Yoram Zilberman

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.

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.

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.

Ultrasound-based nonviral gene delivery induces bone formation in vivo

Nonviral gene delivery is a promising, safe, therapeutic tool in regenerative medicine. This study is the first to achieve nonviral, ultrasound-based, osteogenic gene delivery that leads to bone tissue formation, in vivo. We hypothesized that direct in vivo sonoporation of naked DNA encoding for the osteogenic gene, recombinant human bone morphogenetic protein-9 (rhBMP-9) would induce bone formation. A luciferase plasmid (Luc), encoding rhBMP-9 or empty pcDNA3 vector mixed with microbubbles, was injected into the thigh muscles of mice. After injection, noninvasive sonoporation was applied. Luc activity was monitored noninvasively, and quantitatively using bioluminescence imaging in vivo, and found for 14 days with a peak expression on day 7. To examine osteogenesis in vivo, rhBMP-9 plasmid was sonoporated into the thigh muscles of transgenic mice that express the Luc gene under the control of a human osteocalcin promoter. Following rhBMP-9 sonoporation, osteocalcin-dependent Luc expression lasted for 24 days and peaked on day 10. Bone tissue was formed in the site of rhBMP-9 delivery, as was shown by micro-computerized tomography and histology. The sonoporation method was also compared with previously developed electrotransfer-based gene delivery and was found significantly inferior in its efficiency of gene delivery. We conclude that ultrasound-mediated osteogenic gene delivery could serve as a therapeutic solution in conditions requiring bone tissue regeneration after further development that will increase the transfection efficiency.

Nonvirally Engineered Porcine Adipose Tissue‐Derived Stem Cells: Use in Posterior Spinal Fusion

Multiple factors alter intervertebral disc volume, structure, shape, composition, and biomechanical properties, often leading to low back pain. Spinal fusion is frequently performed to treat this problem. We recently published results of our investigation of a novel system of in vivo bone formation, in which we used nonvirally nucleofected human mesenchymal stem cells that overexpress a bone morphogenetic protein gene. We hypothesized that primary porcine adipose tissue‐derived stem cells (ASCs) nucleofected with plasmid containing recombinant human bone morphogenetic protein‐6 (rhBMP‐6) could induce bone formation and achieve spinal fusion in vivo. Primary ASCs were isolated from freshly harvested porcine adipose tissue. Overexpression of rhBMP‐6 was achieved ex vivo by using a nucleofection technique. Transfection efficiency was monitored by assessing a parallel transfection involving an enhanced green fluorescent protein reporter gene and flow cytometry analysis. rhBMP‐6 protein secreted by the cells was measured by performing an enzyme‐linked immunosorbent assay. Genetically engineered cells were injected into the lumbar paravertebral muscle in immunodeficient mice. In vivo bone formation was monitored by a quantitative microcomputed tomography (μCT). The animals were euthanized 5 weeks postinjection, and spinal fusion was evaluated using in vitro μCT and histological analysis. We found formation of a large bone mass adjacent to the lumbar area, which produced posterior spinal fusion of two to four vertebrae. Our data demonstrate that efficient bone formation and spinal fusion can be achieved using ex vivo, nonvirally transfected primary ASCs. These results could pave the way to a novel biological solution for spine treatment.

Molecular Imaging of the Skeleton: Quantitative Real‐Time Bioluminescence Monitoring Gene Expression in Bone Repair and Development

Monitoring gene expression in vivo, noninvasively, is a critical issue in effective gene therapy systems. To date, there are no adequate molecular imaging techniques, which quantitatively monitor gene expression in vivo in skeletal development and repair. The aim of this study was to monitor gene expression in skeletal development and repair, using a real‐time molecular imaging system, which quantitatively and noninvasively detects bioluminescence in vivo. Our experimental model consisted of transgenic mice harboring the luciferase marker gene under the regulation of the human osteocalcin (hOC) promoter. A new light detection cooled charge coupled device (CCCD) camera was applied to monitor luciferase expression. In vitro, mesenchymal stem cells (MSCs) isolated from bone marrow of transgenic mice exhibited hOC promoter regulation, detected by luciferase expression that correlated with their osteogenic differentiation. During development from 1 week to 1.5 years, transgenic mice exhibited transgene expression in a wide spectrum of skeletal organs, including calvaria, vertebra, tail, and limbs, reaching a peak at 1 week in most of the skeletal organs. In two skeletal repair models, bone fracture and marrow ablation, the noninvasive CCCD system revealed a peak of luciferase expression at 6 days postsurgery. All quantitative, noninvasive, real‐time CCCD measurements correlated with a luciferase biochemical assay and luciferase immunohistochemistry, which demonstrated luciferase expression in hypertrophic chondrocytes and trabecular osteoblasts. Our studies show for the first time (1) the CCCD detection system is a reliable quantitative gene detection tool for the skeleton in vivo, (2) expression of luciferase regulated by the hOC promoter is significantly decreased with age in most skeletal sites, and (3) the dynamics of hOC regulation during mice skeletal development and repair in real time, quantitatively and noninvasively.

The use of a synthetic oxygen carrier-enriched hydrogel to enhance mesenchymal stem cell-based bone formation in vivo

A major hurdle to surmount in bone-tissue engineering is ensuring a sufficient oxygen supply to newly forming tissue to avoid cell death or delayed development of osteogenic features. We hypothesized that an oxygen-enriched hydrogel scaffold would enhance tissue-engineered bone formation in vivo. To test this, we used a well-characterized mesenchymal stem cell (MSC) line, Tet-off BMP2 MSC, whose cells were engineered to express recombinant human bone morphogenetic protein-2. Cells were suspended in hydrogel supplemented with perfluorotributylamine (PFTBA) and implanted subcutaneously in an ectopic site, a radial bone defect, or a lumbar paravertebral muscle (mouse model of spinal fusion) in C3H/HeN mice. For controls, we used cells suspended in the same gel without PFTBA. In the ectopic site, there were significant increases in bone formation (2.5-fold increase), cell survival, and osteocalcin activity in the PFTBA-supplemented groups. PFTBA supplementation significantly increased structural parameters of bone in radial bone defects and triggered a significant 1.4-fold increase in bone volume in the spinal fusion model. We conclude that synthetic oxygen carrier supplementation of tissue-engineered implants enhances ectopic bone formation and yields better bone quality and volume in bone-repair and spinal fusion models, probably due to increased cell survival.