Abstract: Throughout brain development, neural stem cells self-organise into refined networks, establishing the infrastructure for efficient neuronal communication. Here, we established an in vitro platform to study the formation and perturbation of these networks using a live-cell imaging protocol and bioimage analysis pipeline. Cortical neurons, derived from human embryonic stem cells via Neurogenin-2 and green fluorescent protein (GFP) viral transduction, were imaged over 33 days. To minimise phototoxic stress under repeated fluorescence imaging, we optimised culture conditions by testing three factors: extracellular matrix (human- or murine-derived laminin), culture media (Neurobasal or Brainphys Imaging media), and seeding density (1×105 or 2×105 cells per cm2). Brainphys Imaging medium was observed to support cell survival to a greater extent than Neurobasal medium with either laminin type, as quantified by PrestoBlue viability assay (p = 0.0052) and neurite outgrowth analysis (p < 0.0001). However, a combination of Neurobasal medium and human laminin detrimentally impacted neurite outgrowth (p < 0.0001). Using our optimised protocol, we captured timeseries microscopy images and built a computational pipeline to evaluate multiscale network features. At global resolution, we introduced two measures of population-wide somata clustering and neurite fasciculation. At local resolution, we developed a tool that generates spatially-embedded models of neuron networks, representing cell bodies as points (network nodes) and neurite connections as links between these points (network edges). Application of this pipeline to pharmacologically-induced disease models of schizophrenia (treated with MK-801) and epilepsy (treated with kainic acid) revealed distinct topological profiles. Graph theoretical analysis demonstrated significantly reduced clustering coefficients (p = 0.0398) and small-world indices (p = 0.0268) in schizophrenia relative to epilepsy models. These analytical tools reveal, for the first time, disrupted anatomical connectivity across neuronal networks, extending beyond standard neurite outgrowth measures to capture precise motifs in neuron organisation.
