Glia-enriched human brain organoids for studying developmental alterations in leukodystrophies

Leukodystrophies (LKD) comprise mostly inherited white matter disorders whose developmental origins and underlying pathomechanisms remain poorly understood. Progress has been limited by restricted access to human primary tissue and by the inability of animal models to fully recapitulate human neurodevelopment. Human induced pluripotent stem cell-derived neural organoids provide an alternative approach, but most culture systems do not capture later, glia-rich developmental stages relevant to these diseases. Recent studies have shown that appropriate patterning cues and long in vitro differentiation can generate neuroglial spheroids comprising multiple neuronal and macroglial populations, including mature oligodendrocytes. Here, we adapt the human oligodendrocyte (hOLS) spheroid platform to establish 3D models of globoid cell leukodystrophy (GLD), 4H leukodystrophy (4H), and Canavan disease (CD). Here we adapt this organoid platform to establish 3D models of globoid cell leukodystrophy (GLD), 4H leukodystrophy (4H), and Canavan disease (CD). Across 150 days of differentiation, hOLS from different genetic backgrounds displayed largely comparable growth kinetics and developmental trajectories, comprising multiple neuroglial cell types with predominantly ventral forebrain and midbrain identities. Comparison of LKD and control hOLS revealed both shared and disease-specific alterations, with prominent transcriptional changes in lysosomal, metabolic, and stress-response pathways. Lineage-resolved pathway analysis further identified signatures across individual LKDs, including mitochondrial and translational stress in GLD glial lineages, disruption of RNA metabolic and cell-cycle pathways in 4H, and prominent astrocyte-centered metabolic dysregulation in CD. Our results establish hOLS as a human 3D in vitro platform for studying the impact of LKD-associated genetic variants on early neural development and for dissecting lineage-specific disease vulnerability.