Detailed culture protocol for the generation of human polarized cortical assembloids (polCA) v1

Abstract

Cortical organoids generating all major cortical cell types in distinct compartments are widely used to mechanistically study cortical development, evolution and related disorders. However, the lack of morphogen gradients imparting cortical positional information and topography in current systems hinders the solid investigation of complex cortical phenotypes. Here, we describe an assembloid culture protocol that combines an organizer-like structure expressing the morphogen FGF8 with an elongated organoid, thus introducing a polarized source of FGF8 morphogen within individual organoids. This system, referred to as polarized cortical assembloid (polCA), allows for the establishment of FGF8-dependent positional identity along the longitudinal axis of individual organoids and the generation of spatially oriented populations with distinct transcriptional signatures relevant for the cortical protomap. This protocol can be adapted to include organizer-like structures expressing various morphogens, allowing for a reliable analysis of their effect on cortical positional identity.