Caracterización in vitro de células madre/estromales mesenquimales adiposas caninas modificadas mediante exofucosilación enzimática

Résumé

Mesenchymal stem/stromal cells (MSC) are a population of undifferentiated cells with the capacity for self-renewal and differentiation into cells of different mesodermal lineages. Their identification and therapeutic use is raising great expectations in both human and veterinary medicine due to their ability to secrete anti-inflammatory and immunomodulatory molecules, as well as other biochemical signals that stimulate cell regeneration and repair of damaged tissues. Previous studies have shown that these beneficial trophic effects are closely linked to their ability to migrate and extravasate into inflamed tissues. These tissues express high levels of E-Selectin, whose expression is regulated by proinflammatory cytokines such as IL-1 or TFN-α. However, MSC lack the expression of E-selectin ligands such as CLA (sialofucosylated glycoform of PSGL-1) or HCELL (sialofucosylated glycoform of the CD44 receptor), which limits their ability to migrate to injured tissues and thus affects their therapeutic efficacy after intravenous administration. Nevertheless, several strategies are being developed to improve the in vivo migration capacity of MSC to these tissues, including cell surface glycoengineering, in which enzymatic exofucosylation stands out. This technique consists of the transient modification of the CD44 receptor present on MSC, which is modified by an enzymatic process and transformed into the HCELL molecule, the most potent ligand for E-Selectin. The main objective of this study was to determine whether it is possible to effectively fucosylate canine adipose-derived mesenchymal stem/stromal cells (AT-MSC), and whether this enzymatic exofucosylation process causes any alteration in their properties and/or cellular and functional characteristics, also evaluating their preservation under refrigeration conditions with a view to their possible therapeutic use in veterinary clinical practice. MATERIALS AND METHODS: AT-MSC were isolated from adipose lipoexplants of donor dogs (n=10). For this purpose, once in the laboratory the lipoexplants were subjected to enzymatic mechanical digestion to obtain the primary culture and subsequent subcultures until passages 2-4, at which point they were subjected to enzymatic exofucosylation to perform the planned experiments: 1) Verification of the enzymatic exofucosylation process of canine AT-MSC; 2) Determination of the stability of enzymatic exofucosylation in FUC AT-MSC under culture conditions (37°C) and under refrigeration (4°C); 3) Cellular characterization of FUC AT-MSC according to their cell morphology, immunophenotype and multipotent differentiation capacity SUMMARY/ABSTRACT - 269 - towards adipose, bone and cartilage tissue cells; 4) Functional characterization of FUC AT-MSC by determining their proliferation, migration, secretion and cell immunomodulation capacity; and 5) Study of cell viability under refrigeration conditions (4ºC) of canine AT-MSC after fucosylation. Data were compiled in an Excel© v.19 database and analyzed using GraphPadPrism© v.8 software. RESULTS: Our results showed that canine AT-MSC can be effectively fucosylated (>98%), reversing this process under cell culture conditions in less than 72h under cell culture conditions (6%), but remain stable and functional under refrigerated conditions (4°C) for at least 4 days (>90%). Furthermore, we observed that FUC AT-MSC maintained their cellular properties preserving their morphology, cellular immunophenotype, multipotent differentiation capacity towards adipose, bone and cartilage tissues, and proliferation capacity. However, we found that enzymatic exofucosylation increased their migration capacity 1.7-fold, and that they became more anti-inflammatory and immunomodulatory, with these effects being further enhanced in the presence of proinflammatory cytokines (TNF-α + IFNγ), such as those expressed in inflamed or damaged tissues. Finally, we also observed that enzymatic exofucosylation does not significantly affect the cell viability of canine AT-MSC, which can remain functional in refrigeration at 4°C for at least 4 days,. with a cell viability higher than 90%. CONCLUSIONS: In view of the results obtained, we can conclude that enzymatic exofucosylation is a feasible and safe technique for application in canine AT-MSC, since it does not significantly affect the cellular characteristics of these mesenchymal cells and, importantly, improves their migratory capacity and their therapeutic anti-inflammatory and immunomodulatory effects, and can be used to optimize and improve the properties of canine AT-MSC, to be used in the near future in veterinary clinical practice for the treatment of various pathologies with an inflammatory and/or inmunomediated basis.