors in vitro
AID  - 10.1101/182279
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 182279
4099  - http://biorxiv.org/content/early/2017/08/30/182279.short
4100  - http://biorxiv.org/content/early/2017/08/30/182279.full
AB  - Robust protocols for directed differentiation of human pluripotent cells are needed to establish the extent to which 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 neuromesodermal 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 progenitors. We use 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 (RNA-Seq) was then 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 cellular and molecular mechanisms regulating the generation of human spinal cord, allow scale-up of distinct cell populations for global analyses, including proteomic, biochemical and chromatin interrogation and open up translational opportunities.