Specification of human brain regions with orthogonal gradients of WNT and SHH in organoids reveals patterning variations across cell lines

The repertoire of neurons depends on the location of their progenitors along antero-posterior and dorso-ventral axes of the neural tube. To model these axes, we designed the Duo-MAPS diffusion device to expose spheres of induced pluripotent stem cells (iPSC) to concomitant orthogonal gradients of a posteriorizing and a ventralizing morphogen, activating WNT and SHH signaling, respectively. Comparison with single-cell transcriptomes from the fetal human brain revealed that Duo-MAPS-patterned organoids generated an extensive diversity of neuronal lineages from the forebrain, midbrain, and hindbrain. WNT and SHH crosstalk translated into early patterns of gene expression programs associated with the generation of specific brain lineages. Human iPSC lines showed substantial differences in regional specification driven both by interindividual and line-to-line variations in response to morphogens, highlighting that donor-specific and line-specific genetic and epigenetic variations may impact regional specification. Morphogen gradients promise to be a key approach to model the brain in its entirety.