Development of brain organoids from human induced pluripotent stem cells for studying the rare leukodystrophy megalencephalic leukoencephalopathy with subcortical cysts

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital disease characterized by macrocephaly, brain oedema, cysts, myelin vacuolation, and astrocyte swelling leading to severe motor disabilities, seizures, and cognitive difficulties. This disorder belongs to astrocytopathies, a group of leukodystrophies distinguished by progressive cystic degeneration of myelin and mainly due to astrocyte dysfunctions. In most patients, MLC is caused by mutations in the MLC1 gene, encoding for the MLC1 protein, which in the brain is almost exclusively expressed at perivascular astrocyte end-feet and astrocyte-astrocyte contacts. The role of MLC1 and the pathological mechanism underlining MLC disease are not completely known, but experimental and clinical evidence suggests that the MLC1 protein could be involved in controlling astrocyte activation and volume regulation following different physiological and pathological stimuli. Abnormally activated/swollen astrocytes incapable of properly supporting brain development might be the cause of the defects observed in MLC. The astrocytes perform several important functions during neurodevelopment; they control neurogenesis/synaptogenesis, oligodendrocyte differentiation, myelination, and blood-brain barrier formation. To investigate the effects of MLC1 mutations on neurodevelopment, in this study, we generated cerebral organoids from human induced pluripotent stem cells derived from 3 healthy individuals and 4 MLC patients carrying mutations in the MLC1 gene. The preliminary characterization of 53-day-old and 70-day-old organoids by western blotting, quantitative PCR, and immunostaining revealed the expression of markers of astrocytes (MLC1, Vimentin, GFAP, EAAT2, Cx43, and AQP-4), neurons (NEFH and MAP2), and oligodendrocytes (OLIG1, OLIG2 and MBP), confirming that these organoids contain the major cell populations of the central nervous system. At day 53, most patient-derived organoids showed a lower expression of the astrocyte-specific markers MLC1, Vimentin, SOX9, GFAP, and Cx43 compared to control organoids, suggesting that MLC1 mutations may cause astrocyte differentiation/maturation defects. Moreover, in patient-derived organoids cultured for 70 days, a lower expression of oligodendrocyte maturation markers compared to controls suggests that the defects in MLC astrocyte maturation could affect oligodendrocyte differentiation. In conclusion, the brain organoids we generated can represent a new powerful system for elucidating the pathophysiological mechanism of MLC, a crucial step towards the development of effective pharmacological treatments for patients.