Mesenchymal stem cells as cellular factories for targeted P2X7 blockade after traumatic brain injury.

BACKGROUND

Traumatic brain injury (TBI) causes long-term disability, with inflammation contributing to both primary and secondary brain damage. Extracellular adenosine triphosphate (ATP) and its receptor purinergic receptor X7 (P2X7) drive neuroinflammation and represent promising therapeutic targets. Therefore, we developed a strategy to mitigate acute post-TBI inflammation using mesenchymal stem cells (MSCs) engineered to deliver a P2X7-blocking nanobody (P2X7nb).

METHODS

Primary MSCs were transfected in vitro with bicistronic messenger RNA (mRNA) encoding P2X7nb and a haloalkane dehalogenase Tag reporter, with nanobody secretion confirmed by enzyme-linked immunosorbent assay (ELISA). In vivo, TBI was induced in mice using the closed-head injury model. Twenty-4 hours post-TBI, MSCs labeled with iron oxide nanoparticles were administered via intra-arterial injection into the internal carotid artery. Dynamic 9.4 T magnetic resonance imaging (MRI) using susceptibility-weighted imaging (SWI) allowed real-time monitoring of cellular accumulation. Behavioral performance was assessed using the beam walk test, while mice were euthanized on Day 4 post-TBI for immunofluorescence analysis of cell localization. Data were analyzed using either an unpaired t-test or Mann-Whitney U test, depending on data distribution, with statistical significance set at p < 0.05.

RESULTS

Transfection (38.9% efficiency) and release of P2X7nb were confirmed, with a peak secretion of 17.29 ± 1.84 ng/mL. SWI MRI revealed accumulation of labeled MSCs in the brain post-injection, with a persistent hypointense signal ranging from 2.45% ± 2.93% to 0.77% ± 0.88% of cell area (% of right hemisphere) over 3 days. Behavioral assessment using the beam walk test revealed no cell transplantation-related motor impairment, while histology confirmed the presence of transfected MSCs in injured brain tissue.

CONCLUSION

MSCs expressing a P2X7nb are detectable in the injured brain following intra-arterial delivery for at least 3 days, without producing adverse effects. These findings provide insights into cell-based immunomodulatory therapies targeting neuroinflammation in TBI and highlight the utility of noninvasive MRI to monitor therapeutic cell biodistribution and persistence.