TIME DIFFERENCES OF BONE MARROW MESENCHYMAL STEM CELLS AND NUCLEUS PULPOSUS-LIKE CELLS IN A NON CONTACT CO-CULTURE SYSTEM_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CHEN Lei ¹ , HU Xiunian ¹ , FU Lengxi ² , YE Junjian ¹

1Department of Orthopedics, 2Central Laboratory, the First Affiliated Hospital of Fujian Medical University, Fuzhou Fujian, 350004, P.R.China. Corresponding author: YE Junjian, E-mail: yejunjian@sohu.com;

Corresponding author：

Keywords：

Bone marrow mesenchymal stem cells; Nucleus pulposus-like cells; Non contact co-culture; Rabbit

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Objective To investigate time differences in directional differentiation of bone marrow mesenchymal stem cells (BMSCs) into nucleus pulposus-like cells (NPCs) in a non contact co-culture system so as to search for the best time for transplantation in vivo. Methods Six New Zealand white rabbits (aged 6 weeks, weighing 1.5-2.0 kg) were selected. BMSCs were collected and cultured for immunocytochemistry identification of CD34, CD44, CD45, and CD90; NPCs were isolated and identified immunocytochemically by RT-PCR. The 2nd passage BMSCs and the primary NPCs were co-cultured in a non contact co-culture system. The cell morphological changes were observed and the cell growth curves were made at 1, 3, and 5 passages after co-culture. The expressions of the aggrecan and collagen type II genes were detected by RT-PCR in BMSCs at 5, 10, and 15 days after co-culture; the expressions of the aggrecan and collagen type II proteins were detected by Western blot at 5, 10, 15, 20, 25, and 30 days after co-culture. Results The expressions of CD44 and CD90 were positive, CD34 and CD45 were negative in BMSCs. The expressions of the collagen type II and aggrecan were positive in NPCs. At 2 weeks after co-culture, the morphology of BMSCs changed obviously, the cells were polygonal and irregular shape. The cell growth rate showed no difference within 3 passages, but decreased obviously after 3 passages. RT-PCR showed that the expressions of collagen type II and aggrecan genes at 10 and 15 days were significantly higher than those at 5 days (P lt; 0.05), no significant difference was found between at 10 days and at 15 days (P gt; 0.05). Western blot showed that the expressions of collagen type II and aggrecan proteins gradually increased with time, and there was significant difference within 5, 10, and 15 days (P lt; 0.05), but no significant difference was found after 15 days of co-culture (P gt; 0.05). Conclusion In a non contact co-culture system, BMSCs can differentiate into the NPCs. The expression of collagen type II and aggrecan can reach a stable level at 15 days after co-culture, and it is the suitable time for transplantation in vivo.

Citation： CHEN Lei,HU Xiunian,FU Lengxi,YE Junjian. TIME DIFFERENCES OF BONE MARROW MESENCHYMAL STEM CELLS AND NUCLEUS PULPOSUS-LIKE CELLS IN A NON CONTACT CO-CULTURE SYSTEM. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(11): 1369-1374. doi: Copy

1.	Zhang Y, An HS, Tannoury C, et al. Biological treatment for degenerative disc disease: implications for the field of physical medicine and rehabilitation. Am J Phys Med Rehabil, 2008, 87(9): 694-702.
2.	Yang X, Li X. Nucleus pulposus tissue engineering: a brief review. Eur Spine J , 2009, 18(11): 1564-1572.
3.	Cappello R, Bird JL, Pfeiffer D, et al. Notochordal cell produce and assemble extracellular matrix in a distinct manner, which may be tesponsible for the maintenance of healthy nucleus pulposus. Spine (Phila Pa 1976), 2006, 31(8): 873-883.
4.	Leung VY, Chan D, Cheung KM. Regeneration of intervertebral disc by mesenchymal stem cells: potentials, limitations, and future direction. Eur Spine J, 2006, 15 Suppl 3: S406-413.
5.	Iwashina T, Mochida J, Sskai D, et al. Feasibility of using a human nucleus pulposus cell line as a cell source in cell transplantation therapy for intervertebral disc degeneration. Spine (Phila Pa 1976), 2006, 31(11): 1177-1186.
6.	Strassburg S, Richardson SM, Freemont AJ, et al. Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype. Regen Med, 2010, 5(5): 701-711.
7.	Risbud MV, Albert TJ, Guttapalli A, et al. Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro: implications for cell-based transplantation therapy. Spine (Phila Pa 1976), 2004, 29(23): 2627-2632.
8.	Steck E, Bettram H, Abel R, et al. Induction of intervertebral disc-like cells from adult mesenchymal stem cells. Stem Cells, 2005, 23(3): 403-411.
9.	Luo W, Xiong W, Qiu M, et al. Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype utilizing simulated microgravity In vitro. J Huazhong Univ Sci Technolog Med Sci, 2011, 31(2): 199-203.
10.	Sakai D, Mochida J, Iwashina T, et al. Regenerative effects of transplanting mesenchymal stem cells embedded in atelocollagen to the degenerated intervertebral disc. Biomaterials, 2006, 27(3): 335-345.
11.	Richardson SM, Hoyland JA. Stem cell regeneration of degenerated intervertebral discs: current status. Curr Pain Headache Rep, 2008, 12(2): 83-88.
12.	Noël D, Gazit D, Bouquet C, et al. Short-term BMP-2 expression is sufficient for in vivo osteochondral differentiation of mesenchymal stem cells. Stem Cells, 2004, 22(1): 74-85.
13.	Yang M, Ma QJ, Dang GT, et al. In vitro and in vivo induction of bone formation based on ex vivo gene therapy using rat adipose-derived adult. stem cells expressing BMP-7. Cytotherapy, 2005, 7(3): 273-281.
14.	Li X, Lee JP, Balian G, et al. Modulation of chondrocytic properties of fat-derived mesenchymal cells in co-cultures with nucleus pulposus. Connect Tissue Res, 2005, 46(2): 75-82.
15.	Richardson SM, Walker RV, Parker S, et al. Intervertebral disc cell-mediated mesenchymal stem cell differentiation. Stem Cells, 2006, 24(3): 707-716.
16.	Leung VY, Chan D, Cheung KM. Regeneration of intervertebral disc by mesenchymal stem cells: potentials, limitations, and future direction. Eur Spine J, 2006, 15 Suppl 3: S406-413.
17.	Strassburg S, Richardson SM, Freemont AJ, et al. Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype. Regen Med, 2010, 5(5): 701-711.
18.	Pattison ST, Melrose J, Ghosh P, et al. Regulation of gelatinase-A (MMP-2) production by ovine intervertebral disc nucleus pulposus cells grown in alginate bead culture by transforming growth factor-β1 and insulin like growth factor-I. Cell Biol Int, 2001, 25(7): 679-689.
19.	Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining mμltipotent mesenchymal stromal cells. The International Society for Cellμlar Therapy position statement. Cytotherapy, 2006, 8(4): 315-317.
20.	Horwitz EM, Le Blanc K, Dominici M, et al. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy, 2005, 7(5): 393-395.

1. Zhang Y, An HS, Tannoury C, et al. Biological treatment for degenerative disc disease: implications for the field of physical medicine and rehabilitation. Am J Phys Med Rehabil, 2008, 87(9): 694-702.
2. Yang X, Li X. Nucleus pulposus tissue engineering: a brief review. Eur Spine J , 2009, 18(11): 1564-1572.
3. Cappello R, Bird JL, Pfeiffer D, et al. Notochordal cell produce and assemble extracellular matrix in a distinct manner, which may be tesponsible for the maintenance of healthy nucleus pulposus. Spine (Phila Pa 1976), 2006, 31(8): 873-883.
4. Leung VY, Chan D, Cheung KM. Regeneration of intervertebral disc by mesenchymal stem cells: potentials, limitations, and future direction. Eur Spine J, 2006, 15 Suppl 3: S406-413.
5. Iwashina T, Mochida J, Sskai D, et al. Feasibility of using a human nucleus pulposus cell line as a cell source in cell transplantation therapy for intervertebral disc degeneration. Spine (Phila Pa 1976), 2006, 31(11): 1177-1186.
6. Strassburg S, Richardson SM, Freemont AJ, et al. Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype. Regen Med, 2010, 5(5): 701-711.
7. Risbud MV, Albert TJ, Guttapalli A, et al. Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro: implications for cell-based transplantation therapy. Spine (Phila Pa 1976), 2004, 29(23): 2627-2632.
8. Steck E, Bettram H, Abel R, et al. Induction of intervertebral disc-like cells from adult mesenchymal stem cells. Stem Cells, 2005, 23(3): 403-411.
9. Luo W, Xiong W, Qiu M, et al. Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype utilizing simulated microgravity In vitro. J Huazhong Univ Sci Technolog Med Sci, 2011, 31(2): 199-203.
10. Sakai D, Mochida J, Iwashina T, et al. Regenerative effects of transplanting mesenchymal stem cells embedded in atelocollagen to the degenerated intervertebral disc. Biomaterials, 2006, 27(3): 335-345.
11. Richardson SM, Hoyland JA. Stem cell regeneration of degenerated intervertebral discs: current status. Curr Pain Headache Rep, 2008, 12(2): 83-88.
12. Noël D, Gazit D, Bouquet C, et al. Short-term BMP-2 expression is sufficient for in vivo osteochondral differentiation of mesenchymal stem cells. Stem Cells, 2004, 22(1): 74-85.
13. Yang M, Ma QJ, Dang GT, et al. In vitro and in vivo induction of bone formation based on ex vivo gene therapy using rat adipose-derived adult. stem cells expressing BMP-7. Cytotherapy, 2005, 7(3): 273-281.
14. Li X, Lee JP, Balian G, et al. Modulation of chondrocytic properties of fat-derived mesenchymal cells in co-cultures with nucleus pulposus. Connect Tissue Res, 2005, 46(2): 75-82.
15. Richardson SM, Walker RV, Parker S, et al. Intervertebral disc cell-mediated mesenchymal stem cell differentiation. Stem Cells, 2006, 24(3): 707-716.
16. Leung VY, Chan D, Cheung KM. Regeneration of intervertebral disc by mesenchymal stem cells: potentials, limitations, and future direction. Eur Spine J, 2006, 15 Suppl 3: S406-413.
17. Strassburg S, Richardson SM, Freemont AJ, et al. Co-culture induces mesenchymal stem cell differentiation and modulation of the degenerate human nucleus pulposus cell phenotype. Regen Med, 2010, 5(5): 701-711.
18. Pattison ST, Melrose J, Ghosh P, et al. Regulation of gelatinase-A (MMP-2) production by ovine intervertebral disc nucleus pulposus cells grown in alginate bead culture by transforming growth factor-β1 and insulin like growth factor-I. Cell Biol Int, 2001, 25(7): 679-689.
19. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining mμltipotent mesenchymal stromal cells. The International Society for Cellμlar Therapy position statement. Cytotherapy, 2006, 8(4): 315-317.
20. Horwitz EM, Le Blanc K, Dominici M, et al. Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy, 2005, 7(5): 393-395.

Chinese Journal of Reparative and Reconstructive Surgery

TIME DIFFERENCES OF BONE MARROW MESENCHYMAL STEM CELLS AND NUCLEUS PULPOSUS-LIKE CELLS IN A NON CONTACT CO-CULTURE SYSTEM

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