We investigated, by immunofluorescence, neuronal and glial cell differentiation capacity as well as the expression of different proliferation/differentiation regulators in c-organoids derived from patients with the different subtypes of MS and healthy controls at day 42, 120 and 200.

Glial cell analysis revealed a decrease in Olig2⁺ oligodendrocytes in MS organoids compared to control at day 42. Further analysis at day 200 showed a marked reduction in Olig2⁺/APC⁺ and O4⁺ mature oligodendrocytes and a decrease of MBP⁺ myelin. In parallel, a decline of GFAP⁺ astrocytes was detected, indicating a dysregulation in oligodendrocyte and astrocyte differentiation. Neuronal analysis revealed a significant decrease of GAD67⁺ GABAergic neurons in MS organoids, while vGlut1⁺ Glutamatergic neurons remained unchanged, which mirrors an imbalance of excitatory and inhibitory neurons observed in MS patient brains. These results were associated with a decrease of E2F1 expression in MS organoids, which controls oligodendrocyte precursor transition from proliferation to differentiation and GABAergic neuron differentiation, and an increase of PAK1 expression in MS organoids, which can block myelin formation. Interestingly both proteins control p21 expression which is necessary for oligodendrocyte differentiation and was described as a protein of interest in MS pathogenesis in our previous work.

Using MS organoids, we demonstrated a loss of oligodendrocyte and astrocyte differentiation/maturation associated with a defect of myelination capacity as well as an imbalance in GABAergic and glutamatergic neurons, potentially due to a defect of p21 expression and its regulators, PAK1 and E2F1.