Speaker
Description
Globoid Cell Leukodystrophy (GLD) is a lysosomal storage disorder due to mutations in the galactosylceramidase (GALC) gene, which is essential for sphingolipid metabolism. GALC deficiency leads to the accumulation of toxic psychosine, causing demyelination, neurodegeneration, and neuroinflammation in the central and peripheral nervous systems. While dysfunctional oligodendrocytes (OLs) were traditionally seen as the leading cause of GLD, new evidence suggests that neurons and astrocytes may play a significant role in white matter damage.
In previous studies, we used patient-specific human induced pluripotent stem cells (hiPSCs) to model GLD and explore gene therapy strategies. Using hiPSC-derived neural stem/progenitor cells and 2D neuronal/ glial cultures, We identify previously unrecognized early pathogenic events, uncovering mutation-dependent defects in the differentiation of neurons and OLs. Nevertheless, the 2D culture conditions do not support the full maturation of neurons and glial cells and lack the complex architecture needed to study cell-to-cell interactions in this disease.
To address these issues, we generated OL-enriched 3D spheroids (hOLS) from hiPSCs of healthy donors (HD), GLD patients, and isogenic GALC knock-out (KO) and knock-in (KI) lines generated via CRISPR-Cas9 editing. This research investigates the early neurodevelopmental defects associated with GLD pathology and the impact of GALC deficiency on glial and neuronal maturation. We demonstrated that hOLS were efficiently generated from all hiPSC lines and were composed by multiple populations of glial and neuronal progenitor cells and their differentiated progeny. Immunofluorescence data reflected the maturity of the evolving cell types, which included MBP-positive oligodendrocytes. Additionally, through single-cell RNA sequencing, we identified gene expression alterations and changes in cellular composition that were explicitly linked to GLD. The integration of transcriptomics with the phenotypic analysis of hOLS provides new insights into the molecular signature associated to GLD, offering mechanistic insight into the early pathogenesis of this disorder.
| Author(s) | E. Mangiameli*, C. Rosato, F. Cupaioli, R. Alfieri, I. Merelli, A. Gritti. |
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| Affiliation(s) | "E. Mangiameli* 1, C. Rosato1,2, F. Cupaioli 3, R. Alfieri 3, I. Merelli1,3, A. Gritti 1,2 1 San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy, 2 Universita’ Vita-Salute San Raffaele, Milan, Italy, 3 National Research Council (CNR), Institute for Biomedical Technologies, Segrate (MI), Italy." |
