Protocol for assessing myelination by human iPSC-derived oligodendrocytes in Shiverer mouse ex vivo brain slice cultures

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Human induced pluripotent stem cell (iPSC)-derived oligodendrocytes are a powerful tool for studying aberrant myelination in neurodegenerative and neurodevelopmental disorders; however, they often fail to myelinate in vitro . Here, we present a protocol for axonal ensheathment and perinodal segmentation using an ex vivo model. We describe steps for preparing Shiverer mouse brain slice cultures, oligodendrocyte transplantation, visualization, and analysis. This approach suits multiple culture formats, highlighting the potential for the screening of myelin-modulating drugs and compounds in a cost- and time-effective manner, while reducing animal use. • Instructions for the generation and culture of mouse organotypic brain sections • Procedures for the transplantation of human iPSC-derived oligodendrocytes • Steps for analyzing myelin and oligodendrocyte complexity via confocal microscopy • Compatibility of the approach with human, mouse, and rat oligodendroglia Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Human induced pluripotent stem cell (iPSC)-derived oligodendrocytes are a powerful tool for studying aberrant myelination in neurodegenerative and neurodevelopmental disorders; however, they often fail to myelinate in vitro . Here, we present a protocol for axonal ensheathment and perinodal segmentation using an ex vivo model. We describe steps for preparing Shiverer mouse brain slice cultures, oligodendrocyte transplantation, visualization, and analysis. This approach suits multiple culture formats, highlighting the potential for the screening of myelin-modulating drugs and compounds in a cost- and time-effective manner, while reducing animal use.