Monocyte activation drives preservation of membrane thiols by promoting release of oxidised membrane moieties via extracellular vesicles.

The redox state of cellular exofacial molecules is reflected by the amount of available thiols. Furthermore, surface thiols can be considered as indicators of immune cell activation. One group of thiol containing proteins, peroxiredoxins, in particular, have been associated with inflammation. In this study, we assessed surface thiols of the U937 and Thp1 monocyte cell lines and primary monocytes in vitro upon inflammatory stimulation by irreversibly labelling the cells with a fluorescent derivative of maleimide. We also investigated exofacial thiols on circulating blood mononuclear cells in patients with rheumatoid arthritis and healthy controls. When analysing extracellular vesicles, we combined thiol labelling with the use of antibodies to specific CD markers to exclude extracellular vesicle mimicking signals from thiol containing protein aggregates. Furthermore, differential detergent lysis was applied to confirm the vesicular nature of the detected extracellular events in blood plasma. We found an increase in exofacial thiols on monocytes upon in vitro stimulation by LPS or TNF, both in primary monocytes and monocytic cell lines (p<0.0005). At the same time, newly released extracellular vesicles showed a decrease in their exofacial thiols compared with those from unstimulated cells (p<0.05). We also found a significant elevation of surface thiols on circulating monocytes in rheumatoid arthritis patients (p<0.05) and newly released extracellular vesicles of isolated CD14+ cells from rheumatoid arthritis patients had decreased thiol levels compared with healthy subjects (p<0.01). Exofacial peroxiredoxin 1 was demonstrated on the surface of primary and cultured monocytes, and the number of peroxiredoxin 1 positive extracellular vesicles was increased in rheumatoid arthritis blood plasma (p<0.05). Furthermore, an overoxidised form of peroxiredoxin was detected in extracellular vesicle-enriched preparations from blood plasma. Our data show that cell surface thiols play a protective role and reflect oxidative stress resistance state in activated immune cells. Furthermore, they support a role of extracellular vesicles in the redox regulation of human monocytes, possibly representing an antioxidant mechanism.

Iron-loaded PLLA nanoparticles as highly efficient intracellular markers for visualization of mesenchymal stromal cells by MRI.

Monitoring of the fate of cells after injection appears paramount for the further development of cell therapies. In this context magnetic resonance imaging (MRI) is increasing in relevance owing to its unique tissue visualization properties. For assessment of cell trafficking and homing, the cells have to be labeled to become MR visible. The rather low sensitivity of MRI demands dedicated intracellular markers with high payloads of MR contrast agents for ensuring sensitive detection of local cell aggregations. In the presented work the application of custom-designed nanometer-sized iron oxide loaded poly-(l-lactide) (iPLLA) nanoparticles was investigated. The particles were synthesized by the mini-emulsion process and evaluated for labeling of mesenchymal stromal cells (MSCs). The efficient cellular uptake and long intracellular retention times of the particles as well as their nontoxicity are demonstrated. The average cellular iron content was 55 pg iron per cell. Further incorporation of, for example, fluorescent dye enables the generation of multireporter particles, providing the great potential for multimodal imaging. The efficiency of these nanoparticles as MRI contrast agent was evaluated in vitro using relaxation rate mapping, yielding relaxivities r2 = 273.3, r2 (*) = 545.1 mm(-1) s(-1) at 3 T and r2 = 415.7, r2 (*) = 872.3 mm(-1) s(-1) at 11.7 T. The high r2 (*) relaxivity of the iPLLA nanoparticles enabled visualization of a single labeled cell in vitro at 50-µm spatial resolution. In vivo evaluation in a rat injury model revealed the potential of the iPLLA particles to efficiently label MSCs for MRI monitoring of ~20 000-40 000 injected cells at 11.7 T. In conclusion the presented work demonstrates the applicability of iPLLA particles as efficient intracellular marker for MSC labeling for monitoring the fate of the cells by MRI.

GMP-adapted overexpression of CXCR4 in human mesenchymal stem cells for cardiac repair.

BACKGROUND: Human mesenchymal stem cells (MSC) have been utilized for cardiac regeneration after myocardial damage. Their clinical effects are marginal and only a minority of administered cells could make their way into the myocardium. The chemokine receptor CXCR4 has been identified as crucial for migration and homing of stem cells. In this study we overexpressed CXCR4 on human MSC to improve cell trafficking and tissue repair. METHODS: Human MSC were isolated from the spongiosa of tibia and femur as well as from pelvic bone marrow. MSC were characterized by differentiation assays and FACS analysis. CXCR4 was overexpressed by mRNA-nucleofection. Intracellular signaling was analyzed to demonstrate functionality of CXCR4. The modified Boyden chamber, wounding assays and time lapse microscopy were utilized to investigate MSC migration. RESULTS: MSC did not express relevant amounts of CXCR4 spontaneously. CXCR4 could be overexpressed in 93% of MSC with a cell viability of 62%. Functionality of the overexpressed CXCR4 was demonstrated by a significant cytosolic Ca(2+) increase and activation of different MAP kinases followed by SDF-1α stimulation. In contrast no improvement of cell migration could be observed. There was a strong basal MSC chemokinesis independent from CXCR4 expression. CONCLUSIONS: CXCR4 could be effectively overexpressed in human MSC by mRNA-nucleofection. Despite functionality of CXCR4 MSC were characterized by a strong basal chemokinesis that could not be further enhanced by CXCR4 overexpression. As isolation, culture and nucleofection of pelvic bone marrow-derived MSC basically fulfill the GMP-requirements our approach seems suited for an in vivo application in patients.