Local source of FGF8 specifies a fronto-temporal axis in engineered human brain organoids (PolarOids)

The establishment of the fronto-temporal axis in the developing human cortex is achieved by temporary signaling activity coming from secondary organizers. However, studying this process remains challenging due to the limited access to fetal material and inaccuracy of animal models in mimicking human-specific aspects. Although human brain organoids provide a potential solution to study this, their lack of axial organization does not supply the appropriate positional information needed to recapitulate detailed patterning processes in situ. Here, we develop a method that combines a cell line expressing the morphogen FGF8 fused to organoids engineered with PDMS moulds representing the exact length of the developing brain, resulting in the formation of polarized cortical organoids, or PolarOids. Thanks to a dose-sensitive reporter for anterior cortical differentiation, we carefully titrate and time the fusion dynamics to elicit a gradient in the patterned tissue while preserving neural activity as assessed through electrophysiology. We show that our localized FGF8 source is sufficient to trigger a distance-dependent transcriptional signature that matches fronto-temporal cortical fates in vivo. Lastly, we use these PolarOids to model a disease-relevant FGFR3 mutation and describe the transcriptional loss of axial gradient formation underlying a dramatic cortical phenotype observed in patients. As such, Polaroids provide a powerful tool to model hitherto neglected human diseases caused by defects in antero-posterior patterning in the brain In order to explore the position-dependent transcriptional changes in PolarOids, we performed bulk RNA-sequencing analysis on consecutive tissue segments of D60 PolarOids (3 replica, 1 is an outlier) and compared to unpatterned controls without fgf8 OrEB. We defined these sections based on their relative distance from the OrEB (when present) as proximal (P), medial (M) and distal (D) to the source. Elongated organoids without OrEB were used to generate three control segments (C1, C2 and C3). Further, FGF3 mutant PolarOids (3 replica) were sequenced.