HUMAN INDUCED PLURIPOTENT STEM CELLS-DERIVED CEREBRAL ORGANOIDS AS A MODEL FOR C9ORF72-LINKED FRONTO-TEMPORAL DEMENTIA: INSIGHTS INTO NEURONAL AND GLIAL DIFFERENTIATION DYNAMICS

Ferrero C^1,2,3, Pacca P^1,2, Vercelli A^1,2 and Boido M^1,2 | ¹Department of Neuroscience “Rita Levi Montalcini”, University of Turin, Italy; ²Neuroscience Institute Cavalieri Ottolenghi, Italy ³University School for Advanced Studies IUSS Pavia, Pavia, Italy

Fronto-temporal dementia (FTD) is a progressive neurodegenerative disorder characterized by selective degeneration of the frontal and temporal lobes, leading to profound alterations in personality, behavior, and language. Among its genetic causes, the hexanucleotide repeat expansion in the C9ORF72 gene represents one of the most frequent mutations, also implicated in amyotrophic lateral sclerosis (ALS). In this study, we explored the potential of cerebral organoids derived from human induced pluripotent stem cells (hiPSCs) as a dynamic and physiologically relevant model to investigate FTD pathophysiology associated with C9ORF72 mutations. Organoids were generated from hiPSCs obtained from both healthy donors and FTD patients and cultured up to 120 days in vitro (DIV). Their development was monitored at 30, 60, 90, and 120 DIV through morphometric and immunohistochemical analyses targeting key neural and glial markers. The neural markers SOX2 and Ki67 were employed to assess stemness and proliferation, PAX6 to identify neural progenitors, and TBR1, CTIP2, SATB2, and MAP2 to evaluate neuronal differentiation and cortical organization. In parallel, the astrocytic markers GFAP and S100B were analyzed to investigate glial maturation and temporal dynamics of differentiation. Preliminary results demonstrated a time-dependent growth for both control and FTD organoids, with diameters increasing from approximately 400 μm at early stages to over 2 mm by day 90. Marker expression analysis revealed an expected progressive transition from progenitor to neuronal identity: SOX2⁺ cells decreased from ~50% to 30%, and Ki67⁺ cells from ~30% to <10%, indicating reduced proliferative activity. SATB2 expression, associated with upper-layer cortical neurons, increased significantly between day 60 and 90, reaching ~30% in control organoids, but was markedly reduced or absent in FTDderived organoids, suggesting altered cortical layer formation. Furthermore, analysis of glial markers revealed consistent differentiation of astrocytic populations after approximately four months in culture in both control and FTD organoids, with clear expression of GFAP and S100B indicating astrocyte maturation. This temporal emergence of glial identity complements neuronal findings, underscoring the complex cellular interplay underlying cortical development. Overall, these results highlight the potential of cerebral organoids as a robust and versatile platform for modeling FTD, enabling the investigation of disease mechanisms and the testing of novel therapeutic strategies beyond traditional animal models.