Dan Gazit

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.

Osteogenic differentiation of noncultured immunoisolated bone marrow-derived CD105+ cells.

The culture expansion of human mesenchymal stem cells (hMSCs) may alter their characteristics and is a costly and time‐consuming stage. This study demonstrates for the first time that immunoisolated noncultured CD105‐positive (CD105+) hMSCs are multipotent in vitro and exhibit the capacity to form bone in vivo. hMSCs are recognized as promising tools for bone regeneration. However, the culture stage is a limiting step in the clinical setting. To establish a simple, efficient, and fast method for applying these cells for bone formation, a distinct population of CD105+ hMSCs was isolated from bone marrow (BM) by using positive selection based on the expression of CD105 (endoglin). The immunoisolated CD105+ cell fraction represented 2.3% ± 0.45% of the mononuclear cells (MNCs). Flow cytometry analysis of freshly immunoisolated CD105+ cells revealed a purity of 79.7% ± 3.2%. In vitro, the CD105+ cell fraction displayed significantly more colony‐forming units‐fibroblasts (CFU‐Fs; 6.3 ± 1.4) than unseparated MNCs (1.1 ± 0.3; p < .05). Culture‐expanded CD105+ cells expressed CD105, CD44, CD29, CD90, and CD106 but not CD14, CD34, CD45, or CD31 surface antigens, and these cells were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages. In addition, freshly immunoisolated CD105+ cells responded in vivo to recombinant bone morphogenetic protein‐2 by differentiating into chondrocytes and osteoblasts. Genetic engineering of freshly immunoisolated CD105+ cells was accomplished using either adenoviral or lentiviral vectors. Based on these findings, it is proposed that noncultured BM‐derived CD105+ hMSCs are osteogenic cells that can be genetically engineered to induce tissue generation in vivo.

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.

Evidence for skeletal progenitor cells in the degenerate human intervertebral disc.

Study Design. To identify and characterize endogenous progenitor cell population from intervertebral disc. Objective. To determine if progenitor cells exist in degenerate human discs. Summary of Background Data. Back pain, a significant source of morbidity in our society, is directly linked to the pathology of the intervertebral disc. Because disc disease is accompanied by a loss of cellularity, there is considerable interest in regeneration of cells of both the anulus fibrosus (AF) and nucleus pulposus (NP). Methods. To determine if skeletal progenitor cells are present in the disc, samples were obtained from the degenerate AF and NP of 5 patients (Thompson grade 2 and 3, mean age 34 ± 7.6 years) undergoing anterior cervical discectomy and fusion procedures as well as adult rat lumbar spine. Results. Cells isolated from degenerate human tissues expressed CD105, CD166, CD63, CD49a, CD90, CD73, p75 low affinity nerve growth factor receptor, and CD133/1, proteins that are characteristic of marrow mesenchymal stem cells. In osteogenic media, there was an induction of alkaline phosphatase activity and expression of alkaline phosphatase, osteocalcin, and Runx-2 mRNA. When maintained in adipogenic media, a small percentage of cells displayed evidence of adipogenic differentiation: accumulation of cytosolic lipid droplets and increased expression of peroxisome proliferator-activated receptor-&ggr;2 and lipoporotein lipase mRNA. AF- and NP-derived cells also evidenced chondrogenic differentiation. CD133 (+) cells in the AF were able to commit to either the chondrogenic or adipogenic lineages. The results of the human disc studies were confirmed using cell derived from the NP and AF tissue of the mature rat disc. Conclusion. The analytical data indicated that the pathologically degenerate human disc contained populations of skeletal progenitor cells. These findings suggest that these endogenous progenitors may be used to orchestrate the repair of the intervertebral disc.

Human Parathyroid Hormone 1–34 Reverses Bone Loss in Ovariectomized Mice

The experimental work characterizing the anabolic effect of parathyroid hormone (PTH) in bone has been performed in nonmurine ovariectomized (OVX) animals, mainly rats. A major drawback of these animal models is their inaccessibility to genetic manipulations such as gene knockout and overexpression. Therefore, this study on PTH anabolic activity was carried out in OVX mice that can be manipulated genetically in future studies. Adult Swiss‐Webster mice were OVX, and after the fifth postoperative week were treated intermittently with human PTH(1–34) [hPTH(1–34)] or vehicle for 4 weeks. Femoral bones were evaluated by microcomputed tomography (μCT) followed by histomorphometry. A tight correlation was observed between trabecular density (BV/TV) determinations made by both methods. The BV/TV showed >60% loss in the distal metaphysis in 5‐week and 9‐week post‐OVX, non‐PTH‐treated animals. PTH induced a ∼35% recovery of this loss and a ∼40% reversal of the associated decreases in trabecular number (Tb.N) and connectivity. PTH also caused a shift from single to double calcein‐labeled trabecular surfaces, a significant enhancement in the mineralizing perimeter and a respective 2‐ and 3‐fold stimulation of the mineral appositional rate (MAR) and bone formation rate (BFR). Diaphyseal endosteal cortical MAR and thickness also were increased with a high correlation between these parameters. These data show that OVX osteoporotic mice respond to PTH by increased osteoblast activity and the consequent restoration of trabecular network. The Swiss‐Webster mouse model will be useful in future studies investigating molecular mechanisms involved in the pathogenesis and treatment of osteoporosis, including the mechanisms of action of known and future bone antiresorptive and anabolic agents.

Estrogen modulates estrogen receptor alpha and beta expression, osteogenic activity, and apoptosis in mesenchymal stem cells (MSCs) of osteoporotic mice.

In the mouse, ovariectomy (OVX) leads to significant reductions in cancellous bone volume while estrogen (17β‐estradiol, E2) replacement not only prevents bone loss but can increase bone formation. As the E2‐dependent increase in bone formation would require the proliferation and differentiation of osteoblast precursors, we hypothesized that E2 regulates mesenchymal stem cells (MSCs) activity in mouse bone marrow. We therefore investigated proliferation, differentiation, apoptosis, and estrogen receptor (ER) α and β expression of primary culture MSCs isolated from OVX and sham‐operated mice. MSCs, treated in vitro with 10−7 M E2, displayed a significant increase in ERα mRNA and protein expression as well as alkaline phosphatase (ALP) activity and proliferation rate. In contrast, E2 treatment resulted in a decrease in ERβ mRNA and protein expression as well as apoptosis in both OVX and sham mice. E2 up‐regulated the mRNA expression of osteogenic genes for ALP, collagen I, TGF‐β1, BMP‐2, and cbfa1 in MSCs. In a comparison of the relative mRNA expression and protein levels for two ER isoforms, ERα was the predominant form expressed in MSCs obtained from both OVX and sham‐operated mice. Cumulatively, these results indicate that estrogen in vitro directly augments the proliferation and differentiation, ERα expression, osteogenic gene expression and, inhibits apoptosis and ERβ expression in MSCs obtained from OVX and sham‐operated mice. Co‐expression of ERα, but not ERβ, and osteogenic differentiation markers might indicate that ERα function as an activator and ERβ function as a repressor in the osteogenic differentiation in MSCs. These results suggest that mouse MSCs are anabolic targets of estrogen action, via ERα activation. J. Cell. Biochem. Suppl. 36: 144–155, 2001.

Histone H4-related osteogenic growth peptide (OGP): a novel circulating stimulator of osteoblastic activity.

It has been established that regenerating marrow induces an osteogenic response in distant skeletal sites and that this activity is mediated by factors released into the circulation by the healing tissue. In the present study we have characterized one of these factors, a 14 amino acid peptide named osteogenic growth peptide (OGP). Synthetic OGP, identical in structure to the native molecule, stimulates the proliferation and alkaline phosphatase activity of osteoblastic cells in vitro and increases bone mass in rats when injected in vivo. Immunoreactive OGP in high abundance is present physiologically in the serum, mainly in the form of an OGP‐OGP binding protein complex. A marked increase in serum bound and unbound OGP accompanies the osteogenic phase of post‐ablation marrow regeneration and associated systemic osteogenic response. Authentic OGP is identical to the C‐terminus of histone H4 and shares a five residue motif with a T‐cell receptor beta‐chain V‐region and the Bacillus subtilis outB locus. Since these latter proteins have not been implicated previously in the control of cell proliferation or differentiation, OGP may belong to a novel, heretofore unrecognized family of regulatory peptides. Perhaps more importantly, OGP appears to represent a new class of molecules involved in the systemic control of osteoblast proliferation and differentiation.

Stem cells as vehicles for orthopedic gene therapy.

Adult stem cells reside in adult tissues and serve as the source for their specialized cells. In response to specific factors and signals, adult stem cells can differentiate and give rise to functional tissue specialized cells. Adult mesenchymal stem cells (MSCs) have the potential to differentiate into various mesenchymal lineages such as muscle, bone, cartilage, fat, tendon and ligaments. Adult MSCs can be relatively easily isolated from different tissues such as bone marrow, fat and muscle. Adult MSCs are also easy to manipulate and expand in vitro. It is these properties of adult MSCs that have made them the focus of cell-mediated gene therapy for skeletal tissue regeneration. Adult MSCs engineered to express various factors not only deliver them in vivo, but also respond to these factors and differentiate into skeletal specialized cells. This allows them to actively participate in the tissue regeneration process. In this review, we examine the recent achievements and developments in stem-cell-based gene therapy approaches and their applications to bone, cartilage, tendon and ligament tissues that are the current focus of orthopedic medicine.