The Effect of Serial Passaging on the Proliferation and Differentiation of Bovine Adipose-Derived Stem Cells

Abstract

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.

Effects of Serial Passage on the Characteristics and Chondrogenic Differentiation of Canine Umbilical Cord Matrix Derived Mesenchymal Stem Cells

“…This observation supports that serial passage also influenced transcriptional levels of differentiation-associated markers in MSCs. Similarly, our gene expression results corroborate with the previous studies that lower expression of Sox9 (Hargus et al, 2008), collagen II and aggrecan were found in the P5-induced pellets (Zhao, 2011). …”

“…In addition to the slower growth, changes in response to chemical stimuli were observed after passage 4 (Wall et al, 2007). Recent studies reported by Danoviz et al (2011) and Zhao et al (2011) showed that increasing doubling times were found in human and bovine adipose stem cells (ASCs) at later passage.…”

“…Interestingly, recent research has demonstrated that adipose derived stem cells are capable of long-term self-renewal in culture, while maintaining multipotency in several species, such as human, mouse, canine and bovine (Zuk et al, 2001; Zheng et al, 2006; Neupane et al, 2008; Zhao et al, 2011). However, the presence of a multipotent stem cell population in canine tissues has not yet been fully investigated.…”

Adipose-derived stem cells retain their regenerative potential after methotrexate treatment

“…Groups have reported that monolayer expansion can influence many characteristics of ASCs including surface marker expression, proliferation, and differentiation potential (Mitchell, et al, 2006; Schellenberg, et al, 2011; Zhao, et al, 2012). However, increased CFU counts and uninhibited differentiation potential of MTX-treated, freshly isolated SVF cells suggests that monolayer expansion does not cause MTX resistance of ASCs.…”

Opportunities and challenges in three-dimensional brown adipogenesis of stem cells

“…For example, long-term in vitro expansion may not affect the cell proliferation of ASCs until passage 5, but has a negative effect on adipogenic and chondrogenic differentiation while potentially favoring osteogenesis (Zhao et al, 2012b). Additionally, the long-term culture of MSCs could cause cellular senescence, resulting in growth arrest and reduction in differentiation (Cheng et al, 2011) Therefore, a pluripotent stem cell population would be more beneficial to overcome these limitations.…”