Postdoctoral Fellow UMG Goettingen, Institute for Pharmacology and Toxicology Göttingen, United States
Abstract: Myelin disorders, including Pelizaeus-Merzbacher Disease (PMD), are marked by oligodendrocyte dysfunction and impaired myelination. Despite their severity, therapeutic progress is limited by the lack of human-specific models. Bioengineered neuronal organoids (BENOs) derived from human iPSCs provide a relevant platform for studying these disorders. BENOs are forebrain organoids with dorsal and ventral identities, containing interconnected networks of neurons, astrocytes, and oligodendrocytes. In this study, 120-day-old BENOs derived from a PMD patient iPSC line (PLP1-C33Y, c.98G>A) showed reduced gene expression of key oligodendrocyte markers (MBP, PLP1, OLIG2) and impaired neuronal network formation with increased excitability as demonstrated by multielectrode array (MEA) analysis (N=2 independent differentiations, n=6-8 BENO) in comparison to isogenic control PLP1-Y33C. Notably, this hyperexcitable phenotype represents a previously unreported feature of PMD. While functional and gene expression studies provided valuable insights into the PMD phenotype, a deeper understanding of the underlying pathomechanism required the investigation of oligodendrocyte morphology within the 3D environment of BENOs. Current imaging methods, limited to 2D analysis or sliced organoids, fail to capture the 3D complexity of oligodendrocyte architecture. To address this, we developed 3D Oligodendrocyte Morphometrics (3DOM), a machine-learning tool for whole-organoid analysis. Using 3DOM and a refined, oligodendrocyte-enhancing BENO protocol, we phenotyped PLP1-C33Y and isogenic control BENOs on Day 120 (N=2, n=10). BCAS1 immunostaining revealed reduced oligodendrocyte soma counts, larger and less circular somas, and less mature, fragmented processes compared to isogenic control. Results were validated in CRISPR knock-in lines carrying the c.98G>A mutation (N=2, n=8). To study dynamic oligodendrocyte behavior, we are adapting 3DOM for live imaging. With an established fluorescent reporter iPSC line (f-PLP1), 3DOM will enable real-time tracking of demyelination and remyelination processes. This work establishes BENOs and 3DOM as powerful tools for modeling myelin disorders, providing new opportunities to investigate disease mechanisms and identify therapeutic targets.
