Stroke lesion environments alter wound repair functions of cell grafts

Introduction:: Stroke is one of the leading causes of death worldwide with time-limited treatment options.¹ Strokes can either be ischemic or hemorrhagic¹, with ischemic strokes evoked by occlusion of blood vessels in the brain while hemorrhagic strokes due to direct blood vessel rupture¹. In the mammalian adult, both stroke injury modes provoke formation of lesions with cores that are devoid of neural elements and dominated by fibrotic scarring that impairs neurological recovery². In neonates, glia-based repair mechanisms coordinate a scar-free wound-healing response after injury that aids neural circuit regeneration³. Similar glia-based repair can be replicated in the adult using cell grafting⁴. However, cell graft outcomes are dramatically altered by injury mode (ischemic vs hemorrhagic) and lesion size resulting in ineffective glia repair in the most severe injuries⁴. For example, Neural Progenitor Cell (NPC) grafts differentiate into wound repair astrocytes in small ischemic lesions, but these phenotypes, and overall graft survival, are perturbed in large ischemic lesions as well as hemorrhagic injuries (Fig1A). For this reason, we are developing cell conditioning paradigms that will improve graft survival and retainment of astrocyte phenotype when applied to these more severe injuries. We hypothesize that grafting engineered immature astroglia at sub-acute time points after large ischemic and hemorrhagic strokes will improve glia repair outcomes by cell grafts to enhance recovery of vasculature with glial barrier functions and minimize fibrotic scarring.


Materials and Methods:: We have engineered immature astrocytes with glia repair competency similar to neonate glia using hysteretic conditioning (HC). Mouse embryonic stem cells (mESC) that constitutively express the ribosomal protein Rp122 with a hemagglutinin (HA) tag (RiboTag) were used to generate passagable Neural Progenitor Cells (NPC) that were differentiated into either post mitotic astrocytes (PmA) or engineered into immature astrocytes (ImA). ImA were derived by transient and cyclical exposure of NPC to differentiation media and NPC media. Expression of astrocyte (Gfap, S100b), proliferation (Top2a, Ki67) and immaturity (Igfbp2) phenotypes were determined by qPCR and immunocytochemistry. The glia repair competency of cell graft candidates was evaluated in vitro using high serum exposure testing to simulate lesion environments and scratch assay. NPC, ImA and PmA were transplanted into striatal ischemic- (L-NIO injection) or hemorrhagic (Collagenase injection) strokes at 48 hours post injury and tissue was collected at two weeks. Tissue was evaluated by immunohistochemistry with proliferation (Top2a), astrocyte (s100b, Gfap), myeloid lineage (Cd13) and neuronal (NeuN) antibody staining. Image analysis and quantification was conducted on the astroglia border, immune response, graft survival and lesion size.


Results, Conclusions, and Discussions:: ImA show simultaneous expression of astrocyte and proliferation markers characteristic of immature astrocytes (Fig1B). ImA had attenuated EMT (Epithelial to Mesenchymal Transition)-like transitions compared to NPC upon exposure to simulated wound environments in vitro by a multidimensional analysis EMT score incorporating quantifications for nuclear size, cytoplasmic area, Gfap protein expression and F-actin fiber density (Fig1C). ImA show enhanced cell survival and retention of wound repair astrocyte phenotype compared to PmA and significant decrease fibrotic scarring at lesions (Fig1D).

Conclusion: We developed and validated a new method for deriving immature astrocytes by HC. ImA show enhanced resistance to EMT-like transitions, which is promising for improving graft outcomes in hemorrhagic lesions in vivo. Despite being at different cell states prior to grafting both NPC and ImA adopt wound repair astrocyte phenotypes to minimize lesion size when grafted into ischemic strokes. We are currently evaluating graft outcomes in hemorrhagic strokes. This work will shed further light on cell and non-cell autonomous regulation of cell graft outcomes in stroke.


Acknowledgements (Optional): : We are grateful to Boston University Core Facilities and Staff. This work was supported by funding from Boston University (T.M.O), Craig H. Neilsen Foundation (T.M.O.) and Schlumberger Foundation (H.A).


References (Optional): : 1. Burda, J.E & Sofroniew, M.V. (2014). Neuron. 2. Dorrier, C. E. et al. (2021). Neuroscience. 3. Li, Y et al., (2020). Nature. 4. O’Shea T.M., et al., (2022). Nature Comm.