ROS-responsive exogenous functional mitochondria can rescue neural cells post-ischemic stroke

Background: The transfer of mitochondria from healthy mesenchymal stem cells (MSCs) to injured MSCs has been shown to have potential therapeutic benefits for neural cell post-ischemic stroke. Specifically, functional mitochondria can perform their normal functions after being internalized by stressed cells, leading to host cell survival. However, while this approach shows promise, there is still a lack of understanding regarding which neural cells can internalize functional mitochondria and the regulatory mechanisms involved. To address this gap, we investigated the ability of different neural cells to internalize exogenous functional mitochondria extracted from MSCs. Methods: Functional mitochondria (F-Mito) isolated from umbilical cord derived-MSCs (UCMSCs) were labeled with lentivirus of HBLV-mito-dsred-Null-PURO vector. The ability of stressed cells to internalize F-Mito was analyzed using a mouse (C57BL/6 J) middle cerebral artery occlusion (MCAO) model and an oxygen-glucose deprivation/reoxygenation (OGD/R) cell model. The cell viability was measured by CCK-8 kit. Time-course of intracellular ROS levels in stressed cells were analyzed by DCFH-DA staining after OGD/R and F-Mito treatment. MitoSOX, Mitotracker and WGA labeling were used to assess the relationship between ROS levels and the uptake of F-Mito at the single-cell level. Pharmacological modulation of ROS was performed using acetylcysteine (ROS inhibitor). Results: Our findings demonstrate that neurons and endothelial cells are more effective at internalizing mitochondria than astrocytes, both in vitro and in vivo, using an ischemia-reperfusion model. Additionally, internalized F-Mito decreases host cell reactive oxygen species (ROS) levels and rescues survival. Importantly, we found that the ROS response in stressed cells after ischemia is a crucial determinant in positively mediating the internalization of F-Mito by host cells, and inhibiting the generation of ROS chemicals in host cells may decrease the internalization of F-Mito. These results offer insight into how exogenous mitochondria rescue neural cells via ROS response in an ischemic stroke model. Overall, our study provides solid evidence for the translational application of MSC-derived mitochondria as a promising treatment for ischemic stroke.


(B). The Fluorescent images demonstrated the internalization of F-Mito into endothelial cells
within normal area (red rectangle) and the lesion area (yellow rectangle).

(C). The Fluorescent images demonstrated the internalization of F-Mito into astrocytes within
normal area (red rectangle) and the lesion area (yellow rectangle).

(D).
The Fluorescent images demonstrated the internalization of F-Mito into microglial cells within normal area (red rectangle) and the lesion area (yellow rectangle).
(E). The proportion of cells with F-Mito in brain was represented, indicating statistically significant differences in comparisons made between the normal and injured brain tissues. The level of significance was denoted by *p < 0.05, **p < 0.01, and ***p < 0.001, as determined by Student t-test. The data were presented as means ± standard deviation (s.d.), and the study was conducted on three animals in each group.

Supplementary Fig 9. The treatment of F-Mito reduced intracellular ROS and restored cell viability after OGD/R in neurons.
The study was performed on primary neurons, and the results were reported for a period of 24 hours after OGD/R. The treatment with F-Mito led to a restoration of cell viability and a reduction in intracellular ROS levels.
(A-B). The intracellular ROS levels and cell viability in mice primary neurons over time after OGD/R. Statistical analysis was conducted using One-way ANOVA with Bonferroni correction, and significance was considered when samples were compared to the control group at their respective time points (*p < 0.05, **p < 0.01, ***p < 0.001). The data were presented as means ± s.d., n = 3 independent experiments. 11

Supplementary Fig 10. The treatment of F-Mito reduced intracellular ROS and restored cell viability after OGD/R in astrocytes.
The study was performed on astrocytes, and the results were reported for a period of 24 hours after OGD/R. The treatment with F-Mito led to a restoration of cell viability and a reduction in intracellular ROS levels.

(A-B). The intracellular ROS levels and cell viability in astrocytes over time after OGD/R.
Statistical analysis was conducted using One-way ANOVA with Bonferroni correction, and significance was considered when samples were compared to the control group at their respective time points (*p < 0.05, **p < 0.01, ***p < 0.001). The data were presented as means ± s.d., n = 3 independent experiments. Bonferroni correction, and the significance levels were set at *p < 0.05, **p < 0.01, and ***p < 0.001. The data were presented as means ± s.d. and were obtained across three independent experiments. Bonferroni correction, and significance was considered when samples were compared to the OGD/R group or in comparisons between two different samples under a given line (*p < 0.05, **p < 0.01, ***p < 0.001). The data were presented as means ± s.d., n = 3 independent experiments.

Supplementary
(B-C). Fluorescent images were captured to illustrate the entry of F-Mito into PC-12 and bEnd.3 cells, respectively, after 6 hours in the OGD/R-NAC-F-Mito or OGD/R-F-Mito group.
The statistics of the comparison of the F-Mito positive cell proportion in PC-12 and bEnd.3 cells were presented on the right side of the panels. All statistical analyses were performed