engleski

Variations of stemness genes expression of canine mesenchymal stem cells during in vitro cultivation

The ultimate goal of successful expansion in vitro is to maintain the stemness of adipose-derived mesenchymal stem cells (AD-MSCs), reflected in their ability to maintain self-renewal, i.e., undergo extensive proliferation while maintaining their multipotent differentiation potential. Since stem cell therapy involves cellular manipulation and in vitro cultivation, it is necessary to reveal its impact on the expression of stemness genes during cultivation and consequently stem cell desirable properties. We analysed changes in expression of several genes (FGF, INS, LIF, OCT4, SOX2 and WNT3A) responsible for stemness in eight healthy young female canine donors of different breeds (poodle, Rhodesian ridgeback, dachshund, beagle, Jack Russell terrier, Belgian shepherd, and two mix breed) referred to elective surgical procedure as it is the opportunity to collect the sample and bank the cells for use in adult age. Changes in the expression of stemness genes were studied, using validated RT2 Profiler PCR Array Format R with SYBR Green-optimized primer assays, in cells obtained by collagenase digestion of adipose tissue samples cultivated and analyzed in two-time points ; passage (P) P3 and P6. Variations of expression occurred between early (P3) and late (P6) passages, along with increased FGF and LIF expression, stable INS, SOX2 and OCT4, and decreased expression of WNT3A. The decreased expression of WNT3A, despite the stable expression of other stemness markers, explains the proliferation arrest and occurrence of senescence. It was already shown that blocking WNT signalling arrests growth, and a decline in WNT3A signalling is necessary for mesenchymal stem cells to proceed with replicative senescence. Though, stable expression of other stemness markers in cells cultivated in standard condition between P3 and P6 indicate their preserved stemness properties prior and optimal timeframe for transplantation. These results will be further validated on protein level and more canine donors’ cells.

stem cells, genes, stemness, canine, expression

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