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Expression of exogenous or endogenous green fluorescent protein in adipose tissue-derived stromal cells during chondrogenic differentiation

Molecular and Cellular Biochemistry volume 277, pages 181–190 (2005)Cite this article

Abstract

Pluripotent stem cells within the adipose stromal compartment, termed adipose-derived stromal cells (ASCs), have the potential to differentiate into a variety of cell lineages both in vitro and in vivo. Imaging with expression of exogenous or endogenous green fluorescent protein (GFP) reporters facilitates the detailed research on ASCs’ physiological behavior during differentiation in vivo. This study was aimed to confirm whether ASCs expressing GFP still could be induced to chondrogenesis, and to compare the expression of exogenous or endogenous GFP in ASCs during chondrogenic differentiation. ASCs were harvested from inguinal fat pads of normal nude mice or GFP transgenic mice. Monolayer cultures of ASCs from normal mice were passaged three times and then infected with replication-incompetent adenoviral vectors carrying GFP genes. Allowed to recover for 5 days, Ad/GFP infected ASCs were transferred to chondrogenic medium as well as the ASCs from transgenic mice cultured in vitro over the same passages. The level of GFP in transgenic ASCs maintained stable till 3 months after chondrogenic induction. Whereas, high level of GFP expression in Ad/GFP infected ASCs could last for only 8 weeks and then declined stepwise. Important cartilaginous molecules such as SOX9, collagen type I, collagen type II, aggrecan, collagen type X were assessed using immunocytochemistry, RT-PCR, and Western Blot. The results indicated that no matter the GFP was exogenous or endogenous, it did not influence the chondrogenic potential of ASCs in comparison with the normal controls. Moreover, chondrogenic lineages from ASCs also underwent phenotypic modulation called dedifferentiation as a result of long-term culture in monolayers similar to normal chondrocytes.

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References

  1. 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 cell-based therapies. Tissue Eng 7(2): 211–228, 2001

    Article  PubMed  Google Scholar 

  2. Saladin R, Fajas L, Dana S, Halvorsen YD, Auwerx J, Briggs M: Differential regulation of peroxisome proliferator activated receptor gamma1 (PPAR-gamma1) and PPARgamma2 messenger RNA expression in the early stages of adipogenesis. Cell Growth Differ 10(1): 43–48, 1999

    PubMed  Google Scholar 

  3. 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. Metabolism 50(4): 407–413, 2001

    Article  PubMed  Google Scholar 

  4. Fuller JA, Ghadially FN: Ultrastructural observations on surgically produced partial-thickness defects in articular cartilage. Clin Orthop 86: 193–205, 1972

    PubMed  Google Scholar 

  5. Risbud M, Ringe J, Bhonde R, Sittinger M: In vitro expression of cartilage-specific markers by chondrocytes on a biocompatible hydrogel: Implications for engineering cartilage tissue. Cell Transplant 10(8): 755–763, 2001

    PubMed  Google Scholar 

  6. Ehlers EM, Fuss M, Rohwedel J, Russlies M, Kuhnel W, Behrens P: Development of a biocomposite to fill out articular cartilage lesions: light, scanning and transmission electron microscopy of sheep chondrocytes cultured on a collagen I/III sponge. Anat Anz 181(6): 513–518, 1999

    Google Scholar 

  7. Milgram JW: Morphologic alterations of the subchondral bone in advanced degenerative arthritis. Clin Orthop 173: 293–312, 1983

    PubMed  Google Scholar 

  8. Hayes AJ, Benjamin M, Ralphs JR: Extracellular matrix in development of the intervertebral disc. Matrix Biol 20(2): 107–121, 2001

    Article  PubMed  Google Scholar 

  9. Hutton WC, Ganey TM, Elmer WA, Kozlowska E, Ugbo JL, Doh ES, Whitesides TE Jr: Does long-term compressive loading on the intervertebral disc cause degeneration? Spine 25(23): 2993–3004, 2000

    Article  PubMed  Google Scholar 

  10. Morales TI, Roberts AB: Transforming growth factor β regulates the metabolism of proteoglycans in bovine cartilage organ cultures. J Biol Chem 263(26): 12828–12831, 1998

    Google Scholar 

  11. van Beuningen HM, Glansbeek HL, van der Kraan PM, van den Berg WB: Differential effects of local application BMP-2 or TGF-β1 on both articular cartilage composition and osteophyte formation. Osteoarthritis Cartilage 6(5): 306–317, 1998

    Article  PubMed  Google Scholar 

  12. Bianco P, Cossu G: Uno, nessuno e centomila: searching for the identity of mesodermal progenitors. Exp Cell Res 251(2): 257–263, 1999

    Article  PubMed  Google Scholar 

  13. Iwasaki M, Nakata K, Nakahara H, Nakase T, Kimura T, Kimata K, Caplan AI, Ono K: Transforming growth factor-beta 1 stimulates chondrogenesis and inhibits osteogenesis in high density culture of periosteum-derived cells. Endocrinology 132(4): 1603–1608, 1993

    Article  PubMed  Google Scholar 

  14. 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 13(12): 4279–4295, 2002

    Article  PubMed  Google Scholar 

  15. Erickson GR, Gimble JM, Franklin DM, Rice HE, Awad H, Guilak F: Chondrogenic potential of adipose tissue-derived stromal cells in vitro and in vivo. Biochem Biophys Res Commun 290(2): 763–769, 2002

    Article  PubMed  Google Scholar 

  16. 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 113(2): 585–594, 2004

    Article  PubMed  Google Scholar 

  17. Denk W, Strickler JH, Webb WW: Two-photon laser scanning fluorescence microscopy. Science 248(4951): 73–76, 1990

    PubMed  Google Scholar 

  18. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC: Green fluorescent protein as a marker for gene expression. Science 263(5148): 802–805, 1994

    PubMed  Google Scholar 

  19. Lippincott-Schwartz J, Patterson GH: Development and use of fluorescent protein markers in living cells. Science 300(5616): 87–91, 2003

    Article  PubMed  Google Scholar 

  20. Megason SG, Fraser SE: Digitizing life at the level of the cell: high-performance laser-scanning microscopy and image analysis for in toto imaging of development. Mech Dev 120(11): 1407–1420, 2003

    Article  PubMed  Google Scholar 

  21. Hadjantonakis AK, Dickinson ME, Fraser SE, Papaioannou VE: Technicolour transgenics: imaging tools for functional genomics in the mouse. Nat Rev Genet 4(8): 613–625, 2003

    Article  PubMed  Google Scholar 

  22. Wakefield LM, Winokur TS, Hollands RS, Christopherson K, Levinson AD, Sporn MB: Recombinant latent transforming growth factor beta 1 has a longer plasma half-life in rats than active transforming growth factor beta 1, and a different tissue distribution. J Clin Invest 86(6): 1976–1984, 1990

    PubMed  Google Scholar 

  23. Walton PE, Gopinath R, Burleigh BD, Etherton TD: Administration of recombinant human insulin-like growth factor I to pigs: determination of circulating half-lives and chromatographic profiles. Horm Res 31(3): 138–142, 1989

    PubMed  Google Scholar 

  24. International Guiding Principles for Animal Research: Geneva: Council for International Organization of Medical Sciences, 1985

  25. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B: A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 95(5): 2509–2514, 1998

    Article  PubMed  Google Scholar 

  26. Bjornsson S: Simultaneous preparation and quantitation of proteoglycans by precipitation with alcian blue. Anal Biochem 210(2): 282–291, 1993

    Article  PubMed  Google Scholar 

  27. Tanaka H, Murphy CL, Murphy C, Kimura M, Kawai S, Polak JM: Chondrogenic differentiation of murine embryonic stem cells: effects of culture conditions and dexamethasone. J Cell Biochem 93(3): 454–462, 2004

    Article  PubMed  Google Scholar 

  28. Schwartz NB, Pirok EW III, 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 62: 177–225, 1999

    PubMed  Google Scholar 

  29. 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 275(9): 6321–6327, 2000

    Article  PubMed  Google Scholar 

  30. Knudson CB, Knudson W: Cartilage proteoglycans. Semin Cell Dev Biol 12(2): 69–78, 2001

    Article  PubMed  Google Scholar 

  31. Watanabe H, Yamada Y, Kimata K: Roles of aggrecan, a large chondroitin sulfate proteoglycan, in cartilage structure and function. J Biochem (Tokyo) 124(4): 687–693, 1998

    Google Scholar 

  32. 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 55(2): 91–97, 2002

    Article  PubMed  Google Scholar 

  33. 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 99(7): 4397–4402, 2002

    Article  PubMed  Google Scholar 

  34. Lefebvre V, Huang W, Harley VR, Goodfellow PN, de Crombrugghe B: SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1 (II) collagen gene. Mol Cell Biol 17(4): 2336–2346, 1997

    PubMed  Google Scholar 

  35. Dessau W, von der Mark H, von der Mark K, Fischer S: Changes in the patterns of collagens and fibronectin during limb-bud chondrogenesis. J Embryol Exp Morphol 57: 51–60, 1980

    PubMed  Google Scholar 

  36. Linsenmayer TF, Toole BP, Trelstad RL: Temporal and spatial transitions in collagen types during embryonic chick limb development. Dev Biol 35(2): 232–239, 1973

    Article  PubMed  Google Scholar 

  37. Poliard A, Nifuji A, Lamblin D, Plee E, Forest C, Kellermann O: Controlled conversion of an immortalized mesodermal progenitor cell towards osteogenic, chondrogenic, or adipogenic pathways. J Cell Biol 130(6): 1461–1472, 1995

    Article  PubMed  Google Scholar 

  38. Kosher RA, Solursh M: Widespread distribution of type II collagen during embryonic chick development. Dev Biol 131(2): 558–566, 1989

    PubMed  Google Scholar 

  39. Cheah KS, Lau ET, Au PK, Tam PP: Expression of the mouse alpga1 (II) collagen gene is not restricted to cartilage during development. Development 111(4): 945–953, 1991

    PubMed  Google Scholar 

  40. Kirsch T, von der Mark K: Remodelling of collagen types I, II and X and calcification of human fetal cartilage. Bone Miner 18(2): 107–117, 1992

    Article  PubMed  Google Scholar 

  41. Kosher RA, Kulyk WM, Gay SW: Collagen gene expression during limb cartilage differentiation. J Cell Biol 102(4): 1151–1156, 1986

    Article  PubMed  Google Scholar 

  42. Linsenmayer TF, Eavey RD, Schmid TM: Type X collagen: a hypertrophic cartilage-specific molecule. Pathol Immunopathol Res 7(1–2): 14–19, 1988

    PubMed  Google Scholar 

  43. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y: ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 407(3): 313–319, 1997

    Article  PubMed  Google Scholar 

  44. Langer R, Vacanti JP: Tissue engineering. Science 260(5110): 920–926, 1993

    PubMed  Google Scholar 

  45. Evans CH, Ghivizzani SC, Lechman ER, Mi Z, Jaffurs D, Robbins PD: Lessons learned from gene therapy approaches. Arthritis Res 1(1): 21–24, 1999

    Article  PubMed  Google Scholar 

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Affiliations

  1. Department of Oral and Maxillofacial Surgery, West China College of Stomatology, Sichuan University, Chengdu, 610041, P.R. China

    Yunfeng Lin, Weidong Tian, Xizhe Chen, Zhengbin Yan, Zhiyong Li, Ju Qiao, Lei Liu, Wei Tang & Xiaohui Zheng

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  1. Yunfeng Lin
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Lin, Y., Tian, W., Chen, X. et al. Expression of exogenous or endogenous green fluorescent protein in adipose tissue-derived stromal cells during chondrogenic differentiation. Mol Cell Biochem 277, 181–190 (2005). https://doi.org/10.1007/s11010-005-5996-2

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  • DOI: https://doi.org/10.1007/s11010-005-5996-2

Keywords

  • adipose tissue-derived stromal cells
  • chondrogenic differentiation
  • green fluorescent protein