Modelling rostro-caudal neural tube regionalization from human embryonic stem cells with a microfluidic morphogenic gradient

Studies in animal models have been fundamental for understanding brain development but only a fraction of these findings have been validated in a human context. Here, we tissue-engineered a model (MiSTR) of human neural tube regionalization based on human embryonic stem cells (hESCs) and microfluidic cell culturing. By exposing differentiating hESCs to a WNT signalling gradient we mimicked early rostro-caudal neural patterning, and with >80% reproducibility generated a coherent tissue with progressive caudalization from forebrain over midbrain to hindbrain, including formation of isthmic organiser characteristics. Single-cell transcriptomics revealed that rostro-caudal organization was established already at 24 hours of differentiation, before expression of neural markers. Moreover, the transcriptomic hallmarks of rostro-caudal MiSTR organization accurately recapitulated gene expression patterns of the early rostro-caudal neural plate in mouse embryos. MiSTR thereby represents a novel in vitro model of human neurodevelopment to deconstruct and systematically analyze factors responsible for rostro-caudal neural tube patterning. Overall design: Single-cell transcriptomics of an in vitro model of early human neural patterning, at various time points and dorsal and ventral locations.