Overexpression of human DKK1 via rAAV vector-mediated gene transfer stimulates chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells

Jagadeesh Kumar Venkatesan, Gertrud Schmitt, Susanne Speicher-Mentges, Henning Madry, Magali Cucchiarini


Enhancing the chondroregenerative activities of mesenchymal stem cells via therapeutic gene transfer as reinforced sources of implantable cells in sites of cartilage injury is a promising tool to improve the natural processes of cartilage repair. In the present study, we show that overexpression of the Dickkopf-related protein 1 (DKK1) via clinically adapted recombinant adeno-associated viral (rAAV) vectors is capable of significantly stimulating proliferative, anabolic, and chondrodifferentiation events in primary human mesenchymal stem cells compared with control (reporter rAAV lacZ) transduction over an extended period of time in vitro (21 days). Strikingly, administration of the rAAV DKK1 candidate vector concomitantly restrained unwanted osteogenic and hypertrophic differentiation outcomes in these cells. These findings reveal the possible future benefits of such an approach to treat articular cartilage lesions in relevant experimental models in vivo.


cartilage repair; MSCs; rAAV; DKK1; chondrogenic differentiation


Akiyama H, Lyons JP, Mori-Akiyama Y, Yang X, Zhang R, Zhang Z, Deng JM, Taketo MM, Nakamura T, Behringer RR, McCrea PD & de Crombrugghe B 2004 Interactions between Sox9 and beta-catenin control chondrocyte differentiation. Genes Dev 18 1072-1087

Amano K, Hata K, Sugita A, Takigawa Y, Ono K, Wakabayashi M, Kogo M, Nishimura R & Yoneda T 2009 Sox9 family members negatively regulate maturation and calcification of chondrocytes through up-regulation of parathyroid hormone-related protein. Mol Biol Cell 20 4541-4551

Apparailly F, Millet V, Noël D, Jacquet C, Sany J & Jorgensen C 2002 Tetracycline-inducible interleukin-10 gene transfer mediated by an adeno-associated virus: application to experimental arthritis. Hum Gene Ther 13 1179-1188

Baksh D, Song L & Tuan RS 2004 Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med 8 301-316

Cucchiarini M, Ren XL, Perides G & Terwilliger EF 2003 Selective gene expression in brain microglia mediated via adeno-associated virus type 2 and type 5 vectors. Gene Ther 10 657-667

Cucchiarini M & Madry H 2005 Gene therapy for cartilage defects. J Gene Med 7 1495-1509

Cucchiarini M & Madry H 2010 Genetic modification of mesenchymal stem cells for cartilage repair. Biomed Mater Eng 20 135-143

Cucchiarini M, Ekici M, Schetting S, Kohn D & Madry H 2011 Metabolic activities and chondrogenic differentiation of human mesenchymal stem cells following recombinant adeno-associated virus-mediated gene transfer and overexpression of fibroblast growth factor 2. Tissue Eng Part A 17 1921-1933

Evans CH, Ghivizzani SC, Smith P, Shuler FD, Mi Z & Robbins PD 2000 Using gene therapy to protect and restore cartilage. Clin Orthop Relat Res 379 Suppl S214-S219

Evans CH & Huard J 2015 Gene therapy approaches to regenerating the musculoskeletal system. Nat Rev Rheumatol 11 234-242

Fedi P, Bafico A, Nieto Soria A, Burgess WH, Miki T, Bottaro DP, Kraus MH & Aaronson SA 1999 Isolation and biochemical characterization of the human Dkk-1 homologue, a novel inhibitor of mammalian Wnt signaling. J Biol Chem 274 19465-19472

Frisbie DD, Oxford JT, Southwood L, Trotter GW, Rodkey WG, Steadman JR, Goodnight JL & McIlwraith CW 2003 Early events in cartilage repair after subchondral bone microfracture. Clin Orthop Relat Res 407 215-227

Frisch J, Venkatesan JK, Rey-Rico A, Schmitt G, Madry H & Cucchiarini M 2014a Influence of insulin-like growth factor I overexpression via recombinant adeno-associated vector gene transfer upon the biological activities and differentiation potential of human bone marrow-derived mesenchymal stem cells. Stem Cell Res Ther 5 103-114

Frisch J, Venkatesan JK, Rey-Rico A, Schmitt G, Madry H & Cucchiarini M 2014b Determination of the chondrogenic differentiation processes in human bone marrow-derived mesenchymal stem cells genetically modified to overexpress transforming growth factor-b via recombinant adeno-associated viral vectors. Hum Gene Ther 25 1050-1060

Funck-Brentano T, Bouaziz W, Marty C, Geoffroy V, Hay E & Cohen-Solal M 2014 Dkk-1-mediated inhibition of Wnt signaling in bone ameliorates osteoarthritis in mice. Arthritis Rheumatol 66 3028-3039

Glinka A, Wu W, Delius H, Monaghan AP, Blumenstock C & Niehrs C 1998 Dickkopf-1 is a member of a new family of secreted proteins and functions in head induction. Nature 391 357-362

Goldring MB, Fukuo K, Birkhead JR, Dudek E & Sandell LJ 1994 Transcriptional suppression by interleukin-1 and interferon-gamma of type II collagen gene expression in human chondrocytes. J Cell Biochem 54 85-99

Gregory CA, Singh H, Perry AS & Prockop DJ 2003 The Wnt signaling inhibitor dickkopf-1 is required for reentry into the cell cycle of human adult stem cells from bone marrow. J Biol Chem 278 28067-28078

Haleem-Smith H, Calderon R, Song Y, Tuan RS & Chen FH 2012 Cartilage oligomeric matrix protein enhances matrix assembly during chondrogenesis of human mesenchymal stem cells. J Cell Biochem 113 1245-1252

Hattori T, Müller C, Gebhard S, Bauer E, Pausch F, Schlund B, Bösl MR, Hess A, Surmann-Schmitt C, von der Mark H, de Crombrugghe B & von der Mark K 2010 SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification. Development 137 901-911

Ikeda T, Kamekura S, Mabuchi A, Kou I, Seki S, Takato T, Nakamura K, Kawaguchi H, Ikegawa S & Chung UI 2004 The combination of SOX5, SOX6, and SOX9 (the SOX trio) provides signals sufficient for induction of permanent cartilage. Arthritis Rheum 50 3561-3573

Im GI & Quan Z 2010 The effects of Wnt inhibitors on the chondrogenesis of human mesenchymal stem cells. Tissue Eng Part A 16 2405-2413

Im GI, Lee JM & Kim HJ 2011 Wnt inhibitors enhance chondrogenesis of human mesenchymal stem cells in a long-term pellet culture. Biotechnol Lett 33 1061-1068

Jeon SY, Park JS, Yang HN, Woo DG & Park KH 2012 Co-delivery of SOX9 genes and anti-Cbfa-1 siRNA coated onto PLGA nanoparticles for chondrogenesis of human MSCs. Biomaterials 33 4413-4423

Johnstone B, Alini M, Cucchiarini M, Dodge GR, Eglin D, Guilak F, Madry H, Mata A, Mauck RL, Semino CE & Stoddart MJ 2013 Tissue engineering for articular cartilage repair--the state of the art. Eur Cell Mater 25 248-267

Kim HJ & Im GI 2011 Electroporation-mediated transfer of SOX trio genes (SOX-5, SOX-6, and SOX-9) to enhance the chondrogenesis of mesenchymal stem cells. Stem Cells Dev 20 2103-2114

Krause U, Ryan DM, Clough BH & Gregory CA 2014 An unexpected role for a Wnt-inhibitor: Dickkopf-1 triggers a novel cancer survival mechanism through modulation of aldehyde-dehydrogenase-1 activity. Cell Death Dis 5 e1093- e1104

Leijten JC, Emons J, Sticht C, van Gool S, Decker E, Uitterlinden A, Rappold G, Hofman A, Rivadeneira F, Scherjon S, Wit JM, van Meurs J, van Blitterswijk CA & Karperien M 2012 Gremlin 1, frizzled-related protein, and Dkk-1 are key regulators of human articular cartilage homeostasis. Arthritis Rheum 64 3302-3312

Leung VY, Gao B, Leung KK, Melhado IG, Wynn SL, Au TY, Dung NW, Lau JY, Mak AC, Chan D & Cheah KS 2011 SOX9 governs differentiation stage-specific gene expression in growth plate chondrocytes via direct concomitant transactivation and repression. PLoS Genet 7 e1002356-e1002371

Liu TM, Guo XM, Tan HS, Hui JH, Lim B & Lee EH 2011 Zinc-finger protein 145, acting as an upstream regulator of SOX9, improves the differentiation potential of human mesenchymal stem cells for cartilage regeneration and repair. Arthritis Rheum 63 2711-2720

Liu S, Zhang E, Yang M & Lu L 2014 Overexpression of Wnt11 promotes chondrogenic differentiation of bone marrow-derived mesenchymal stem cells in synergism with TGF-b. Mol Cell Biochem 390 123-131

Liu P, Sun L, Chen H, Sun S, Zhou D, Pang B & Wang J 2015 Lentiviral-mediated multiple gene transfer to chondrocytes promotes chondrocyte differentiation and bone formation in rabbit bone marrow-derived mesenchymal stem cells. Oncol Rep 34 2618-2626

Madry H, van Dijk CN & Mueller-Gerbl M 2010 The basic science of the subchondral bone. Knee Surg Sports Traumatol Arthrosc 18 419-433

Madry H & Cucchiarini M 2016 Gene therapy for human osteoarthritis: principles and clinical translation. Expert Opin Biol Ther 16 331-346

Mason JB, Gurda BL, Hankenson KD, Harper LR, Carlson CS, Wilson JM & Richardson DW 2017 Wnt10b and Dkk-1 gene therapy differentially influenced trabecular bone architecture, soft tissue integrity, and osteophytosis in a skeletally mature rat model of osteoarthritis. Connect Tissue Res 58 542-552

Oh H, Chun CH & Chun JS 2012 Dkk-1 expression in chondrocytes inhibits experimental osteoarthritic cartilage destruction in mice. Arthritis Rheum 64 2568-2578

Pagnotto MR, Wang Z, Karpie JC, Ferretti M, Xiao X & Chu CR 2007 Adeno-associated viral gene transfer of transforming growth factor-beta1 to human mesenchymal stem cells improves cartilage repair. Gene Ther 14 804-813

Samulski RJ, Chang LS & Shenk T 1987 A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication. J Virol 61 3096-3101

Samulski RJ, Chang LS & Shenk T 1989 Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol 63 3822-3828

Sandell LJ & Adler P 1999 Developmental patterns of cartilage. Front Biosci 4 D731-D742

Skowroński J & Rutka M 2013 Osteochondral lesions of the knee reconstructed with mesenchymal stem cells - results. Ortop Traumatol Rehabil 15 195-204

Steinert AF, Palmer GD, Pilapil C, Nöth U, Evans CH & Ghivizzani SC 2009 Enhanced in vitro chondrogenesis of primary mesenchymal stem cells by combined gene transfer. Tissue Eng Part A 15 1127-1139

Steinert AF, Weissenberger M, Kunz M, Gilbert F, Ghivizzani SC, Göbel S, Jakob F, Nöth U & Rudert M 2012. Indian hedgehog gene transfer is a chondrogenic inducer of human mesenchymal stem cells. Arthritis Res Ther 14 R168-R180

Venkatesan JK, Ekici M, Madry H, Schmitt G, Kohn D & Cucchiarini M 2012 SOX9 gene transfer via safe, stable, replication-defective recombinant adeno-associated virus vectors as a novel, powerful tool to enhance the chondrogenic potential of human mesenchymal stem cells. Stem Cell Res Ther 3 22-36

Venkatesan JK, Frisch J, Rey-Rico A, Schmitt G, Madry H & Cucchiarini M 2017 Impact of mechanical stimulation on the chondrogenic processes in human bone marrow aspirates modified to overexpress sox9 via rAAV vectors. J Exp Orthop 4 22-34

Wakitani S, Mitsuoka T, Nakamura N, Toritsuka Y, Nakamura Y & Horibe S 2004 Autologous bone marrow stromal cell transplantation for repair of full-thickness articular cartilage defects in human patellae: two case reports. Cell Transplant 13 595-600

Weng LH, Wang CJ, Ko JY, Sun YC & Wang FS 2010 Control of Dkk-1 ameliorates chondrocyte apoptosis, cartilage destruction, and subchondral bone deterioration in osteoarthritic knees. Arthritis Rheum 62 1393-1402

Xiao X, Li J & Samulski RJ 1996 Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J Virol 70 8098-8108

Yoo JU, Mandell I, Angele P & Johnstone B 2000 Chondrogenitor cells and gene therapy. Clin Orthop Relat Res 379 S164-S170

Zhong L, Huang X, Rodrigues ED, Leijten JC, Verrips T, El Khattabi M, Karperien M & Post JN 2016 Endogenous DKK1 and FRZB regulate chondrogenesis and hypertrophy in three-dimensional cultures of human chondrocytes and human mesenchymal stem cells. Stem Cells Dev 25 1808-1817

Full Text: PDF


  • There are currently no refbacks.

Copyright © 2018 Lorem Ipsum Press