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. 2018 Sep 30:2018:8478953.
doi: 10.1155/2018/8478953. eCollection 2018.

Dorsal Root Ganglion Maintains Stemness of Bone Marrow Mesenchymal Stem Cells by Enhancing Autophagy through the AMPK/mTOR Pathway in a Coculture System

Shuaishuai Zhang  1 , Junqin Li  1 , Huijie Jiang  1 , Yi Gao  1 , Pengzhen Cheng  1 , Tianqing Cao  1 , Donglin Li  2 , Jimeng Wang  3 , Yue Song  1 , Bin Liu  1 , Hao Wu  1 , Chunmei Wang  1 , Liu Yang  1 , Guoxian Pei  1
Affiliations

Dorsal Root Ganglion Maintains Stemness of Bone Marrow Mesenchymal Stem Cells by Enhancing Autophagy through the AMPK/mTOR Pathway in a Coculture System

Shuaishuai Zhang et al. Stem Cells Int. .

Abstract

Our previous studies found that sensory nerve tracts implanted in tissue-engineered bone (TEB) could result in better osteogenesis. To explore the mechanism of the sensory nerve promoting osteogenesis in TEB in vitro, a transwell coculture experiment was designed between dorsal root ganglion (DRG) cells and bone marrow mesenchymal stem cells (BMSCs). BMSC proliferation was determined by CCK8 assay, and osteo-, chondro-, and adipogenic differentiation were assessed by alizarin red, alcian blue, and oil red staining. We found that the proliferation and multipotent differentiation of BMSCs were all enhanced in the coculture group compared to the BMSCs group. Crystal violet staining showed that the clone-forming ability of BMSCs in the coculture group was also enhanced and mRNA levels of Sox2, Nanog, and Oct4 were significantly upregulated in the coculture group. Moreover, the autophagy level of BMSCs, regulating their stemness, was promoted in the coculture group, mediated by the AMPK/mTOR pathway. In addition, AMPK inhibitor compound C could significantly downregulate the protein expression of LC3 and the mRNA level of stemness genes in the coculture group. Finally, we found that the NK1 receptor antagonist, aprepitant, could partly block this effect, which indicated that substance P played an important role in the effect. Together, we conclude that DRG could maintain the stemness of BMSCs by enhancing autophagy through the AMPK/mTOR pathway in a transwell coculture system, which may help explain the better osteogenesis after implantation of the sensory nerve into TEB.

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Figures

Figure 1
Figure 1
Characterization of GFP BMSCs. (a) Morphology of P3 GFP BMSCs. (b) Alizarin red staining of BMSCs after osteogenic induction for 14 days. (c) Oil red staining of BMSCs after adipogenic induction for 10 days. (d) Alcian blue staining of BMSCs after chondrogenic induction for 20 days. (e) Representative results of flow cytometry analysis of P3 BMSCs indicating abundant expression of CD90 and the absence of CD 34, CD11b/c, and CD45.
Figure 2
Figure 2
Coculturing with DRG promoted proliferation and enhanced multipotential differentiation of BMSCs. (a) Morphology and density of GFP BMSCs at days 3, 5, and 8. (b) Proliferation curves of BMSCs after coculture with DRG by the CCK8 assay. (c) Osteogenic, adipogenic, and chondrogenic differentiation of BMSCs were significantly promoted after coculture with DRG for 8 days.
Figure 3
Figure 3
Coculturing with DRG promoted the self-renewal ability and stem cell-related gene expression of BMSCs. (a) CFU assay of the BMSCs and BMSCs + DRG groups. Purple dots refer to cell colonies. (b) Stem cell-related gene expression after coculture for 3, 5, and 8 days. ∗ denotes that differences are statistically significant between BMSCs + DRG and BMSCs groups.
Figure 4
Figure 4
Coculturing with DRG enhanced BMSCs autophagy. (a) LC3 immunofluorescence images after coculture for 8 days. (b) Western blot analysis of LC3II and LC3I and the LC3II/LC3I ratio analysis after coculture for 8 days. MOD: mean optical density. ∗ denotes that differences are statistically significant between BMSCs + DRG and BMSCs groups.
Figure 5
Figure 5
AMPK signaling pathway, not the AKT signaling pathway, was activated in BMSCs after coculture for 8 days.
Figure 6
Figure 6
Compound C treatment downregulated autophagy and stem cell-related genes in the coculture group. (a) Western blot analysis of LC3II and LC3I after treatment with compound C. (b) Analysis of LC3II/LC3I among the three groups. (c) Stem cell-related gene expression after treatment with compound C. ∗ denotes that differences are statistically significant between two groups. Compound C alone did not change the autophagy level or stemness genes of BMSCs (Fig S1).
Figure 7
Figure 7
Effect of aprepitant on autophagy and stemness genes in the coculture system. (a) Western blot analysis of LC3I and LC3II after treatment with aprepitant. (b) Analysis of LC3II/LC3I among the three groups. (c) Stemness gene expression after treatment with aprepitant. ∗ denotes that differences are statistically significant between two groups.
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References

    1. Dumic-Cule I., Pecina M., Jelic M., et al. Biological aspects of segmental bone defects management. International Orthopaedics. 2015;39(5):1005–1011. doi: 10.1007/s00264-015-2728-4. - DOI - PubMed
    1. Nair M. B., Kretlow J. D., Mikos A. G., Kasper F. K. Infection and tissue engineering in segmental bone defects—a mini review. Current Opinion in Biotechnology. 2011;22(5):721–725. doi: 10.1016/j.copbio.2011.02.005. - DOI - PMC - PubMed
    1. Pneumaticos S. G., Triantafyllopoulos G. K., Basdra E. K., Papavassiliou A. G. Segmental bone defects: from cellular and molecular pathways to the development of novel biological treatments. Journal of Cellular and Molecular Medicine. 2010;14(11):2561–2569. doi: 10.1111/j.1582-4934.2010.01062.x. - DOI - PMC - PubMed
    1. Chen S. Y., Qin J. J., Wang L., et al. Different effects of implanting vascular bundles and sensory nerve tracts on the expression of neuropeptide receptors in tissue-engineered bone in vivo . Biomedical Materials. 2010;5(5, article 055002) doi: 10.1088/1748-6041/5/5/055002. - DOI - PubMed
    1. Fan J. J., Mu T. W., Qin J. J., Bi L., Pei G. X. Different effects of implanting sensory nerve or blood vessel on the vascularization, neurotization, and osteogenesis of tissue-engineered bone in vivo. BioMed Research International. 2014;2014:10. doi: 10.1155/2014/412570.412570 - DOI - PMC - PubMed