title: Analyses of chondrogenic induction of adipose mesenchymal stem cells by combined co-stimulation mediated by adenoviral gene transfer.
abstract: BACKGROUND
  Adipose-derived stem cells (ASCs) have the potential to differentiate into cartilage under stimulation with some reported growth and transcriptional factors, which may constitute an alternative for cartilage replacement approaches. In this study, we analyzed the in vitro chondrogenesis of ASCs transduced with adenoviral vectors encoding insulin-like growth factor-1 (IGF-1), transforming growth factor beta-1 (TGF-β1), fibroblast growth factor-2 (FGF-2), and sex-determining region Y-box 9 (SOX9) either alone or in combinations.
  
  
  METHODS
  Aggregate cultures of characterized ovine ASCs were transduced with 100 multiplicity of infections of Ad.IGF-1, Ad.TGF-β1, Ad.FGF-2, and Ad.SOX9 alone or in combination. These were harvested at various time points for detection of cartilage-specific genes expression by quantitative real-time PCR or after 14 and 28 days for histologic and biochemical analyses detecting proteoglycans, collagens (II, I and X), and total sulfated glycosaminoglycan and collagen content, respectively.
  
  
  RESULTS
  Expression analyses showed that co-expression of IGF-1 and FGF-2 resulted in higher significant expression levels of aggrecan, biglycan, cartilage matrix, proteoglycan, and collagen II (all P ≤0.001 at 28 days). Aggregates co-transduced with Ad.IGF-1/Ad.FGF-2 showed a selective expression of proteoglycans and collagen II, with limited expression of collagens I and × demonstrated by histological analyses, and had significantly greater glycosaminoglycan and collagen production than the positive control (P ≤0.001). Western blot analyses for this combination also demonstrated increased expression of collagen II, while expression of collagens I and × was undetectable and limited, respectively.
  
  
  CONCLUSIONS
  Combined overexpression of IGF-1/FGF-2 within ASCs enhances their chondrogenic differentiation inducing the expression of chondrogenic markers, suggesting that this combination is more beneficial than the other factors tested for the development of cell-based therapies for cartilage repair.
authors.forename: Idalia
authors.initials: I
authors.lastname: Garza-Veloz
authors.forename: Viktor J
authors.initials: VJ
authors.lastname: Romero-Diaz
authors.forename: Margarita L
authors.initials: ML
authors.lastname: Martinez-Fierro
authors.forename: Ivan A
authors.initials: IA
authors.lastname: Marino-Martinez
authors.forename: Manuel
authors.initials: M
authors.lastname: Gonzalez-Rodriguez
authors.forename: Herminia G
authors.initials: HG
authors.lastname: Martinez-Rodriguez
authors.forename: Marcela A
authors.initials: MA
authors.lastname: Espinoza-Juarez
authors.forename: Dante A
authors.initials: DA
authors.lastname: Bernal-Garza
authors.forename: Rocio
authors.initials: R
authors.lastname: Ortiz-Lopez
authors.forename: Augusto
authors.initials: A
authors.lastname: Rojas-Martinez
mesh_terms: D000256:Adenoviridae; D000273:Adipose Tissue / Q000166:cytology* / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology*; D002470:Cell Survival; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D020219:Chondrogenesis / Q000502:physiology*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D018014:Gene Transfer Techniques; D006801:Humans; D007150:Immunohistochemistry; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D055758:SOX9 Transcription Factor / Q000378:metabolism; D012756:Sheep; D014161:Transduction, Genetic; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
chemical_list: D055758:SOX9 Transcription Factor; D053773:Transforming Growth Factor beta1; D016222:Fibroblast Growth Factor 2; D007334:Insulin-Like Growth Factor I
references.citation: Injury. 2005 Nov;36 Suppl 4:S14-23
references.pmid: 16291320
references.citation: J Cell Physiol. 2006 Dec;209(3):987-95
references.pmid: 16972251
references.citation: J Orthop Res. 2009 May;27(5):612-9
references.pmid: 18985688
references.citation: J Biomed Mater Res A. 2009 Mar 15;88(4):889-97
references.pmid: 18381637
references.citation: Methods Mol Biol. 2008;449:27-44
references.pmid: 18370081
references.citation: Gene Ther. 2004 Feb;11(4):351-9
references.pmid: 14724680
references.citation: Nat Protoc. 2010 Jul;5(7):1294-311
references.pmid: 20595958
references.citation: Nat Protoc. 2007;2(5):1236-47
references.pmid: 17546019
references.citation: Arthritis Rheum. 2005 Dec;52(12):3910-7
references.pmid: 16320338
references.citation: Methods. 2001 Dec;25(4):402-8
references.pmid: 11846609
references.citation: J Bone Joint Surg Am. 2010 Apr;92(4):994-1009
references.pmid: 20360528
references.citation: Int J Med Sci. 2010 Dec 27;8(1):16-29
references.pmid: 21209798
references.citation: J Gene Med. 2006 Jan;8(1):100-11
references.pmid: 16097039
references.citation: Mol Ther. 2005 Aug;12(2):219-28
references.pmid: 16043093
references.citation: J Orthop Res. 2010 May;28(5):677-86
references.pmid: 19918893
references.citation: Tissue Eng. 2005 Jan-Feb;11(1-2):120-9
references.pmid: 15738667
references.citation: Methods Mol Biol. 2008;449:69-79
references.pmid: 18370084
references.citation: Osteoarthritis Cartilage. 2001;9 Suppl A:S69-75
references.pmid: 11680692
references.citation: Arthritis Rheum. 2003 Feb;48(2):430-41
references.pmid: 12571853
references.citation: Arthritis Rheum. 2002 Feb;46(2):404-19
references.pmid: 11840443
references.citation: Gene Ther. 2007 May;14(10):804-13
references.pmid: 17344902
references.citation: Iowa Orthop J. 2000;20:1-10
references.pmid: 10934618
references.citation: Tissue Cell. 2010 Oct;42(5):282-92
references.pmid: 20810142
references.citation: Cell Physiol Biochem. 2004;14(4-6):311-24
references.pmid: 15319535
references.citation: Biomed Mater Eng. 2010;20(3):145-58
references.pmid: 20930322
references.citation: Arthritis Res Ther. 2007;9(3):213
references.pmid: 17561986
references.citation: Biomaterials. 2007 Jul;28(19):2994-3003
references.pmid: 17399784
references.citation: J Orthop Res. 2006 Aug;24(8):1791-8
references.pmid: 16779832
references.citation: Drug Discov Today. 2001 Mar 1;6(5):259-267
references.pmid: 11182599
references.citation: Arch Orthop Trauma Surg. 2010 Oct;130(10):1311-22
references.pmid: 20532898
references.citation: J Cell Physiol. 2012 May;227(5):1796-804
references.pmid: 21732349
references.citation: J Bone Joint Surg Am. 2003;85-A Suppl 2:111-6
references.pmid: 12721353
references.citation: J Cell Physiol. 2010 Apr;223(1):84-93
references.pmid: 20049852
references.citation: Mol Ther. 2005 Aug;12(2):229-38
references.pmid: 16043094
references.citation: Tissue Eng. 2006 Oct;12(10):2853-62
references.pmid: 17518654
references.citation: PLoS One. 2011;6(7):e22887
references.pmid: 21818404
references.citation: Methods Mol Biol. 2008;449:93-107
references.pmid: 18370086
references.citation: J Orthop Res. 2010 Jun;28(6):834-40
references.pmid: 20058274
references.citation: Methods Mol Med. 2004;100:1-14
references.pmid: 15280583
references.citation: J Orthop Res. 2004 Sep;22(5):1114-9
references.pmid: 15304287
references.citation: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2006 Nov;20(11):1114-8
references.pmid: 17191580
references.citation: Exp Hematol. 2005 Aug;33(8):865-72
references.pmid: 16038778
references.citation: Cytotherapy. 2007;9(8):712-6
references.pmid: 17917888
references.citation: Biomaterials. 2011 Jun;32(16):3910-20
references.pmid: 21377725
references.citation: Osteoarthritis Cartilage. 2003 Jan;11(1):55-64
references.pmid: 12505488
references.citation: Exp Cell Res. 1997 Dec 15;237(2):318-25
references.pmid: 9434627
references.citation: Biochem Biophys Res Commun. 2004 Jul 30;320(3):914-9
references.pmid: 15240135
references.citation: Exp Cell Res. 2001 Aug 15;268(2):189-200
references.pmid: 11478845