Integrative single cell analysis of progressive multiple sclerosis induced neural stem cells reveals epigenetically dysregulated type-I-interferon signaling
Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system, which currently lacks effective therapies that provide regeneration and stop disease progression. A suggested link is anticipated between the development of progressive MS (PMS) and aging, as suggested by recent work identifying hallmarks of cellular senescence in numerous cell types both in vitro with patient cell lines and in vivo in the post-mortem MS brain, including neural stem cells (NSCs). Using an inducible system, directly reprogramming human fibroblasts into induced NSCs (iNSCs), we generated control and PMS iNSC cell lines, which was found to maintain epigenetic age. Here we performed multi-omics, including bulk RNA, single-cell (sc) RNA, single-nucleus (sn) RNA and ATAC, and whole genome bisulfite sequencing. Here, we show that PMS iNSCs have overall increases in senescence and inflammatory associated genes using bulk sequencing. We next identified a unique subcluster of cells within the PMS iNSCs that have a pro-inflammatory and senescent phenotype, associated with interferon gene activation using sc-RNA seq. Within this specific subcluster there is higher expression of genes associated with inflammatory protein secretion, as well as the senescence associated secretory phenotype (SASP). A similar clustering pattern was observed in the ATAC-seq data, a large main cluster and a small inflammatory cluster primarily comprising of PMS iNSCs. Differentially accessible regions within the PMS cluster revealed promoter sites of inflammatory-associated genes to be more accessible correlating with gene expression. Our results highlight a novel cellular mechanism in PMS wherein NSCs are interferon-responsive and senescent-like which may impact other cells within the lesion leading to chronic inflammation. Overall design: iNSC lines, between passages 25-30, were plated at 500,000 cells per well in a growth factor reduced matrigel coated 6 well plate. After 24 hours, the media was changed to fresh media. Cells were harvested in RLT lysis buffer 72 hours after plating then frozen at -80°C until extraction. RNA extraction was performed according to the RNeasy kit followed by DNase treatment (Qiagen). This accession refers to the bulk mRNAseq data.