Enhancement of angiogenic effect of co-transfection human NGF and VEGF genes in rat bone marrow mesenchymal stem cells

The current study explored the feasibility and efficacy of co-transfection of the human nerve growth factor (NGF) and vascular endothelial growth factor 165 (VEGF165) genes in rat bone marrow mesenchymal stem cells (BMSCs). The obtained hNGF and vascular endothelial growth factor (VEGF) cDNAs were cloned into the pEGFP-C1 expression vector to construct the recombinant vectors. Co-transfection in rat BMSCs was performed and the expressions of both genes were detected by RT-PCR, Western blot, and enzyme-linked immunospecific assay. The biological activity of recombinant NGF and VEGF proteins was confirmed using the Chick Chorioallantoic Membrane (CAM) assay. NGF and VEGF genes could be expressed successfully in rat BMSCs. The recombinant NGF and VEGF from the rat BMSCs showed a more significant synergetic biological activity compared with single recombinant NGF or VEGF. These findings demonstrate that the co-transfection of hNGF + VEGF genes can enhance the angiogenic effect in vivo.     

BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high-density culture

Human bone marrow-derived mesenchymal stem cells (MSCs) have been shown to differentiate into distinct mesenchymal tissues including bone and cartilage. The capacity of MSCs to replicate undifferentiated and to mature into cartilaginous tissues suggests these cells as an attractive cell source for cartilage tissue engineering. Here we show that the stimulation of human bone marrow-derived MSCs with recombinant bone morphogenetic protein-2 (BMP2) results in chondrogenic lineage development under serum-free conditions. Histological staining of proteoglycan with Alcian blue and immunohistochemical staining of cartilage-specific type II collagen revealed the deposition of typical cartilage extracellular matrix components. Semi-quantitative real-time gene expression analysis of characteristic chondrocytic matrix genes, such as cartilage link protein, cartilage oligomeric matrix protein, aggrecan, and types I, II, and IX collagen, confirmed the induction of the chondrocytic phenotype in high-density culture upon stimulation with BMP2 and transforming growth factor-beta3 (TGFbeta3). Histologic staining of mineralized extracellular matrix with von Kossa, immunostaining of type X collagen (typical for hypertrophic chondrocytes), and gene expression analysis of osteocalcin and adipocyte-specific fatty acid binding protein (aP2) further documented that BMP2 induced chondrogenic lineage development and not osteogenesis and/or adipogenesis in human MSCs. These results suggest BMP2 as a promising candidate for tissue engineering approaches regenerating articular cartilage on the basis of mesenchymal progenitors from bone marrow.     

Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro

We developed and used real-time RT-PCR assays to investigate how the expression of typical osteoblast-related genes by human bone marrow stromal cells (BMSC) is regulated by (i) the culture time in medium inducing osteogenic differentiation and (ii) the previous expansion in medium enhancing cell osteogenic commitment. BMSC from six healthy donors were expanded in medium without (CTR) or with fibroblast growth factor-2 and dexamethasone (FGF/Dex; these factors are known to increase BMSC osteogenic commitment) and further cultivated for up to 20 days with ascorbic acid, beta-glycerophosphate and dexamethasone (these factors are typically used to induce BMSC osteogenic differentiation). Despite a high variability in the gene expression levels among different individuals, we identified the following statistically significant patterns. The mRNA levels of bone morphogenetic protein-2 (BMP-2), bone sialo protein-II (BSP), osteopontin (OP) and to a lower extent cbfa-1 increased with culture time in osteogenic medium (OM), both in CTR- and FGF/Dex-expanded BMSC, unlike levels of alkaline phosphatase, collagen type I, osteocalcin, and osteonectin. After 20 days culture in OM, BMP-2, BSP, and OP were more expressed in FGF/Dex than in CTR-expanded BMSC (mRNA levels were, respectively, 9.5-, 14.9-, and 5.8-fold higher), unlike all the other investigated genes. Analysis of single-colony-derived strains of BMSC further revealed that after 20 days culture in OM, only a subset of FGF/Dex-expanded clones expressed higher mRNA levels of BMP-2, BSP, and OP than CTR-expanded clones. In conclusion, we provide evidence that mRNA levels of BMP-2, BSP, and OP, quantified using real-time RT-PCR, can be used as markers to monitor the extent of BMSC osteogenic differentiation in vitro; using those markers, we further demonstrated that only a few subpopulations of BMSC display enhanced osteogenic differentiation following FGF/Dex expansion.     

Osteogenesis versus chondrogenesis by BMP-2 and BMP-7 in adipose stem cells

Bone morphogenetic proteins (BMPs) initiate, promote, and maintain chondrogenesis and osteogenesis. We hypothesize that BMP-2 induces an osteogenic, and BMP-7 a chondrogenic phenotype in adipose tissue-derived mesenchymal stem cells (AT-MSCs). We compared the effects of a short 15min BMP-2 or BMP-7 (10ng/ml) treatment on osteogenic and chondrogenic differentiation of AT-MSCs. Gene expression was studied 4 and 14 days after BMP-treatment. At day 4 BMP-2, but not BMP-7, stimulated runx-2 and osteopontin gene expression, and at day 14 BMP-7 down-regulated expression of these genes. At day 4 BMP-2 and BMP-7 stimulated biglycan gene expression, which was down-regulated by BMP-7 at day 14. BMP-7 stimulated aggrecan gene expression at day 14. Our data indicate that BMP-2 treatment for 15min induces osteogenic differentiation, whereas BMP-7 stimulates a chondrogenic phenotype of AT-MSCs. Therefore, AT-MSCs triggered for only 15min with BMP-2 or BMP-7 provide a feasible tool for bone and cartilage tissue engineering.     

Molecular hydrogen inhibits lipopolysaccharide/interferon γ-induced nitric oxide production through modulation of signal transduction in macrophages

Adipose-derived stem cells (ASCs) have been successfully applied in treating bone defects both in animals and humans and promoted osteogenesis in vivo significantly. However, the mechanism of in vivo osteogenesis of ASCs was still little known, we hypothesized that this was mediated in part by interaction between implanted ASCs and local vein endothelial cells. In this study, human adipose-derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVEC) were isolated and characterized. Cells were then either cultured alone or cocultured. Alkaline phosphatase (ALP) staining, quantitative measurement of ALP activity and Alizarin staining of hASCs cultured alone, HUVEC cultured alone and cells cocultured demonstrated that osteogenic differentiation of cocultured cells increased obviously. Osteocalcin (OC) expression of hASCs cocultured with HUVEC showed an obvious raise than hASCs cultured alone. HUVEC cultured alone showed BMP-2 secretion and increased with culturing time. Real-time PCR of the cocultured cells showed four osteogenic differentiation related genes raised with culturing time, while two adipogenic differentiation related genes showed a slightly decrease with culturing time. Results of our study with different culture models showed that in vitro osteogenesis of hASCs was enhanced by coculture with HUVEC which secreted BMP-2. This study not only provided us with an in vitro model of studying interaction between cells, but also helped us to understand the in vivo therapeutic mechanisms of ASCs.     

Human mesenchymal stem cells (hMSCs) expressing truncated soluble vascular endothelial growth factor receptor (tsFlk-1) following lentiviral-mediated gene transfer inhibit growth of Burkitt's lymphoma in a murine model

BACKGROUND: Efficient gene transfer to bone marrow derived mesenchymal stem cells (MSC) would provide an important opportunity to express potent anticancer agents in the tumour microenvironment because of their contribution to the tumour stroma. METHODS: HIV-based lentiviral vectors were pseudotyped with four different envelope proteins; amphotropic murine leukaemia virus (ampho), murine leukaemia virus (10A1), feline endogenous virus (RD114), and the vesicular stomatitis virus glycoprotein (VSVG). These pseudotypes were examined for transduction efficiency in human bone marrow derived MSC. The effect of lentiviral expression of truncated soluble vascular endothelial growth factor decoy receptor (tsFlk-1) in MSC on growth of Raji cells was determined, both in vitro and in vivo. RESULTS: All lentiviral vectors produced significant levels of transduction at an multiplicity of infection (MOI) of 1, those bearing the RD114 envelope glycoprotein consistently produced higher transduction levels (mean 70 +/- 6%) compared with the other pseudotyped lentiviral vectors, although there was significant inter-donor variation. Stable transgene expression was achieved after multiple rounds of transduction with VSVG-pseudotyped particles, without alteration in the differentiative capacity of transduced cells. Co-injection of MSC stably expressing tsFlk-1 with Raji Burkitt's lymphoma cells significantly impaired subcutaneous tumour growth in immunodeficient mice when compared to controls where either unmanipulated MSC or GFP-expressing MSC were used. CONCLUSIONS: Human MSC are easily transduced by pseudotyped lentiviral particles but there is inter-donor variation in transduction efficiency. Gene-modified MSC expressing a gene of therapeutic potential can moderate growth of haematological malignancies.     

Osteogenic differentiation effects on rat bone marrow-derived mesenchymal stromal cells by lentivirus-mediated co-transfection of human BMP2 gene and VEGF165 gene

We proposed a novel combined gene therapy of human vascular endothelial growth factor 165 gene (hVEGF165) and human bone morphogenetic protein 2 gene (hBMP2) for bone regeneration by lentivirus-mediated co-transfection of both genes into rat bone marrow-derived mesenchymal stromal cells (MSCs). Both genes were successfully co-expressed in MSCs confirmed by real-time PCR and ELISA. And the alkaline phosphatase activity of MSCs was significantly augmented by the co-transfection with both genes than any single gene transfection (P < 0.01). These results demonstrated the feasibility of the combined gene therapy by using MSCs lentivirally co-transfected with hVEGF165 and hBMP2 for bone regeneration.     

Wnt and BMP signaling pathways co-operatively induce the differentiation of multiple myeloma mesenchymal stem cells into osteoblasts by upregulating EMX2

Osteoblast differentiation, defined as the process whereby a relatively unspecialized cell acquires the specialized features of an osteoblast, is directly linked to multiple myeloma (MM) bone disease. Wnt and bone morphogenetic protein (BMP) are proved to be implicated in the pathological or defective osteoblast differentiation process. This study aims to test the involvement of Wnt, bone morphogenetic proteins (BMP) pathways, and empty spiracles homeobox 2 (EMX2) in osteoblast differentiation and MM development. Initially, differentially expressed genes in bone marrow mesenchymal stem cells (MSCs) from MM patients and healthy donors were identified using microarray-based gene expression profiling. The functional role of Wnt and BMP in MM was determined. Next, we focused on the co-operative effects of Wnt and BMP on calcium deposition, alkaline phosphatase (ALP) activity, the number of mineralized nodules, and osteocalcin (OCN) content in MSCs. The expression patterns of Wnt and BMP pathway–related genes, EMX2 and osteoblast differentiation-related factors were determined to assess their effects on osteoblast differentiation. Furthermore, regulation of Wnt and BMP in ectopic osteogenesis was also investigated in vivo. An integrated genomic screen suggested that Wnt and BMP regularly co-operate to regulate EMX2 and affect MM. EMX2 was downregulated in MSCs. The activated Wnt and BMP resulted in more calcium salt deposits, mineralized nodules, and a noted increased in ALP activity and OCN content by upregulating EMX2, leading to induced differentiation of MSCs into osteoblasts. Collectively, this study demonstrated that Wnt and BMP pathways could co-operatively stimulate differentiation of MSCs into osteoblasts and inhibit MM progression, representing potential targets for MM treatment.     

Matrix remodeling associated 7 promotes differentiation of bone marrow mesenchymal stem cells toward osteoblasts

The matrix remodeling associated 7 (MXRA7) gene had been ill-studied and its biology remained to be discovered. Inspired by our previous findings and public datasets concerning MXRA7, we hypothesized that the MXRA7 gene might be involved in bone marrow mesenchymal stem cells (BMSCs) functions related to bone formation, which was checked by utilizing in vivo or in vitro methodologies. Micro-computed tomography of MXRA7-deficient mice demonstrated retarded osteogenesis, which was reflected by shorter femurs, lower bone mass in both trabecular and cortical bones compared with wild-type (WT) mice. Histology confirmed the osteopenia-like feature including thinner growth plates in MXRA7-deficient femurs. Immunofluorescence revealed less osteoblasts in MXRA7-deficient femurs. Polymerase chain reaction or western blot analysis showed that when WT BMSCs were induced to differentiate toward osteoblasts or adipocytes in culture, MXRA7 messenger RNA or protein levels were significantly increased alongside osteoblasts induction, but decreased upon adipocytes induction. Cultured MXRA7-deficient BMSCs showed decreased osteogenesis upon osteogenic differentiation induction as reflected by decreased calcium deposition or lower expression of genes responsible for osteogenesis. When recombinant MXRA7 proteins were supplemented in a culture of MXRA7-deficient BMSCs, osteogenesis or gene expression was fully restored. Upon osteoblast induction, the level of active β-catenin or phospho-extracellular signal-regulated kinase in MXRA7-deficient BMSCs was decreased compared with that in WT BMSCs, and these impairments could be rescued by recombinant MXRA7 proteins. In adipogenesis induction settings, the potency of MXRA7-deficient BMSCs to differentiate into adipocytes was increased over the WT ones. In conclusion, this study demonstrated that MXRA7 influences bone formation via regulating the balance between osteogenesis and adipogenesis in BMSCs.     

In vitro chondrogenesis of human synovium-derived mesenchymal stem cells: optimal condition and comparison with bone marrow-derived cells

There are increasing reports that mesenchymal stem cells (MSCs) are present in various tissues other than bone marrow, including synovium. Here we investigated the optimal conditions for in vitro chondrogenesis of human synovium-derived MSCs and compared these cells with bone marrow-derived MSCs, especially in terms of their chondrogenesis potential. Synovium and bone marrow were harvested from six donors during knee operations for ligament injuries. Digested synovium cells or nucleated cells from bone marrow were expanded clonally. A pellet culture system was used for chondrogenesis, and the best combination of up to three cytokines of the seven assessed. Synovium-derived MSCs plated at a lower density expanded more rapidly. Contrary to previous reports, a combination of TGFbeta and dexamethasone was not sufficient to induce chondrogenesis. However, addition of BMP2 to TGFbeta and dexamethasone dramatically increased cartilage pellet size and the synthesis of cartilage matrix. The cartilage pellets were also analyzed by electron microscopy and immunohistology. DNA content per pellet decreased during chondrogenesis, indicating the pellet increased its size through the accumulation of newly synthesized extracellular matrix. Sequential chondrogenic gene expression was demonstrated by RT-PCR. Synovium-derived MSCs looked similar to the bone marrow-derived MSCs in their surface epitopes and proliferation potential; however, cartilage pellets from synovium were significantly larger than those from bone marrow in patient-matched comparisons. We demonstrated that the combination of TGFbeta, dexamethasone, and BMP2 was optimal for in vitro chondrogenesis of synovium-derived MSCs and that the synovium-derived MSCs have a greater chondrogenesis potential than bone marrow-derived MSCs.     

Focal adhesion kinase promotes BMP2-induced osteogenic differentiation of human urinary stem cells via AMPK and Wnt signaling pathways

Human urine-derived stem cells (hUSCs) serve as favorable candidates for bone transplants due to their efficient proliferative and multipotent differentiation abilities, as well as the capacity to secrete a variety of vasoactive agents to facilitate tissue engineering. The present study aimed to explore the role of focal adhesion kinase (FAK) in bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation of hUSCs and to investigate the underlying mechanism. The degree of osteogenic differentiation and the correlated signals, following BMP2 overexpression and siRNA-mediated silencing of FAK, were determined in vitro. Moreover, hUSCs induced bone formation in a rat model with cranial defects, in vivo. Our findings revealed that alkaline phosphatase production, calcium deposits, osteocalcin and osteopontin expression, and bone formation were upregulated in vitro and in vivo following BMP2-induced osteogenic differentiation, and AMPK and Wnt signaling pathway activation by FAK could effectively regulate BMP2-enhanced osteogenic differentiation of hUSCs. Taken together, these findings indicated that FAK could mediate BMP2-enhanced osteogenic differentiation of hUSCs through activating adenosine 5’-monophosphate-activated protein kinase and Wnt signaling pathways.     

Brain from bone: Efficient "meta-differentiation" of marrow stroma-derived mature osteoblasts to neurons with Noggin or a demethylating agent

Bone marrow stromal cells are able to differentiate into adipogenic, chondrogenic, myogenic, osteogenic, and cardiomyogenic lineages, all of which are limited to a mesoderm-derived origin. In this study, we showed that neurons, which are of an ectoderm-origin, could be generated from marrow-derived stromal cells by specific inducers, fibronectin/ornithine coating, and neurosphere formation. The neurons generated from marrow stroma formed neurites, expressed neuron-specific markers and genes, and started to respond to depolarizing stimuli as functional mature neurons. Among stromal cells, isolated mature osteoblasts which had strong in vivo osteogenic activity could be efficiently converted into functional neurons. This transdifferentiation or meta-differentiation was enhanced by Noggin, an inhibitor of bone morphogenetic proteins, in comparison with 5-azacytidine, a demethylating agent capable of altering the gene expression pattern. Marrow stroma is therefore a potential source of cells for neural cell transplantation.     

Expression of Human BMP-2 Gene in Different Tissues of Tobacco Plants

The bone morphogenetic proteins (BMPs) are a family of growth factors that regulate the development of bone. BMP-2 is the most effective in the induction of bone tissue. A large amount of BMP-2 is needed for both bone tissue engineering research and clinical application. Thus, an effective way is necessary to produce sufficient BMP-2 protein. With the advance in plant biotechnology, transgenic plants have been targeted as a bioreactor to produce desired recombinant proteins. Here, the expression of recombinant human bmp-2 gene (rhbmp-2) was studied in tobacco plants using gus as a reporter gene. The difference of expression levels in root, stem and leaf tissues was analyzed by GUS activity assay, semi-quantitive RT-PCR and western blotting. The results indicated that the expression levels of fusion protein in root and stem tissues were significantly higher than those in leaf tissue. For the protein compositions in root and stem tissues were simpler than those in leaf tissue, this suggested that the purification process with root and stem tissues would potentially be easier.     

Osteogenic Potency of Nacre on Human Mesenchymal Stem Cells

Nacre seashell is a natural osteoinductive biomaterial with strong effects on osteoprogenitors, osteoblasts, and osteoclasts during bone tissue formation and morphogenesis. Although nacre has shown, in one study, to induce bridging of new bone across large non-union bone defects in 8 individual human patients, there have been no succeeding human surgical studies to confirm this outstanding potency. But the molecular mechanisms associated with nacre osteoinduction and the influence on bone marrow-derived mesenchymal stem cells (BMSC’s), skeletal stem cells or bone marrow stromal cells remain elusive. In this study we highlight the phenotypic and biochemical effects of Pinctada maxima nacre chips and the global nacre soluble protein matrix (SPM) on primary human bone marrow-derived stromal cells (hBMSCs) in vitro. In static co-culture with nacre chips, the hBMSCs secreted Alkaline phosphatase (ALP) at levels that exceeded bone morphogenetic protein (rhBMP-2) treatment. Concentrated preparation of SPM applied to Stro-1 selected hBMSC’s led to rapid ALP secretions, at concentrations exceeding the untreated controls even in osteogenic conditions. Within 21 days the same population of Stro-1 selected hBMSCs proliferated and secreted collagens I–IV, indicating the premature onset of an osteoblast phenotype. The same SPM was found to promote unselected hBMSC differentiation with osteocalcin detected at 7 days, and proliferation increased at 7 days in a dose-dependent manner. In conclusion, nacre particles and nacre SPM induced the early stages of human bone cell differentiation, indicating that they may be promising soluble factors with osteoinductive capacity in primary human bone cell progenitors such as, hBMSC’s.     

重组人BMP-2在烟草不同组织中的表达

骨形态发生蛋白（BMPs）是一类调节骨组织发育的生长因子。BMP-2是BMP家族中诱骨活性最强的。在骨组织工程研究和临床应用中需要大量的BMP-2。因此,研究出一种能够有效地大量生产BMP-2的方法是十分必要的。随着植物分子生物学的进展,转基因植物被用作一种生物反应器来生产目的蛋白。以gus作为报告基因,研究了重组人bmp-2基因在烟草中的表达。通过GUS活性检测、半定量PCR和Western blotting分析了根、茎、叶组织中基因表达的水平,结果显示融合蛋白在根和茎组织中表达量显著高于叶组织。由于根和茎组织中蛋白组成与叶组织相比相对简单,提示其更易于进行目的蛋白的纯化。