Altered Properties of Feline Adipose Tissue-Derived Mesenchymal Stem Cells During Continuous In Vitro Cultivation

Abstract

Cytotherapy with mesenchymal stem cells (MSCs) has been studied in many species, requiring in vitro cell expansion in order to obtain therapeutic doses of stem cells. Since characteristics of MSCs such as self-renewal and multi-lineage differentiation can be altered during long-term culture, it is important to ensure that stemness is maintained during cultivation. This study assessed changes in the characteristics of feline adipose tissue-derived MSCs (fAT-MSCs) during in vitro passaging. Stem cells isolated from the adipose tissue of a donor cat were cultured for seven sub-passages. Proliferation capacity was analyzed by calculating the cell doubling time and by colorimetric assay. Stem cell-specific marker expression was confirmed by quantitative real-time PCR (qRT-PCR) and immunophenotyping. Expression of adipogenic and osteogenic differentiation markers was also confirmed with qRT-PCR. Histochemical staining and measurement of β-galactosidase activity were used to detect cellular senescence. The proliferation rate of the cells was found to decrease significantly at passage 5 (P5). Gene expression levels of pluripotency markers (SOX2, NANOG, and KLF4) and stem cell surface markers (CD9, CD44, CD90, and CD105) all decreased during continuous culture

for most assays, statistically significant reductions were observed at P5. The ability of cells to undergo adipogenic or osteogenic differentiation was inversely proportional to the number of passages. Percentages of senescent cells identified via histochemical staining increased with the number of passages. These results suggest that increasing the number of passages affects the proliferation and multipotency of fAT-MSCs. In clinical trials, early-passage cells should be used for maximum therapeutic effect.