Gadi Turgeman

Engineered pluripotent mesenchymal cells integrate and differentiate in regenerating bone: a novel cell-mediated gene therapy.

Among the approximately 6.5 million fractures suffered in the United States every year, about 15% are difficult to heal. As yet, for most of these difficult cases there is no effective therapy. We have developed a mouse radial segmental defect as a model experimental system for testing the capacity of Genetically Engineered Pluripotent Mesenchymal Cells (GEPMC, C3H10T1/2 clone expressing rhBMP‐2), for gene delivery, engraftment, and induction of bone growth in regenerating bone.

Engineered human mesenchymal stem cells: a novel platform for skeletal cell mediated gene therapy

Human mesenchymal stem cells (hMSCs) are pluripotent cells that can differentiate to various mesenchymal cell types. Recently, a method to isolate hMSCs from bone marrow and expand them in culture was described. Here we report on the use of hMSCs as a platform for gene therapy aimed at bone lesions.

Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells

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.

Short-Term BMP-2 Expression Is Sufficient for In Vivo Osteochondral Differentiation of Mesenchymal Stem Cells

Currently available murine models to evaluate mesenchymal stem cell (MSC) differentiation are based on cell injection at ectopic sites such as muscle or skin. Due to the importance of environmental factors on the differentiation capacities of stem cells in vivo, we investigated whether the peculiar synovial/cartilaginous environment may influence the lineage specificity of bone morphogenetic protein (BMP)‐2‐engineered MSCs. To this aim, we used the C3H10T1/2‐derived C9 MSCs that express BMP‐2 under control of the doxycycline (Dox)‐repressible promoter, Tet‐Off, and showed in vitro, using the micropellet culture system that C9 MSCs kept their potential to differentiate toward chondrocytes. Implantation of C9 cells, either into the tibialis anterior muscles or into the joints of CB17‐severe combined immunodeficient bg mice led to the formation of cartilage and bone filled with bone marrow as soon as day 10. However, no differentiation was observed after injection of naïve MSCs or C9 cells that were repressed to secrete BMP‐2 by Dox addition. The BMP‐2‐induced differentiation of adult MSCs is thus independent of soluble factors present in the local environment of the synovial/cartilaginous tissues. Importantly, we demonstrated that a short‐term expression of the BMP‐2 growth factor is necessary and sufficient to irreversibly induce bone formation, suggesting that a stable genetic modification of MSCs is not required for stem cell‐based bone/cartilage engineering.

Systemically administered rhBMP‐2 promotes MSC activity and reverses bone and cartilage loss in osteopenic mice

Osteoporosis is a disease manifested in drastic bone loss resulting in osteopenia and high risk for fractures. This disease is generally divided into two subtypes. The first, post‐menopausal (type I) osteoporosis, is primarily related to estrogen deficiency. The second, senile (type II) osteoporosis, is mostly related to aging. Decreased bone formation, as well as increased bone resorption and turnover, are thought to play roles in the pathophysiology of both types of osteoporosis. In this study, we demonstrate in murine models for both type I (estrogen deficiency) and type II (senile) osteopenia/osteoporosis that reduced bone formation is related to a decrease in adult mesenchymal stem cell (AMSC) number, osteogenic activity, and proliferation. Decreased proliferation is coupled with increased apoptosis in AMSC cultures obtained from osteopenic mice. Recombinant human bone morphogenetic protein (rhBMP‐2) is a highly osteoinductive protein, promoting osteogenic differentiation of AMSCs. Systemic intra‐peritoneal (i.p.) injections of rhBMP‐2 into osteopenic mice were able to reverse this phenotype in the bones of these animals. Moreover, this change in bone mass was coupled to an increase in AMSCs numbers, osteogenic activity, and proliferation as well as a decrease in apoptosis. Bone formation activity was increased as well. However, the magnitude of this response to rhBMP‐2 varied among different stains of mice. In old osteopenic BALB/c male mice (type II osteoporosis model), rhBMP‐2 systemic treatment also restored both articular and epiphyseal cartilage width to the levels seen in young mice. In summary, our study shows that AMSCs are a good target for systemically active anabolic compounds like rhBMP‐2. J. Cell. Biochem. 86: 461–474, 2002.

Recombinant TGF‐β1 stimulates bone marrow osteoprogenitor cell activity and bone matrix synthesis in osteopenic, old male mice

We have previously hypothesized that the osteopenic changes seen in the skeletons of old male BALB/c mice are due to reductions in the availability and/or synthesis of bone TGF‐β which results in fewer, less osteogenic marrow osteoprogenitor cells (CFU‐f; OPCs) and lower levels of bone formation. Among other things, this hypothesis would predict that introducing exogenous TGF‐β into old mice (growth factor replacement) should stimulate marrow CFU‐f and increase bone formation. In the present study, we have tested this prediction and, indirectly the hypothesis, by injecting human recombinant TGF‐β1, i.p., into both young adult (4 month) and old mice (24 month). The effects of the growth factor on the skeleton were then assessed by measurements of trabecular bone volume, bone formation, fracture healing, and the number, proliferative, apoptotic, and alkaline phosphatase activity of marrow CFU‐f/OPCs. Our data show that the introduction of 0.5 or 5.0 ug/day of TGF‐β1 into old mice for 20 days 1) increases trabecular bone volume, bone formation and the mineral apposition rate, 2) augments fracture healing, 3) increases the number and size of CFU‐f colonies, and 4) increases proliferation and diminishes apoptosis of CFU‐f in primary bone marrow cultures. Importantly, these stimulatory effects of injected growth factor are apparently age‐specific, i.e., they are either not seen in young animals or, if seen, are found at much lower levels. While these observations do not exclude other possible mechanisms for the osteopenia of old mice, they provide further support for the hypothesis that, with age, diminished TGF‐β synthesis or availability results in a reduction in the marrow osteoprogenitor pool and bone formation. The findings also demonstrate that the latter changes can be reversed, at least transiently, by introducing exogenous TGF‐β1. J. Cell. Biochem. 73:379–389, 1999.