Stroke, predominantly ischemic stroke (80% of cases), is a major cause of mortality and long-term disability globally. Current treatments like thrombectomy and thrombolysis are time-sensitive, limiting their effectiveness. The need for novel therapeutic strategies is urgent. Stem cell-based therapies have shown promise, with extracellular vesicles (EVs) secreted by stem cells playing a crucial role in paracrine effects and tissue regeneration. These EVs, nano-sized vesicles containing RNA, proteins, and other molecules, can cross the blood-brain barrier (BBB). Mesenchymal stem cells (MSCs) are attractive candidates for stem cell therapy, but their limited proliferation and age-related decline in functionality pose challenges. Induced pluripotent stem cells (iPSCs) offer an alternative, providing a plentiful source of early-passage MSCs with enhanced proliferation and differentiation capacity and eliminating ethical concerns. iPSC-derived MSCs (iPSC-MSCs) combine the benefits of both MSCs and iPSCs. However, concerns remain about immunological rejection and chromosomal variation. Therefore, hiPS-MSC-EVs may offer a cell-free therapeutic strategy that circumvents some of the limitations of cell transplantation, including immunogenicity and tumorigenicity, while retaining the therapeutic benefits.

Extensive research supports the therapeutic potential of stem cells in stroke recovery, with a growing focus on the paracrine effects mediated by EVs. Studies have shown that MSC-derived EVs can reduce infarct size, improve neurological function, and enhance angiogenesis in animal models. Allogeneic MSCs from fetal tissues are of interest due to their reduced immunogenicity and potential for off-the-shelf therapies. Previous work demonstrates that EVs derived from various MSC sources, including bone marrow and Wharton's Jelly, can enhance neurovascular regeneration and motor function post-ischemia. However, the application of hiPS-MSC-EVs in enhancing angiogenesis within mouse ischemic stroke models remains unexplored. This necessitates further research into the efficacy and mechanisms of action of these EVs.

This study employed a comprehensive approach involving both in vitro and in vivo models of ischemic stroke. **In vitro:** Human iPSCs were differentiated into hiPS-MSCs using a one-step induction protocol, characterized by morphology, multipotency (osteogenic, chondrogenic, and adipogenic differentiation), and surface marker expression (flow cytometry). hiPS-MSC-EVs were isolated using ultrafiltration and characterized by transmission electron microscopy (TEM), RNA analysis (bioanalyzer), and western blotting to confirm the presence of exosomal markers (CD9, CD63, CD81) and absence of a negative marker (calnexin). The effects of hiPS-MSC-EVs on HT22 hippocampal cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) were assessed using MTT assays (cell proliferation), flow cytometry (apoptosis), and RNA sequencing (RNA-seq) to examine the expression profile and pathways involved in ischemia. **In vivo:** Male C57BL/6 mice underwent middle cerebral artery occlusion (MCAO) to induce ischemic stroke. Mice were intravenously injected with hiPS-MSC-EVs at three different time points. Neurological function was assessed using the modified neurological severity score (mNSS) and Zea Longa score. Infarct volume was determined using H&E staining. Angiogenesis was evaluated by immunohistochemistry (IHC) for VEGF, SDF-1α, and CXCR4, and western blot analysis confirmed protein expression levels. Statistical analyses were performed using appropriate parametric tests (t-tests and ANOVA).

The in vitro studies demonstrated that hiPS-MSC-EVs effectively promoted HT22 cell proliferation, significantly reduced apoptosis, and improved cellular morphology after OGD/R injury. RNA sequencing and IPA analysis revealed differentially expressed genes associated with various biological functions and pathways related to ischemia. In the in vivo MCAO model, treatment with hiPS-MSC-EVs led to a significant reduction in infarct volume compared to the control group (MCAO+PBS). Moreover, hiPS-MSC-EVs significantly improved spontaneous movement abilities and neurological deficits, as evidenced by the Zea Longa and mNSS scores, at day 28 post-treatment. Immunohistochemical and Western blot analyses revealed a significant increase in VEGF and CXCR4 expression in the infarcted hemisphere of the MCAO+EV group compared to the MCAO+PBS group, indicating enhanced angiogenesis. These data suggest a dose-dependent effect of hiPS-MSC-EVs on cell viability and proliferation.

The findings of this study strongly support the neuroprotective effects of hiPS-MSC-EVs in both in vitro and in vivo models of ischemic stroke. The ability of hiPS-MSC-EVs to reduce apoptosis, promote cell proliferation, and enhance angiogenesis contributes to their neuroprotective mechanism. The improved neurological outcome observed in MCAO mice following hiPS-MSC-EV treatment highlights their therapeutic potential. This cell-free approach offers advantages over cell-based therapies, circumventing challenges associated with immune rejection and tumorigenicity. The results are consistent with previous studies demonstrating the beneficial effects of MSC-derived EVs in stroke models, providing further validation for this therapeutic strategy. However, some discrepancies exist between the current results and findings from other studies concerning post-stroke outcomes following stem cell-derived exosome administration, potentially attributed to variations in species, treatment protocols, and stem cell origins.

This study demonstrates the significant neuroprotective effects of hiPS-MSC-EVs in both in vitro and in vivo ischemic stroke models. hiPS-MSC-EVs effectively reduce infarct size, improve neurological function, and promote angiogenesis. These findings support the development of hiPS-MSC-EVs as a promising cell-free therapeutic strategy for ischemic stroke. Future research should focus on larger-scale studies, including female subjects, to validate these findings and investigate the long-term effects and potential side effects of this therapy. Further mechanistic studies are needed to fully elucidate the complex pathways involved in the neuroprotective actions of hiPS-MSC-EVs.

Several limitations should be considered when interpreting the results. The relatively small sample size might limit the statistical power and generalizability of the findings. The study only used male C57BL/6 mice, limiting the extrapolation to female subjects and potentially excluding gender-specific effects. The mouse model may not fully replicate the complexities of human ischemic stroke. The in vitro OGD/R model simplifies the in vivo environment. A broader spectrum of angiogenesis markers would provide a more comprehensive understanding. Lastly, the observation period could be extended to assess long-term effects and potential side effects.