Mesenchymal stem/stromal cells (MSC) are well known for immunomodulation; however, the mechanisms involved in their benefits in the ischemic retina are unknown. This study tested the hypothesis that MSC induces upregulation of transcription factor forkhead box protein P3 (Foxp3) in T cells to elicit immune modulation, and thus, protect against retinal damage. Induced MSCs (iMSCs) were generated by differentiating the induced pluripotent stem cells (iPSC) derived from urinary epithelial cells through a noninsertional reprogramming approach. In in-vitro cultures, iMSC transferred mitochondria to immune cells via F-actin nanotubes significantly increased oxygen consumption rate (OCR) for basal respiration and ATP production, suppressed effector T cells, and promoted differentiation of CD4+CD25+ T regulatory cells (Tregs) in coculture with mouse splenocytes. In in-vivo studies, iMSCs transplanted in ischemia-reperfusion (I/R) injured eye significantly increased Foxp3+ Tregs in the retina compared to that of saline-injected I/R eyes. Furthermore, iMSC injected I/R eyes significantly decreased retinal inflammation as evidenced by reduced gene expression of IL1β, VCAM1, LAMA5, and CCL2 and improved b-wave amplitudes compared to that of saline-injected I/R eyes. Our study demonstrates that iMSCs can transfer mitochondria to immune cells to suppress the effector T cell population. Additionally, our current data indicate that iMSC can enhance differentiation of T cells into Foxp3 Tregs in vitro and therapeutically improve the retina’s immune function by upregulation of Tregs to decrease inflammation and reduce I/R injury-induced retinal degeneration in vivo.

Identifying the specific bioactive molecules produced by mesenchymal stem cells (MSCs) and the signaling pathways and cell types upon which they act is critical to developing MSC-based therapeutics for inflammatory diseases with high unmet needs. Our study aimed to investigate the impact of extracellular vesicle (EV)-derived TNF-Stimulated Gene-6 (TSG-6, from adipose tissue-derived mesenchymal stem cell concentrated conditioned medium, ASC-CCM or TSG-6 overexpression in ASC using ORF expression-ready clone) on microglia and its potential anti-inflammatory effects. EV but not non-vesicular secretome prepared by ultracentrifugation confirmed the expression of TSG-6 exclusively in the small EV (sEV) fraction. sEV ranged from 50–150 nm as determined by Zetasizer, demonstrated bilipid membrane evidenced by transmission electron microscopy, expressed positive exosomal (e.g. CD63) markers, and were endocytosed by BV2 cells confirmed by DiI fluorescently labeled exosomes. BV2 microglia cultured under serum-free conditions stimulated with TLR4 agonists (LPS and IFNγ) for 12 h in the presence of p-ASC-EV (sEV derived from ASC after cytokine stimulation) and TSG-6-ORF-EV significantly reduced nitrite release (p < 0.001), phagocytic activity (p < 0.001) and reduced CD44 expression (p < 0.05). CD44 knockdown in BV2 cells ablated TSG-6-ORF-EV mediated nitrite release, IL1β downregulation, and phagocytosis with TLR4 agonists. Our results revealed that under cytokine stimulation, the EV portion of ASC-CCM becomes enriched with TSG-6. Overexpressing TSG-6 in ASC leads to an increased concentration of TSG-6 in sEVs. This enriched EV fraction, containing TSG-6, regulates microglial dynamics through a feedback loop with CD44. EV-associated TSG-6 can influence immune cell behavior and signaling, mitigating excessive inflammation or immune dysfunction. Graphical Abstract Exosomal TSG-6 affects microglial activity This study examines TNF-Stimulated Gene-6 (TSG-6), a bioactive protein from mesenchymal stem cells that segregates into the small extracellular vesicle portion of the secretome and influences microglia behavior, reducing inflammation. Additionally, it highlights CD44’s role in mediating these effects and suggests that TSG-6-enriched exosomes suppress TLR signaling to regulate phagocytosis and inflammation.

Traumatic Brain Injury (TBI) affects millions of people each year, causing loss of workforce, a significant burden on the healthcare system. Research into the biological effects of TBI has focused on cellular and tissue responses in the order of hours to days after exposure. Unfortunately, treatments that address these secondary processes after TBI have not been effective. Addressing earlier events and identifying first responders is necessary to prevent or limit irreversible damage. Here, we analyze molecular and cellular changes that occur during and immediately after injury in the somatosensory cortex of mice after an airblast TBI using various techniques. Early changes recorded within 5 min of TBI injury show persistent calcium activation, followed by unique phosphorylation patterns activating specific protein binding and calcium-activated pathways. Imaging mass lipidomics and Raman microspectroscopy identified conformational changes of lipid membranes, pointing towards membrane damage as the first response to mechanical pressure. Real-time monitoring of blood vessels shows rapid constriction followed by reduced blood flow within minutes of injury. The present study uncovers the early sequence of events in TBI: ionic calcium imbalance occurs within seconds, activating subcellular phosphorylation patterns, which in turn trigger downstream pathways and a parallel destabilization of membrane phospholipids.

Blast‐induced traumatic brain injury (bTBI) was reported in 125,000 U.S. service men and women from 2000 to 2018. With no prophylactic treatments having been granted FDA approval, there is a clear need for further understanding of the impact of blasts on the central nervous system. The biological response of brain cells due to the near‐instantaneous overpressure of blast onset remains unresolved. Laser‐induced pressures isolate high‐amplitude, short‐duration pressure transients, similar to the initial peak of blast pressures. In this study, we uncover the effects of high‐amplitude, short‐duration pressure transients on monocultures of astrocytes, microglia, and neurons through intracellular calcium imaging, cell viability assays, and quantifying intracellular and extracellular immune signaling proteins. The results indicate that while all three cell types follow a similar activation curve for induced intracellular calcium transients, the downstream impact of high‐amplitude, short‐duration pressures on neurons and glia deviate. Neurons are particularly susceptible to non‐reversible damage, while glia activates traditionally neuroprotective pathways rather than neurodegenerative pathways in response to high‐amplitude, short‐duration pressures. This work has important implications for developing countermeasures for bTBI with either the high‐frequency component of a blast wave or the effects of that component being potential targets for prevention.