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Small extracellular vesicles (EVs) are among the most frequently investigated EVs and play major roles in intercellular communication by delivering various cargo molecules to target cells. They could potentially represent an alternative delivery strategy to treat ocular toxoplasmosis, a parasitosis affecting the retinal pigment epithelium (RPE). To date, the uptake of human small EVs by RPE cells has never been reported. In this study, we report on the intracellular uptake of fluorescently labelled human urine and fibroblast-derived small EVs by human RPE cells. In summary, both dye-labelled urinary small EVs and small EVs obtained from fibroblasts stably expressing membrane-bound green fluorescent protein were successfully internalized by RPE cells as revealed by immunohistochemistry. In recipient ARPE19 cells, BODIPY-labelled small EVs were found in close vicinity to the parasite Toxoplasma gondii. Additionally, an ultrastructural method was enabled to distinguish between labelled exogenous and endogenous small EVs within target cells.

1SummaryIn the last decades, the scientific community spared no effort to elucidate the therapeutic potential of mesenchymal stromal cells (MSCs). Unfortunately, in vitro cellular senescence occurring along with a loss of proliferative capacity is a major drawback in view of future therapeutic applications of these cells in the field of regenerative medicine. Even though insight into the mechanisms of replicative senescence in human medicine has evolved dramatically, knowledge about replicative senescence of canine MSCs is still scarce. Thus, we developed a high-content analysis workflow to simultaneously investigate three important characteristics of senescence in canine adipose-derived MSCs (cAD-MSCs): morphological changes, activation of the cell cycle arrest machinery and increased activity of the senescence-associated β-galactosidase. We took advantage of this tool to demonstrate that passaging of cAD-MSCs results in the appearance of a senescence phenotype and proliferation arrest. This was partially prevented upon immortalization of these cells using a newly designed PiggyBac™ Transposon System, which allows for the expression of the human polycomb ring finger proto-oncogene BMI1 and the human telomerase reverse transcriptase under the same promotor. Our results indicate that cAD-MSCs immortalized with this new vector maintain their proliferation capacity and differentiation potential for a longer time than untreated cAD-MSCs. This study not only offers a workflow to investigate replicative senescence in eukaryotic cells with a high-content analysis approach but also paves the way for a rapid and effective generation of immortalized MSC lines. This promotes a better understanding of these cells in view of future applications in regenerative medicine.