Morphologic, Proliferative, and Cytogenetic Changes during In Vitro Propagation of Cat Adipose Tissue-Derived Mesenchymal Stromal/Stem Cells

Simple Summary Stem cell therapy for veterinary patients such as cats often requires a significant quantity of mesenchymal stromal/stem cells (MSCs), typically expanded in laboratory conditions. However, this expansion process can alter the characteristics and genetic stability of these cells. This study focused on assessing the characteristics of MSCs derived from the adipose tissue of cats (cAT-MSCs) during culture conditions. For this purpose, cell morphological features, growth behavior, and cytogenetic stability were examined in passages 2, 4, and 6. Additionally, MSCs' multipotency and surface markers were assessed. The cAT-MSCs exhibited a spindle-shaped morphology, typical of MSCs. As the cells were cultured over successive passages, a reduction in their growth rate was observed after passage 4, accompanied by abnormalities in the nuclei. cAT-MSCs showed multipotency and their surface marker expression aligned with the expected immunophenotype of MSCs. Cytogenetic studies revealed some structural abnormalities in the chromosomes of the cAT-MSCs, such as gaps, breaks, deletions, duplications, and early chromatid segregation. Nevertheless, these alterations did not show a significant increase over subsequent passages. In conclusion, cAT-MSCs decreased their proliferative capacity after passage 4, accompanied by morphological alterations and signs of structural instability.Abstract Stem cell therapy in cat patients needs a high quantity of mesenchymal stromal/stem cells (MSCs) requiring in vitro propagation under culture conditions which may potentially impact cellular characteristics and genetic stability. This study aimed to assess the in vitro characteristics and cytogenetic stability of cat adipose tissue-derived MSCs (cAT-MSCs). For this purpose, morphological features, clonogenic potential, and proliferative capacity of cAT-MSCs were assessed at passages 2 (P2), P4, and P6. Multipotency and immunophenotype were evaluated. Cytogenetic analyses were conducted up to P6. The cAT-MSCs exhibited a spindle-shaped morphology in early passages. The doubling time increased from 2.5 days at P2 to 9.4 at P4 and 10.5 at P6, accompanied by the observation of nuclear abnormalities such as cluster formation, karyorrhexis, karyolysis, and a decline in the mitotic index at P4. Cells demonstrated multipotency capacity and were CD45-, CD90+, and CD44+. Metaphase analysis at P2 and P4 revealed some indications of structural instability such as gaps, breaks, deletions, duplications, and early chromatid segregation, but these alterations did not show an increase across passages. In conclusion, cAT-MSCs decreased their proliferative capacity after P4, accompanied by morphological alterations and signs of structural instability.