(E-196) Morphological response of activated microglia upon treatment with mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) is indicative of their functional changes

Introduction:: Mesenchymal stromal cells (MSCs), widely known for their regenerative effects, can exert their therapeutic response through the secretion of extracellular vesicles (MSC-EVs). Furthermore, the function of MSCs and MSC-EVs can be enhanced through priming MSCs with proinflammatory cytokines (e.g. Interferon-gamma (IFN-ү) and Tumor necrosis factor-alpha (TNF-α))(1). However, MSC-EVs exhibit functional heterogeneity i.e. different MSC lines as well as manufacturing methods yield MSC-EVs with inconsistent functional properties. Lack of characterization tools that can identify differences between MSC-EV batches poses a challenge in their clinical translation. To address this, we need a targeted, therapeutically-relevant approach that can assess MSC-EV potency.

In vitro and in vivo research has shown that MSC-EVs modulate neuroinflammation, a hallmark of most neurodegenerative diseases such as Alzheimer’s Disease or Traumatic Brain Injury. They do so by interacting with microglia, a brain resident immune cell that plays a critical role in maintaining the homeostatic environment. Microglia have multiple projections scavenging the brain for exterior insults and display a ramified morphology. Under chronic activation, they become highly mobile and phagocytic adopting an amoeboid morphology. This shift in microglia morphology and function are interlinked and also indicate changes in the secretion of cytokines/chemokines and metabolism(2). Due to this relationship, we hypothesized that microglia morphological response could be used as a readout of MSC-EV potency. 


Materials and Methods:: Primed MSC-EVs (pEVs) were manufactured using MSCs (RoosterBio) cultured in serum-free media supplemented with IFN-γ and TNF-α (50 ng/mL each) for 48 hours. We used ultracentrifugation to harvest the MSC-EVs from the conditioned media. Centrifuged pellets were resuspended in PBS++ and stored in -80°C for further use.

An immortalized human microglia cell line (C20) was used for morphology, secretion and metabolomic studies. The negative control (-CTL) group represented microglia grown in growth media. An activated microglia phenotype (+CTL) was induced using 5ng/mL each of IFN-γ and TNF-α. 10uL of EV solution was added to each microglia culture (100 uL total volume) concurrently with IFN-γ/TNF-α. 24 hours post-treatment with MSC-EVs the media was collected and stored in -80°C to assess secreted factors. The cells were fixed and stained for their morphological assessment. A similar experiment was conducted wherein the cells were flash frozen in liquid nitrogen and stored in -80°C for lipidomic studies. Using CellProfiler, 2000+ features on a single cell level were evaluated to understand the overall shift in microglia morphology upon activation and post-treatment. For secretion profiling, we used a Raybiotech human antibody array that measured the difference in secretome based on 200 targets. Lipidomic profiling was performed using harvested microglia analyzed with ion mobility mass spectrometry. 


Results, Conclusions, and Discussions:: Results and Discussion: Microglia morphology significantly changes upon activation with IFN-γ/TNF-α. Specifically, they become larger (increased area) as well as more complex (increased form factor) upon activation (Figure 1A). Using principal component analysis (PCA) on 21 cellular and nuclear features, we calculate the overall morphological score represented by principal component 1 (PC1), which accounts for 70.6% of the variance. The addition of pEVs results in the overall morphology shifting away from the +CTL (and towards -CTL) suggesting a therapeutic effect of MSC-EVs on microglia(Figure 1B).

Secretion studies reveal an increased expression of various angiogenic (PDGF-AA, uPAR) and inflammatory markers (IL-17, IL-6R) with activation. Treatment with pEVs causes a decrease in these factors indicative of immunomodulation. PCA on 85 secretion targets reveals distinct secretion profiles for +CTL and primed MSC-EV (+CTL + pEVs) treated groups (Figure 2A). Pathway analysis conducted using proteins that are significantly different between the +CTL and pEV-treated groups, reveals significantly enriched cellular and metabolic pathways. Regulation of factors associated with cell adhesion and motility (PAI-1, ICAM-1, M-CSF) directly impacts the cell morphology, establishing the link between morphological and functional changes observed(3,4). A similar trend was observed with lipidomic profiling wherein the overall lipid profile changes between the +CTL and pEV treated groups (Figure 2B).



Conclusion: These insights suggest that microglia morphology can be used as a quantitative readout of their function. This work provides a framework for comprehensively profiling the morphological response of activated microglia upon MSC-EV treatment. The high throughput, single-cell resolution of our approach enables the screening of cell lines and manufacturing conditions to enhance MSC-EV function and address their heterogeneity. Furthermore, this information can be used towards probing into the different mechanisms of action that the MSC-EVs employ in immunomodulation.
This high-throughput approach can be extended to other target cell types of MSC-EVs and help in building disease-specific models. It also enables screening of multiple therapeutics simultaneously which could eventually reduce the burden on large-scale animal studies.


Acknowledgements (Optional): :

References (Optional): : 1. Noronha Nc NDC, Mizukami A, Caliári-Oliveira C, Cominal JG, Rocha JLM, Covas DT, et al. Priming approaches to improve the efficacy of mesenchymal stromal cell-based therapies. Stem Cell Research and Therapy. 2019.

2. 2. Orihuela R, Mcpherson CA, Harry GJ. Microglial M1/M2 polarization and metabolic states Correspondence Themed Section: Inflammation: maladies, models, mechanisms and molecules linked articles. British Journal of Pharmacology 649 British Journal of Pharmacology [Internet]. 2016;665. Available from: www.brjpharmacol.orghttps://dx.doi.org/10.1111/bph.2016.173.issue-4

3. 3. Dietrich JB. The adhesion molecule ICAM-1 and its regulation in relation with the blood–brain barrier. J Neuroimmunol. 2002 Jul 1;128(1–2):58–68.

4. 4. Laflamme N, Cisbani G, Préfontaine P, Srour Y, Bernier J, St-Pierre MK, et al. mCSF-induced microglial activation prevents myelin loss and promotes its repair in a mouse model of multiple sclerosis. Front Cell Neurosci. 2018;12.