Modeling Long-Range Connectivity Deficits in Autism Spectrum Disorders using Cerebral Organoids

Introduction: Autism spectrum disorders (ASDs) are highly prevalent, exerting a profound impact on the quality of life for affected individuals and generating substantial socioeconomic challenges. Existing therapies have limited effectiveness in addressing these conditions, primarily due to our incomplete understanding of the underlying pathophysiology. A growing body of evidence suggests that individuals with ASDs exhibit long-range connectivity deficits in the brain, directly impairing information transfer between brain regions during crucial developmental stages. Notably, there has been limited exploration in the realm of in vitro platforms for modeling and assessing long-range axonal connectivity, a process which is vital for integrating distal neural circuits within the brain. Furthermore, the limited availability of viable human brain tissue for research underscores the pressing need to develop dependable, physiologically relevant human brain models. This research focuses on developing a system aimed at recapitulating ASD-relevant pathophysiology in a Brain on a Chip (BOC) model using three-dimensional (3D) cerebral organoids (COs) derived from human induced pluripotent stem cells (hiPSCs) carrying genetic mutations associated with ASDs. In particular, the effect of R451C substitution in the NLGN3 gene, which is the first identified mutation associated with ASDs and responsible for encoding postsynaptic adhesion molecule Neuroligin-3, has been investigated in human CO cultures for the first time. This work combines hiPSC-derived CO cultures and microfabrication bioengineering to recapitulate the long-range projections connecting brain regions. The presented platform represents a powerful model of disrupted connectivity underlying ASD pathophysiology and could provide insights into potential mechanisms and therapeutic strategies.

Materials and

Methods: Microfabrication techniques (CAD modeling, photolithography, physical vapor deposition) were used to create Cerebral Organoid Connectivity Apparatus with Microelectrode Array (COCAMEA) microfluidic platforms to culture CO slice pairs and guide the formation of interconnecting cerebral tracts. Soft lithography techniques were used to fabricate polydimethylsiloxane (PDMS) microfluidic platforms with microchannel arrays to enable extension of neurite tracts between the two CO compartments. The electrodes (60 Ti/Pt recording traces) enable longitudinal electrophysiological characterization and stimulation of axonal bundles along the connecting channels. COs were generated from hiPSCs using reported protocols, characterized via morphological analyses, sliced to a 300-m thickness, and transduced with EGFP and mCherry adeno-associated viruses (AAVs) to distinguish projections. Immunohistochemistry using stem cell, neuronal, thalamic, and cortical markers was performed. Spontaneous, as well as stimulation- and ion channel antagonist-mediated, electrophysiological activity were recorded from CO slices with high fidelity using the fabricated MEAs. Cortical COs derived from CRISPR-engineered NLGN3 R451C-mutant (2 clones) and NLGN3 KO ES (2 clones) hiPSC lines were generated and are being cultured on COCAMEAs for investigation into neurite growth patterns, morphology, and electrophysiology compared to isogenic controls.

Results, Conclusions, and Discussions: Immunostaining highlighted robust expression of essential neuronal protein and cortical markers (e.g. MAP2, Synapsin, TBR1, TBR2) in COs patterned into a cortical lineage, demonstrating that differentiation methods align with reported literature. AAV-transduced COs demonstrated neurite outgrowth between compartments and morphological innervation of CO slices. Cortical COs were successfully generated from CRISPR-engineered lines with NLGN3 R451C and NLGN3 null mutations. MEA devices were equipped with precisely defined Ti/Pt metal electrodes and SU-8 insulation, following an optimized fabrication protocol. The average impedance of devices consisting of 60 60-m Ti/Pt recording electrodes with SU-8 photoresist insulation is 167±44 kΩ, which is comparable to the commercial MEA range. Cortical COs demonstrated consistent neural spiking and network bursting electrical activity. Action potentials, initiated in stimulated neurons traversed communication channels, eliciting responses in CO pairs. The observed average delay indicates functional connectivity through established neurite paths.

An in vitro platform was developed to model long-range neuronal connectivity and functionally validated using calcium imaging, viral tracing, and electrophysiological recording techniques. Non-invasive extracellular recording electrodes directly interface with CO neurons and enable precise electrophysiological recording and stimulation. Microfabricated microfluidic platforms guide neurite extension and the formation of a reciprocal inter-organoid cerebral tract. CRISPR-engineered COs with NLGN3 R451C knockin and NLGN3 knockout mutations have been generated. These are presently being cultured and recorded on COCAMEAs to explore signaling phenotype differences in COs, marking a first in the field.

Due to the human-specific physiological relevance and the intrinsic ability of COs to form functional neural tissues, this novel platform has the potential to be a powerful model of disrupted connectivity underlying ASD pathophysiology and provide insights into potential treatment regimens for patients, serving both basic science and translational research. Furthermore, the in vitro system has longitudinal characterization capabilities which can also be applied to model other ASD genetic etiologies as well as other neuropsychiatric disorders, such as schizophrenia. This research will accelerate the development of effective treatment strategies to improve patient well-being.

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