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Cloning of the minimal functional domain of human LIM mineralization protein-3 able to induce bone mineralization: in vitro and in vivo study
GENE THERAPY FOR MUSCULOSKELETAL DISEASES 383. Cloning of the Minimal Functional Domain of Human Lim Mineralization Protein-3 Able To Induce Bone Mineralization: In Vitro and In Vivo Study Enrico Pola,1,2 Wanda Lattanzi,3 Wentao Go,3 Huijie Sun,3 Kaori Okada,3 Andrea Gambotto,3 Carlo A. Logroscino,1 Paul D. Robbins.2 1 Orthopaedics and Traumatology, Università Cattolica del Sacro Cuore School of Medicine, Rome, Italy; 2Genetics and Biochemistry, University of Pittsburgh Medical Center, Pittsburgh, PA; 3Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA. Human LIM mineralization protein (LMP)-3 is one of the three splice variants of LMP recently identified. LMPs are involved in the osteoblast differentiation program and structurally characterized by the two conserved LIM and PDZ domains. Human LMP-1 (hLMP-1) shows one N-terminal PDZ domain and three C-term LIM domains connected by a non-conserved “Unique” region, deleted in the hLMP-2. hLMP-3 misses almost completely the LIM domains along with part of the unique region, due to a frame shift mutation. The three isoforms are expressed almost ubiquitously but show quantitative differences, hLMP-3 being the less expressed in all the analyzed tissues. Both hLMP-1 and hLMP-3 has been demonstrated to induce bone formation in vitro and ectopic bone formation in vivo, while hLMP-2 is not osteoinductive, suggesting that LIM domains are not essential for this function. Thus it has been hypothesized that the osteoinductive domain could reside in the “Unique” region that is partially conserved in hLMP-3. To examine the osteoinductive properties of this minimal domain we have cloned three different length of the Unique region of the hLMP-3 gene, corresponding to 120, 90 and 60 bp, fused to the enhanced green fluorescent protein (eGFP) and named L40-eGFP, L30-eGFP and L20-eGFP respectively. Thus we tested the ability of these constructs to induce bone specific gene expression and bone mineralization in vitro and ectopic bone formation in vivo in comparison to the full-length gene hLMP-3. Here we demonstrate that adenoviral-mediated gene transfer of all the 3 domains induces expression of certain bone-specific genes in a mouse fibroblasts cell line. The up-regulation of osteo-specific genes was assessed in mouse fibroblasts also by means of biolistic transfection using a plasmid containing a L20-eGFP fusion gene. In addition, we demonstrate that all the domains are able to induce mineralization in fibroblast and mesenchymal stem cells. An experiment to evaluate if direct gene transfer of the three constructs into murine skeletal muscle results in ectopic bone formation as efficiently as using LMP-3 is being performed. Finally in order to propose these new constructs as an effective approach to induce bone formation in vivo for clinical applications, we have synthesized a peptide of 20 aminoacid, corresponding to the fragment of 60 bp of the Unique region (named PTD-OD-1). The peptide enter the cells by a protein transduction domain (PTD-5) and its ability to induce in vitro expression of bone-specific genes and bone mineralization both in fibroblast and in human mesenchymal stem cells will be evaluated. PTD-OD-1 could represent a safe and powerful tool for clinical applications, and merit several analysis to evaluate its ability.
Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright The American Society of Gene Therapy
384. Stem Cells Expressing SMAD 8: A Platform for Tendon Regeneration Gadi Pelled,1 Andrea Hoffman,2 Gadi Turgeman,1 Peter Eberle,2 Yoram Zilberman,1 Hadassah Shinar,3 Keren Keinan-Adamsky,3 Andreas Winkel,2 Gil Navon,3 Gerhard Gross,2 Dan Gazit.1 1 Skeletal Biotech Lab, Hebrew University of Jerusalem, Jerusalem, Israel; 2Signalling and Gene Regulation, GBF, Braunschweig, Germany; 3School of Chemistry, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel. Current reconstructive techniques to repair torn tendons consist of autografts, allografts or synthetic prosthesis. Unfortunately, none of these surgical alternatives provide a long-term adequate solution. In this study, we defined a novel mechanism that mediates the differentiation of MSCs into tendon based on the SMAD8 signaling molecule, which could be used as a regenerative platform. A variant of SMAD8, deficient of the MH1 domain, was transfected to MSCs that express bone morphogenetic protein 2 (BMP2). The in vitro phenotype of the engineered MSCs was evaluated using RT-PCR, Histochemistry and morphology. In addition, the cells were injected into subcutaneous tissue and into the kidney capsule of female C3H/HeN mice in order to assess the tissue formation in vivo. Engineered MSCs, also expressing Lacz or Luciferase marker genes, were implanted on a Collagen sponge into a 3 mm partial defect of nude rats Achilles tendon. 5 weeks post implantation, the area of the injection or implantation was isolated and analyzed by light and electron microscopy, histochemical staining, RT-PCR and double quantum filtered (DQF) MRI. Cell survival in the implantation site was non-invasively and quantitatively demonstrated in vivo by the detection of Luciferase bioluminescence, using a CCCD system. Our results indicated a tenocytic phenotype of the engineered MSCs, based on microscopy and the expression of the tendon characteristic genes, Six 1and Scleraxis. Formation of tendon tissue in vivo was confirmed by the analysis of injected implants, which revealed the dense connective tissue with parallel-organized fibers and spindle shaped cells (A). Electron microscopic analysis showed tightly packed collagen fibers adjacent to fibroblast-shaped cells with active Endoplasmic Reticulum (B). Moreover, we were also able to repair Achilles tendon defects with the engineered MSCs. CCCD imaging quantitatively monitored cells survival in the operated tendon, and imunohistochmical staining detected the labeled implanted cells in site (C &F). DQF MRI analysis proved that highly ordered Collagen fibers were formed in the site of implantation as opposed to the control group (D, E). Since the expression of BMP2 is known to drive MSCs to osteogenic differentiation, we propose that the expression of SMAD8 in MSCs induces an inhibition of the BMP2 signaling pathway, resulting in the differentiation of MSCs into tendon cells. This is the first study showing that a particular SMAD signaling cascade is involved in tendon formation. Moreover, this is the first time a regenerative effect is shown to be exerted by the combination of a secreted factor and a signaling molecule. These findings may have considerable importance for therapeutic avenue in tendon in which SMAD 8 signaling plays a pivotal role.
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Molecular Therapy Volume 9, Supplement 1, May 2004 Copyright The American Society of Gene Therapy
384. Stem Cells Expressing SMAD 8: A Platform for Tendon Regeneration Gadi Pelled,1 Andrea Hoffman,2 Gadi Turgeman,1 Peter Eberle,2 Yoram Zilberman,1 Hadassah Shinar,3 Keren Keinan-Adamsky,3 Andreas Winkel,2 Gil Navon,3 Gerhard Gross,2 Dan Gazit.1 1 Skeletal Biotech Lab, Hebrew University of Jerusalem, Jerusalem, Israel; 2Signalling and Gene Regulation, GBF, Braunschweig, Germany; 3School of Chemistry, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel. Current reconstructive techniques to repair torn tendons consist of autografts, allografts or synthetic prosthesis. Unfortunately, none of these surgical alternatives provide a long-term adequate solution. In this study, we defined a novel mechanism that mediates the differentiation of MSCs into tendon based on the SMAD8 signaling molecule, which could be used as a regenerative platform. A variant of SMAD8, deficient of the MH1 domain, was transfected to MSCs that express bone morphogenetic protein 2 (BMP2). The in vitro phenotype of the engineered MSCs was evaluated using RT-PCR, Histochemistry and morphology. In addition, the cells were injected into subcutaneous tissue and into the kidney capsule of female C3H/HeN mice in order to assess the tissue formation in vivo. Engineered MSCs, also expressing Lacz or Luciferase marker genes, were implanted on a Collagen sponge into a 3 mm partial defect of nude rats Achilles tendon. 5 weeks post implantation, the area of the injection or implantation was isolated and analyzed by light and electron microscopy, histochemical staining, RT-PCR and double quantum filtered (DQF) MRI. Cell survival in the implantation site was non-invasively and quantitatively demonstrated in vivo by the detection of Luciferase bioluminescence, using a CCCD system. Our results indicated a tenocytic phenotype of the engineered MSCs, based on microscopy and the expression of the tendon characteristic genes, Six 1and Scleraxis. Formation of tendon tissue in vivo was confirmed by the analysis of injected implants, which revealed the dense connective tissue with parallel-organized fibers and spindle shaped cells (A). Electron microscopic analysis showed tightly packed collagen fibers adjacent to fibroblast-shaped cells with active Endoplasmic Reticulum (B). Moreover, we were also able to repair Achilles tendon defects with the engineered MSCs. CCCD imaging quantitatively monitored cells survival in the operated tendon, and imunohistochmical staining detected the labeled implanted cells in site (C &F). DQF MRI analysis proved that highly ordered Collagen fibers were formed in the site of implantation as opposed to the control group (D, E). Since the expression of BMP2 is known to drive MSCs to osteogenic differentiation, we propose that the expression of SMAD8 in MSCs induces an inhibition of the BMP2 signaling pathway, resulting in the differentiation of MSCs into tendon cells. This is the first study showing that a particular SMAD signaling cascade is involved in tendon formation. Moreover, this is the first time a regenerative effect is shown to be exerted by the combination of a secreted factor and a signaling molecule. These findings may have considerable importance for therapeutic avenue in tendon in which SMAD 8 signaling plays a pivotal role.
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