Osteogenic differentiation of human perivascular stem cells and its regulation based on Wnt/beta-catenin signaling pathway
Yuan Wei1, Liu Jingdong1, Xu Guanghui1, Kang Jian1, Li Fuping1, Wang Yingjie1, Zhi Zhongzheng1, Li Guanwu2
1Department of Spine Surgery, Shanghai Fourth People’s Hospital, Tongji University, Shanghai 200434, China; 2Department of Radiology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China
Abstract: BACKGROUND: Perivascular stem cells are derived from adult adipose tissue, which can be easily purified. Perivascular stem cells represent a comparatively homogenous population and have the ability of strong proliferation and multidirectional differentiation.
OBJECTIVE: To explore the osteogenic differentiation ability of human perivascular stem cells and the regulation by Wnt/β-catenin signaling pathway by in vitro and in vivo experiments.
METHODS: Perivascular stem cells were extracted from human lipoaspirate via fluorescence-activated cell sorting. Osteogenic differentiation of passage 1 perivascular stem cells was observed. Cells were randomly divided into blank control group, osteogenic differentiation group and Wnt/β-catenin signaling inhibition group. Alkaline phosphatase staining was performed on day 5 of osteogenic induction. Alizarin red staining was performed on day 10. On day 7, the protein expression of Runt related transcription factor 2 was detected by western blot assay. Perivascular stem cells + nano-hydroxyapatite/poly lactic-co-glycolic acid scaffold was prepared. Bone repair effect of perivascular stem cells in vivo was evaluated by using the model of tibia monolayer cortical bone defect in athymic mice.
RESULTS AND CONCLUSION: (1) There were about (1.82±0.32)×107 stromal vascular fraction cells per 100 mL of fat tissue, and of which the living cells accounted for (82.72±5.37)%. Through fluorescence-activated cell sorting, the proportion of adventitial cells (CD34+, CD45-, CD146-) was (17.66±1.05)%; and the proportion of pericytes (CD146+, CD45-, CD34-) was (7.18±0.52)%. Perivascular stem cells proliferated rapidly and still maintained strong proliferative ability within 10 generations. (2) In vitro studies had confirmed that perivascular stem cells had the ability of osteogenic differentiation. In addition, the expression of Runt-related transcription factor 2 (RUNX2) was significantly reduced and osteogenic differentiation of perivascular stem cells was attenuated by inhibition of Wnt signaling pathway. Animal experiments further confirmed the osteogenic effect of perivascular stem cell composite scaffolds. (3) These results indicate that Wnt/β-catenin signaling pathway plays an important role in the osteogenic differentiation of perivascular stem cells, which provides a new therapeutic option for bone repair.
Key words: human fat tissue, perivascular stem cell, fluorescence activated cell sorting, osteogenic differentiation, signaling pathway