Novel model of cortical-meningeal organoid co-culture system improves human cortical brain organoid cytoarchitecture.

Human cortical organoids (hCOs), derived from human induced pluripotent stem cells (iPSCs), provide a platform to interrogate mechanisms of human brain development and diseases in complex three- dimensional tissues. However, current hCO development methods lack important non-neural tissues, such as the surrounding meningeal layer, that have been shown to be essential for normal corticogenesis and brain development. Here, we first generated hCOs from a single rosette to create more homogenous organoids with consistent size around 250 µm by day 5. We then took advantage of a 3D co-culture system to encapsulate brain organoids with a thin layer of meningeal cells from the very early stages of cortical development. Immunostaining analysis was performed to display different cortical layer markers during different stages of development. Real-time monitoring of organoid development using IncuCyte displayed enhanced morphology and increased growth rate over time. We found that meningeal-encapsulated organoids illustrated better laminar organization by exhibiting higher expression of REELIN by Cajal-Retzius neurons. Presence of meningeal cells resulted in a greater expansion of TBR2 intermediate progenitor cells (IPCs), the deep cortical layer (CTIP2) and upper cortical layer (BRN2). Finally, meningeal-encapsulated organoids enhanced outer radial glial and astrocyte formation illustrated by stronger expression of HOPX and GFAP markers, respectively. This study presents a novel 3D co-culture platform to more closely mimic the in vivo cortical brain structure and enable us to better investigating mechanisms underlying the neurodevelopmental disorders during embryonic development.