Novel immortalization approach defers senescence of cultured canine adipose-derived mesenchymal stromal cells.

In 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 ß-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.

Isolation of Human Small Extracellular Vesicles and Tracking of their Uptake by Retinal Pigment Epithelial Cells In Vitro.

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.

Morphometric and functional study of the canine atlantoaxial joint.

The atlantoaxial joint can be affected by instability, in most cases a congenital pathology in young small breed dogs. Causes of atlantoaxial instability (AAI) are variable but are usually attributed to a lack of ligamentous support. The purpose of the present study was to specify the role of the ligamentous structures in the stabilisation of the atlantoaxial joint and to find possible adaptations of the ligaments' internal structure to their specific function. Five Beagle cadavers were included in this study. Each dog was subjected to a computed tomography (CT) and a magnetic resonance imaging (MRI) examination of the upper cervical region. This region was then dissected and the ligamentous structures stabilising the atlantoaxial joint were measured and removed for histological analysis. A ligament to dens ratio (LDR) was established in order to provide a basis for comparison with the measurements taken in other dog breeds. MRI and gross anatomical measurements were very similar, confirming the validity of the results. MRI thus seems reliable for evaluating the ligamentous structures of the canine occipitoatlantoaxial region. The movement exerting the greatest stress on the atlantoaxial ligaments and inducing the greatest distension of the alar ligaments was a head flexion combined with a rotation. A clear adaptation of the ligamentous shape and internal structure to their specific function was observed. Histologically, alar ligaments consisted of wavy collagen fibres and a high proportion of elastic fibres, providing them with a remarkable elasticity compared to the transverse ligament structure which was much more rigid.