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Extracellular Vesicles from Mesenchymal Stromal Cells Modulate Inflammatory Responses on Feline Mixed Glia
Nikolia Darzenta1,2, Emily Davis1,2, Anna Cochran1,2
1Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL, USA.
Abstract:
The extracellular vesicles (EVs) secreted by mesenchymal stromal cells (MSC-EVs) exhibit immunoregulatory functions dependent on their parent cells. MSC-EVs are promising candidates for treating neuroinflammation in neurological diseases due to their acellular nature and their ability to reach the central nervous system. However, the conditions of MSCs for producing EVs with the highest anti-inflammatory efficacy are still unknown. Therefore, the first objective was to study the characteristics of the EVs produced by MSCs cultured in different conditions. The second objective was to evaluate the in vitro anti-inflammatory properties of those EVs in feline stimulated mixed glia. Umbilical cord-derived MSCs were treated with serum-free (SF) media, inflammatory (IF) media, or media supplemented with 5% EV-depleted fetal bovine serum (FBS). The isolated MSC-EVs were characterized by particle size and yield, and their anti-inflammatory ability was evaluated in lipopolysaccharide (LPS) stimulated feline mixed glia. All EV isolates were <160 nm, and the primary mixed glia consisted of microglia, astrocytes, neurons, and endothelial cells. Our results indicate that IF-EVs statistically significantly decreased the production of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) and downregulated the transcription of the, nuclear factor kappa B p65 subunit in inflammatory mixed glia after 48 hours. In addition, SF- and FBS-EVs significantly reduced in vitro the secretion of IL-6 after 48 hours, but only SF-EVs achieved a significant effect on inhibiting the expression of p65 at 48 hours. Moreover, messenger RNA (mRNA) levels of inducible nitric oxide synthase (iNOS) were significantly decreased following treatment with SF-EV for 24 hours. This study demonstrates that MSC culture conditions affect the therapeutic potential of the secreted EVs in feline mixed glia.
Insights
Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) show varied anti-inflammatory effects based on culture conditions. Inflammatory media-derived EVs most effectively reduced inflammatory markers in feline glial cells.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) are investigated for treating neuroinflammation due to their immunomodulatory properties and ability to cross the blood-brain barrier.
- Optimizing MSC culture conditions is crucial for maximizing the anti-inflammatory efficacy of secreted EVs for neurological disease therapies.
- Current knowledge gaps exist regarding the specific MSC culture parameters that yield MSC-EVs with superior therapeutic potential.
Purpose of the Study:
- To characterize extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) cultured under distinct conditions.
- To evaluate the *in vitro* anti-inflammatory efficacy of these MSC-EVs in lipopolysaccharide (LPS)-stimulated feline mixed glial cells.
Main Methods:
- Umbilical cord-derived MSCs were cultured in serum-free (SF), inflammatory (IF), or EV-depleted fetal bovine serum (FBS) supplemented media.
- Isolated MSC-EVs were characterized for particle size and yield.
- Anti-inflammatory effects were assessed by measuring cytokine production (IL-6, TNF-α) and gene expression (NF-κB p65, iNOS) in LPS-stimulated feline mixed glia.
Main Results:
- All tested MSC-EV isolates were below 160 nm in size.
- IF-EVs significantly reduced interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) production and downregulated NF-κB p65 transcription.
- SF-EVs and FBS-EVs reduced IL-6 secretion, with SF-EVs also significantly inhibiting p65 expression and iNOS mRNA levels.
Conclusions:
- MSC culture conditions significantly influence the immunomodulatory potential of secreted EVs.
- EVs derived from MSCs cultured in inflammatory media demonstrate potent anti-inflammatory effects in a feline glial cell model.
- These findings highlight the importance of optimizing MSC culture environments for developing effective EV-based therapies for neuroinflammatory conditions.
