Midline Assembloids Reveal Regulators of Human Axon Guidance.

Organizers are specialized cell populations that orchestrate cell patterning and axon guidance in the developing nervous system. Although non-human models have led to fundamental discoveries about the organization of the nervous system midline by the floor plate, an experimental model of human floor plate would enable broader insights into regulation of human neurodevelopment and midline connectivity. Here, we have developed stem cell-derived organoids resembling human floor plate (hFpO) and assembled them with spinal cord organoids (hSpO) to generate midline assembloids (hMA). We demonstrate that hFpO promote Sonic hedgehog-dependent ventral patterning of human spinal progenitors and Netrin-dependent guidance of human commissural axons, paralleling non-human models. To investigate evolutionary-divergent midline regulators, we profiled the hFpO secretome and identified 27 evolutionarily divergent genes between human and mouse. Utilizing the hMA platform, we targeted these candidates in an arrayed CRISPR knockout screen and reveal that GALNT2 , a gene involved in O-linked glycosylation, impairs floor plate-mediated guidance of commissural axons in humans. This novel platform extends prior axon guidance discoveries into human-specific neurobiology with implications for mechanisms of nervous system evolution and neurodevelopmental disorders.

Kirigami electronics for long-term electrophysiological recording of human neural organoids and assembloids.

Realizing the full potential of organoids and assembloids to model neural development and disease will require improved methods for long-term, minimally invasive recording of electrical activity. Current technologies, such as patch clamp, penetrating microelectrodes, planar electrode arrays and substrate-attached flexible electrodes, do not allow chronic recording of organoids in suspension, which is necessary to preserve architecture. Inspired by kirigami art, we developed flexible electronics that transition from a two-dimensional to a three-dimensional basket-like configuration with either spiral or honeycomb patterns to accommodate the long-term culture of organoids in suspension. Here we show that this platform, named kirigami electronics (KiriE), integrates with and enables chronic recording of cortical organoids for up to 120 days while preserving their morphology, cytoarchitecture and cell composition. We demonstrate integration of KiriE with optogenetic and pharmacological manipulation and modeling phenotypes related to a genetic disease. Moreover, KiriE can capture corticostriatal connectivity in assembloids following optogenetic stimulation. Thus, KiriE will enable investigation of disease and activity patterns underlying nervous system assembly.

Kirigami electronics for long-term electrophysiological recording of human neural organoids and assembloids.

Organoids and assembloids have emerged as a promising platform to model aspects of nervous system development. Longterm, minimally-invasive recordings in these multi-cellular systems are essential for developing disease models. Current technologies, such as patch-clamp, penetrating microelectrodes, planar electrode arrays and substrate-attached flexible electrodes, do not, however, allow chronic recording of organoids in suspension, which is necessary to preserve their architecture. Inspired by the art of kirigami, we developed flexible electronics that transition from a 2D pattern to a 3D basketlike configuration to accommodate the long-term culture of organoids in suspension. This platform, named kirigami electronics (KiriE), integrates with and enables chronic recording of cortical organoids while preserving morphology, cytoarchitecture, and cell composition. KiriE can be integrated with optogenetic and pharmacological stimulation and model disease. Moreover, KiriE can capture activity in cortico-striatal assembloids. Moving forward, KiriE could reveal disease phenotypes and activity patterns underlying the assembly of the nervous system.