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

Neural organoids derived from human-induced pluripotent stem cells (iPSCs) provide a model to study the earliest stages of human brain development, including 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 instead been cocultured with non-neural structures and cell types to promote their maturation and model interactions with neuronal cells. One component of the brain that does not form de novo in neural organoids is the meninges, a trilayered structure that surrounds the central nervous system and secretes key signaling molecules required for mammalian brain development. Most studies of meninges-brain signaling have been performed in mice or using two-dimensional cultures of human cells, which do not accurately recapitulate the architecture and cellular diversity of the tissue. To overcome this, we developed a coculture system of neural organoids generated from human iPSCs fused with fetal leptomeninges (LPM) from mice with fluorescently labeled meninges (Col1a1-GFP), which we call leptomeningeal neural organoid (LMNO) fusions. This proof-of-concept study tests the stability of the different cell types in the LPM (fibroblasts and macrophages) and the fused neural organoid (progenitors and neurons), as well as the interface between the organoid and meningeal tissue. We test the longevity of the fusion pieces after 30 and 60 days in culture, 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.