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Development. 2018 Jul 12;145(16). pii: dev166215. doi: 10.1242/dev.166215.

Neural differentiation, selection and transcriptomic profiling of human neuromesodermal progenitor-like cells in vitro.

Author information

1
Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
2
Human Pluripotent Cell Facility, Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
3
Data analysis group, Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.
4
Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK k.g.storey@dundee.ac.uk.

Abstract

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 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. Using CRISPR-Cas9 to engineer human embryonic stem cells with a GFP-reporter for neuromesodermal progenitor-associated gene Nkx1.2 we facilitate selection of this cell population. RNA-sequencing was then used to identify human and conserved neuromesodermal progenitor transcriptional signatures, to validate this differentiation protocol and to reveal new pathways/processes in human neural differentiation. This optimised protocol, novel reporter line and transcriptomic data are useful resources with which to dissect molecular mechanisms regulating human spinal cord generation and allow the scaling-up of distinct cell populations for global analyses, including proteomic, biochemical and chromatin interrogation.

KEYWORDS:

CRISPR-Cas9; Dual SMAD inhibition; Human ES cells; Human neural development; Human neuromesodermal progenitor transcriptome; Human spinal cord; Neuromesodermal progenitor-like cells; Nkx1.2 reporter

PMID:
29899136
PMCID:
PMC6124542
DOI:
10.1242/dev.166215
[Indexed for MEDLINE]
Free PMC Article

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