IN VITRO DIFFERENTIATION OF SYNOVIAL-DERIVED MESENCHYMAL STEM CELLS INFECTED BY ADENOVIRUS VECTOR MEDIATED BY BONE MORPHOGENETIC PROTEIN 2/7 GENES INTO FIBROCARTILAGE CELLS IN RABBITS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

FU Peiliang ¹ , ZHANG Lei ¹ , WU Haishan ¹ , CONG Ruijun ¹ , CHEN Song ¹ , DING Zheru ¹ , HU Kaimeng ²

1Department of Joint Surgery, Shanghai Changzheng Hospital, Shanghai, 200003, P.R.China;;
2Section of Histology &;
Embryology, Basic Research Department, Second Military Medical University. Corresponding author: WU Haishan, E-mail: wuhaishan@vip.sina.com;

Corresponding author：

Keywords：

Adenovirus vector; Bone morphogenetic protein 2/7 Synovial-derived mesenchymal stem cells; Fibrocartilage cells; Rabbit

DOI：

10.7507/1002-1892.20130079

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Objective To investigate the feasibility of rabbit synovial-derived mesenchymal stem cells (SMSCs) differentiating into fibrocartilage cells by the recombinant adenovirus vector mediated by bone morphogenetic protein 2/7 (BMP-2/7) genes in vitro. Methods SMSCs were isolated and purified from 3-month-old New Zealand white rabbits [male or female, weighing (2.1 ± 0.3) kg]; the morphology was observed; the cells were identified with immunocytological fluorescent staining, flow cytometry, and cell cycles. The adipogenic, osteogenic, and chondrogenic differentiations were detected. The recombinant plasmid of pAdTrack-BMP-2-internal ribosome entry site (IRES)-BMP-7 was constructed and then was used to infect SMSCs. The cell DNA content and the oncogenicity were tested to determine the safety. Then infected SMSCs were cultured in incomplete chondrogenic medium in vitro. Chondrogenic differentiation of infected SMSCs was detected by RT-PCR, immunofluorescent staining, and toluidine blue staining. Results SMSCs expressed surface markers of stem cells, and had multi-directional potential. The transfection efficiency of SMSCs infected by recombinant plasmid of pAdTrack-BMP-2-IRES-BMP-7 was about 70%. The safety results showed that infected SMSCs had normal double time, normal chromosome number, and normal DNA content and had no oncogenicity. At 21 days after cultured in incomplete chondrocyte medium, RT-PCR results showed SMSCs had increased expressions of collegan type I and collegan type II, particularly collegan type II; the expressions of RhoA and Sox-9 increased obviously. Immunofluorescent staining and toluidine blue staining showed differentiation of SMSCs into fibrocartilage cells. Conclusion It is safe to use pAdTrack-BMP-2-IRES-BMP-7 for infecting SMSCs. SMSCs infected by pAdTrack-BMP-2-IRES-BMP-7 can differentiate into fibrocartilage cells spontaneously in vitro.

Citation： FU Peiliang,ZHANG Lei,WU Haishan,CONG Ruijun,CHEN Song,DING Zheru,HU Kaimeng. IN VITRO DIFFERENTIATION OF SYNOVIAL-DERIVED MESENCHYMAL STEM CELLS INFECTED BY ADENOVIRUS VECTOR MEDIATED BY BONE MORPHOGENETIC PROTEIN 2/7 GENES INTO FIBROCARTILAGE CELLS IN RABBITS. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(3): 345-352. doi: 10.7507/1002-1892.20130079 Copy

1.	赵文君, 邢国胜, 于顺禄. 滑膜间充质干细胞及其在组织工程中的应用. 中国修复重建外科杂志, 2011, 25(12): 1504-1507.
2.	De Bari C, Dell’Accio F, Tylzanowski P, et al. Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum, 2001, 44(8): 1928-1942.
3.	Djouad F, Bony C, Haupl T, et al. Transcriptional profiles discriminate bone marrow-derived and synovium-derived mesenchymal stem cells. Arthritis Res Ther, 2005, 7(6): 1304-1315.
4.	Jo CH, Ahn HJ, Kim HJ, et al. Surface characterization and chondrogenic differentiation of mesenchymal stromal cells derived from synovium. Cytotherapy, 2007, 9(4): 316-327.
5.	Morito T, Muneta T, Hara K, et al. Synovial fluid-derived mesenchymal stem cells increase after intra-articular ligament injury in humans. Rheumatology (Oxford), 2008, 47(8): 1137-1143.
6.	Zhang S, Muneta T, Morito T, et al. Autologous synovial fluid enhances migration of mesenchymal stem cells from synovium of osteoarthritis patients in tissue culture system. J Orthop Res, 2008, 26(10): 1413-1418.
7.	Horie M, Sekiya I, Muneta T, et al. Intra-articular injected synovial stem cells differentiate into meniscal cells directly and promote meniscal regeneration without mobilization to distant organs in rat massive meniscal defect. Stem Cells, 2009, 27(4): 878-887.
8.	Sakaguchi Y, Sekiya I, Yagishita K, et al. Comparison of human stem cells derived from various mesenchymal tissues. Arthritis Rheum, 2005, 52(8): 2521-2529.
9.	Shirasawa S, Sekiya I, Sakaguchi Y, et al. In vitro chondrogenesis of human synovium-derived mesenchynlal stem cells: optimal condition and cmnparison with bone morrow-derived cells. J Cell Biochem, 2006, 97(1): 84-97.
10.	Park Y, Suginoto M, Watrin A, et al. BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel. Osteoarthritis Cartilage, 2005, 13(6): 527-536.
11.	Kurth T, Hedbom E, Shintani N, et al. Chondrogenic potential of human synovial mesenchymal stem cells in alginate. Osteparthritis Cartilage, 2007, 15(10): 1178-1189.
12.	Miyamoto C, Matsumoto T, Sakimura K, et al. Osteogenic protein-1 with transforming growth factor-beta 1: potent inducer of chondrogenesis of synovial mesenchymal stem cells in vitro. J Orthop Sci, 2007, 12(6): 555-561.
13.	张正政, 李卫平, 杨睿, 等. 滑膜间充质干细胞分离培养、免疫表型鉴定及在混合淋巴反应体系中的抑制效应. 中国组织工程研究, 2012, 16(19): 3531-3519.
14.	Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell, 2003, 113(6): 685-700.
15.	Nagase T, Muneta T, Ju YJ, et al. Analysis of the chondrogenic potential of human synovial stem cells according to harvest site and culture parameters in knees with medial compartment osteoarthritis. Arthritis Rheum, 2008, 58(5): 1389-1398.
16.	Maniatopiulos C, Sidek J, Melcher AH. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res, 1988, 254(2): 317-330.
17.	Wan M, Cao X. BMP signaling in skeletal development. Biochem Biophys Res Commun, 2005, 328(3): 651-657.
18.	Bi W, Huang W, Whitworth DJ, et al. Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization. Proc Natl Acad Sci U S A, 2001, 98(12): 6698-6703.
19.	Nishimura K, Solchaga LA, Caplan AI, et al. Chondroprogenitor cells of synovial tissue. Arthritis Rheum, 1999, 42(12): 2631-2637.
20.	Dell’Accio F, Vanlauwe J, Bellemans J, et al. Expanded phenotypically stable chondrocytes persisit in the repair tissue and contribute to cartilage matrix formation and structural integration in a goat model of autologous chondrocyte implantation. J Orthop Res, 2003, 21(1): 123-131.
21.	Brittberg M, Peterson L, Sjogren-Jansson E, et al. Articular cartilage engineering with autologous chondrocyte transplantation. A review of recent developments. J Bone Joint Surg (Am), 2003, 85-A Suppl 3: 109-115.
22.	Johnstone B, Hering TM, Caplan AI, et al. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res, 1998, 238(1): 265-272.
23.	Sekiya I, Larson BL, Vuoristo JT, et al. Comparison of effect of BMP-2, -4, and -6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma. Cell Tissue Res, 2005, 320(2): 269-276.
24.	Yokoyama A, Sekiya I, Miyazaki K, et al. In vitro cartilage formation of composites of synovium-derived mesenchymal stem cells with collagen gel. Cell Tissue Res, 2005, 322(2): 289-298.
25.	Wu L, Ishikawa Y, Genge B, et al. Effect of osteogenic protein-1 on the development and mineralization of primary cultures of avian growth plate chondrocytes: modulation by retinoic acid. J Cell Biochem, 1997, 67(4): 498-513.
26.	Jones BA, Pei M. Synovium-derived stem cells: a tissue-specific stem cell for cartilage engineering and regeneration. Tissue Eng Part B Rev, 2012, 18(4): 301-311.
27.	Krüger JP, Endres M, Neumann K, et al. Chondrogenic differentiation of human subchondral progenitor cells is affected by synovial fluid from donors with osteoarthritis or rheumatoid arthritis. J Orthop Surg Res, 2012, 7: 10.
28.	Dhinsa BS, Adesida AB. Current clinical therapies for cartilage repair, their limitation and the role of stem cells. Curr Stem Cell Res Ther, 2012, 7(2): 143-148.

1. 赵文君, 邢国胜, 于顺禄. 滑膜间充质干细胞及其在组织工程中的应用. 中国修复重建外科杂志, 2011, 25(12): 1504-1507.
2. De Bari C, Dell’Accio F, Tylzanowski P, et al. Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum, 2001, 44(8): 1928-1942.
3. Djouad F, Bony C, Haupl T, et al. Transcriptional profiles discriminate bone marrow-derived and synovium-derived mesenchymal stem cells. Arthritis Res Ther, 2005, 7(6): 1304-1315.
4. Jo CH, Ahn HJ, Kim HJ, et al. Surface characterization and chondrogenic differentiation of mesenchymal stromal cells derived from synovium. Cytotherapy, 2007, 9(4): 316-327.
5. Morito T, Muneta T, Hara K, et al. Synovial fluid-derived mesenchymal stem cells increase after intra-articular ligament injury in humans. Rheumatology (Oxford), 2008, 47(8): 1137-1143.
6. Zhang S, Muneta T, Morito T, et al. Autologous synovial fluid enhances migration of mesenchymal stem cells from synovium of osteoarthritis patients in tissue culture system. J Orthop Res, 2008, 26(10): 1413-1418.
7. Horie M, Sekiya I, Muneta T, et al. Intra-articular injected synovial stem cells differentiate into meniscal cells directly and promote meniscal regeneration without mobilization to distant organs in rat massive meniscal defect. Stem Cells, 2009, 27(4): 878-887.
8. Sakaguchi Y, Sekiya I, Yagishita K, et al. Comparison of human stem cells derived from various mesenchymal tissues. Arthritis Rheum, 2005, 52(8): 2521-2529.
9. Shirasawa S, Sekiya I, Sakaguchi Y, et al. In vitro chondrogenesis of human synovium-derived mesenchynlal stem cells: optimal condition and cmnparison with bone morrow-derived cells. J Cell Biochem, 2006, 97(1): 84-97.
10. Park Y, Suginoto M, Watrin A, et al. BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel. Osteoarthritis Cartilage, 2005, 13(6): 527-536.
11. Kurth T, Hedbom E, Shintani N, et al. Chondrogenic potential of human synovial mesenchymal stem cells in alginate. Osteparthritis Cartilage, 2007, 15(10): 1178-1189.
12. Miyamoto C, Matsumoto T, Sakimura K, et al. Osteogenic protein-1 with transforming growth factor-beta 1: potent inducer of chondrogenesis of synovial mesenchymal stem cells in vitro. J Orthop Sci, 2007, 12(6): 555-561.
13. 张正政, 李卫平, 杨睿, 等. 滑膜间充质干细胞分离培养、免疫表型鉴定及在混合淋巴反应体系中的抑制效应. 中国组织工程研究, 2012, 16(19): 3531-3519.
14. Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell, 2003, 113(6): 685-700.
15. Nagase T, Muneta T, Ju YJ, et al. Analysis of the chondrogenic potential of human synovial stem cells according to harvest site and culture parameters in knees with medial compartment osteoarthritis. Arthritis Rheum, 2008, 58(5): 1389-1398.
16. Maniatopiulos C, Sidek J, Melcher AH. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res, 1988, 254(2): 317-330.
17. Wan M, Cao X. BMP signaling in skeletal development. Biochem Biophys Res Commun, 2005, 328(3): 651-657.
18. Bi W, Huang W, Whitworth DJ, et al. Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization. Proc Natl Acad Sci U S A, 2001, 98(12): 6698-6703.
19. Nishimura K, Solchaga LA, Caplan AI, et al. Chondroprogenitor cells of synovial tissue. Arthritis Rheum, 1999, 42(12): 2631-2637.
20. Dell’Accio F, Vanlauwe J, Bellemans J, et al. Expanded phenotypically stable chondrocytes persisit in the repair tissue and contribute to cartilage matrix formation and structural integration in a goat model of autologous chondrocyte implantation. J Orthop Res, 2003, 21(1): 123-131.
21. Brittberg M, Peterson L, Sjogren-Jansson E, et al. Articular cartilage engineering with autologous chondrocyte transplantation. A review of recent developments. J Bone Joint Surg (Am), 2003, 85-A Suppl 3: 109-115.
22. Johnstone B, Hering TM, Caplan AI, et al. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res, 1998, 238(1): 265-272.
23. Sekiya I, Larson BL, Vuoristo JT, et al. Comparison of effect of BMP-2, -4, and -6 on in vitro cartilage formation of human adult stem cells from bone marrow stroma. Cell Tissue Res, 2005, 320(2): 269-276.
24. Yokoyama A, Sekiya I, Miyazaki K, et al. In vitro cartilage formation of composites of synovium-derived mesenchymal stem cells with collagen gel. Cell Tissue Res, 2005, 322(2): 289-298.
25. Wu L, Ishikawa Y, Genge B, et al. Effect of osteogenic protein-1 on the development and mineralization of primary cultures of avian growth plate chondrocytes: modulation by retinoic acid. J Cell Biochem, 1997, 67(4): 498-513.
26. Jones BA, Pei M. Synovium-derived stem cells: a tissue-specific stem cell for cartilage engineering and regeneration. Tissue Eng Part B Rev, 2012, 18(4): 301-311.
27. Krüger JP, Endres M, Neumann K, et al. Chondrogenic differentiation of human subchondral progenitor cells is affected by synovial fluid from donors with osteoarthritis or rheumatoid arthritis. J Orthop Surg Res, 2012, 7: 10.
28. Dhinsa BS, Adesida AB. Current clinical therapies for cartilage repair, their limitation and the role of stem cells. Curr Stem Cell Res Ther, 2012, 7(2): 143-148.

Chinese Journal of Reparative and Reconstructive Surgery

IN VITRO DIFFERENTIATION OF SYNOVIAL-DERIVED MESENCHYMAL STEM CELLS INFECTED BY ADENOVIRUS VECTOR MEDIATED BY BONE MORPHOGENETIC PROTEIN 2/7 GENES INTO FIBROCARTILAGE CELLS IN RABBITS

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