Abstract
ABSTRACTRobust 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.
Highlights
Head and trunk nervous systems have distinct developmental origins
The Neuromesodermal progenitor (NMP) generated in this way were differentiated into neural progenitors by day 6, following replating and culture in basal media alone (Gouti et al 2014)
We included all-trans retinoic acid (RA) 100 nM in this differentiation protocol from the beginning of day 4 (D4). This regime did not elicit reproducible expression of Pax6 by day 6 (D6) (Fig. 1D, E). This contrasted with a positive control for Pax6 transcription provided by a protocol for inducing anterior neural progenitors, exposure to Noggin 50 ng/ml and the TGFb receptor type 1 inhibitor SB431542 10 μM following removal of self-renewal conditions (dual SMAD inhibition, (Chambers et al 2009), Fig. 1D)
Summary
Head and trunk nervous systems have distinct developmental origins. Head or anterior neural progenitors are derived from the epiblast rostral to the primitive streak and will form regions of the brain. Mouse and chick embryos as well as in vitro NMPs are identified by co-expression of early neural (Sox2) and mesodermal Brachyury (Bra) proteins, but as yet lack unique molecular markers (Olivera-Martinez et al 2012; Gouti et al 2014; Turner et al 2014; Henrique et al 2015; Tsakiridis and Wilson 2015). Almost all in vitro protocols for making NMPs from mouse and human embryonic stem cells (hESCs) involve exposure to various durations of Wnt agonist with or without FGF (Gouti et al 2014; Tsakiridis et al 2014; Turner et al 2014; Lippmann et al 2015) and one approach has included TGFb inhibition (to promote loss of self-renewal in human ESC and repress mesendoderm differentiation, after (Chambers et al 2009)) (Denham et al 2015). We deploy CRISPR-Cas engineering to make a reporter for enrichment for human NMPs and provide the first transcriptomic profiling of this cell population and derived spinal cord progenitors
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