(G-273) Recovery of Cerebral Ischemia by Application of Extracellular Vesicles from 3D Aggregated Adult Human Mesenchymal Stem Cells as Evaluated by High Field MRI at 21.1 T

Introduction:: Stroke is a leading cause of death and disability worldwide [1]. There are limited treatment options for ischemic strokes. Tissue plasminogen activator (tPA) is the only FDA approved drug for ischemic stroke treatment, which works by dissolving blood clots to restore blood flow to the brain; however, it has a therapeutic window of only 4.5 h [2]. The use of human mesenchymal stem cells (hMSC) has shown promise as a biotherapeutic for ischemic stroke. This work compares the efficacy of extracellular vesicles harvested from 3D hMSC aggregates (Agg-D hMSC) in a female rodent model.


Materials and Methods:: hMSC were aggregated at passage 4 using a WAVE Bioreactor [3]. EV were harvested from aggregates, labeled with 1 mg/mL of ultra-small iron-oxide nanoparticles and purified for injection using ultracentrifugation [4]. A transient middle cerebral artery occlusion model was instituted in female Sprague-Dawley rats for 1 h [5]. Immediately following the occlusion, the animals received the treatment (EV or saline control; n=5) via intra-arterial injection. MR experiments were acquired using the 21.1-T vertical bore magnet at the NHMFL. In vivo assessment utilized a linear birdcage double-tuned ²³Na/¹H radio frequency coil on 0, 1, 3, 7 and 21-d post-ischemia to monitor tissue recovery. Cell administration was confirmed with gradient recalled echo images (50x50-μm in-plane resolution). Traditional lesion volume was evaluated using T₂W-RARE at a 100x100-μm resolution, while functional recovery was assessed with a 3D ²³Na CSI acquired at 1-mm isotropic resolution. Using a 3x3x3-mm³ voxel placed in the ipsilateral and contralateral hemispheres, semi-LASER spectroscopy evaluated metabolic changes.


Results, Conclusions, and Discussions:: 3D ²³Na CSI data were reconstructed in MATLAB to a zero-filled 0.5-mm isotropic resolution. The volumetric and signal analysis for the 3D ²³Na CSI and T₂W were performed in Amira 3D Visualization software. A signal threshold generated from the contralateral hemisphere was used to define the ischemic lesion. MR spectroscopy data was processed in Topsin to monitor metabolites (total choline-Cho, total creatine-Cre, N-acetyl aspartate-NAA and lactate) longitudinally.
3D ²³Na CSI and T₂W RARE data show that lesion volume of the group that received the EV treatment decreased from day 1 to 3, whereas the group that received the saline control treatment had an increase in lesion volume over this period. By day 7, both groups began to exhibit a decrease in the lesion volume (Figure 1A). On Day 1, MR spectroscopy data showed elevated levels of lactate in the ischemic lesions (Figure 1B).

MR data indicate that the lesion reccovery following the transplantation of the 3D-EV resulted in improved recovery compared to the control PBS group in the male rat model.


Acknowledgements (Optional): : All work has been conducted in accordance with the Florida State University Animal Care and Use Committee. This work was supported by the NIH (R01-NS102395 awarded to SCG; R01-NS125016 to YL). The National High Magnetic Field Laboratory is funded by the NSF (DMR-1644779) and the State of Florida.


References (Optional): : 1. Rexrode KM, Kathryn M. Rexrode Correspondence to: Kathryn M. Rexrode, Madsen TE, et al (2022) The impact of sex and gender on stroke. In: Circulation Research. https://www.ahajournals.org/doi/10.1161/CIRCRESAHA.121.319915. Accessed 9 Nov 2022

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3. Xuegang Yuan, Jens T. Rosenberg, Yijun Liu, Samuel C. Grant, Teng Ma, Aggregation of human mesenchymal stem cells enhances survival and efficacy in stroke treatment, Cytotherapy, Volume 21, Issue 10, 2019, Pages 1033-1048, https://doi.org/10.1016/j.jcyt.2019.04.055

4. Yuan X, Sun L, Jeske R, Nkosi D, York SB, Liu Y, Grant SC, Meckes DG Jr, Li Y. Engineering extracellular vesicles by three-dimensional dynamic culture of human mesenchymal stem cells. J Extracell Vesicles. 2022 Jun;11(6):e12235. doi: 10.1002/jev2.12235. PMID: 35716062; PMCID: PMC9206229.

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