Abstract WP380: Integrative Transcriptomic and Secretome Profiling Uncovers CRISPR Targets for Improved Stem Cell Treatment in Chronic Stroke

Stem cell transplantation holds promise for promoting recovery in the sub-acute and chronic phases of stroke. With this promise comes a mystery of the molecular mechanisms underlying stem cell actions, yet illumination of these mechanisms is crucial to enhance stem cell efficacy. In this study, we utilize Translating Ribosome Affinity Purification followed by RNA sequencing (TRAP-seq) in combination with secretome profiling of cell media to identify stem cell-secreted factors that could act as molecular targets for enhancing stem cell-mediated stroke recovery. Our clinically tested human neural stem cells (NR1), modified to express a ribosomal tag, were transplanted into the cortex of adult male Sprague Dawley rats (n=5), one week following stroke induced by distal middle cerebral artery occlusion. Two days following transplantation, brains were harvested and cortical tissue, surrounding the transplantation, excised. TRAP-seq was performed on the excised tissue (i.e. Translating Ribosome Affinity Purification, followed by RNAseq) to identify gene transcripts unique to NR1 transplantation. We identified 175 different NR1 genes encoding for known secreted factors. In parallel, to characterize the NR1 secretome in vitro at the protein level, we conducted a 63-plex Luminex Assays on NR1-conditioned cell media. Integrated transcriptomic and proteomic analysis reveals a subset of stem cell-secreted proteins known to be involved in pathways of synaptic plasticity, axonal remodeling, and neuroinflammation. These are all pathways that have been previously characterized in stroke brain recovery. Four novel gene candidates[GKS1] LGALS1, SPARC, VEGFA, and CXCL12,were prioritized for further characterization and functional screening using in vitro CRISPR-based knockout strategies. Overall, our findings identify key stem cell-secreted factors with potential roles in stem cell-based neural repair enhancing stroke recovery. Utilizing gene manipulation we will further characterize stem cell-based repair.