Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells
Andrea Hoffmann, … , Gerhard Gross, Dan Gazit
Andrea Hoffmann, … , Gerhard Gross, Dan Gazit
Published April 3, 2006
Citation Information: J Clin Invest. 2006;116(4):940-952. https://doi.org/10.1172/JCI22689.
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Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells

Abstract

Tissue regeneration requires the recruitment of adult stem cells and their differentiation into mature committed cells. In this study we describe what we believe to be a novel approach for tendon regeneration based on a specific signalling molecule, Smad8, which mediates the differentiation of mesenchymal stem cells (MSCs) into tendon-like cells. A biologically active Smad8 variant was transfected into an MSC line that coexpressed the osteogenic gene bone morphogenetic protein 2 (BMP2). The engineered cells demonstrated the morphological characteristics and gene expression profile of tendon cells both in vitro and in vivo. In addition, following implantation in an Achilles tendon partial defect, the engineered cells were capable of inducing tendon regeneration demonstrated by double quantum filtered MRI. The results indicate what we believe to be a novel mechanism in which Smad8 inhibits the osteogenic pathway in MSCs known to be induced by BMP2 while promoting tendon differentiation. These findings may have considerable importance for the therapeutic replacement of tendons or ligaments and for engineering other tissues in which BMP plays a pivotal developmental role.

Authors

Andrea Hoffmann, Gadi Pelled, Gadi Turgeman, Peter Eberle, Yoram Zilberman, Hadassah Shinar, Keren Keinan-Adamsky, Andreas Winkel, Sandra Shahab, Gil Navon, Gerhard Gross, Dan Gazit

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Figure 1

Tenogenic phenotype in C3H10T1/2-BMP2 cells due to forced expression of Smad8 L+MH2.

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Tenogenic phenotype in C3H10T1/2-BMP2 cells due to forced expression of ...
The parental C3H10T1/2 and C3H10T1/2 constitutively expressing the BMP2 gene were stably transfected with different Smad molecules as described in Methods. (A) Histological analysis of ALP-positive cells at 6 and 10 days after confluence (dac). Note the elongated tenocyte-like appearance of BMP2/Smad8 L+MH2–expressing cells compared with BMP2/Smad8WT–expressing cells. (B) Recombinant expression of FLAG-tagged Smad8 variants in C3H10T1/2-BMP2 cells. Western immunoblotting of cellular extracts of C3H10T1/2-BMP2 cells. Open arrowhead indicates a nonspecific band; filled arrowhead indicates Smad8WT and Smad8 L+MH2 domains. WB, Western blot. (C) Smad8 L+MH2, but not Smad1 or Smad5 L+MH2, elicited a tenocyte-like morphology in C3H10T1/2-BMP2. Histological analysis (ALP) and phase-contrast microscopy. All cells represent day 10 after confluence. Top panels: In the presence of BMP2 all C3H10T1/2 cells underwent mesenchymal differentiation and extensive matrix development resulting in multilayer growth. At this relatively late stage of cultivation residual ALP-positive cells were observed embedded in matrix and on the top of the layer. The presence of coexpressed L+MH2 domains from Smad1 and Smad5 did not influence this morphology. The presence of Smad8 L+MH2 dramatically changed this morphology to an elongated cell phenotype. Bottom panels: Parental C3H10T1/2 cells expressing Smad1 and Smad5 L+MH2 developed an osteoblast-like appearance. Smad8 L+MH2–expressing cells did not possess a comparable osteogenic capacity. Also, development of an elongated cell phenotype was not observed. Magnification, ×40.