Mesenchymal stem cell-derived extracellular vesicles ameliorate Alzheimer's disease-like phenotypes in a preclinical mouse model

Abstract

Alzheimer's disease (AD) is an irreversible neurodegenerative disorder that affects more than 44 million people worldwide. Despite the high disease burden, there is no effective treatment for people suffering from AD. Mesenchymal stem cells (MSCs) are multipotent stromal cells that have been widely studied due to their therapeutic potential. However, administration of cells has been found to have a multitude of limitations. Recently, extracellular vesicles (EVs) derived from MSCs have been studied as a therapeutic candidate, as they exhibit similar immunoprotective and immunomodulatory abilities as the host human MSCs. Methods: To test the potential therapeutic effects of MSC EVs, human bone-marrow derived MSCs were grown in three-dimensional (3D) cell culture, and small EVs were harvested using differential ultracentrifugation. These small EVs were given to non-transgenic (NT) or 5XFAD (5 familial Alzheimer's disease mutations) mice intranasally (IN) every 4 days for 4 months. The mice were then required to perform a variety of behavioral assays to measure changes in learning and memory. Afterwards, immunohistochemistry was performed on brain slices to measure amyloid beta (Aβ) and glial fibrillary acidic protein (GFAP) levels. Results: The data revealed that 5XFAD mice that received hMSC-EV treatment behaved significantly better in cognitive tests than saline treated 5XFAD mice, with no significant change between EV-treated 5XFAD mice and NT mice. Additionally, we found lower Aβ plaque load in the hippocampus of the EV-treated mice. Finally, less colocalization between GFAP and Aβ plaques was found in the brain of EV-treated mice compared to saline. Conclusions: Taken together, these data suggest that IN administration of MSC-derived EVs can slow down AD pathogenesis.

Keywords: Alzheimer's disease; exosomes; extracellular vesicles; mesenchymal stem cells; microvesicles.

Figure 1

Characterization of 3D hMSC small EVs. (A) Image of 2D versus 3D cell growth. Scale bar = 100 μm. (B) Immunoblot analysis of hBM MSC-derived sEVs. (C and D) Nanoparticle tracking analysis of hMSC-EVs. (E) Electron microscopy of sEVs.

Figure 2

Treatment with hMSC-derived EVs ameliorate cognitive decline of 5XFAD mice. (A) Mice were tested for memory using novel object recognition test (NORT). hMSC-EV treated 5XFAD mice performed significantly better than saline treated 5XFAD mice. N=6. (B) Y-maze test was used to determine spatial working memory. hMSC-EV treated 5XFAD mice performed significantly better than saline treated 5XFAD mice. N=6. Finally, mice were tested for spatial learning and memory using the Barnes maze. hMSC-EV treated 5XFAD mice performed significantly better on both (C) primary escape time and (D) primary escape error. One-way ANOVA was used to determine the statistical significance of A and B. Two-way ANOVA was used to assess the significance of C and D. N=5. *P < 0.05; **P < 0.01.

Figure 3

Intranasal administration of hMSC-EVs significantly decreases amyloid plaques in the HPC of 5XFAD mice. (A) Representative staining of brains with thioflavin S (ThioS), NeuN, and DAPI. Scale bar = 1 mm. (B) 20 one mm² images were taken from the HPC of 4 different mice and plaque density was quantified using ImageJ. ****P < 0.0001.

Figure 4

Levels of amyloid beta are significantly decreased in the brain of 5XFAD mice after treatment with hMSC-EVs. Three mice brains had the HPC dissected out, then HPC lysate or the rest of the brain lysate was run and analyzed by immunoblot assay. *P < 0.05.

Figure 5

Treatment of 5XFAD mice with hMSC-EVs decreases the amount of GFAP in the brain. Three mice brains had the HPC dissected out, then HPC lysate or the rest of the brain lysate was run and analyzed by immunoblot assay. *P < 0.05; **P < 0.01.

Figure 6

hMSC-EV treatment decreases colocalization between reactive astrocytes and amyloid plaques. (A) Representative immunohistochemistry images of brain slices stained with ThioS, GFAP, and DAPI. Scale bar = 1 mm. (B) We took 25 one mm² images throughout the brain, and Pearson's correlation coefficient (PCC) was obtained through ImageJ analysis to determine colocalization of GFAP and ThioS. **P < 0.01.