The effect of serial passaging on the proliferation and differentiation of bovine adipose-derived stem cells.

Adipose-derived stem cells (ASCs) represent an excellent cell source for the development of regenerative therapies for a broad variety of tissue disorders. Commonly, in vitro expansion is necessary to obtain sufficient cell populations for research purposes and clinical applications. Although it has been demonstrated that human ASCs can maintain their adipogenic, chondrogenic and osteogenic potential in long-term culture (up to 15 passages), it is not guaranteed that a satisfactory level of differentiation is achievable in later passages. In this study, we investigated the self-renewal and multilineage differentiation capacity of bovine ASCs, isolated from the interdigital fat pad, and explored how serial passaging influences the cells. A proliferation study examined the changes in growth kinetics from passage 1 to 5, and multilineage (adipogenesis, chondrogenesis and osteogenesis) differentiation studies were conducted to compare the potential between passage 2 (P2) and passage 5 (P5). From the proliferation study, a statistically significant change in the doubling time did not appear until P5. In the differentiation study, both P2 and P5 ASCs could be stimulated to undergo multilineage differentiation under specific culturing conditions. However, adipogenic and chondrogenic cultures showed significantly lower levels of differentiation in the P5-induced cultures. In contrast, P5-induced osteogenic cultures had higher alkaline phosphatase enzyme activity than P2-induced cultures, suggesting an increase in the osteogenic response with serial passaging. Overall, bovine ASCs are capable of self-renewal and multilineage differentiation; however, long-term in vitro expansion has a negative effect on adipogenic and chondrogenic differentiation, while potentially favoring osteogenesis.