Molecular and Cellular Biochemistry

, Volume 301, Issue 1–2, pp 83–92 | Cite as

Osteogenic differentiation of adipose derived stem cells promoted by overexpression of osterix

  • Ling Wu
  • Yao Wu
  • Yunfeng Lin
  • Wei Jing
  • Xin Nie
  • Ju Qiao
  • Lei Liu
  • Wei Tang
  • Weidong Tian


Adipose-derived stem cells (ASCs) are considered to be multipotent mesenchymal stem cells that are easily induced to differentiate into functional osteoblasts both in vitro and in vivo. Osterix (Osx) is a zinc finger-containing transcription factor of Sp gene family, which plays important roles in bone development and mineralization. In this study, we hypothesized that overexpression of Osx in murine ASCs would promote their osteogenic differentiation in vitro. A plasmid expressing Osx (pcDNA3.1-Osx) was constructed and applied to transfect monolayers of murine ASCs. Then expression of bone-related genes, nodule formation, proliferation rate, and alkaline phosphatase activity were examined to evaluate the osteogenic potential of ASCs with pcDNA3.1-Osx transfection. Results of RT-PCR and immunohistochemistry showed that pcDNA3.1-Osx transfection enhanced the expression of bone matrix proteins, such as bone sialoprotein, osteocalcin, osteopontin, and Collagen type I in ASCs. At the same time, overexpression of Osx in ASCs enhanced alkaline phosphatase activity and capability to form mineralized nodules, while not inhibited their proliferation rate. These results indicated that pcDNA3.1-Osx transfection promoted the osteogenic differentiation of ASCs, while not affecting their proliferative ability. Since they can be easily isolated and genetically modified, ASCs are hopeful cell sources in the further application of hard tissue engineering.


Adipose derived stem cells Osterix Osteogenic differentiation Osteoblasts Overexpression 



We are grateful to Prof. Ganns, University of Toronto, for kindly providing the plasmid containing the cDNA of human Osterix. We also thank Hao Yang, Key Laboratory of Transplant Immunology (Sichun University) for helpful discussion. This work was funded by the Chinese National Natural Science Foundation (30200318), the Special Project of National Grand Fundamental Research Program of China (2002CCC00700) and Teaching & Research Award for Outstanding Young Teachers in Higher Education Institutions of PR China (2003682)


  1. 1.
    Grove JE, Bruscia E, Krause DS (2004) Plasticity of bone marrow-derived stem cells. Stem Cells 22:487–500PubMedCrossRefGoogle Scholar
  2. 2.
    Pomerantz J, Blau HM (2004) Nuclear reprogramming: a key to stem cell function in regenerative medicine. Nat Cell Biol 6:810–816PubMedCrossRefGoogle Scholar
  3. 3.
    Kofron MD, Laurencin CT (2006) Bone tissue engineering by gene delivery. Adv Drug Deliv Rev 58:555–576PubMedCrossRefGoogle Scholar
  4. 4.
    Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7:211–228PubMedCrossRefGoogle Scholar
  5. 5.
    Lin Y, Liu L, Li Z, Qiao J, Wu L, Tang W, Zheng X, Chen X, Yan Z, Tian W (2006) Pluripotency potential of human adipose-derived stem cells marked with exogenous green fluorescent protein. Mol Cell Biochem 291:1–10PubMedCrossRefGoogle Scholar
  6. 6.
    Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH (2002) Human adipose tissue is a source of multipotent stem cells. Mol Cell Biol 13:4279–4295CrossRefGoogle Scholar
  7. 7.
    Reyes M, Lund T, Lenvik T, Aguiar D, Koodie L, Verfaillie CM (2001) Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 98:2615–2625PubMedCrossRefGoogle Scholar
  8. 8.
    Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP (1997) Osteogenic differentiation of purified, culture expanded human mesenchymal stem cells in vitro. J Cell Biochem 64:295–312PubMedCrossRefGoogle Scholar
  9. 9.
    Majumdar MK, Thiede MA, Haynesworth SE, Bruder SP, Gerson SL (2000) Human marrow-derived mesenchymal stem cells (MSCs) express hematopoietic cytokines and support long-term hematopoiesis when differentiated toward stromal and osteogenic lineages. J Hematother Stem Cell Res 9:841–848PubMedCrossRefGoogle Scholar
  10. 10.
    Zhang X, Yang M, Lin L, Chen P, Ma KT, Zhou CY, Ao YF (2006) Runx2 overexpression enhances osteoblastic differentiation and mineralization in adipose-derived stem cells in vitro and in vivo. Calcif Tissue Int 79:169–78PubMedCrossRefGoogle Scholar
  11. 11.
    Wan DC, Shi YY, Nacamuli RP, Quarto N, Lyons KM, Longaker MT (2006) Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor type IB signaling. Proc Natl Acad Sci USA 103:12335–12340PubMedCrossRefGoogle Scholar
  12. 12.
    Quarto N, Longaker MT (2006) FGF-2 inhibits osteogenesis in mouse adipose tissue-derived stromal cells and sustains their proliferative and osteogenic potential state. Tissue Eng 12:1405–1418PubMedCrossRefGoogle Scholar
  13. 13.
    Hattori H, Sato M, Masuoka K, Ishihara M, Kikuchi T, Matsui T, Takase B, Ishizuka T, Kikuchi M, Fujikawa K, Ishihara M (2004) Osteogenic potential of human adipose tissue-derived stromal cells as an alternative stem cell source. Cells Tissues Organs 178:2–12PubMedCrossRefGoogle Scholar
  14. 14.
    Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B (2002) The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108:17–29PubMedCrossRefGoogle Scholar
  15. 15.
    Philipsen S, Suske G (1999) A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res 27:2991–3000PubMedCrossRefGoogle Scholar
  16. 16.
    Milona MA, Gough JE, Edgar AJ (2003) Expression of alternatively spliced isoforms of human Sp7 in osteoblast-like cells. BMC Genomics 4:43–53PubMedCrossRefGoogle Scholar
  17. 17.
    Yagi K, Tsuji K, Nifuji A, Shinomiya K, Nakashima K, DeCrombrugghe B, Noda M (2003) Bone morphogenetic protein-2 enhances osterix gene expression in chondrocytes. J Cell Biochem 88:1077–1083PubMedCrossRefGoogle Scholar
  18. 18.
    Cao Y, Zhou Z, de Crombrugghe B, Nakashima K, Guan H, Duan X, Jia SF, Kleinerman ES (2005) Osterix, a transcription factor for osteoblast differentiation, mediates antitumor activity in murine osteosarcoma. Cancer Res 65:1124–1128PubMedCrossRefGoogle Scholar
  19. 19.
    Tai G, Christodoulou I, Bishop AE, Polak JM (2005) Use of green fluorescent fusion protein to track activation of the transcription factor osterix during early osteoblast differentiation. Biochem Biophys Res Commun 333:1116–1122PubMedCrossRefGoogle Scholar
  20. 20.
    Tu Q, Valverde P, Chen J (2006) Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells. Biochem Biophys Res Commun 341:1257–1265PubMedCrossRefGoogle Scholar
  21. 21.
    Lin Y, Tian W, Chen X, Yan Z, Li Z, Qiao J, Liu L, Tang W, Zheng X (2005) Expression of exogenous or endogenous green fluorescent protein in adipose tissue-derived stromal cells during chondrogenic differentiation. Mol Cell Biochem 277:181–190PubMedCrossRefGoogle Scholar
  22. 22.
    Gao Y, Jheon A, Nourkeyhani H, Kobayashi H, Ganss B (2004) Molecular cloning, structure, expression, and chromosomal localization of the human Osterix (SP7) gene. Gene 341:101–110PubMedCrossRefGoogle Scholar
  23. 23.
    Lin Y, Chen X, Yan Z, Liu L, Tang W, Zheng X, Li Z, Qiao J, Li S, Tian W (2006) Multilineage differentiation of adipose-derived stromal cells from GFP transgenic mice. Mol Cell Biochem 285:69–78PubMedCrossRefGoogle Scholar
  24. 24.
    Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63PubMedCrossRefGoogle Scholar
  25. 25.
    Johnson RL, Tabin CJ (1997) Molecular models for vertebrate limb development. Cell 90:979–990PubMedCrossRefGoogle Scholar
  26. 26.
    Olsen BR, Reginato AM, Wang W (2000) Bone development. Annu Rev Cell Dev Biol 16:191–220PubMedCrossRefGoogle Scholar
  27. 27.
    Harada S, Rodan GA (2003) Control of osteoblast function and regulation of bone mass. Nature 423:349–355PubMedCrossRefGoogle Scholar
  28. 28.
    Ganss B, Kim RH, Sodek J (1999) Bone sialoprotein. Crit Rev Oral Biol Med 10:79–98PubMedCrossRefGoogle Scholar
  29. 29.
    Viereck V, Siggelkow H, Tauber S, Raddatz D, Schutze N, Hufner M (2002) Differential regulation of Cbfa1/Runx2 and osteocalcin gene expression by vitamin-D3, dexamethasone, and local growth factors in primary human osteoblasts. J Cell Biochem 86:348–356PubMedCrossRefGoogle Scholar
  30. 30.
    Standal T, Borset M, Sundan A (2004) Role of osteopontin in adhesion, migration, cell survival and bone remodeling. Exp Oncol 26:179–184PubMedGoogle Scholar
  31. 31.
    Beck GR Jr, Sullivan EC, Moran E, Zerler B (1998) Relationship between alkaline phosphatase levels, osteopontin expression, and mineralization in differentiating MC3T3-E1 osteoblasts. J Cell Biochem 68:269–280PubMedCrossRefGoogle Scholar
  32. 32.
    Tai G, Polak JM, Bishop AE, Christodoulou I, Buttery LD (2004) Differentiation of osteoblasts from murine embryonic stem cells by overexpression of the transcriptional factor osterix. Tissue Eng 10:1456–1466PubMedGoogle Scholar
  33. 33.
    Kim YJ, Kim HN, Park EK, Lee BH, Ryoo HM, Kim SY, Kim IS, Stein JL, Lian JB, Stein GS, van Wijnen AJ, Choi JY (2006) The bone-related Zn finger transcription factor Osterix promotes proliferation of mesenchymal cells. Gene 366:145–151PubMedCrossRefGoogle Scholar
  34. 34.
    Huang W, Rudkin GH, Carlsen B, Ishida K, Ghasri P, Anvar B, Yamaguchi DT, Miller TA (2002) Overexpression of BMP-2 modulates morphology, growth, and gene expression in osteoblastic cells. Exp Cell Res 274:226–234PubMedCrossRefGoogle Scholar
  35. 35.
    Cho HH, Park HT, Kim YJ, Bae YC, Suh KT, Jung JS (2005) Induction of osteogenic differentiation of human mesenchymal stem cells by histone deacetylase inhibitors. J Cell Biochem 96:533–542PubMedCrossRefGoogle Scholar
  36. 36.
    Igarashi M, Kamiya N, Hasegawa M, Kasuya T, Takahashi T, Takagi M (2004) Inductive effects of dexamethasone on the gene expression of Cbfa1, Osterix and bone matrix proteins during differentiation of cultured primary rat osteoblasts. J Mol Histol 35:3–10PubMedCrossRefGoogle Scholar
  37. 37.
    Lee RH, Kim B, Choi I, Kim H, Choi HS, Suh K, Bae YC, Jung JS (2004) Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem 14:311–324PubMedCrossRefGoogle Scholar
  38. 38.
    Bouwman P, Philipsen S (2002) Regulation of the activity of Sp1-related transcription factors. Mol Cell Endocrinol 195:27–38PubMedCrossRefGoogle Scholar
  39. 39.
    Ganss B, Jheon A (2004) Zinc finger transcription factors in skeletal development. Crit Rev Oral Biol Med 15:282–297PubMedGoogle Scholar
  40. 40.
    Opitz O, Rustgi A (2000) Interaction between Sp1 and cell cycle regulatory proteins is important in transactivation of a differentiation-related gene. Cancer Res, 60:2825–2830PubMedGoogle Scholar
  41. 41.
    Aslam F, Palumbo L, Augenlicht LH, Velcich A (2001) The Sp family of transcription factors in the regulation of the human and mouse MUC2 gene promoters. Cancer Res 61:570–576PubMedGoogle Scholar
  42. 42.
    Kaczynski J, Cook T, Urrutia R (2003) Sp1- and Krüppel-like transcription factors. Genome Biol 4:206–213PubMedCrossRefGoogle Scholar
  43. 43.
    Wu D, Razzano P, Grande DA (2003) Gene therapy and tissue engineering in repair of the musculoskeletal system. J Cell Biochem 88:467–481PubMedCrossRefGoogle Scholar
  44. 44.
    Gazit D, Turgeman G, Kelley P, Wang E, Jalenak M, Zilberman Y, Moutsatsos I (1999) Engineered pluripotent mesenchymal cells integrate and differentiate in regenerating bone: a novel cell-mediated gene therapy. J Gene Med 1:121–133PubMedCrossRefGoogle Scholar
  45. 45.
    Pola E, Gao W, Zhou Y, Pola R, Lattanzi W, Sfeir C, Gambotto A, Robbins PD (2004) Efficient bone formation by gene transfer of human LIM mineralization protein-3. Gene Ther 11:683–693PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Ling Wu
    • 1
  • Yao Wu
    • 1
  • Yunfeng Lin
    • 1
  • Wei Jing
    • 1
  • Xin Nie
    • 1
  • Ju Qiao
    • 2
  • Lei Liu
    • 1
  • Wei Tang
    • 1
  • Weidong Tian
    • 1
  1. 1.Department of Oral and Maxillofacial Surgery, West China College of StomatologySichuan UniversityChengduP.R. China
  2. 2.Department of Othodontics, West China College of StomatologySichuan UniversityChengduP.R. China

Personalised recommendations