EXPRESSION PROFILE OF OSTEOGENIC-RELATED GENES DURING SPONTANEOUS CALCIFICATION OF RAT BONE MARROW MESENCHYMAL STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

JIAOXuefeng ¹ , HUANGYongcan ² , HUANGYizhou ³ , WUChengguang ³ ,  DENGLi ⁴

1. Department of Pharmacology, West China School of Pharmacy, Sichuan University, Chengdu Sichuan, 610041, P. R. China;
2. Department of Orthopaedics and Traumatology, Li Ka Shing Faculty of Medicine, the University of Hong Kong;
3. Regenerative Medicine Research Center, Chengdu Sichuan, 610041, P. R. China;
4. Division of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University;

Corresponding?author：

DENGLi, Email: dengli2000@21cn.com

Keywords：

Bone marrow mesenchymal stem cells; Spontaneous calcification; Gene microarray; Gene expression profile; Bioinformatics; Rat

DOI：

10.7507/1002-1892.20140032

Video：

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Objective To analyze the expression profile changes of osteogenic-related genes during spontaneous calcification of rat bone marrow mesenchymal stem cells (BMSCs). Methods BMSCs were isolated from 3-day-old healthy Sprague Dawley rats;cells at the 4th generation were used to establish the spontaneous calcification model in vitro. Spontaneous calcification process was recorded by inverted phase contrast microscope observation and alizarin red staining after 7 and 14 days of culture. For gene microarray analysis, cell samples were collected at 0, 7, and 14 days after culture; the differentially expressed genes were analyzed by bioinformatics methods and validated by real-time quantitative PCR (RT-qPCR) assay. Results Rat BMSCs calcified spontaneously in vitro. When cultured for 7 days, the cells began to aggregate and were weakly positive for alizarin red staining. After 14 days of culture, obvious cellular aggregation and typical mineralized nodules were observed, the mineralized nodules were brightly positive for alizarin red staining. A total of 576 gene probe-sets expressed differentially during spontaneous calcification, corresponding 378 rat genes. Among them, 359 gene probe-sets expressed differentially between at 0 and 7 days, while only 13 gene probe-sets expressed differentially between at 7 and 14 days. The 378 differentially expressed genes were divided into 6 modes according to their expression profiles. Moreover, according to their biological functions, differentially expressed genes related to bone cell biology could be classified into 7 major groups:angiogenesis, apoptosis, bone-related genes, cell cycle, development, cell communication, and signal pathways related to osteogenic differentiation. In cell cycle group, 12 down-regulated genes were linked with each other functionally. Matrix metalloproteinase 13 (Mmp13), secreted phosphoprotein 1 (Spp1), Cxcl12, Mmp2, Mmp3, Apoe, and Itga7 had more functional connections with other genes. The results of genes Spp1, Mgp, Mmp13, Wnt inhibitory factor 1, Cxcl12, and cyclin A2 by RT-qPCR were consistent with that of gene microarray. Conclusion The first 7 days after rat BMSCs were seeded are a key phase determining the fate of spontaneous calcification. Multiple genes related with cell communication, bone-related genes, cell cycle, transforming growth factor-β signaling pathway, mitogen-activated protein kinase signaling pathway, and Wnt signaling pathway are involved during spontaneous calcification.

Citation： JIAOXuefeng, HUANGYongcan, HUANGYizhou, WUChengguang, DENGLi. EXPRESSION PROFILE OF OSTEOGENIC-RELATED GENES DURING SPONTANEOUS CALCIFICATION OF RAT BONE MARROW MESENCHYMAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(2): 133-141. doi: 10.7507/1002-1892.20140032 Copy

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26.	Mak W,Shao X,Dunstan CR,et al.Biphasic glucocorticoid-dependent regulation of Wnt expression and its inhibitors in mature osteoblastic cells.Calcif Tissue Int,2009,85(6):538-545.
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28.	Vaes BL,Dechering KJ,van Someren EP,et al.Microarray analysis reveals expression regulation of Wnt antagonists in differentiating osteoblasts.Bone,2005,36(5):803-811.
29.	Filipak M,Estervig DN,Tzen CY,et al.Integrated control of proliferation and differentiation of mesenchymal stem cells.Environ Health Perspect,1989,80:117-125.
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31.	G?tz W,Reichert C,Canullo L,et al.Coupling of osteogenesis and angiogenesis in bone substitute healing-a brief overview.Ann Anat,2012,194(2):171-173.

1. Wong RS.Mesenchymal stem cells:angels or demons.J Biomed Biotechnol,2011,2011:459510.
2. Yoon YS,Park JS,Tkebuchava T,et al.Unexpected severe calcification after transplantation of bone marrow cells in acute myocardial infarction.Circulation,2004,109(25):3154-3157.
3. Breitbach M,Bostani T,Roell W,et al.Potential risks of bone marrow cell transplantation into infarcted hearts.Blood,2007,110(4):1362-1369.
4. Bi Y,Ehirchiou D,Kilts TM,et al.Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche.Nat Med,2007,13(10):1219-1227.
5. Huang YC,Zhu HM,Cai JQ,et al.Hypoxia inhibits the spontaneous calcification of bone marrow-derived mesenchymal stem cells.J Cell Biochem,2012,113(4):1407-1415.
6. Zhou Y,Guan XX,Zhu ZL,et al.Caffeine inhibits the viability and osteogenic differentiation of rat bone marrow-derived mesenchymal stromal cells.Br J Pharmacol,2010,161(7):1542-1552.
7. Ernst J,Bar-Joseph Z.STEM:a tool for the analysis of short time series gene expression data.BMC Bioinformatics,2006,7:191.
8. Zhou X,Su Z.EasyGO:Gene Ontology-based annotation and functional enrichment analysis tool for agronomical species.BMC Genomics,2007,8:246.
9. Mao X,Cai T,Olyarchuk JG,et al.Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary.Bioinformatics,2005,21(19):3787-3793.
10. von Mering C,Huynen M,Jaeggi D,et al.STRING:a database of predicted functional associations between proteins.Nucleic Acids Res,2003,31(1):258-261.
11. Allori AC,Sailon AM,Warren SM.Biological basis of bone formation,remodeling,and repair-part I:biochemical signaling molecules.Tissue Eng Part B Rev,2008,14(3):259-273.
12. Allori AC,Sailon AM,Warren SM.Biological basis of bone formation,remodeling,and repair-part Ⅱ:extracellular matrix.Tissue Eng Part B Rev,2008,14(3):275-283.
13. Granchi D,Ochoa G,Leonardi E,et al.Gene expression patterns related to osteogenic differentiation of bone marrow-derived mesenchymal stem cells during ex vivo expansion.Tissue Eng Part C Methods,2010,16(3):511-524.
14. Kulterer B,Friedl G,Jandrositz A,et al.Gene expression profiling of human mesenchymal stem cells derived from bone marrow during expansion and osteoblast differentiation.BMC Genomics,2007,8:70.
15. Schilling AF,Schinke T,Munch C,et al.Increased bone formation in mice lacking apolipoprotein E.J Bone Miner Res,2005,20(2):274-282.
16. Zhang C,Tang W,Li Y.Matrix metalloproteinase 13(MMP13) is a direct target of osteoblast-specific transcription factor osterix (Osx) in osteoblasts.PLoS One,2012,7(11):e50525.
17. Hosogane N,Huang Z,Rawlins BA,et al.Stromal derived factor-1 regulates bone morphogenetic protein 2-induced osteogenic differentiation of primary mesenchymal stem cells.Int J Biochem Cell Biol,2010,42(7):1132-1141.
18. Franceschi RT,Ge C,Xiao G,et al.Transcriptional regulation of osteoblasts.Cells Tissues Organs,2009,189(1-4):144-152.
19. Qin C,D'Souza R,Feng JQ.Dentin matrix protein 1(DMP1):new and important roles for biomineralization and phosphate homeostasis.J Dent Res,2007,86(12):1134-1141.
20. Friedman MS,Long MW,Hankenson KD.Osteogenic differentiation of human mesenchymal stem cells is regulated by bone morphogenetic protein-6.J Cell Biochem,2006,98(3):538-554.
21. Dan H,Simsa-Maziel S,Reich A,et al.The role of matrix gla protein in ossification and recovery of the avian growth plate.Front Endocrinol (Lausanne),2012,3:79.
22. Zebboudj AF,Shin V,Bostr?m K.Matrix GLA protein and BMP-2 regulate osteoinduction in calcifying vascular cells.J Cell Biochem,2003,90(4):756-765.
23. Hamidouche Z,Fromigué O,Nuber U,et al.Autocrine fibroblast growth factor 18 mediates dexamethasone-induced osteogenic differentiation of murine mesenchymal stem cells.J Cell Physiol,2010,224(2):509-515.
24. Kikuchi A,Yamamoto H,Sato A,et al.Wnt5a:its signalling,functions and implication in diseases.Acta Physiol (Oxf),2012,204(1):17-33.
25. Rauner M,Sipos W,Pietschmann P.Age-dependent Wnt gene expression in bone and during the course of osteoblast differentiation.Age (Dordr),2008,30(4):273-282.
26. Mak W,Shao X,Dunstan CR,et al.Biphasic glucocorticoid-dependent regulation of Wnt expression and its inhibitors in mature osteoblastic cells.Calcif Tissue Int,2009,85(6):538-545.
27. Nemoto E,Ebe Y,Kanaya S,et al.Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis.Biochem Biophys Res Commun,2012,422(4):627-632.
28. Vaes BL,Dechering KJ,van Someren EP,et al.Microarray analysis reveals expression regulation of Wnt antagonists in differentiating osteoblasts.Bone,2005,36(5):803-811.
29. Filipak M,Estervig DN,Tzen CY,et al.Integrated control of proliferation and differentiation of mesenchymal stem cells.Environ Health Perspect,1989,80:117-125.
30. Stein GS,Lian JB,Gerstenfeld LG,et al.The onset and progression of osteoblast differentiation is functionally related to cellular proliferation.Connect Tissue Res,1989,20(1-4):3-13.
31. G?tz W,Reichert C,Canullo L,et al.Coupling of osteogenesis and angiogenesis in bone substitute healing-a brief overview.Ann Anat,2012,194(2):171-173.

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EXPRESSION PROFILE OF OSTEOGENIC-RELATED GENES DURING SPONTANEOUS CALCIFICATION OF RAT BONE MARROW MESENCHYMAL STEM CELLS

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