Development and characterization of 3D spinal cord organoids to advance the study of amyotrophic lateral sclerosis

We differentiated iPSCs into neural stem cells (NSCs). NSCs were plated on low-attachment plates and were cultured in floating conditions using an orbital shaker. NSCs were differentiated in these conditions into motor neuron progenitors, immature motor neurons (MNs) and finally mature MNs.

We then characterized cells by phase-contrast and confocal microscopy. We found that SCOs derived from sALS patients were smaller and with irregular morphology compared to healthy controls. Using the GFAP marker, we found that sALS SCOs have a thicker glial layer compared to healthy controls.

We also found that healthy controls show longer neurites compared to sALS SCOs. By RNAseq, we found a ten-fold increase of deregulated gene in SCOs respect to 2D cell models. Moreover, in ALS SCOs we found an extensive deregulation of genes involved in extracellular matrix organization when compared to control.

By deconvolution analysis we found that healthy controls SCOs show a higher amount of differentiated cells compared to sALS SCOs. Our data suggest that brain organoids represent a promising tool for the investigation of pathogenic mechanisms of ALS. In fact, we found typical pathological hallmarks of the pathology, such as the presence of gliosis, the smaller length of neurites, decreased level of mature MNs, and deregulation of genes implicated in ALS.

In conclusion, SCOs represent a promising tool for the investigation of pathogenic mechanisms of ALS.