Rostrocaudal patterning and neural crest differentiation of human pre-neural spinal cord progenitors in vitro

Abstract

14The spinal cord emerges from a niche of neuromesodermal progenitors (NMPs) formed and 15 maintained by Wnt/FGF signals in the posterior end of the embryo. NMPs can be generated from 16 human pluripotent stem cells and hold promise for spinal cord replacement therapies. However, 17NMPs are transient and unable to produce the full range of rostrocaudal spinal cord identities in vitro. 18Here we report the generation of NMP-derived pre-neural progenitors (PNPs) with stem cell-like self-19 renewal capacity. PNPs maintain pre-spinal cord identity by co-expressing the transcription factors 20 SOX2 and CDX2, and they lose the mesodermal potential by downregulating TBXT. Over 10 passages 21 these cells divide to self-renew and to make trunk neural crest, while gradually adopting a more 22 posterior identity by activating colinear HOX gene expression. Rostrocaudal identity can be prolonged

Neuromesodermal progenitor origin of trunk neural crestin vivo

“…We demonstrate that at least a portion of trunk neural crest cells in the developing zebrafish embryo arise from neuromesodermal progenitors rather than from the neural plate border epiblast. Interestingly, these trunk neural crest progenitors form via an intermediary neural progenitor fate, much like in vitro NMps derived from hPSCs that require a passage via pre-neural fate to acquire posterior trunk NC features progressively (Cooper et al, 2020). All human in vitro differentiation protocols that successfully generate trunk NC identities must first generate an intermediate neuromesodermal axial progenitor, drive them to a pro-neural fate, in order to form NC (Frith et al, 2018;Gomez et al, 2019;Hackland et al, 2019).…”

Shaping axial identity during human pluripotent stem cell differentiation to neural crest cells

“…Although RA addition to cranial NC cells results in posteriorisation to a vagal axial identity, it does not lead to the induction of HOX genes posterior to HOX PG 7 [82,83,86] and early attempts to produce trunk NC derivatives from hPSCs have relied on the expression of PHOX2B or ASCL1, which also mark vagal NC derivatives in addition to trunk NC-derived sympathoadrenal progenitors [83,89]. A number of recent studies have demonstrated the efficient generation of NMP-like cells from both mouse and human PSCs following stimulation with Wnt (using the GSK-3 inhibitor CHIR99021 (CHIR) or Wnt3a) and FGF2/8 for 2-4 days [67,85,[90][91][92][93][94][95]. Since then, extensive studies have optimised their generation and utilised these progenitors in vitro (reviewed in [20]).…”

“…PSC-derived NMP-like cells have been shown to give rise to both neural spinal cord and paraxial mesoderm cells [1,93,[96][97][98][99][100]. Interestingly, some studies have demonstrated that trunk NC can be generated via an NMP intermediate reflecting the developmental origin of these cells in vivo [85,91,[101][102][103][104][105]. The earliest example of trunkNC induction and differentiation in vitro found that treating NMP-like cells with BMP resulted in up-regulation of NC markers and generated NC-like cells capable of generating peripheral nervous system (e.g.…”

“…Published studies reporting the generation and axial characterisation, determined by HOX gene expression, of in vitro derived neural crestThe concentration of CHIR used in each experiment has been noted due to the role of Wnt signalling in mediated the cranial-trunk switch[101,105,127]. Only studies which have determined axial identity through HOX gene analysis have been included in this table[74,76,[82][83][84][85][86]91,101,[104][105][106]126].…”

In Vitro Generation of Posterior Motor Neurons from Human Pluripotent Stem Cells

“…During amniote embryonic development, posteriorly located neuromesodermal progenitors have been shown to give rise to cell lineages of the post‐cranial axis (Cambray & Wilson, 2007; Tzouanacou et al., 2009; Wymeersch et al., 2016). Similarly, NMP‐like cells generated in vitro from hPSCs can also be steered towards posterior neural, mesodermal, and neural crest lineages (Cooper et al., 2021; Frith et al., 2018; Gouti et al., 2014; Lippmann et al., 2015; Mouilleau et al., 2021; Turner, Rué, Mackenzie, Davies, & Martinez Arias, 2014; Verrier, Davidson, Gierliński, Dady, & Storey, 2018). Moreover, we have recently shown that the efficient generation of posterior thoracic HOX PG(6‐9) motor neurons is best achieved by differentiating through a neuromesodermal progenitor intermediary state (Wind et al., 2021).…”