AN IN VITRO STUDY ON BIOLOGICAL CHARACTERISTICS OF BONE MARROW MESENCHYMAL STEM CELLS IN MICROENVIRONMENT OF PREMATURE SENESCENCE OF NUCLEUS PULPOSUS CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

 WANGYuntao , WUXiaotao , WANGFeng , HONGXin , BAOJunping , ZHULei

Department of Orthopaedics, Zhongda Hospital, Southeast University, Nanjing Jiangsu, 210009, P. R. China;

Corresponding author：

WANGYuntao, Email: wangyttod@seu.edu.cn

Keywords：

Bone marrow mesenchymal stem cells; Nucleus pulposus cells; Premature senescence; Intervertebral disc degeneration; Biological characteristics

DOI：

10.7507/1002-1892.20140168

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Objective To investigate the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) in microenvironment of premature senescence of nucleus pulposus cells (NPCs) so as to lay a foundation for the repair of intervertebral disc degeneration by BMSCs transplantation. Methods Human degenerative nucleus pulposus and normal bone marrow were collected, and then NPCs and BMSCs were isolated, cultured, and identified. The 3rd passage BMSCs and the 1st passage NPCs with premature senescence were co-cultured without contact in the Transwell culture system. NPCs to BMSCs ratio was 75%:25% (group A), 50%:50% (group B), and 0:100% (group C). The morphological changes of BMSCs were observed by inverted phase contrast microscopy and transmission electron microscopy. At 3 and 6 days after co-culture, cell counting kit 8 was used to detect cell viability, flow cytometry was used to observe the cell cycle and detect DNA metabolism after BrdU labeling. Cell senescence was also evaluated by detecting senescence associated β-galactosidase (SA-β-gal) activity. Results The typical morphology of cell senescence was seen in groups A and B, especially in group A. At 3 and 6 days after co-culture, the cell survival rate of group A was significantly lower than that of group B (P<0.05). At 3 days after co-culture, the proportion of cells in G₁ phase in group A was significantly higher than that in groups B and C (P<0.05), the proportion of cells in S phase in group A was significantly lower than that in groups B and C (P<0.05). At 6 days, the proportion of cells in G₁ phase in group A was about 81.0%, and the proportion of cells in S phase and G₂ phase decreased, showing significant difference when compared with groups B and C (P<0.05); the proportion of cells in G₁ phase in group B was about 74.4%, showing significant difference when compared with group C (P<0.05). BrdU content in group A was significantly lower than that in groups B and C at 3 and 6 days after co-culture (P<0.05), but no significant difference was found between groups B and C at 3 days (P>0.05); Brdu content in group B was also significantly reduced when compared with group C (P<0.05) at 6 days. At 6 days, SA-β-gal activity was significantly increased in groups A and B, and significant difference was shown in SA-β-gal positive cell number between groups (P <0.05). Conclusion Premature senescence of NPCs can down-regulate the proliferation capacity of co-cultured BMSCs by the paracrine effect. The greater proportion of NPCs with premature senescence is, the earlier senescence of BMSCs will be induced.

Citation： WANGYuntao, WUXiaotao, WANGFeng, HONGXin, BAOJunping, ZHULei. AN IN VITRO STUDY ON BIOLOGICAL CHARACTERISTICS OF BONE MARROW MESENCHYMAL STEM CELLS IN MICROENVIRONMENT OF PREMATURE SENESCENCE OF NUCLEUS PULPOSUS CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(6): 758-762. doi: 10.7507/1002-1892.20140168 Copy

1.	Wang YT, Wu XT, Wang F. Regeneration potential and mechanism of bone marrow mesenchymal stem cell transplation for treating intervertebral disc degeneration. J Orthop Sci, 2010, 15(6):707-719.
2.	王锋, 吴小涛, 王运涛, 等. 干细胞移植修复椎间盘退变的研究现状. 中国修复重建外科杂志, 2013, 27(5):568-572.
3.	王锋, 吴小涛, 王运涛, 等. 非接触共培养条件下人BMSCs对氧化应激环境中退变髓核细胞凋亡的保护研究. 中国修复重建外科杂志, 2010, 24(4):391-398.
4.	Sun W, Zhang K, Liu G, et al. Sox9 gene transfer enhanced regenerative effect of bone marrow mesenchymal stem cells on the degenerated intervertebral disc in a rabbit model. PLoS One, 2014, 9(4):e93570.
5.	Tang X, Jing L, Chen J. Changes in the molecular phenotype of nucleus pulposus cells with intervertebral disc aging. PLoS One, 2012, 7(12):e52020.
6.	Ciapetti G, Granchi D, Devescovi V, et al. Ex vivo observation of human intervertebral disc tissue and cells isolated from degenerated intervertebral discs. Eur Spine J, 2012, 21 Suppl 1:S10-19.
7.	Klotz B, Mentrup B, Regensburger M, et al. 1, 25-dihydroxyvitamin D3 treatment delays cellular aging in human mesenchymal stem cells while maintaining their multipotent capacity. PLoS One, 2012, 7(1):e29959.
8.	Kim JS, Kim EJ, Kim HJ, et al. Proteomic and metabolomic analysis of H₂O₂-induced premature senescent human mesenchymal stem cells. Exp Gerontol, 2011, 46(6):500-510.
9.	Cai B, Zhu S, Li J, et al. Bone marrow-derived mesenchymal stem cells protected rat cardiomyocytes from premature senescence. Int J Cardiol, 2012, 154(2):180-182.
10.	Burova E, Borodkina A, Shatrova A, et al. Sublethal oxidative stress induces the premature senescence of human mesenchymal stem cells derived from endometrium. Oxid Med Cell Longev, 2013, 2013:474931.
11.	Wang YT, Wu XT, Chen H, et al. Endoscopy-assisted posterior lumbar interbody fusion in a single segment. J Clin Neurosci, 2014, 21(2):287-292.
12.	Kim KW, Chung HN, Ha KY, et al. Senescence mechanisms of nucleus pulposus chondrocytes in human intervertebral discs. Spine J, 2009, 9(8):658-666.
13.	Jeong SW, Lee JS, Kim KW. In vitro lifespan and senescence mechanisms of human nucleus pulposus chondrocytes. Spine J, 2014, 14(3):499-504.
14.	Alt EU, Senst C, Murthy SN, et al. Aging alters tissue resident mesenchymal stem cell properties. Stem Cell Res, 2012, 8(2):215-225.
15.	Ryu BY, Orwig KE, Oatley JM, et al. Effects of aging and niche microenvironment on spermatogonial stem cell self-renewal. Stem Cells, 2006, 24(6):1505-1511.
16.	Kan CD, Li SH, Weisel RD, et al. Recipient age determines the cardiac functional improvement achieved by skeletal myoblast transplantation. J Am Coll Cardiol, 2007, 50(11):1086-1092.
17.	Choudhery MS, Khan M, Mahmood R, et al. Bone marrow derived mesenchymal stem cells from aged mice have reduced wound healing, angiogenesis, proliferation and anti-apoptosis capabilities. Cell Biol Int, 2012, 36(8):747-753.
18.	Brandl A, Meyer M, Bechmann V, et al. Oxidative stress induces senescence in human mesenchymal stem cells. Exp Cell Res, 2011, 317(11):1541-1547.
19.	Zhao CQ, Wang LM, Jiang LS, et al. The cell biology of intervertebral disc aging and degeneration. Ageing Res Rev, 2007, 6(3):247-261.
20.	Gruber HE, Hoelscher GL, Ingram JA, et al. Senescent vs. non-senescent cells in the human annulus in vivo:cell harvest with laser capture microdissection and gene expression studies with microarray analysis. BMC Biotechnol, 2010, 10:5.
21.	Ren JL, Pan JS, Lu YP, et al. Inflammatory signaling and cellular senescence. Cell Signal, 2009, 21(3):378-383.

1. Wang YT, Wu XT, Wang F. Regeneration potential and mechanism of bone marrow mesenchymal stem cell transplation for treating intervertebral disc degeneration. J Orthop Sci, 2010, 15(6):707-719.
2. 王锋, 吴小涛, 王运涛, 等. 干细胞移植修复椎间盘退变的研究现状. 中国修复重建外科杂志, 2013, 27(5):568-572.
3. 王锋, 吴小涛, 王运涛, 等. 非接触共培养条件下人BMSCs对氧化应激环境中退变髓核细胞凋亡的保护研究. 中国修复重建外科杂志, 2010, 24(4):391-398.
4. Sun W, Zhang K, Liu G, et al. Sox9 gene transfer enhanced regenerative effect of bone marrow mesenchymal stem cells on the degenerated intervertebral disc in a rabbit model. PLoS One, 2014, 9(4):e93570.
5. Tang X, Jing L, Chen J. Changes in the molecular phenotype of nucleus pulposus cells with intervertebral disc aging. PLoS One, 2012, 7(12):e52020.
6. Ciapetti G, Granchi D, Devescovi V, et al. Ex vivo observation of human intervertebral disc tissue and cells isolated from degenerated intervertebral discs. Eur Spine J, 2012, 21 Suppl 1:S10-19.
7. Klotz B, Mentrup B, Regensburger M, et al. 1, 25-dihydroxyvitamin D3 treatment delays cellular aging in human mesenchymal stem cells while maintaining their multipotent capacity. PLoS One, 2012, 7(1):e29959.
8. Kim JS, Kim EJ, Kim HJ, et al. Proteomic and metabolomic analysis of H₂O₂-induced premature senescent human mesenchymal stem cells. Exp Gerontol, 2011, 46(6):500-510.
9. Cai B, Zhu S, Li J, et al. Bone marrow-derived mesenchymal stem cells protected rat cardiomyocytes from premature senescence. Int J Cardiol, 2012, 154(2):180-182.
10. Burova E, Borodkina A, Shatrova A, et al. Sublethal oxidative stress induces the premature senescence of human mesenchymal stem cells derived from endometrium. Oxid Med Cell Longev, 2013, 2013:474931.
11. Wang YT, Wu XT, Chen H, et al. Endoscopy-assisted posterior lumbar interbody fusion in a single segment. J Clin Neurosci, 2014, 21(2):287-292.
12. Kim KW, Chung HN, Ha KY, et al. Senescence mechanisms of nucleus pulposus chondrocytes in human intervertebral discs. Spine J, 2009, 9(8):658-666.
13. Jeong SW, Lee JS, Kim KW. In vitro lifespan and senescence mechanisms of human nucleus pulposus chondrocytes. Spine J, 2014, 14(3):499-504.
14. Alt EU, Senst C, Murthy SN, et al. Aging alters tissue resident mesenchymal stem cell properties. Stem Cell Res, 2012, 8(2):215-225.
15. Ryu BY, Orwig KE, Oatley JM, et al. Effects of aging and niche microenvironment on spermatogonial stem cell self-renewal. Stem Cells, 2006, 24(6):1505-1511.
16. Kan CD, Li SH, Weisel RD, et al. Recipient age determines the cardiac functional improvement achieved by skeletal myoblast transplantation. J Am Coll Cardiol, 2007, 50(11):1086-1092.
17. Choudhery MS, Khan M, Mahmood R, et al. Bone marrow derived mesenchymal stem cells from aged mice have reduced wound healing, angiogenesis, proliferation and anti-apoptosis capabilities. Cell Biol Int, 2012, 36(8):747-753.
18. Brandl A, Meyer M, Bechmann V, et al. Oxidative stress induces senescence in human mesenchymal stem cells. Exp Cell Res, 2011, 317(11):1541-1547.
19. Zhao CQ, Wang LM, Jiang LS, et al. The cell biology of intervertebral disc aging and degeneration. Ageing Res Rev, 2007, 6(3):247-261.
20. Gruber HE, Hoelscher GL, Ingram JA, et al. Senescent vs. non-senescent cells in the human annulus in vivo:cell harvest with laser capture microdissection and gene expression studies with microarray analysis. BMC Biotechnol, 2010, 10:5.
21. Ren JL, Pan JS, Lu YP, et al. Inflammatory signaling and cellular senescence. Cell Signal, 2009, 21(3):378-383.

Chinese Journal of Reparative and Reconstructive Surgery

AN IN VITRO STUDY ON BIOLOGICAL CHARACTERISTICS OF BONE MARROW MESENCHYMAL STEM CELLS IN MICROENVIRONMENT OF PREMATURE SENESCENCE OF NUCLEUS PULPOSUS CELLS

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