ISOLATION, CULTURE AND IDENTIFICATION OF CARTILAGE DERIVED STEM CELLS FROM THREE SUBTYPES OF CARTILAGES_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

XUEKe , ZHANGXiaodie ,  LIUKai

Department of Plastic and Reconstructive Surgery, Shanghai 9 th People's Hospital, Shanghai Jiaotong University School of Medicine, Key Laboratory of Tissue Engineering, Shanghai, 200011, P. R. China;

Corresponding author：

LIUKai, Email: drkailiu@126.com

Keywords：

Cartilage tissue engineering; Cartilage derived stem cells; Cartilage subtype; Fibronectin

DOI：

10.7507/1002-1892.20150104

Video：

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Objective To isolate and culture cartilage derived stem cells from different subtypes of cartilages, and to identify their characteristics. Methods Cartilage derived stem cells were isolated from different subtypes of cartilages (auricle cartilage, articular cartilage, and intervertebral cartilage) by using adhesive method of fibronectin. The expressions of positive surface markers (CD29 and CD90) and negative surface markers (CD34 and CD45) in cartilage derived stem cells were detected via flow cytometry. The single cell colony-forming efficiency of cartilage derived stem cells was determined by clonal formation unit test; the multipotent differentiation capacity was identified by chondrogensis, osteogenesis, and adipogenesis induction. RT-PCR was used to test the expression of osteogenic, chondrogenic, and adipogenic genes; and bone marrow mesenchymal stem cells (BMSCs) served as control. Results Three cell populations were successfully isolated from different subtypes of cartilages, which could express CD29 and CD 90 highly, but did not express CD34 and CD45. After 2 weeks of culture, single cartilage derived stem cell could form single cell colony. In addition, cartilage derived stem cells had high chondrogenesis, osteogenesis, and adipogenesis potentials. After osteogenic induction, the expressions of collagen type Ⅰ and collagen type X in articular and intervertebral cartilage stem cells were significantly higher than those in BMSCs (P<0.05), while there was no significant difference between auricular cartilage stem cells and BMSCs (P>0.05). The expressions of Aggrecan and collagen type Ⅱ in cartilage derived stem cells after chondrogenic induction were significantly higher than those in BMSCs (P<0.05). While the ability of adipogenic differentiation was lower than that in BMSCs, but no significant difference was found (P>0.05). Conclusion Cartilage derived stem cells in different subtypes of cartilages possess typical characteristics of stem cells.

Citation： XUEKe, ZHANGXiaodie, LIUKai. ISOLATION, CULTURE AND IDENTIFICATION OF CARTILAGE DERIVED STEM CELLS FROM THREE SUBTYPES OF CARTILAGES. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(4): 483-489. doi: 10.7507/1002-1892.20150104 Copy

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2.	Pei M, He F, Li J, et al. Repair of large animal partial-thickness cartilage defects through intraarticular injection of matrix-rejuvenated synovium-derived stem cells. Tissue Eng Part A, 2013, 19(9-10):1144-1154.
3.	Reverte-Vinaixa MM, Joshi N, Diaz-Ferreiro EW, et al. Medium-term outcome of mosaicplasty for grade Ⅲ-IV cartilage defects of the knee. J Orthop Surg (Hong Kong), 2013, 21(1):4-9.
4.	Naderi-Meshkin H, Andreas K, Matin MM, et al. Chitosan-based injectable hydrogel as a promising in situ forming scaffold for cartilage tissue engineering. Cell Biol Int, 2014, 38(1):72-84.
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7.	Kreuz PC, Gentili C, Samans B, et al. Scaffold-assisted cartilage tissue engineering using infant chondrocytes from human hip cartilage. Osteoarthritis Cartilage, 2013, 21(12):1997-2005.
8.	Veronesi F, Maglio M, Tschon M, et al. Adipose-derived mesenchymal stem cells for cartilage tissue engineering:state-of-the-art in in vivo studies. J Biomed Mater Res A, 2014, 102(7):2448-2466.
9.	Diekman BO, Christoforou N, Willard VP, et al. Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells. Proc Natl Acad Sci U S A, 2012, 109(47):19172-19177.
10.	Lee HY, Kopesky PW, Plaas A, et al. Adult bone marrow stromal cell-based tissue-engineered aggrecan exhibits ultrastructure and nanomechanical properties superior to native cartilage. Osteoarthritis Cartilage, 2010, 18(11):1477-1486.
11.	Cui JH, Park SR, Park K, et al. Preconditioning of mesenchymal stem cells with low-intensity ultrasound for cartilage formation in vivo. Tissue Eng, 2007, 13(2):351-360.
12.	Pelttari K, Winter A, Steck E, et al. Premature induction of hypertrophy during in vitro chondrogenesis of human mesenchymal stem cells correlates with calcification and vascular invasion after ectopic transplantation in SCID mice. Arthritis Rheum, 2006, 54(10):3254-3266.
13.	De Bari C, Dell'Accio F, Luyten FP. Failure of in vitro-differentiated mesenchymal stem cells from the synovial membrane to form ectopic stable cartilage in vivo. Arthritis Rheum, 2004, 50(1):142-150.
14.	Peng H, Huard J. Muscle-derived stem cells for musculoskeletal tissue regeneration and repair. Transpl Immunol, 2004, 12(3-4):311-319.
15.	Park J, Gelse K, Frank S, et al. Transgene-activated mesenchymal cells for articular cartilage repair:a comparison of primary bone marrow-, perichondrium/periosteum- and fat-derived cells. J Gene Med, 2006, 8(1):112-125.
16.	Dowthwaite GP, Bishop JC, Redman SN, et al. The surface of articular cartilage contains a progenitor cell population. J Cell Sci, 2004, 117(Pt 6):889-897.
17.	Kobayashi S, Takebe T, Zheng YW, et al. Presence of cartilage stem/progenitor cells in adult mice auricular perichondrium. PLoS One, 2011, 6(10):e26393.
18.	Jiang YZ, Tuan RS. Origin and function of cartilage stem/progenitor cells in osteoarthritis. Nat Rev Rheumatol, 2014.[Epub ahead of print]. doi:10.1038/nrrheum.2014.200.
19.	Paebst F, Piehler D, Brehm W, et al. Comparative immunophenotyping of equine multipotent mesenchymal stromal cells:an approach toward a standardized definition. Cytometry A, 2014, 85 (8):678-687.
20.	Dueñas F, Becerra V, Cortes Y, et al. Hepatogenic and neurogenic differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses. BMC Vet Res, 2014, 10:154.
21.	Wang F, Scoville D, He XC, et al. Isolation and characterization of intestinal stem cells based on surface marker combinations andcolony-formation assay. Gastroenterology, 2013, 145(2):383-395.e1-21.
22.	Yoon DS, Choi Y, Jang Y, et al. SIRT1 directly regulates SOX2 to maintain self-renewal and multipotency in bone marrow-derived mesenchymal stem cells. Stem Cells, 2014, 32(12):3219-3231.

1. Jungmann PM, Kraus MS, Nardo L, et al. T(2) relaxation time measurements are limited in monitoring progression, once advanced cartilage defects at the knee occur:longitudinal data from the osteoarthritis initiative. J Magn Reson Imaging, 2013, 38(6):1415-1424.
2. Pei M, He F, Li J, et al. Repair of large animal partial-thickness cartilage defects through intraarticular injection of matrix-rejuvenated synovium-derived stem cells. Tissue Eng Part A, 2013, 19(9-10):1144-1154.
3. Reverte-Vinaixa MM, Joshi N, Diaz-Ferreiro EW, et al. Medium-term outcome of mosaicplasty for grade Ⅲ-IV cartilage defects of the knee. J Orthop Surg (Hong Kong), 2013, 21(1):4-9.
4. Naderi-Meshkin H, Andreas K, Matin MM, et al. Chitosan-based injectable hydrogel as a promising in situ forming scaffold for cartilage tissue engineering. Cell Biol Int, 2014, 38(1):72-84.
5. Liu M, Yu X, Huang F, et al. Tissue engineering stratified scaffolds for articular cartilage and subchondral bone defects repair. Orthopedics, 2013, 36(11):868-873.
6. Viti F, Scaglione S, Orro A, et al. Guidelines for managing data and processes in bone and cartilage tissue engineering. BMC Bioinformatics, 2014, 15 Suppl 1:S14.
7. Kreuz PC, Gentili C, Samans B, et al. Scaffold-assisted cartilage tissue engineering using infant chondrocytes from human hip cartilage. Osteoarthritis Cartilage, 2013, 21(12):1997-2005.
8. Veronesi F, Maglio M, Tschon M, et al. Adipose-derived mesenchymal stem cells for cartilage tissue engineering:state-of-the-art in in vivo studies. J Biomed Mater Res A, 2014, 102(7):2448-2466.
9. Diekman BO, Christoforou N, Willard VP, et al. Cartilage tissue engineering using differentiated and purified induced pluripotent stem cells. Proc Natl Acad Sci U S A, 2012, 109(47):19172-19177.
10. Lee HY, Kopesky PW, Plaas A, et al. Adult bone marrow stromal cell-based tissue-engineered aggrecan exhibits ultrastructure and nanomechanical properties superior to native cartilage. Osteoarthritis Cartilage, 2010, 18(11):1477-1486.
11. Cui JH, Park SR, Park K, et al. Preconditioning of mesenchymal stem cells with low-intensity ultrasound for cartilage formation in vivo. Tissue Eng, 2007, 13(2):351-360.
12. Pelttari K, Winter A, Steck E, et al. Premature induction of hypertrophy during in vitro chondrogenesis of human mesenchymal stem cells correlates with calcification and vascular invasion after ectopic transplantation in SCID mice. Arthritis Rheum, 2006, 54(10):3254-3266.
13. De Bari C, Dell'Accio F, Luyten FP. Failure of in vitro-differentiated mesenchymal stem cells from the synovial membrane to form ectopic stable cartilage in vivo. Arthritis Rheum, 2004, 50(1):142-150.
14. Peng H, Huard J. Muscle-derived stem cells for musculoskeletal tissue regeneration and repair. Transpl Immunol, 2004, 12(3-4):311-319.
15. Park J, Gelse K, Frank S, et al. Transgene-activated mesenchymal cells for articular cartilage repair:a comparison of primary bone marrow-, perichondrium/periosteum- and fat-derived cells. J Gene Med, 2006, 8(1):112-125.
16. Dowthwaite GP, Bishop JC, Redman SN, et al. The surface of articular cartilage contains a progenitor cell population. J Cell Sci, 2004, 117(Pt 6):889-897.
17. Kobayashi S, Takebe T, Zheng YW, et al. Presence of cartilage stem/progenitor cells in adult mice auricular perichondrium. PLoS One, 2011, 6(10):e26393.
18. Jiang YZ, Tuan RS. Origin and function of cartilage stem/progenitor cells in osteoarthritis. Nat Rev Rheumatol, 2014.[Epub ahead of print]. doi:10.1038/nrrheum.2014.200.
19. Paebst F, Piehler D, Brehm W, et al. Comparative immunophenotyping of equine multipotent mesenchymal stromal cells:an approach toward a standardized definition. Cytometry A, 2014, 85 (8):678-687.
20. Dueñas F, Becerra V, Cortes Y, et al. Hepatogenic and neurogenic differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses. BMC Vet Res, 2014, 10:154.
21. Wang F, Scoville D, He XC, et al. Isolation and characterization of intestinal stem cells based on surface marker combinations andcolony-formation assay. Gastroenterology, 2013, 145(2):383-395.e1-21.
22. Yoon DS, Choi Y, Jang Y, et al. SIRT1 directly regulates SOX2 to maintain self-renewal and multipotency in bone marrow-derived mesenchymal stem cells. Stem Cells, 2014, 32(12):3219-3231.

Chinese Journal of Reparative and Reconstructive Surgery

ISOLATION, CULTURE AND IDENTIFICATION OF CARTILAGE DERIVED STEM CELLS FROM THREE SUBTYPES OF CARTILAGES

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