Skip to main content
Journal of Cellular and Molecular Medicine logoLink to Journal of Cellular and Molecular Medicine
. 2007 May 1;9(4):929–939. doi: 10.1111/j.1582-4934.2005.tb00389.x

Molecular and cellular characterization during chondrogenic differentiation of adipose tissue‐derived stromal cells in vitro and cartilage formation in vivo

Yunfeng Lin 1, En Luo 1, Xizhe Chen 1, Lei Liu 1, Ju Qiao 2, Zhengbin Yan 1, Zhiyong Li 1, Wei Tang 1, Xiaohui Zheng 1, Weidong Tian 1,
PMCID: PMC6740126  PMID: 16364200

Abstract

Human adipose tissue is a viable source of mesenchymal stem cells (MSCs) with wide differentiation potential for musculoskeletal tissue engineering research. The stem cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and expanded in vitro easily. This study was to determine molecular and cellular characterization of PLA cells during chondrogenic differentiation in vitro and cartilage formation in vivo. When cultured in vitro with chondrogenic medium as monolayers in high density, they could be induced toward the chondrogenic lineages. To determine their ability of cartilage formation in vivo, the induced cells in alginate gel were implanted in nude mice subcutaneously for up to 20 weeks. Histological and immunohistochemical analysis of the induced cells and retrieved specimens from nude mice at various intervals showed obviously cartilaginous phenotype with positive staining of specific extracellular matrix (ECM). Correlatively, results of RT‐PCR and Western Blot confirmed the expression of characteristic molecules during chondrogenic differentiation namely collagen type II, SOX9, cartilage oligomeric protein (COMP) and the cartilage‐specific proteoglycan aggrecan. Meanwhile, there was low level synthesis of collagen type X and decreasing production of collagen type I during induction in vitro and formation of cartilaginous tissue in vivo. These cells induced to form engineered cartilage can maintain the stable phenotype and indicate no sign of hypertrophy in 20 weeks in vivo, however, when they cultured as monolayers, they showed prehypertrophic alteration in late stage about 10 weeks after induction. Therefore, it is suggested that human adipose tissue may represent a novel plentiful source of multipotential stem cells capable of undergoing chondrogenesis and forming engineered cartilage.

Keywords: chondrogenic differentiation, lipoaspirate cells, collagen, cartilage oligomeric protein

References

  • 1. Homminga, GN , Bulstra, SK , Bouwmeester, PS , van der Linden, AJ . Perichondral grafting for cartilage lesions of the knee. J Bone Joint Surg Br. 1990; 72: 1003–7. [DOI] [PubMed] [Google Scholar]
  • 2. Hangody, L , Kish, G , Karpati, Z , Udvarhelyi, I , Szigeti, I , Bely, M . Mosaicplasty for the treatment of articular carti lage defects: Application in clinical practice. Orthopedics 1998; 21: 751–6. [DOI] [PubMed] [Google Scholar]
  • 3. Fortier, LA , Nixon, AJ , Williams, J , Cable, CS . Isolation and chondrocytic differentiation of equine bone marrow‐derived mesenchymal stem cells. Am J Vet Res. 1998; 59: 1182–7. [PubMed] [Google Scholar]
  • 4. Gronthos, S , Zannettino, AC , Hay, SJ , Shi, S , Graves, SE , Kortesidis, A , Simmons, PJ . Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow. J Cell Sci. 2003; 116: 1827–35. [DOI] [PubMed] [Google Scholar]
  • 5. Reyes, M , Lund, T , Lenvik, T , Aguiar, D , Koodie, L , Verfaillie, CM . Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 2001; 98: 2615–25. [DOI] [PubMed] [Google Scholar]
  • 6. Jaiswal, N , Haynesworth, SE , Caplan, AI , Bruder, SP . Osteogenic differentiation of purified, culture expanded human mesenchymal stem cells in vitro . J Cell Biochem. 1997; 64: 295–312. [PubMed] [Google Scholar]
  • 7. Zuk, PA , Zhu, M , Mizuno, H , Huang, J , Futrell, JW , Katz, AJ , Benhaim, P , Lorenz, HP , Hedrick, MH . Multilineage cells from human adipose tissue: Implications for cellbased therapies. Tissue Eng. 2001; 7: 211–28. [DOI] [PubMed] [Google Scholar]
  • 8. Saladin, R , Fajas, L , Dana, S , Halvorsen, YD , Auwerx, J , Briggs, M . Differential regulation of peroxisome proliferator activated receptor gamma1 (PPAR‐γ1) and PPARγ2 messenger RNA expression in the early stages of adipogenesis. Cell Growth Differ. 1999; 10: 43–8. [PubMed] [Google Scholar]
  • 9. Halvorsen, YD , Bond, A , Sen, A , Franklin, DM , Lea‐Currie, YR , Sujkowski, D , Ellis, PN , Wilkison, WO , Gimble, JM . Thiazolidinediones and glucocorticoids synergistically induce differentiation of human adipose tissue stromal cells: Biochemical, cellular, and molecular analysis. Metab Clin Exp. 2001; 50: 407–13. [DOI] [PubMed] [Google Scholar]
  • 10. Huang, JI , Zuk, PA , Jones, NF , Zhu, M , Lorenz, HP , Hedrick, MH , Benhaim, P . Chondrogenic potential of multipotential cells from human adipose tissue. Plast Reconstr Surg. 2004; 113: 585–94. [DOI] [PubMed] [Google Scholar]
  • 11. Wickham, MQ , Erickson, GR , Gimble, JM , Vail, TP , Guilak, F . Multipotent stromal cells derived from the infrapatellar fat pad of the knee. Clin Orthop. 2003; 412: 196–212. [DOI] [PubMed] [Google Scholar]
  • 12. Lin, Y , Tian, W , Chen, X , Yan, Z , Li, Z , Qiao, J , Liu, L , Tang, W , Zheng, X . Expression of exogenous or endogenous green fluorescent protein in adipose tissue‐derived stromal cells during chondrogenic differentiation. Mol Cell Biochem. 2005; 277:181–90. [DOI] [PubMed] [Google Scholar]
  • 13. Zuk, PA , Zhu, M , Ashjian, P , De Ugarte, DA , Huang, JI , Mizuno, H , Alfonso, ZC , Fraser, JK , Benhaim, P , Hedrick, MH . Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002; 13: 4279–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. Sive, JI , Baird, P , Jeziorsk, M , Watkins, A , Hoyland, JA , Freemont, AJ . Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs. Mol Pathol. 2002; 55: 91–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15. Sekiya, I , Vuoristo, JT , Larson, BL , Prockop, DJ . In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci USA. 2002; 99: 4397–402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. Lefebvre, V , Huang, W , Harley, VR , Goodfellow, PN , de Crombrugghe, B . SOX9 is a potent activator of the chondrcyte‐specific enhancer of the pro α1 (II) colagen gene. Mol Cell Biol. 1997; 17: 2336–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. Tanaka, H , Murphy, CL , Murphy, C , Kimura, M , Kawai, S , Polak, JM . Chondrogenic differntiation of murine embryonic stem cells: effects of culture conditions and dexamethasone. J Cell Biochem. 2004; 93: 454–62. [DOI] [PubMed] [Google Scholar]
  • 18. Schwartz, NB , Pirok, EW 3rd , Mensch, JR Jr , Domowicz, MS . Domain organization, genomic structure, evolution, and regulation of expression of the aggrecan gene family. Prog Nucleic Acid Res Mol Biol. 1999; 62: 177–225. [DOI] [PubMed] [Google Scholar]
  • 19. Bayliss, MT , Howat, S , Davidson, C , Dudhia, J . The organization of aggrecan in human articular cartilage. Evidence for age‐related changes in the rate of aggregation of newly synthesized molecules. J Biol Chem. 2000; 275: 6321–7. [DOI] [PubMed] [Google Scholar]
  • 20. Skioldebrand, E , Heinegard, D , Eloranta, ML , Nilsson, G , Dudhia, J , Sandgren, B , Ekman, S . Enhanced concentration of COMP (cartilage oligomeric matrix protein) in osteochondral fractures from racing Thoroughbreds. J Orthop Res. 2005; 23: 156–63. [DOI] [PubMed] [Google Scholar]
  • 21. Crnkic, M , Mansson, B , Larsson, L , Geborek, P , Heinegard, D , Saxne, T . Serum cartilage oligomeric matrix protein (COMP) decreases in rheumatoid arthritis patients treated with infliximab or etanercept. Arthritis Res Ther. 2003; 5: R181–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22. Watanabe, H , Yamada, Y , Kimata, K . Roles of aggrecan, a large chondroitin sulfate proteoglycan, in cartilage structure and function. J Biochem. (Tokyo) 1998; 124: 687–93. [DOI] [PubMed] [Google Scholar]
  • 23. Knudson, CB , Knudson, W . Cartilage proteoglycans. Semin Cell Dev Biol. 2001; 12: 69–78. [DOI] [PubMed] [Google Scholar]
  • 24. Tchetina, E , Mwale, F , Poole, AR . Distinct phases of coordinated early and late gene expression in growth plate chondrocytes in relationship to cell proliferation, matrix assembly, remodeling, and cell differentiation. J Bone Miner Res. 2003; 18: 844–51. [DOI] [PubMed] [Google Scholar]
  • 25. Goldring, MB . Human chondrocyte cultures as models of cartilage‐specific gene regulation. Methods Mol Med. 2004; 107: 69–96. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Paige, KT , Cima, LG , Yaremchuk, MJ , Schloo, BL , Vacanti, JP , Vacanti, CA . De novo cartilage generation using calcium alginate‐chondrocyte constructs. Plastic Reconstruct Surg. 1996; 97: 168–80. [DOI] [PubMed] [Google Scholar]
  • 27. Dragoo, JL , Choi, JY , Lieberman, JR , Huang, J , Zuk, PA , Zhang, J , Hedrick, MH , Benhaim, P . Bone induction by BMP‐2 transduced stem cells derived from human fat. J Orthop Res. 2003; 21: 622–9. [DOI] [PubMed] [Google Scholar]
  • 28. Martin, JA , Mitchell, CJ , Klingelhutz, AJ , Buckwalter, JA . Effects of telomerase and viral oncogene expression on the in vitro growth of human chondrocytes. J Gerontol A Biol Sci Med Sci. 2002; 57: B48–53. [DOI] [PubMed] [Google Scholar]
  • 29. Barbero, A , Grogan, S , Schafer, D , Heberer, M , Mainil‐Varlet, P , Martin, I . Age related changes in human articular chondrocyte yield, proliferation and post‐expansion chondrogenic capacity. Osteoarthritis Cartilage 2004; 12: 476–84. [DOI] [PubMed] [Google Scholar]
  • 30. Ariga, K , Miyamoto, S , Nakase, T , Okuda, S , Meng, W , Yonenobu, K , Yoshikawa, H . The relationship between apoptosis of endplate chondrocytes and aging and degeneration of the intervertebral disc. Spine 2001; 26: 2414–20. [DOI] [PubMed] [Google Scholar]
  • 31. Benya, PD , Shaffer, JD . Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell 1982; 30: 215–24. [DOI] [PubMed] [Google Scholar]
  • 32. Binette, F , McQuaid, DP , Haudenschild, DR , Yaeger, PC , McPherson, JM , Tubo, R . Expression of a stable articular cartilage phenotype without evidence of hypertrophy by adult human articular chondrocytes in vitro . J Orthopaed Res. 1998; 16: 207–16. [DOI] [PubMed] [Google Scholar]
  • 33. Miyazaki, Y , Tsukazaki, T , Hirota, Y , Yonekura, A , Osaki, M , Shindo, H , Yamashita, S . Dexamethasone inhibition of TGF β‐induced cell growth and type II collagen mRNA expression through ERK‐integrated AP‐1 activity in cultured rat articular chondrocytes. Osteoarthritis Cartilage 2000; 8: 378–85. [DOI] [PubMed] [Google Scholar]
  • 34. Joyce, ME , Roberts, AB , Sporn, MB , Bolander, ME . Transforming growth factor‐β and the initiation of chondrogenesis and osteogenesis in the rat femur. J Cell Biol. 1990; 110: 2195–207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Ponticiello, MS , Schinagl, RM , Kadiyala, S , Barry, FP . Gelatin‐based resorbable sponge as a carrier matrix for human mesenchymal stem cells in cartilage regeneration therapy. J Biomed Mater Res. 2000; 52: 246–55. [DOI] [PubMed] [Google Scholar]
  • 36. Kramer, J , Hegert, C , Guan, K , Wobus, AM , Muller, PK , Rohwedel, J . Embryonic stem cell‐derived chondrogenic differentiation in vitro: activation by BMP‐2 and BMP‐4. Mech Dev 2000; 92: 193–205. [DOI] [PubMed] [Google Scholar]
  • 37. Stockwell, RA , Sprinz, R . Glycosaminoglycan content and cell density of rabbit articular cartilage in experimental lipoarthrosis. J Anat. 1981; 133: 309–15. [PMC free article] [PubMed] [Google Scholar]
  • 38. Goessler, UR , Bugert, P , Bieback, K , Baisch, A , Sadick, H , Verse, T , Kluter, H , Hormann, K , Riedel, F . Expression of collagen and fiber‐associated proteins in human septal cartilage during in vitro dedifferentiation. Int J Mol Med. 2004; 14: 1015–22. [PubMed] [Google Scholar]
  • 39. Korbling, M , Estrov, Z , Champlin, R . Adult stem cells and tissue repair. Bone Marrow Transplant. 2003; 32 Suppl 1: S23–4. [DOI] [PubMed] [Google Scholar]
  • 40. Glowacki, J . Engineered cartilage, bone, joints, and menisci. Potential for temporomandibular joint reconstruction. Cells Tissues Organs 2001; 169: 302–8. [DOI] [PubMed] [Google Scholar]
  • 41. Wu, D , Razzano, P , Grande, DA . Gene therapy and tissue engineering in repair of the musculoskeletal system. J Cell Biochem. 2003; 88: 467–81. [DOI] [PubMed] [Google Scholar]
  • 42. Trippel, SB , Ghivizzani, SC , Nixon, AJ . Gene‐based approaches for the repair of articular cartilage. Gene Ther. 2004; 11: 351–9. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Cellular and Molecular Medicine are provided here courtesy of Blackwell Publishing

RESOURCES