Leptomeningeal Neural Organoid (LMNO) Fusions as Models to Study Meninges-Brain Signaling

Abstract Neural organoids derived from human induced pluripotent stem cells provide a model to study the earliest stages of neurogenesis, neural differentiation, and synaptogenesis. However, neural organoids lack supportive tissues and some non-neural cell types that are key regulators of brain development. Neural organoids have been co-cultured with non-neural structures and cell types to promote their maturation and model interactions with neuronal cells. One structure that does not form de novo with neural organoids is the meninges, a tri-layered structure that surrounds the CNS and secretes key signaling molecules required for mammalian brain development. We developed a co-culture system of neural organoids fused with fetal leptomeninges from mice with fluorescently labeled meninges. These proof-of-concept studies test the stability of the different cell types in the leptomeninges and the fused brain organoid, as well as the interface between the organoid and meningeal tissue. We test the longevity of the fusion pieces, describe best practices for preparing the meninges sample before fusion, and examine the feasibility of single or multiple meninges pieces fused to a single organoid. We discuss potential uses of the current version of the LMNO fusion model and opportunities to improve the system.