Human spinal cord in vitro differentiation pace is initially maintained in heterologous embryonic environments

Abstract

Species-specific differentiation pace in-vitro indicates that some aspects of neural differentiation are governed by cell intrinsic properties. Here we describe a novel in-vitro human neural-rosette assay that recapitulates dorsal spinal cord differentiation but proceeds more rapidly than in the human embryo, suggesting that it lacks endogenous signalling dynamics. To test whether in-vitro conditions represent an intrinsic differentiation pace, human iPSC-derived neural rosettes were challenged by grafting into the faster differentiating chicken embryonic neural tube iso-chronically, or hetero-chronically into older embryos. In both contexts in-vitro differentiation pace was initially unchanged, while long-term analysis revealed iso-chronic slowed and hetero-chronic conditions promoted human neural differentiation. Moreover hetero-chronic conditions did not alter the human neural differentiation programme, which progressed to neurogenesis, while the host embryo advanced into gliogenesis. This study demonstrates that intrinsic properties limit human differentiation pace, and that timely extrinsic signals are required for progression through an intrinsic human neural differentiation programme.

Engineering fluorescent reporters in human pluripotent cells and strategies for live imaging human neurogenesis

“…To facilitate analysis of cell behaviour in live tissue derived from human pluripotent stem cells we generated a series of fluorescent reporter hESC or iPSC lines (Dady, et al, 2022)(Figures 1A,B). To monitor cell shape and dynamics we used a plasma membrane (pm) localised protein tagged with eGFP or mKate2 (pm-eGFP or pm-mKate2) (Sanders, et al, 2013).…”

“…To generate these reporters, we used the hESC line H9 (for pm-eGFP, pm-mKate2, H2B-mEos3.2), or hiPSC line ChiPS4 for F-Tractin-mKate2 and a further pm-eGFP line (Dady, et al, 2022) and we deployed the piggyBac transposon-mediated stable integration strategy (Sanders, et al, 2013, Sarkar, et al, 2003, Yusa, et al, 2009, Yusa, et al, 2011). The piggyBac vectors are composed of the specific fusion protein of interest whose expression is driven by a CAG promoter (Figures 1A,B).…”

“…Here we first focussed on capturing fundamental parameters of human neurogenesis using our established spinal cord rosette assay (Dady, et al, 2022, Verrier, et al, 2018)(Figure 2A). This involves differentiating hPSCs into Neuro-Mesodermal Progenitor-like cells (NMP-like cells) and their subsequent differentiation into SOX2 expressing neural progenitors, which self-organise into neural rosettes exhibiting localised expression of apico-basal polarity markers such as NCAD/CDH2 by Day 6 (D6) following NMP-L cell plating (Dady, et al, 2022). This differentiation assay generates neural crest progenitors and dorsal neural progenitors, with the first neurons apparent from D8, peak neurogenesis at D14 and gliogenesis onset by ∼ D20.…”

ERK1/2 signalling dynamics promote neural differentiation by regulating chromatin accessibility and the polycomb repressive complex

“…We reasoned that the use of human ESCs, with the slower progression to neural progenitor cell identity, would provide a better temporal resolution of mechanisms mediating neural differentiation. To this end, we directed the differentiation of human ESCs towards spinal cord using a protocol established previously [ 91 , 92 ] ( Fig 2A and see Methods ). This involved the generation of NMP-like cells on day 3 (NMP-L D3) (characterised by the coexpression of BRA and SOX2, which are analogous to NMP cells in the mouse CLE) and their differentiation into PAX6 expressing neural progenitors by day 8 (NP D8), which also transcribed the known PRC target HOXD11 ( Fig 2B–2B” and S2 Data ).…”

“…We reasoned that the use of human ESCs, with the slower progression to neural progenitor cell identity, would provide a better temporal resolution of mechanisms mediating neural differentiation. To this end, we directed the differentiation of human ESCs towards spinal cord using a protocol established previously [91,92] To elucidate how chromatin compaction is regulated at the PAX6 locus, we interrogated PRC occupancy over developmental time using ChIP-qPCR in NMP-L (D3) cells versus NPs (D8). We assessed the occupancy of the core PRC1 protein Ring1B (which is required in the embryonic mouse epiblast that contains NMPs, while Ring1A loss affects only later development) [74] and for PRC2, Jarid2 (as the latter is highly expressed in the NMP containing caudal lateral epiblast and newly forming neural progenitors in chick [93] and mouse embryos (S1 Fig) ).…”