ALS is a neurodegenerative disorder marked by progressive motor neuron loss, leading to paralysis and death. Rodent models have limitations in recapitulating ALS pathophysiology, contributing to clinical trial failures. Human iPSCs provide a promising alternative, enabling patient-specific models. However, their immaturity in vitro poses a challenge for modeling ALS, where pathological abnormalities are time-dependent and spread through a complex neural network, the CST.

We developed an in vitro platform combining microfluidic devices and multielectrode array (MEA) to model the corticospinal tract (CST) using regionally specific human induced pluripotent stem cell-derived motor neurons (hiPSC-MN) and astrocytes (hiPSC-A). We hypothesize that this system promotes the maturation of both hiPSC-MN and hiPSC-A, providing a physiologically relevant model for studying Amyotrophic lateral sclerosis (ALS).

We adapted protocols to generate regionally specific hiPSC cortical (hiPSC-CSMN, hiPSC-cA) and spinal cord (hiPSC-SMN, hiPSC-scA) neurons and astrocytes. These were co-cultured in microfluidic compartments and assessed using immunocytochemistry (synaptic density, neurite complexity, maturation/regional identity markers) and MEA (spike rate, bursting activity) over 10 weeks.

Axonal projections emerged between cortical and spinal compartments, mimicking CST connections. In all co-cultures, MEA recordings showed increased electrophysiological activity over time, indicating network maturation. Cortical astrocytes promoted motor neuron maturation, reflected by enhanced synaptic density and neurite complexity, independent of the motor neuron subtype (CSMN or SMN). This led to greater spike rates and robust bursting activity. Cortical neurons outperformed spinal neurons in morphological complexity and electrophysiological activity. CST-like co-cultures (CSMN/cA with SMN/scA) exhibited superior maturation compared to spinal-only, cortical-only, or mixed cultures (CSMN/scA with SMN/cA).

The cortical regional identity of neurons and astrocytes promotes the formation of complex neuronal networks, resembling their in vivo counterparts. The arrangement of these cells within a CST-like network promotes higher degrees of maturation, both morphologically and electrophysiologically, offering an accurate and functional platform for ALS modeling.