Opportunities and challenges for the application of single-cell RNA sequencing and spatial transcriptomics in...

Ischemic stroke (IS) is characterized by high rates of morbidity, disability and mortality. The pathological process underlying IS involves spatiotemporal dynamic responses of multiple cell types and marked cellular heterogeneity which cannot be fully elucidated using traditional bulk sequencing technologies. Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) have provided high-resolution tools for investigating pathological mechanisms and developing precise diagnostic and therapeutic strategies for IS. In this review, we systematically summarize the core principles, mainstream platform characteristics and applicability of these two technologies in IS research, focusing particularly on how they have helped to reveal dynamic cellular evolution following ischemia, spatial molecular heterogeneity between the infarct core and penumbra, and key differences between human and mouse models. We also review key breakthroughs, including the LGALS9-CD44 repair signaling axis, the optimization of oligodendrocyte precursor cell (OPC) transplantation, and the molecular classification of IS. In addition, we discuss the potential for this technology to be translated clinically, and analyze the levels of accompanying evidence for relevant research findings from four dimensions: the screening of early diagnostic biomarkers, the development of novel therapeutic targets, improving molecular understanding for cell therapy, and the construction of disease classifications at the molecular level. We also highlight potential core challenges including the low capture efficiency of brain cells, inherent contradiction between spatial resolution and sensitivity, scarcity and insufficient standardization of human samples, and prominent obstacles in translating preclinical results to clinical practice. In future, the optimization of technologies that are specific to brain tissue, the construction of multicenter large-sample human stroke databases, and the implementation of cross-species validation studies, will help to promote the application of scRNA-seq and ST from basic mechanistic analysis to precise clinical application in the field of IS, thereby providing scientific support for improving the diagnosis and treatment of IS.