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 leukodystrophy caused by primary astrocyte dysfunction and characterized by macrocephaly, cerebral edema, subcortical cysts, and myelin vacuolation. The disease is mainly associated with mutations in the gene encoding MLC1, an astrocyte-specific protein highly expressed at perivascular astrocytic end-feet. Although the MLC1 precise function remains unclear, several studies demonstrated its critical role in regulating astrocyte volume, proliferation, and activation in response to injurious stimuli. Using astrocytes differentiated from patient-derived induced pluripotent stem cells (iPSCs), we recently showed that MLC1 mutations lead to astrocyte maturation defects. In this study, we generated iPSC-derived brain organoids to investigate whether impaired astrocyte maturation also affects oligodendrocyte differentiation/maturation, potentially contributing to the myelin abnormalities observed in MLC. Brain organoids were generated from two healthy controls and four MLC patients and analyzed at days 50 and 70 of differentiation using Western blotting, quantitative PCR, and immunofluorescence to assess the expression of astrocytic, oligodendrocytic, and neuronal markers. Both control and MLC-derived organoids successfully developed the major brain cellular populations. However, MLC organoids exhibited a reduced expression of mature astrocyte markers at both time points compared to controls. In addition, immunofluorescence at day 50 revealed a marked downregulation of oligodendrocyte markers in MLC organoids. These findings confirm impaired terminal differentiation of MLC astrocytes and suggest that this dysfunction may negatively affect oligodendrocyte development, a process essential for proper myelination. iPSC-derived brain organoids therefore represent a valuable model to study MLC pathophysiology and a robust platform for future therapeutic screening.