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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.
Curr Opin Mol Ther 2002 Aug
PMID:Cell-mediated gene therapy for bone formation and regeneration. 1222 77

Bone morphogenetic proteins (BMPs) delivered on scaffolds can induce ectopic bone formation after subcutaneous injection. Adenoviral vectors (Ad) carrying BMP2, BMP7, and BMP9 cDNAs have been shown to produce bone through endochondral ossification. The present study was performed to elucidate the histological events leading to ectopic ossification for two novel first-generation adenoviral constructs encoding BMPs, AdBMP4 and AdBMP6. In vitro, the viral constructs produced and secreted the mature BMP4 and BMP6 proteins. In vivo, the calf muscles of athymic nude rats were injected with AdBMP4, AdBMP6, AdBMP2, or AdlacZ. Rats were sacrificed 3, 6, 9, 16, 21, 60, and 90 days postinjection. Whereas AdBMP4 produced ectopic bone through mechanisms similar to endochondral ossification, AdBMP6 seemed to induce bone by way of mechanisms similar to both intramembranous and endochondral ossification pathways. At the relatively low vector dose used in this study, AdBMP2 caused an initial recruitment of primitive mesenchymal cells, without further development to bone. From computed tomographic analysis, AdBMP6 produced the most rapid tissue calcification. The ultimate density of ectopic bone formed by AdBMP4 and AdBMP6 was comparable. The current study demonstrates that AdBMP4 and AdBMP6 are more potent than the prototypical osteogenic adenoviral vector AdBMP2 and seem to induce ectopic bone by different mechanisms.
Mol Ther 2002 Oct
PMID:Ectopic osteogenesis using adenoviral bone morphogenetic protein (BMP)-4 and BMP-6 gene transfer. 1237 87

Much of the work conducted on adult stem cells has focused on mesenchymal stem cells (MSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.
Mol Biol Cell 2002 Dec
PMID:Human adipose tissue is a source of multipotent stem cells. 1247 52

Strategies using mesenchymal stem cell (MSC)-mediated gene therapy have been developed to improve bone healing. However, transduction efficiency into MSCs by each vector is not always high. To overcome this problem, we used a modified adenoviral vector (Adv-F/RGD) with an RGD-containing peptide in the HI loop of the fiber knob domain of adenovirus type 5 (Ad5). Transduction efficiency into bone marrow-derived MSCs with Adv-F/RGD increased 12-fold compared with a vector containing the wild-type fiber (Adv-F/wt) by beta-galactosidase chemiluminescent assay. As a next step, we constructed AxCAhBMP2-F/RGD and AxCAhBMP2-F/wt carrying human bone morphogenetic protein 2 (BMP2). At the same multiplicity of infection, MSCs infected with AxCAhBMP2-F/RGD produced higher amounts of BMP2 than cells infected with AxCAhBMP2-F/wt, and also differentiated towards the osteogenic lineage more efficiently in vitro. Furthermore, using ex vivo gene transduction, we evaluated the potential for ectopic bone formation by the transduced MSCs in vivo. Transduction with AxCAhBMP2-F/RGD exhibited greatly enhanced new bone formation. These data suggest that Adv-F/RGD is useful for introducing foreign genes into MSCs and that it will be a powerful gene therapy tool for bone regeneration and other tissue engineering.
Mol Ther 2003 Mar
PMID:Efficient BMP2 gene transfer and bone formation of mesenchymal stem cells by a fiber-mutant adenoviral vector. 1266 31

Calcium induces transcriptional activation of the fos promoter by activation of the cyclic AMP response element (CRE)-binding protein (CREB), and in some cells its effect is enhanced synergistically by cyclic GMP (cGMP) through an unknown mechanism. We observed calcium-cGMP synergism in neuronal and osteogenic cells which express type II cGMP-dependent protein kinase (G-kinase); the effect on the fos promoter was mediated by the CRE and proportional to G-kinase activity. Dominant negative transcription factors showed involvement of CREB- and C/EBP-related proteins but not of AP-1. Expression of C/EBP-beta but not C/EBP-alpha or -delta enhanced the effects of calcium and cGMP on a CRE-dependent reporter gene. The transactivation potential of full-length CREB fused to the DNA-binding domain of Gal4 was increased synergistically by calcium and cGMP, and overexpression of C/EBP-beta enhanced the effect, while a dominant negative C/EBP inhibited it. With a mammalian two-hybrid system, coimmunoprecipitation experiments, and in vitro binding studies, we demonstrated that C/EBP-beta and CREB interacted directly; this interaction involved the C terminus of C/EBP-beta but occurred independently of CREB's leucine zipper domain. CREB Ser(133) phosphorylation was stimulated by calcium but not by cGMP; in cGMP-treated cells, (32)PO(4) incorporation into C/EBP-beta was decreased and C/EBP-beta/CRE complexes were increased, suggesting regulation of C/EBP-beta functions by G-kinase-dependent dephosphorylation. C/EBP-beta and CREB associated with the fos promoter in intact cells, and the amount of promoter-associated C/EBP-beta was increased by calcium and cGMP. We conclude that calcium and cGMP transcriptional synergism requires cooperation of CREB and C/EBP-beta, with calcium and cGMP modulating the phosphorylation states of CREB and C/EBP-beta, respectively.
Mol Cell Biol 2003 Jun
PMID:Synergism between calcium and cyclic GMP in cyclic AMP response element-dependent transcriptional regulation requires cooperation between CREB and C/EBP-beta. 1277 52

Strategies for genetic prenatal diagnosis on fetal cells in the maternal circulation have been limited by lack of a cell type present only in fetal blood. However, the recent identification of mesenchymal stem cells (MSC) in first trimester fetal blood offers the prospect of targeting MSC for non-invasive prenatal diagnosis. We developed protocols for fetal MSC enrichment from maternal blood and determined sensitivity and specificity in mixing experiments of male fetal MSC added to female blood, in dilutions from 1 in 10(5) to 10(8). We then used the optimal protocol to isolate fetal MSC from maternal blood in the first trimester, using blood taken after surgical termination of pregnancy as a model of increased feto-maternal haemorrhage. In model mixtures, we could amplify one male fetal MSC in 2.5 x 10(7) adult female nucleated cells, yielding a 100% pure population of fetal cells, but not one fetal MSC in 10(8) nucleated cells. Fetal MSC were identified in one of 20 post-termination maternal blood samples and confirmed as fetal MSC by XY fluorescence in-situ hybridization (FISH), immunophenotyping and osteogenic and adipogenic differentiation. We report the isolation of fetal MSC from maternal blood; however, their rarity in post-termination blood suggests they are unlikely to have a role in non-invasive prenatal diagnosis. Failure to locate these cells routinely may be attributed to their low frequency in maternal blood, to sensitivity limitations of enrichment technology, and/or to their engraftment in maternal tissues soon after transplacental passage. We speculate that gender microchimerism in post-reproductive maternal tissues might result from feto-maternal trafficking of MSC in early pregnancy.
Mol Hum Reprod 2003 Aug
PMID:Identification of fetal mesenchymal stem cells in maternal blood: implications for non-invasive prenatal diagnosis. 1283 27

The purpose of the present study was to examine the process of bone formation in the regenerating cranial appendages of roe deer (Capreolus capreolus) and fallow deer (Dama dama) during the early postcasting period. After the antlers are cast, osteoclastic and osteoblastic activities lead to a smoothing of the pedicle's separation surface, a strengthening of the pedicle bone, and a partial restoration of the distal pedicle portion that was lost along with the cast antler. Initially, bone formation occurs by intramembranous ossification, but early during the regeneration process cartilage is formed at the tips of the cranial appendages, and is subsequently replaced by bone in a process of endochodral ossification. Shortly after the antlers are cast, the cambium layer of the periosteum in the distal pedicle is markedly enlarged, which suggests that the periosteum serves as a cell source for the bone-forming tissue covering the exposed pedicle bone. The histological findings of our study are consistent with the view that the bony component of the regenerating cranial appendages of deer is largely derived from the pedicle periosteum. Based on findings in other bone systems, we speculate that stem cells that can undergo both osteogenic and chondrogenic differentiation are present in the pedicle periosteum. The early onset of chondrogenesis in the regeneration process is regarded as an adaptation to the necessity of producing a huge volume of bone within a short period. This parallels the situation in other cases of chondrogenesis in membrane bones.
Anat Rec A Discov Mol Cell Evol Biol 2003 Aug
PMID:Histological studies of bone formation during pedicle restoration and early antler regeneration in roe deer and fallow deer. 1284 10

It is well established that core binding factor Runx2/Cbfa1 is required for osteoblast recruitment and differentiation from mesenchymal stem cells. Transcriptional regulation of the Runx2/Cbfa1 gene by osteogenic factors such as bone morphogenetic proteins (BMPs) plays an important role in the stimulation of bone formation by these cytokines. BMP7 (also termed OP-1) is a member of the transforming growth factor beta (TGF-beta) superfamily and induces osteoblast differentiation from mesenchymal precursor stem cells in vitro as well as bone formation in vivo. This study examines the effects of BMP7 on markers of osteoblast differentiation and specifically on human Runx2/Cbfa1 gene transcription in a mouse C2C12 myoblast cell line where it induces expression of both alkaline phosphatase (ALP) and endogenous Runx2/Cbfa1. To further understand the mechanisms of human Runx2/Cbfa1 transcriptional regulation by BMP7, we cloned 3.0 kb of the human Runx2/Cbfa1 gene 5'-upstream flanking region and created a series of promoter deletions cloned into luciferase-based reporter vectors (Runx2/Cbfa1/Luc). Sequence data revealed six copies of the osteoblastic cis-acting element (OSE2) in the proximal promoter region. In C2C12 cells transiently transfected with Runx2/Cbfa1/Luc deletion constructs, transcriptional activity of Runx2/Cbfa1 was upregulated up to 2-fold after 24 h of BMP7 treatment. Mutational analysis demonstrated that the minimal responsive promoter region for BMP7-regulated transcription maps to a proximal -74 OSE2 site. Electromobility shift assays with C2C12 cellular extracts indicate that BMP7 increases binding of OSE2 promoter sequences, and supershift assays with anti-Runx2/Cbfa1 antibodies demonstrate that Runx2/Cbfa1 is part of the nucleoprotein complex binding OSE2. Together, these data indicate BMP7 can upregulate Runx2/Cbfa1 gene expression in C2C12 myoblast cells, and suggest that Runx2/Cbfa1 may bind to OSE2 elements within its own promoter to autoregulate gene transcription in differentiating osteoblasts.
Mol Cell Endocrinol 2003 Jul 31
PMID:Transcriptional regulation of the human Runx2/Cbfa1 gene promoter by bone morphogenetic protein-7. 1289 May 74

Different animal strains have different genetic backgrounds that influence their physiological function and pathological process. The differences in genetic background may affect the efficiency of adenoviral infection and target gene expression and further cause different gene therapy results when target genes are delivered with adenoviral vectors. In this study, ectopic bone was not seen in ADCMVBMP4 injection sites, but was formed in ADCMVBMP9 injection sites in all rat strains. The mean volumes of bone induced with ADCMVBMP9 were 0.87 +/- 0.2 cm3 in Wistar, 0.26 +/- 0.1 cm3 in Long-Evans, 0.34 +/- 0.2 cm3 in Sprague-Dawley, 0.44 +/- 0.1 cm3 in ACI, 0.66 +/- 0.2 cm3 in PVG, and 0.58 +/- 0.1 cm3 in Fischer 344 rats. This indicates that ADCMVBMP9 has different bone formation potentials in different immunocompetent rat strains (P = 0.02). The basic levels of CD4+ and CD8+ T cells in blood before viral infection and titers of adenoviral neutralizing antibodies 30 days post-viral infection were significantly different among rat strains (P < 0.01). The efficiencies of target gene expression delivered with adenovirus were also significantly different in primary muscle cell cultures from different rat strains (P < 0.01). The different osteogenic potentials of ADCMVBMP9 among rat strains may be, in part, due to the differences in immune factors and target gene expression efficiency in muscle tissue.
Mol Ther 2003 Nov
PMID:Rat strain differences in the ectopic osteogenic potential of recombinant human BMP adenoviruses. 1459 16

We explored the transduction kinetics of HIV-1-derived lentiviral vectors containing the CMV, EF1alpha, or PGK promoter expressing EGFP in fetal rhesus monkey bone marrow-derived mesenchymal stem cells (rhMSC). Studies included the effects of transduction (MOI 0-100) on growth, cell cycle, and differentiation toward an osteogenic lineage. Flow cytometric analysis indicated an approximate 8- to 10-fold greater quantity of EGFP-expressing rhMSC when cells were transduced with the CMV or EF1alpha promoter compared to PGK, although quantitative PCR revealed no differences at the DNA level. The CMV promoter initially expressed 10- to 100-fold higher levels of EGFP compared to EF1alpha or PGK, respectively, at increasing MOI, although a significant decline in transgene expression was observed posttransduction and with advancing passage (P < 0.01), whereas a significant increase in the level of expression was observed over time with the EF1alpha promoter. At an MOI of 100, a transient arrest at the S phase of the cell cycle was observed for both vector constructs. Transduced rhMSC differentiated toward an osteogenic lineage comparable to untransduced rhMSC and showed equivalent levels of alkaline phosphatase activity. These findings suggest that the SIN HIV-1-derived lentiviral vectors used in these studies can efficiently transduce rhMSC in vitro (CMV > EF1alpha > PGK) without inhibiting differentiation potential, although the cell cycle was transiently altered at high MOI
Mol Ther 2004 Jan
PMID:Morphological analysis and lentiviral transduction of fetal monkey bone marrow-derived mesenchymal stem cells. 1474 84


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