Background/Objectives
Mesenchymal stromal cells (MSCs) exert therapeutic effects, primarily mediated through extracellular vesicles (EVs) with immunomodulatory properties. CD73, expressed on MSCs, catalyzes the conversion of extracellular AMP to adenosine, a potent anti-inflammatory mediator. However, the absence of CD39, which hydrolyzes extracellular ATP (eATP) and ADP to AMP, restricts the full activation of the purinergic signaling cascade. Given that eATP acts as a pro-inflammatory damage-associated molecular pattern (DAMP) post-stroke, this study investigates whether ectopic CD39 expression in immortalized MSCs (ciMSCs) enhances the therapeutic efficacy of their EVs in neuroinflammatory conditions.
Methods
ciMSCs were transduced with lentivirus to express CD39. EVs were isolated by ultracentrifugation and characterized per MISEV guidelines. CD39+-eGFP EVs were quantified via image flow cytometry, and ATP hydrolysis was measured calorimetrically. Immunomodulatory potential was tested via in vitro multi-donor mixed lymphocyte reaction (mdMLR) with mononuclear cells from 12 donors to evaluate responses with and without eATP. Therapeutic efficacy was evaluated in a murine middle cerebral artery occlusion (MCAO) model. Neuromuscular coordination was assessed with tightrope test, while infarct volume and cerebral edema were analyzed through Nissl and cresyl violet staining.
Results
Image flow cytometry and western blot analysis confirmed that ciMSC-EVs carried CD39. CD39+ ciMSC-EVs demonstrated efficient ATP-to-adenosine conversion, a capability not observed in unmodified ciMSC-EVs. In mdMLR assays, while both EV types suppressed T cell activation in the absence of eATP, CD39+ ciMSC-EVs exhibited markedly enhanced suppression in the presence of eATP, supporting the role of adenosine pathways in modulating immune responses. In the ischemic stroke model, administration of CD39+ ciMSC-EVs significantly improved neuromuscular coordination, reduced infarct volume, and decreased edema compared to EVs from unmodified ciMSCs, highlighting their superior therapeutic potential.
Conclusion
CD39 expression enhances the immunomodulatory and therapeutic potential of ciMSC-EVs under inflammatory conditions. CD39+ ciMSC-EVs represent a promising candidate for treating neuroinflammatory disorders like ischemic stroke.
Keywords
CD39, extracellular vesicles, ischemic stroke, immunomodulation, mesenchymal stromal cells
Funding/Acknowledgments
This project is supported by funding from the Deutsche Forschungsgemeinschaft (DFG).
