Abstract WP136: Grafted Human Neural Stem Cell-Secreted Matrilin-2 Improves Functional Recovery in Ischemic-Stroke Rats

Abstract

Identifying the secretome of grafted stem cells is an important first step to elucidate how the graft communicates with the host brain, at the molecular level , to enhance post-stroke recovery. Here we use Translating Ribosome Affinity Purification (TRAP) to uncover the secretome of grafted human neural stem cells (hNSCs) and identify potential efficacious stem cell factors. Nude rats were subjected to permanent distal cerebral artery occlusion; 7d later hNSCs expressing GFP under a ribosomal-specific promoter were transplanted into the cortex of stroke (S) and naive (N) rats. The graft site was dissected 7d later and processed using TRAP to isolate ribosome-bound RNA from the grafted hNSCs. TRAP-isolated RNA was subjected to RNA sequencing and 479 differentially expressed genes were found between hNSC grafts in S and N brains. Of these, 21 genes encode secreted proteins, 14 of which were upregulated in hNSCs grafts in the stroke-injured brain. Pearson Correlation analysis revealed that the expression level of 9 of the 14 genes positively correlated with functional recovery. Gene Ontology (GO) analysis of the 14 genes identified the top enriched biological process as Response to Wounding which is related (i.e. a direct ancestor in the GO tree) to inflammatory response and axon injury. Three genes were present in this GO term: NOG, MATN2 and WNT1. MATN2 encodes matrilin-2, a protein known to be involved in axon regeneration and inflammation. In situ hybridization and immunohistochemistry showed high expression of matrilin-2 in grafted hNSCs. To test if matrilin-2 could affect post-stroke recovery we infused human recombinant matrilin-2 or vehicle into the cortex of stroke-injured rats. Seven days post-infusion, rats receiving matrilin-2 showed greater behavior recovery than the vehicle group. As matrilin-2 promotes axon outgrowth we investigated if it activated Rac-1 GTPase, which is associated with axon sprouting. Recombinant matrilin-2 increased activation of Rac-1 in primary mouse neural cultures, compared to the control. Our data show that grafted hNSCs in the stroke-injured brain express MATN2 and that secreted matrilin-2 may be involved in hNSC-induced stroke recovery potentially via activation of Rac-1 GTPase and axon sprouting.