Periodic Reporting for period 1 - SCIENSC (Identification of Transiently Formed Immune-Endogenous Neural Stem Cell Niches in Spinal Cord Injury)
Reporting period: 2023-09-01 to 2025-08-31
Spinal cord injury (SCI) is a devastating condition of the nervous system, often leading to lifelong paralysis with no effective treatment. After an injury, immune cells rapidly invade the spinal cord, and at the same time a small pool of endogenous neural stem cells (eNSCs) becomes activated. These eNSCs have an intrinsic potential to aid the injured spinal cord repair, but due to the restrictive spinal cord cellular and molecular environment they mostly turn into glial scar-forming cells that in the long run restrict repair.
The SCIENSC project was designed to uncover how the immune system communicates with neural stem cells, with the aim of determining if and which immune signals block or promote regeneration and establish new experimental models that allow these interactions to be studied in a reproducible, ethical and high-throughput manner.
Three objectives guided the project:
1. Map spatiotemporal immune-cell dynamics after spinal-cord injury and identify cell populations that interact with eNSCs.
2. Characterise molecular communication pathways between immune cells and eNSCs that influence stem cell cellular behaviour.
3. Lay the groundwork for identifying DNA enhancer elements that are specifically activated by injury in key immune cells, paving the way for targeted, tissue and context-dependent therapeutic modulation.
The project resulted in generating new biological insights into neuro-immune regulation of repair and also delivered a modular 3D spinal cord injury organoid model that can reduce animal use and accelerate therapeutic discovery. These advances support the EU’s strategic goals in health research, open science, and the 3Rs principle (replacement, reduction and refinement of animal experimentation).
The SCIENSC project was designed to uncover how the immune system communicates with neural stem cells, with the aim of determining if and which immune signals block or promote regeneration and establish new experimental models that allow these interactions to be studied in a reproducible, ethical and high-throughput manner.
Three objectives guided the project:
1. Map spatiotemporal immune-cell dynamics after spinal-cord injury and identify cell populations that interact with eNSCs.
2. Characterise molecular communication pathways between immune cells and eNSCs that influence stem cell cellular behaviour.
3. Lay the groundwork for identifying DNA enhancer elements that are specifically activated by injury in key immune cells, paving the way for targeted, tissue and context-dependent therapeutic modulation.
The project resulted in generating new biological insights into neuro-immune regulation of repair and also delivered a modular 3D spinal cord injury organoid model that can reduce animal use and accelerate therapeutic discovery. These advances support the EU’s strategic goals in health research, open science, and the 3Rs principle (replacement, reduction and refinement of animal experimentation).
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.
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.
The SCIENSC project produced several advances that go beyond current knowledge and methodology:
- Discovery of rapid adaptive-immune cell engagement: the identification of an early, spatially directed T-cell infiltration into the spinal cord where they directly contact endogenous neural stem cells.
- Mechanistic insight into regeneration control: single-cell and functional assays linked IFN-γ signalling to the suppression of eNSC proliferation, providing a new explanation for limited regenerative capacity.
- Development of a novel spinal-cord organoid model: this is the first system derived from injury-activated eNSCs that reproduces fibrosis, inflammation and stem cell dynamics in a controlled setting, enabling 3Rs-compliant mechanistic and drug-screening studies.
- Foundation for enhancer-based precision modulation: the definition of IFN-γ⁺ TRM-like T cells as a specific target population and the establishment of single-cell enhancer-mapping pipelines set the stage for future enhancer-driven tools to fine-tune immune activity in tissue repair.
To ensure further success and completion, next steps include high-throughput enhancer mapping, validation of candidate regulatory elements in the organoid and in vivo models with a long-future goal of developing novel regenerative/immunomodulatory strategies.
- Discovery of rapid adaptive-immune cell engagement: the identification of an early, spatially directed T-cell infiltration into the spinal cord where they directly contact endogenous neural stem cells.
- Mechanistic insight into regeneration control: single-cell and functional assays linked IFN-γ signalling to the suppression of eNSC proliferation, providing a new explanation for limited regenerative capacity.
- Development of a novel spinal-cord organoid model: this is the first system derived from injury-activated eNSCs that reproduces fibrosis, inflammation and stem cell dynamics in a controlled setting, enabling 3Rs-compliant mechanistic and drug-screening studies.
- Foundation for enhancer-based precision modulation: the definition of IFN-γ⁺ TRM-like T cells as a specific target population and the establishment of single-cell enhancer-mapping pipelines set the stage for future enhancer-driven tools to fine-tune immune activity in tissue repair.
To ensure further success and completion, next steps include high-throughput enhancer mapping, validation of candidate regulatory elements in the organoid and in vivo models with a long-future goal of developing novel regenerative/immunomodulatory strategies.