Background: The clinical application of canine mesenchymal stem cells (MSCs) necessitates efficient and safe culture methods to produce large quantities of cells. Traditionally, fetal bovine serum (FBS) has been used for MSC expansion, but it carries risks such as contamination and adverse immune responses. Methods: In this study, we investigate the efficacy and efficiency of canine allogeneic serum as an effective alternative to FBS for the in vitro culture of canine MSCs. We measured the population doubling time of canine MSCs in allogeneic serum conditions and utilized qRT-PCR, flowcytometric analysis, and cellular staining/color-metric assay for investigating its effects on cellular senescence during long-term culture and the expression of key pluripotency-related transcriptomes. Results: Our findings demonstrate that canine MSCs cultured with allogeneic serum exhibited enhanced proliferation rates, reduced cellular senescence, and lower apoptosis levels compared to those cultured with FBS. Additionally, the expression of key pluripotency-related transcription factors, including Oct4, Sox2, and Nanog, was increased in canine MSCs cultured with allogeneic serum. Conclusions: These results highlight the potential of canine allogeneic serum to provide a safer and more effective culture environment, supporting the large-scale expansion and maintenance of canine MSCs for clinical applications.

Background: In healthy dentin conditions, odontoblasts have an important role such as protection from invasion of pathogens. In mammalian teeth, progenitors such as mesenchymal stem cells (MSCs) can migrate and differentiate into odontoblast-like cells, leading to the formation of reparative dentin. For differentiation using stem cells, it is crucial to provide conditions similar to the complex and intricate in vivo environment. The purpose of this study was to evaluate the potential of differentiation into odonto/ osteoblasts, and compare co-culture with/without epithelial cells. Methods: MSCs and epithelial cells were successfully isolated from dental tissues. We investigated the influences of epithelial cells on the differentiation process of dental pulp stem cells into odonto/osteoblasts using co-culture systems. The differentiation potential with/without epithelial cells was analyzed for the expression of specific markers and calcium contents. Results: Differentiated odonto/osteoblast derived from dental pulp tissue-derived mesenchymal stem cells with/without epithelial cells were evaluated by qRT-PCR, immunostaining, calcium content, and ALP staining. The expression of odonto/ osteoblast-specific markers, calcium content, and ALP staining intensity were significantly increased in differentiated cells. Moreover, the odonto/osteogenic differentiation capacity with epithelial cells co-culture was significantly higher than without epithelial cells co-culture. Conclusions: These results suggest that odonto/osteogenic differentiation co-cultured with epithelial cells has a more efficient application.