Zulma Gazit

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.

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.

Advanced Molecular Profiling in Vivo Detects Novel Function of Dickkopf‐3 in the Regulation of Bone Formation

A bioinformatics‐based analysis of endochondral bone formation model detected several genes upregulated in this process. Among these genes the dickkopf homolog 3 (Dkk3) was upregulated and further studies showed that its expression affects in vitro and in vivo osteogenesis. This study indicates a possible role of Dkk3 in regulating bone formation.

Bioluminescent Imaging in Bone

Monitoring gene expression in vitro and in vivo, is crucial when analyzing osteogenesis and developing effective bone gene therapy protocols. Until recently, molecular analytical tools were only able to detect protein expression either in vitro or in vivo. These systems include histology and immunohistochemistry, fluorescent imaging, PET (micro-PET), CT (micro-CT), and bioluminescent imaging. The last is the only system to date that can enable efficient quantitative monitoring of gene expression both in vitro and in vivo. Effective bioluminescent imaging in bone can be achieved by using transgenic mice harboring the luciferase reporter gene, downstream of an osteogenesis specific promoter. The aim of this chapter is to comprehensively describe the various protocols needed for the detection of bioluminescence in bone development and repair.