Abstract: BACKGROUND: Mesenchymal stem cells have shown good therapeutic effects in ischemic stroke, while the role of multilineage-differentiating stress-enduring (Muse) cells isolated from adipose-derived mesenchymal stem cells in ischemic stroke needs further study. 
OBJECTIVE: To explore the neurorestorative effect of intravenous administration of adipose Muse cells on ischemic stroke in rats.
METHODS: The Muse cells expressing stage-specific embryonic antigen 3 were sorted by magnetic beads after long-term (4 hours) trypsin incubation of human adipose-derived mesenchymal stem cells. The middle cerebral artery occlusion model was established in rats. After successful modeling, the adipose-derived mesenchymal stem cell group and adipose-derived Muse group were injected with 200 μL adipose-derived mesenchymal stem cell suspension or adipose-derived Muse cell suspension (containing 2×105 cells) via the tail vein. The saline group was injected with 200 μL saline. Behavioral scores of rats were evaluated at 3 and 7 days after injection. At 3 days after injection, hematoxylin-eosin staining was used to detect brain tissue damage. Immunofluorescence was used to detect the expression of microtubule-associated protein 2 and Ki67 in the brain tissue injury area. TUNEL staining was used to observe cell apoptosis in the injury area. Western blot assay was used to detect the protein expression of growth-associated protein 43, Bcl2, and Bax. 
RESULTS AND CONCLUSION: (1) After magnetic bead sorting, the flow cytometry showed that the expression rate of stage-specific embryonic antigen 3 was as high as 80%. (2) At 7 days after injection, the neurological function injury scores of the adipose mesenchymal stem cell group and the adipose Muse cell group were reduced compared with those of the saline group (P < 0.05). At 3 days after cell transplantation, the neurological function injury scores of the adipose Muse cell group were reduced compared with those of the adipose mesenchymal stem cell group (P < 0.05). (3) Hematoxylin-eosin staining showed that the inflammatory response and vacuolation in the cerebral cortex of rats in the adipose mesenchymal stem cell group and the adipose Muse cell group were alleviated. Microtubule-associated protein 2 fluorescence staining showed that the adipose Muse cell group inhibited neuronal loss more significantly. (4) TUNEL staining and Ki67 fluorescence staining results showed that apoptosis of cells in the injured area of the adipose mesenchymal stem cell group and the adipose Muse cell group was reduced, and cell proliferation was increased. (5) Western blot assay results showed that the Bcl-2/Bax ratio and growth-associated protein 43 protein expression in the adipose mesenchymal stem cell group and the adipose Muse cell group were increased, and the adipose Muse cell group was superior to the adipose mesenchymal stem cell group in inhibiting cell apoptosis. The results showed that the nerves of rats transplanted with adipose mesenchymal stem cells and adipose Muse cells were well repaired, and Muse cells played a better role in inhibiting cell apoptosis.

Key words: adipose mesenchymal stem cell, Muse cell, ischemic stroke, stage-specific embryonic antigen 3 (SSEA3), multilineage-differentiating stress-enduring cells