Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the progressive degeneration of both upper and lower motor neurons (MNs), which results in paralysis and death within two to five years of diagnosis. Induced pluripotent stem cells (iPSCs) derived from ALS patients serve as a useful tool for disease modelling, but existing approaches lack sufficient cellular maturity and fail to replicate the complexity of in vivo-like environments. To overcome these limitations, a spinal cord chip (SC-chip) was developed, a microfluidic organ-on-chip model that combines brain microvascular endothelial-like cells (iBMECs) with spinal cord (SC) produced from iPSCs to create a system that replicates the blood-brain barrier (BBB).
This platform aims to improve neuronal maturation and uncover early molecular signals unique to the disease that give a more physiologically accurate model of early-onset sporadic ALS (sALS). Increased MN maturation in the chip environment is proven by the SC-chip model with the use of media flow, which detects changes in neurofilaments (NFs) and early molecular abnormalities in glutamate signalling in the MNs of ALS patients.
The SC-chip consists of two parallel microfluidic channels divided by a porous membrane. After 14 days of culture, iBMECs are added to the “vascular” channel, while iPSC-derived spinal motor neuron progenitor cells (spNPCs) are seeded into the “neural” channel. Media in the neural channel are completely replaced every 3.2 hours, maintaining a steady flow at around 5.88 µL/h.
To examine cellular composition and disease-specific molecular changes, the experimental approach comprised transcriptomics, proteomics, and single-nuclei RNA sequencing (snRNA-seq). Compared to static culture conditions, SC-chips under perfusion exhibited significantly improved tissue development and increased thickness of the neural layer. Transcriptomic profiling revealed that genes linked to DNA replication, cell proliferation, and neuronal differentiation were increased in perfused chips, while antibody staining gave a high number of ISL1+/SMI32+ motor neurons.
The capacity of the SC-chip environment to support neuronal maturation was highlighted by gene set enrichment analysis with accelerated neuron and synapse growth. There was no significantly cell death was observed as all cultures showed consistent tissue growth with no variation in the total number of motor neurons or lactate dehydrogenase (LDH) levels.
These results align with previous studies connecting NF dysregulation to the pathophysiology of ALS and its application as a biomarker of disease progression. A total of six cell types were found by snRNA-seq profiling of more than 49,000 nuclei from SC-chips. With the expected downregulation of progenitor markers and overexpression of neuronal and MN-specific genes, trajectory analysis explains the gradual differentiation from neural stem cells (NSCs) to mature MN-specific genes.
Comparative analysis with fetal and adult motor neurons in vivo verified that MNs produced from SC-chip matched native MNs better than MNs from 96-well plates. Additionally, the incorporation of TDP-43 expression in both cell lines and iPSC-derived MNs was used to provide important insights into mRNA dysregulation.
The MN populations derived from SC-chips exhibited hindbrain or cervical identity instead of caudal lumbar signatures, which indicates that the chip environment does not promote caudalization of the cultures. Furthermore, a selective downregulation of genes associated with inhibitory Îł-aminobutyric acid (GABA) receptors and synapse formation was observed, alongside an increased expression of genes involved in glutamatergic signalling across various subsets of MNs.
Reference: Lall D, Workman MJ, Sances S, et al. An organ-chip model of sporadic ALS using iPSC-derived spinal cord motor neurons and an integrated blood-brain-like barrier. Cell Stem Cell. 2025. doi:10.1016/j.stem.2025.05.015
