Three-dimensional dynamic culture and hypoxic preconditioning synergistically enhance the therapeutic effects of GMSCs on intracerebral hemorrhage

Intracerebral hemorrhage (ICH) is a common cerebrovascular disorder. There is still a high risk of severe neurological deficits even when patients survive ICH. The application of new treatment techniques can reduce mortality, but reversing neurological damage caused by ICH remains challenging. Although cell-based therapies have shown promise in treating cerebral hemorrhage, recurring challenges are premature apoptosis of transplanted cells accompanied by insufficient numbers and lack of engraftment in the target tissue, thus limiting therapeutic efficacy. Here, we show that hypoxic preconditioning combined with three-dimensional (3D) dynamically cultured gingival lamina propria-derived MSCs (GMSCs) significantly improves cell viability, anti-apoptosis ability, and therapeutic efficacy compared with cells cultured in traditional monolayers. Hypoxia-preconditioned 3D-GMSCs also exhibit notable neuroprotective and promote extracellular vesicles (EVs) biogenesis potential as evidenced by transwell in vitro intervention experiments and RNA-seq; further confirmed by characterization analysis of EVs. Furthermore, 3D-GMSCs-EVs derived from hypoxic preconditioning not only had better cell and tissue uptake capabilities but also reduced bleeding in mice with cerebral hemorrhage and improved motor function. Subsequent in vitro experiments on cerebral hemorrhage found that hypoxia-preconditioned 3D-GMSCs-EVs promoted the reduction of neuronal oxidative stress levels and mitochondrial function recovery. This study innovated the culture method of GMSCs, demonstrated effective modification of EVs, and provided new ideas for establishing a cell-free treatment for cerebral hemorrhage.