(E-195) Pericyte morphological response to Tumor Necrosis Factor-alpha is modulated by mesenchymal stromal cell extracellular vesicles.

Introduction:: A key attribute of neurodegenerative disease is a chronic inflammatory response that is driven by the neuroinflammatory cascade. The neuroinflammatory cascade is characterized by disruption of the blood brain barrier (BBB), activated cells such as microglia, and increased secretion of inflammatory cytokines and chemokines. Within the BBB, pericytes are a heterogeneous cells located in the basement membrane and line the endothelium. Pericytes help regulate BBB disruption, inflammation, leukocyte extravasation, and adaptive immunity (Rustenhoven, et al., 2017). Although the role of pericytes in regulating key aspects of the neural immune environment are known, there are no effective pericyte-targeting therapies for the treatment of neurodegenerative diseases such as Alzheimer’s Disease.

Mesenchymal stromal cells (MSCs) have well-established immunomodulatory function; however, there are issues with MSC localization to brain tissue and intravenously administered MSCs become localized to the lungs (Fischer, et al., 2019). MSC-derived extracellular vesicles (MSC-EVs) have emerged as a promising alternative to MSCs for modulating neuroinflammation as they can modulate different immune cells (e.g. T cells and microglia), and can cross the BBB (Branscome, et al., 2020). However, the mechanisms and target cells through which MSC-EVs exert their therapeutic function in the context of neuroinflammation are still poorly understood. Due to their perivascular localization, and their similar phenotypic behavior to MSCs, pericytes are an attractive target for MSC-EVs. Morphology has been used previously to illustrate the functional changes after inflammatory stimulus with MSCs. As pericytes respond to inflammatory signals, we hypothesized that pericyte morphology can be used to assess MSC-EV function.


Materials and Methods:: Human brain pericytes (ScienCell) were first identified through immunofluorescence staining of NG2, NFκB, and α-SMA. Human brain pericytes were seeded in a 96-well plate at 2500 cells/cm^2. They were then activated with the inflammatory cytokine Tumor Necrosis Factor Alpha (TNF-α) (50ng/mL) 24 hours. Adipose derived MSCs (RoosterBio) were seeded in T175 flasks for 24 hours. After, each flask is primed under different conditions (Table 1) for 48 hours in serum free media (RoosterBio). After priming, the EVs were harvested from the media using ultracentrifugation and characterized for content, size, and yield. For treatment with MSC-EVs, the EVs were added to the pericytes concurrent with TNF-α stimulation and incubated for 24 hours. After 24 hours of treatment, pericytes were stained for cell and nuclear morphology using Hoechst and fluorescein-maleimide, respectively. Single cell morphological data was obtained using automated high-throughput imaging and automated image analysis software – CellProfiler. CellProfiler extracted over 96 different morphological parameters on a single-cell basis, which were further analyzed on a median per-well basis.


Results, Conclusions, and Discussions:: After treatment with TNF-α, pericyte morphology exhibited several distinct morphological changes. The pericytes became larger and more complex as represented by an increase in major axis length and a decrease in form factor, respectively, after TNF-α treatment (Figure 1). This was evident across multiple experiments (n=4 total). Differences in pericyte morphological response can occur due to specific priming and manufacturing methods of MSCs to produce MSC-EVs. Addition of MSC-EVs to TNF-α stimulated pericytes induced an alternative pericyte morphology from that of the unstimulated and TNF-α stimulated pericytes. The MSC-EVs increase both complexity and size of the pericytes reflected in form factor and major axis length, respectively. In addition, pericyte morphology identifies differences in priming conditions of the MSCs (Figure 2). Some priming conditions significantly change the morphology compared to both the controls and other priming conditions. For example, the priming condition 2 in Figure 2, significantly lowered form factor and increased pericyte major axis length compared to both the controls and other priming conditions.

Although we hypothesized TNF-α stimulated pericytes would obtain a morphology similar to unstimulated pericytes with MSC-EV treatment, we see a further increase in size and complexity beyond TNF-α treatment alone. Although this may suggest MSC-EVs result in an increased inflammatory response, our results agree with published work from our group showing MSCs, which are phenotypically similar to pericytes, exhibit a significant increase in aspect ratio and decreased form factor with cytokine treatment (Andrews, et al., 2021). Therefore, our current data could suggest that this shift in pericyte morphology could be indicative of a shift towards a ‘primed MSC’ morphological state and be evidence that they can secrete desired immunomodulatory factors. Furthermore, these experiments underscore that pericyte morphology can be indicative of differences in MSC-EV priming and manufacturing conditions. Therefore, implementation of this high throughput screening approach could represent a rapid, low cost, and readily implemented method to assess MSC function.


Acknowledgements (Optional): :

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Branscome, H., Paul, S., Yin, D., El-Hage, N., Agbottah, E., Zadeh, M. A., . . . Kashanchi, F. (2020, June). Use of Stem Cell Extracellular Vesicles as a “Holistic” Approach to CNS Repair. Frontiers in Cell and Developmental Biology, 8, 455. doi:10.3389/fcell.2020.00455

Fischer, U. M., Harting, M. T., Jimenez, F., Monzon-Posadas, W. O., Xue, H., Savitz, S. I., . . . Cox, Jr., C. S. (2009, June). Pulmonary Passage is a Major Obstacle for Intravenous Stem Cell Delivery: The Pulmonary First-Pass Effect. Stem Cells and DEvelopment, 18(5), 683-691. doi:10.1089/scd.2008.0253

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