A Human iPSC-derived Organoid System to Model Myelin Destruction and Repair

We engineered a novel human iPSC-derived organoid system enriched with mature, myelinating oligodendrocytes and functionally reactive microglia. This model enables the study of human CNS remyelination following a demyelinating insult, encapsulating: myelin fragmentation, microglial clearance of myelin debris and oligodendrocyte genesis, differentiation, and axon ensheathment. This system provides a powerful and unparalleled capacity to interrogate complex human cell behaviour, relevant to those studying glial biology and neurological disease mechanisms. Dissociation of organoids into single cells for transcriptomic analysis was performed as per manufacturer’s protocol using a Papain Dissociation System (Worthington Biochemical). Briefly, 16 organoids per group were incubated in 20 U/mL papain enzyme solution at 37°C for 60 min. Every 15 min, gentle trituration was applied to each sample to break up cell clusters into a single cell suspension. After the digestion, DNase-protease inhibitor solution was added to single cell suspensions followed by centrifugation at 300 x g for 5 min. Cell pellets were then processed with the Chromium Gene Expression Flex Library Prep kit (10x Genomics) as per manufacturer’s protocol. Briefly, single cells were fixed overnight at 4 °C using 4 % PFA (ProSciTech) diluted in Concentrated Fix & Permealisation buffer (from Chromium Gene Expression Flex Library Prep kit). After fixation, cells were pelleted at 850 x g for 5 min. Cell pellets were quenched with Quenching and Enhancer buffer (from Chromium Gene Expression Flex Library Prep kit). Glycerol was added to each sample to a final concentration of 25 % prior to storing in -80 °C. Library preparation and sequencing were performed by AGRF sequencing facility (Australia). A single-cell suspension (~24,000 cells) was captured from all isolated cells per sample, without selection. Data was generated with the Illumina BCL Convert 4.1.5 pipeline.