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

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.