3D culture-derived MSC EV facilitate repair by activating microglial autophagy in spinal cord injury

Spinal cord injury (SCI) is characterized by persistent neuroinflammation, neuronal loss, and limited tissue repair. Extracellular vesicles derived from human umbilical cord mesenchymal stromal cells (hUC-MSC-EVs) represent a promising cell-free therapeutic approach, although strategies for improving their bioactivity and regulating their cargo composition remain incompletely defined. Here, a gelatin microcarrier-based three-dimensional (3D) culture system was used to generate hUC-MSC-EVs. Compared with EVs generated under conventional two-dimensional (2D) culture conditions, 3D EVs reduced lesion severity, improved functional recovery, and promoted reparative-like changes in ionized calcium-binding adaptor molecule 1(Iba1)-positive microglia/macrophages in a rat SCI model. Transcriptomic, tandem-reporter, and pharmacological analyses indicated that intact autophagic processing contributes to the reparative-like phenotypic effects of 3D EVs in microglial cells. Comparative microRNA (miRNA) profiling identified miRNA-99a-5p as one of the enriched candidates, and gain- and loss-of-function experiments supported its contribution to autophagy-related and phenotype-associated responses in recipient microglia. The 3D culture condition was accompanied by increased nuclear localization of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), increased expression of Y-box binding protein 1 (YBX1), TEA domain transcription factor 4 (TEAD4) occupancy at selected YBX1 promoter regions, and higher EV-associated miR-99a-5p abundance. RNA immunoprecipitation (RIP) analysis further demonstrated an association between YBX1 and miR-99a-5p. Overall, these findings indicate that 3D culture is associated with EV cargo remodeling and improved therapeutic effects after SCI, while miR-99a-5p represents one functional EV-associated cargo contributing to selected microglial responses.