Human spinal cord differentiation proceeds rapidly in vitro and only initially maintains differentiation pace in a heterologous environment

Abstract

Species-specific differentiation pace in in vitro assays indicates that some aspects of neural differentiation are driven by cell-autonomous processes. Here we describe a novel in vitro human neural rosette assay that recapitulates the temporal sequence of dorsal spinal cord differentiation but proceeds more rapidly than in the human embryonic spinal cord, suggesting that in vitro conditions lack endogenous signalling dynamics. To test the extent to which this in vitro assay represents a cell intrinsic differentiation programme, human iPSC-derived neural rosettes were homo-chronically grafted into the faster differentiating chicken embryonic neural tube. Strikingly, in vitro human differentiation pace was not accelerated, even in single host-integrated cells. Moreover, rosette differentiation eventually stalled in a neural progenitor cell state. These findings demonstrate the requirement for timely extrinsic signalling to accurately recapitulate human neural differentiation tempo, and suggest that while intrinsic properties limit differentiation pace, such signals are also required to maintain differentiation progression

Single-cell transcriptome profiling of the human developing spinal cord reveals a conserved genetic programme with human-specific features

“…S2A " type="url"/> ). This is consistent with immunohistochemical analysis of PAX7 in the human spinal cord from CS12 to CS15 ( Betters et al, 2010 ; Dady et al, 2021 preprint). Inspection of genes correlating with PAX7 in human FP cells highlighted several, including CDH7 , TXLNB , CDHR3 , PIFO and RRAD , that were expressed in human FP cells but not in mouse FP cells ( Fig.…”

“…The expression of SOX9 and NFIA/B in progenitors and neurons was consistent with immunohistochemical assays of the embryonic human spinal cord ( Betters et al, 2010 ; Dady et al, 2021 preprint; Deneen et al, 2006 ; Rayon et al, 2020 ). The expression of NFIA correlates with the onset of gliogenesis and is observed at CS15 in ventral progenitors but delayed until ∼CS18 in dorsal regions ( Betters et al, 2010 ; Dady et al, 2021 preprint; Deneen et al, 2006 ; Rayon et al, 2020 ). In addition, we identified a small number of cells ( n =77), present from CS12 onwards, expressing SOX10 , SOX9 , PDGFRA and S100B , characteristic of oligodendrocytes ( Fig.…”

“…Although recent attention has focused on profiling the transcriptomes of cells in several regions of the developing human brain ( Eze et al, 2021 ), the embryonic human spinal cord and PNS have been less well described. The available molecular characterisation has shown that the identity and overall organisation of progenitors and neurons is, in the main, similar between humans and other vertebrates ( Dady et al, 2021 preprint; Marklund et al, 2014 ; Rayon et al, 2020 ). Nevertheless, the extent of the similarity in the molecular composition of cells in mouse and human neural tubes has not been determined.…”

Modular derivation of diverse, regionally discrete human posterior CNS neurons enables discovery of transcriptomic patterns

“…Together, differences emergent in our hPSC-derived scRNA-seq dataset reveal the power of this differentiation platform to generate novel, region-specific spinal subpopulations detectable by standard DEG analysis. Whether novel markers are bona fide, evidence of accelerated maturation of cells in vitro compared to in vivo ( 65 ) or artifacts of in vitro differentiation is subject to future investigation.…”

Modular Derivation and Unbiased Single-cell Analysis of Regional Human Hindbrain And Spinal Neurons Enables Discovery of Nuanced Transcriptomic Patterns along Developmental Axes

“…Altogether, differences emergent in our hPSC-derived scRNAseq dataset reveal the power of this differentiation platform to generate novel, region-specific spinal subpopulations detectable by standard DEG analysis. Whether novel markers are bona fide, evidence of accelerated maturation of cells in vitro compared to in vivo (Dady et al, 2021), or artifacts of in vitro differentiation is subject to future investigation.…”