During the fellowship reporting period, the project combined in-vivo mapping, single-cell genomics and advanced organoid modelling to dissect and reconstruct how immune signals shape neural stem cell behaviour.
Objective 1: Mapping spatiotemporal immune responses after spinal cord injury. Comprehensive immunophenotyping of injured and control spinal cords at several time points (1-, 3-, 5- and 7-days post-injury) revealed expected early neutrophil and macrophage responses and, unexpectedly, an early T-cell infiltration. Immunohistochemistry showed that these T cells entered from the ventral side via sulcal arteries and Virchow-Robin spaces and made direct contact with eNSCs around the spinal cord central canal providing evidence of a rapid adaptive-immune interaction with the stem-cell niche.
Objective 2: Decoding molecular pathway contributing to immune and neural stem cell interactions. To capture molecular interactions at single-cell, high resolution, initially proposed spatial transcriptomics was replaced by single-cell RNA sequencing and multi-omic (RNA+ATAC) analyses. Computational models predicted that IFN-γ produced by T cells inhibits eNSC proliferation, whereas TGF-β from myeloid cells promotes astroglial cell proliferation. These predictions were tested in a newly established eNSC-derived spinal-cord organoid system. Cytokine-screen assays have so far confirmed some of these predictions: IFN-γ suppressed, while TGF-β stimulated organoid growth. Multi-omic analyses on eNSC and their lineages within a newly developed organoid identified transcription factors such as Ascl1 and Sox9, AP-1 that drive neurogenesis or astrogliosis.
Objective 3: Groundwork for immune cell of interest enhancer discovery. Using optimised tissue dissociation method and a flow-cytometry panel, T cells were isolated from spinal cord and spleen and profiled using Smart-seq3. Analysis revealed an IFN-γ-producing tissue-resident-memory-like (TRM-like) T-cell population in the spinal cord that likely limits early eNSC proliferation to control excessive cell proliferation. Computational pipelines for single-cell ATAC-seq enhancer mapping were benchmarked and are ready for immediate application to this defined T-cell subset.
Together, the project delivered an in-vivo and in-vitro framework to dissect immune control of neural stem cells and provided a reproducible 3D organoid model for future mechanistic and screening studies.
