Genetically engineered mesenchymal stem cells: applications in spine therapy.

Spine disorders and intervertebral disc degeneration are considered the main causes for the clinical condition commonly known as back pain. Spinal fusion by implanting autologous bone to produce bony bridging between the two vertebrae flanking the degenerated-intervertebral disc is currently the most efficient treatment for relieving the symptoms of back pain. However, donor-site morbidity, complications and the long healing time limit the success of this approach. Novel developments undertaken by regenerative medicine might bring more efficient and available treatments. Here we discuss the pros and cons of utilizing genetically engineered mesenchymal stem cells for inducing spinal fusion. The combination of the stem cells, gene and carrier are crucial elements for achieving optimal spinal fusion in both small and large animal models, which hopefully will lead to the development of clinical applications.

Maxillofacial-derived stem cells regenerate critical mandibular bone defect.

Stem cell-based bone tissue regeneration in the maxillofacial complex is a clinical necessity. Genetic engineering of mesenchymal stem cells (MSCs) to follow specific differentiation pathways may enhance the ability of these cells to regenerate and increase their clinical relevance. MSCs isolated from maxillofacial bone marrow (BM) are good candidates for tissue regeneration at sites of damage to the maxillofacial complex. In this study, we hypothesized that MSCs isolated from the maxillofacial complex can be engineered to overexpress the bone morphogenetic protein-2 gene and induce bone tissue regeneration in vivo. To demonstrate that the cells isolated from the maxillofacial complex were indeed MSCs, we performed a flow cytometry analysis, which revealed a high expression of mesenchyme-related markers and an absence of non-mesenchyme-related markers. In vitro, the MSCs were able to differentiate into osteogenic, chondrogenic, and adipogenic lineages. Gene delivery of the osteogenic gene BMP2 via an adenoviral vector revealed high expression levels of BMP2 protein that induced osteogenic differentiation of these cells in vitro and induced bone formation in an ectopic site in vivo. In addition, implantation of genetically engineered maxillofacial BM-derived MSCs into a mandibular defect led to regeneration of tissue at the site of the defect; this was confirmed by performing micro-computed tomography analysis. Histological analysis of the mandibles revealed osteogenic differentiation of implanted cells as well as bone tissue regeneration. We conclude that maxillofacial BM-derived MSCs can be genetically engineered to induce bone tissue regeneration in the maxillofacial complex and that this finding may be clinically relevant.

Molecular targets for tendon neoformation.

Tendons and ligaments are unique forms of connective tissue that are considered an integral part of the musculoskeletal system. The ultimate function of tendon is to connect muscles to bones and to conduct the forces generated by muscle contraction into movements of the joints, whereas ligaments connect bone to bone and provide joint stabilization. Unfortunately, the almost acellular and collagen I-rich structure of tendons and ligaments makes them very poorly regenerating tissues. Injured tendons and ligaments are considered a major clinical challenge in orthopedic and sports medicine. This Review discusses the several factors that might serve as molecular targets that upon activation can enhance or lead to tendon neoformation.

Advanced molecular profiling in vivo detects novel function of dickkopf-3 in the regulation of bone formation.

UNLABELLED: 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. INTRODUCTION: Endochondral bone formation is a complex biological process involving numerous chondrogenic, osteogenic, and angiogenic proteins, only some of which have been well studied. Additional key genes may have important roles as well. We hypothesized that to identify key genes and signaling pathways crucial for bone formation, a comprehensive gene discovery strategy should be applied to an established in vivo model of osteogenesis. MATERIALS AND METHODS: We used in vivo implanted C3H10T1/2 cells that had been genetically engineered to express human bone morphogenetic protein-2 (BMP2) in a tetracycline-regulated system that controls osteogenic differentiation. Oligonucleotide microarray data from the implants (n = 4 repeats) was analyzed using coupled two-way clustering (CTWC) and statistical methods. For studying the effects of dickkopf homolog 3 (Dkk3) in chondrogenesis and osteogenesis, C3H10T1/2 mesenchymal progenitors were used. RESULTS: The CTWC revealed temporal expression of Dkk3 with other chondrogenesis-, osteogenesis-, and Wnt-related genes. Quantitative RT-PCR confirmed the expression of Dkk3 in the implants. C3H10T1/2 cells that expressed Dkk3 in the presence of BMP2 displayed lower levels of alkaline phosphatase and collagen I mRNA expression than control C3H10T1/2 cells that did not express Dkk3. Interestingly, the levels of collagen II mRNA expression, Alcian blue staining, and glucose aminoglycans (GAGs) production were not influenced by Dkk3 expression. In vivo microCT and bioluminescence imaging revealed that co-expression of Dkk3 and BMP2 by implanted C3H10T1/2 cells induced the formation of significantly lower quantities of bone than cells expressing only BMP2. CONCLUSIONS: A bioinformatics analysis enabled the identification of Dkk3 as a pivotal gene with a novel function in endochondral bone formation. Our results showed that Dkk3 might have inhibitory effects on osteogenesis, but no effect on chondrogenesis, indicating that Dkk3 plays a regulatory role in endochondral bone formation. Further mechanistic studies are required to reveal the mechanism of action of Dkk3 in endochondral bone formation.

Nucleofection-based ex vivo nonviral gene delivery to human stem cells as a platform for tissue regeneration.

There are several gene therapy approaches to tissue regeneration. Although usually efficient, virusbased approaches may elicit an immune response against the viral proteins. An alternative approach, nonviral transfer, is safer, and can be controlled and reproduced. We hypothesized that in vivo bone formation could be achieved using human mesenchymal stem cells (hMSCs) nonvirally transfected with the human bone morphogenetic protein-2 (hBMP-2) or -9 (hBMP-9) gene. Human MSCs were transfected using nucleofection, a unique electropermeabilization-based technique. Postnucleofection, cell viability was 53.6 +/- 2.5% and gene delivery efficiency was 51% to 88% (mean 68.2 +/- 4.1%), as demonstrated by flow cytometry in enhanced green fluorescent protein (EGFP)-nucleofected hMSCs. Transgene expression lasted longer than 14 days and was very low 21 days postnucleofection. Both hBMP-2- and hBMP-9-nucleofected hMSCs in culture demonstrated a significant increase in calcium deposition compared with EGFP-nucleofected hMSCs. Human BMP-2- and hBMP-9-nucleofected hMSCs transplanted in ectopic sites in NOD/SCID mice induced bone formation 4 weeks postinjection. We conclude that in vivo bone formation can be achieved by using nonvirally nucleofected hMSCs. This could lead to a breakthrough in the field of regenerative medicine, in which safer, nonviral therapeutic strategies present a very attractive alternative.

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.

Human mesenchymal stem cells alter antigen-presenting cell maturation and induce T-cell unresponsiveness.

Infusion of either embryonic or mesenchymal stem cells prolongs the survival of organ transplants derived from stem cell donors and prevents graft-versus-host-disease (GVHD). An in-depth mechanistic understanding of this tolerization phenomenon could lead to novel cell-based therapies for transplantation. Here we demonstrate that while human mesenchymal stem cells (hMSCs) can promote superantigen-induced activation of purified T cells, addition of antigen-presenting cells (APCs; either monocytes or dendritic cells) to the cultures inhibits the T-cell responses. This contact- and dose-dependent inhibition is accompanied by secretion of large quantities of interleukin (IL)-10 and aberrant APC maturation, which can be partially overridden by the addition of factors that promote APC maturation (ie, lipopolysaccharide [LPS] or anti-CD40 monoclonal antibody [mAb]). Thus, our data support an immunoregulatory mechanism wherein hMSCs inhibit T cells indirectly by contact-dependent induction of regulatory APCs with T-cell-suppressive properties. Our data may reveal a physiologic phenomenon whereby the development of a distinct APC population is regulated by the tissue's cellular microenvironment.

Cell-mediated gene therapy for bone formation and regeneration.

Cell-mediated gene therapy is one of the new modalities branching out from the wide-ranging field of gene transfer and therapy. When applied to bone formation and regeneration, it has particular advantages depending on the type of cell used as a platform for gene delivery. When utilizing adult mesenchymal stem cells or osteoprogenitor cells for the expression of bone-promoting osteogenic factors, the cells not only express the factors promoting bone growth, but can respond, differentiate and participate in the bone formation process. The ability of engineered cells to respond to the transgene, as well as to other local signals in vivo, confers on them special properties that enable the formation and regeneration of large-scale bone tissue. This approach is a paradigm for the development of gene therapy strategies for other skeletal tissues. Here, we review the most recent studies related to cell-mediated gene therapy for bone formation and regeneration.