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. Overall design: 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.