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

Laure Verrier, Lindsay Davidson, Marek Gierlinski, Alwyn Dady, Kate Storey (Lead / Corresponding author)

Research output: Contribution to journalArticle

9 Citations (Scopus)
101 Downloads (Pure)

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.

Original languageEnglish
Article numberdev166215
Number of pages10
JournalDevelopment
Volume145
Issue number16
Early online date13 Jun 2018
DOIs
Publication statusPublished - 12 Jul 2018

Keywords

  • neuromesodermal progenitor-like cells
  • human neural development
  • human spinal cord
  • dual SMAD inhibition
  • CRISPR-Cas9
  • human ES cells
  • Nkx1.2 reporter
  • human neuromesodermal progenitor transcriptome
  • Human neuromesodermal progenitor transcriptome
  • Human neural development
  • Neuromesodermal progenitor-like cells
  • Human ES cells
  • Human spinal cord
  • Dual SMAD inhibition
  • CRISPR-cas9
  • Embryo, Mammalian
  • Humans
  • Gene Expression Profiling
  • Body Patterning/genetics
  • Gene Expression Regulation, Developmental
  • Neural Stem Cells/cytology
  • Stem Cells/cytology
  • Cell Lineage/genetics
  • Cells, Cultured
  • Spinal Cord/cytology
  • Animals
  • Cell Differentiation/genetics
  • Mice
  • Mesoderm/cytology
  • Neurogenesis/genetics

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