Human Umbilical Cord Blood Endothelial Progenitor Cell-Derived Extracellular Vesicles Control Important Endothelial Cell Functions.

Circulating endothelial progenitor cells (EPCs) play a pivotal role in the repair of diseases in which angiogenesis is required. Although they are a potentially valuable cell therapy tool, their clinical use remains limited due to suboptimal storage conditions and, especially, long-term immune rejection. EPC-derived extracellular vesicles (EPC-EVs) may be an alternative to EPCs given their key role in cell-cell communication and expression of the same parental markers. Here, we investigated the regenerative effects of umbilical cord blood (CB) EPC-EVs on CB-EPCs in vitro. After amplification, EPCs were cultured in a medium containing an EVs-depleted serum (EV-free medium). Then, EVs were isolated from the conditioned medium with tangential flow filtration (TFF). The regenerative effects of EVs on cells were investigated by analyzing cell migration, wound healing, and tube formation. We also analyzed their effects on endothelial cell inflammation and Nitric Oxide (NO) production. We showed that adding different doses of EPC-EVs on EPCs does not alter the basal expression of the endothelial cell markers nor change their proliferative potential and NO production level. Furthermore, we demonstrated that EPC-EVs, when used at a higher dose than the physiological dose, create a mild inflammatory condition that activates EPCs and boosts their regenerative features. Our results reveal for the first time that EPC-EVs, when used at a high dose, enhance EPC regenerative functions without altering their endothelial identity.

Producing vesicle-free cell culture additive for human cells extracellular vesicles manufacturing.

A new paradigm has emerged recently, which consists in shifting from cell therapy to a more flexible acellular "extracellular vesicle (EV) therapy" approach, thereby opening a new and promising field in nanomedicine. Important technical limitations have still to be addressed for the large-scale production of clinical-grade EV. Cells are cultured in media supplemented with human platelet lysate (hPL) (xenogenic-free) or GMP-grade fetal calf serum (FCS). However, these additives contain high amounts of EV that cannot be separated from cell-secreted -EV. Therefore, cells are generally maintained in additive-free medium during the EV secretion phase, however this can substantially limit their survival. In the present work, we developed a method to prepare vesicle-free hPL (EV-free hPL) or vesicle-free FCS (EV-free FCS) using tangential flow filtration (TFF). We show a very efficient EV depletion (>98%) for both pure hPL and FCS, with a highly conserved protein content. Culture medium containing our EV-free additives supported the survival of human bone marrow MSC (BM-MSC). MSC could survive at least 216 h, their conditioned medium being collected and changed every 72 h. Both the cell survival and the cumulative EV production were substantially higher than in the starving conditions classically used for EV production. In EV-free hPL containing medium, we show that purified EV kept their morphologic and molecular characteristics throughout the production. Finally, we tested our additives with 3 other cell types, human primary Endothelial Colony Forming Cells (ECFC) and two non-adherent human cell lines, Jurkat and THP-1. We confirmed that both EV-free hPL and FCS were able to maintain cell survival and EV production for at least 216 h. Our method provides therefore a new option to help producing large amounts of EV from virtually any mammalian cells, particularly those that do not tolerate starvation. This method can apply to any animal serum for research and development purpose. Moreover, EV-free hPL is clinical-grade compatible and allows preparing xenobiotic-free media for massive therapeutic EV production in both 2D (cell plates) and 3D (bioreactor) setting.

Two New Potential Therapeutic Approaches in Radiation Cystitis Derived from Mesenchymal Stem Cells: Extracellular Vesicles and Conditioned Medium.

Background: Radiation cystitis (RC) results from chronic inflammation, fibrosis, and vascular damage. The urinary symptoms it causes have a serious impact on patients' quality of life. Despite the improvement in irradiation techniques, the incidence of radiation cystitis remains stable over time, and the therapeutic possibilities remain limited. Mesenchymal stem/stromal cells (MSC) appear to offer2 a promising therapeutic approach by promoting tissue repair through their paracrine action via extracellular vesicles (MSC-EVs) or conditioned medium from human mesenchymal stromal cells (MSC-CM). We assess the therapeutic potential of MSC-EVs or MSC-CM in an in vitro model of RC. Methods:in vitro RC was induced by irradiation of human bladder fibroblasts (HUBF) with the small-animal radiation research platform (SARRP). HUBF were induced towards an RC phenotype after 3 × 3.5 Gy irradiation in the presence of either MSC-EVs or MSC-CM, to assess their effect on fibrosis, angiogenesis, and inflammatory markers. Results: Our data revealed in vitro a higher therapeutic potential of MSC-EVs and MSC-CM in prevention of RC. This was confirmed by down-regulation of α-SMA and CTGF transcription, and the induction of the secretion of anti-fibrotic cytokines, such as IFNγ, IL10 and IL27 and the decrease in the secretion of pro-fibrotic cytokines, IGFBP2, IL1ß, IL6, IL18, PDGF, TNFα, and HGF, by irradiated HUBFs, conditioned with MSC-EVs or MSC-CM. The secretome of MSC (MSC-CM) or its subsecretome (MSC-EVs) are proangiogenic, with the ability to induce vessels from HUVEC cells, ensuring the management of bladder vascular lesions induced by irradiation. Conclusion: MSC-EVs and MSC-CM appear to have promising therapeutic potential in the prevention of RC in vitro, by targeting the three main stages of RC: fibrosis, inflammation and vascular damage.

Transplantation of genetically modified hepatocytes after liver preconditioning in Watanabe heritable hyperlipidemic rabbit.

BACKGROUND: Hepatocyte transplantation is a potentially less invasive alternative to liver transplantation for treating inherited metabolic liver diseases. We developed an autotransplantation protocol of ex vivo genetically modified hepatocytes combining lentiviral transduction and transplantation after liver preconditioning by partial portal vein embolization. We investigated the metabolic efficiency of this approach in Watanabe rabbits, animal model of familial hypercholesterolemia. METHODS: Our autotransplantation experimental protocol was used in two groups of rabbits (n = 10), experimental and sham, receiving transduced and control hepatocytes, respectively. Isolated hepatocytes from left liver lobes were transduced using recombinant lentiviruses. Median lobe portal branches were embolized under fluoroscopic control. Functional measurement of low-density lipoprotein (LDL) receptor expression was assessed by LDL internalization assays. Cholesterol level evolution was monitored. Rabbits were killed 20 wk after the procedure. RESULTS: Three rabbits of each group died several hours after hepatocyte transplantation; autopsy revealed portal vein thrombosis in two rabbits from each group. The protocol was therefore modified with hepatocytes being transplanted through splenic injection. Lentiviral hepatocyte transduction efficacy was 64.5%. Fluorescence microscopy revealed Dil-LDL internalization of transduced hepatocytes. Seven rabbits in each group were considered for lipid analysis. Four weeks after autotransplantation, median total cholesterol level decreased in the experimental group, without reaching statistical significance (8.9 [8.0-9.8] g/L versus 6.3 [0.5-8.3]; P = 0.171). In the experimental group, enzyme-linked immunosorbent assay detected significant antibody expression against human low-density lipoprotein receptor. CONCLUSIONS: Autotransplantation protocol allowed a nonstatistically significant improvement of the lipid profile in Watanabe rabbits. Further experiments involving a larger number of animals are necessary to confirm or refute our findings.