7 results on '"Powell, Barry C."'
Search Results
2. Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3
- Author
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Dwivedi Prem P, Anderson Peter J, and Powell Barry C
- Subjects
Transfection ,Nucleofection ,Skull ,Bone ,Primary cell culture ,Mesenchymal ,BMP2 ,luciferase ,Biotechnology ,TP248.13-248.65 - Abstract
Abstract Background Achieving efficient introduction of plasmid DNA into primary cultures of mammalian cells is a common problem in biomedical research. Human primary cranial suture cells are derived from the connective mesenchymal tissue between the bone forming regions at the edges of the calvarial plates of the skull. Typically they are referred to as suture mesenchymal cells and are a heterogeneous population responsible for driving the rapid skull growth that occurs in utero and postnatally. To better understand the molecular mechanisms involved in skull growth, and in abnormal growth conditions, such as craniosynostosis, caused by premature bony fusion, it is essential to be able to easily introduce genes into primary bone forming cells to study their function. Results A comparison of several lipid-based techniques with two electroporation-based techniques demonstrated that the electroporation method known as nucleofection produced the best transfection efficiency. The parameters of nucleofection, including cell number, amount of DNA and nucleofection program, were optimized for transfection efficiency and cell survival. Two different genes and two promoter reporter vectors were used to validate the nucleofection method and the responses of human primary suture mesenchymal cells by fluorescence microscopy, RT-PCR and the dual luciferase assay. Quantification of bone morphogenetic protein (BMP) signalling using luciferase reporters demonstrated robust responses of the cells to both osteogenic BMP2 and to the anti-osteogenic BMP3. Conclusions A nucleofection protocol has been developed that provides a simple and efficient, non-viral alternative method for in vitro studies of gene and protein function in human skull growth. Human primary suture mesenchymal cells exhibit robust responses to BMP2 and BMP3, and thus nucleofection can be a valuable method for studying the potential competing action of these two bone growth factors in a model system of cranial bone growth.
- Published
- 2012
- Full Text
- View/download PDF
3. Bone to pick: the importance of evaluating reference genes for RT-qPCR quantification of gene expression in craniosynostosis and bone-related tissues and cells
- Author
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Yang Xianxian, Hatfield Jodie T, Hinze Susan J, Mu Xiongzheng, Anderson Peter J, and Powell Barry C
- Subjects
Osteocalcin ,Alkaline phosphatase ,18 S RNA ,Gapdh ,β-actin ,geNorm ,Normfinder ,Craniosynostosis ,Bone ,Mineralization ,Medicine ,Biology (General) ,QH301-705.5 ,Science (General) ,Q1-390 - Abstract
Abstract Background RT-qPCR is a common tool for quantification of gene expression, but its accuracy is dependent on the choice and stability (steady state expression levels) of the reference gene/s used for normalization. To date, in the bone field, there have been few studies to determine the most stable reference genes and, usually, RT-qPCR data is normalised to non-validated reference genes, most commonly GAPDH, ACTB and 18 S rRNA. Here we draw attention to the potential deleterious impact of using classical reference genes to normalise expression data for bone studies without prior validation of their stability. Results Using the geNorm and Normfinder programs, panels of mouse and human genes were assessed for their stability under three different experimental conditions: 1) disease progression of Crouzon syndrome (craniosynostosis) in a mouse model, 2) proliferative culture of cranial suture cells isolated from craniosynostosis patients and 3) osteogenesis of a mouse bone marrow stromal cell line. We demonstrate that classical reference genes are not always the most ‘stable’ genes and that gene ‘stability’ is highly dependent on experimental conditions. Selected stable genes, individually or in combination, were then used to normalise osteocalcin and alkaline phosphatase gene expression data during cranial suture fusion in the craniosynostosis mouse model and strategies compared. Strikingly, the expression trends of alkaline phosphatase and osteocalcin varied significantly when normalised to the least stable, the most stable or the three most stable genes. Conclusion To minimise errors in evaluating gene expression levels, analysis of a reference panel and subsequent normalization to several stable genes is strongly recommended over normalization to a single gene. In particular, we conclude that use of single, non-validated “housekeeping” genes such as GAPDH, ACTB and 18 S rRNA, currently a widespread practice by researchers in the bone field, is likely to produce data of questionable reliability when changes are 2 fold or less, and such data should be interpreted with due caution.
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- 2012
- Full Text
- View/download PDF
4. Regulation of bone morphogenetic protein signalling and cranial osteogenesis by Gpc1 and Gpc3.
- Author
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Dwivedi, Prem P., Grose, Randall H., Filmus, Jorge, Hii, Charles S.T., Xian, Cory J., Anderson, Peter J., and Powell, Barry C.
- Subjects
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BONE morphogenetic proteins , *CELLULAR signal transduction , *BONE growth , *SKULL , *OSTEOBLASTS , *SUTURES , *CRANIOSYNOSTOSES , *GLYPICANS - Abstract
Abstract: From birth, the vault of the skull grows at a prodigious rate, driven by the activity of osteoblastic cells at the fibrous joints (sutures) that separate the bony calvarial plates. One in 2500 children is born with a medical condition known as craniosynostosis because of premature bony fusion of the calvarial plates and a cessation of bone growth at the sutures. Bone morphogenetic proteins (BMPs) are potent growth factors that promote bone formation. Previously, we found that Glypican-1 (GPC1) and Glypican-3 (GPC3) are expressed in cranial sutures and are decreased during premature suture fusion in children. Although glypicans are known to regulate BMP signalling, a mechanistic link between GPC1, GPC3 and BMPs and osteogenesis has not yet been investigated. We now report that human primary suture mesenchymal cells coexpress GPC1 and GPC3 on the cell surface and release them into the media. We show that they inhibit BMP2, BMP4 and BMP7 activities, which both physically interact with BMP2 and that immunoblockade of endogenous GPC1 and GPC3 potentiates BMP2 activity. In contrast, increased levels of GPC1 and GPC3 as a result of overexpression or the addition of recombinant protein, inhibit BMP2 signalling and BMP2-mediated osteogenesis. We demonstrate that BMP signalling in suture mesenchymal cells is mediated by both SMAD-dependent and SMAD-independent pathways and that GPC1 and GPC3 inhibit both pathways. GPC3 inhibition of BMP2 activity is independent of attachment of the glypican on the cell surface and post-translational glycanation, and thus appears to be mediated by the core glypican protein. The discovery that GPC1 and GPC3 regulate BMP2-mediated osteogenesis, and that inhibition of endogenous GPC1 and GPC3 potentiates BMP2 responsiveness of human suture mesenchymal cells, indicates how downregulation of glypican expression could lead to the bony suture fusion that characterizes craniosynostosis. [Copyright &y& Elsevier]
- Published
- 2013
- Full Text
- View/download PDF
5. Regulation of bone morphogenetic protein signalling and cranial osteogenesis by Gpc1 and Gpc3
- Author
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Charles S. T. Hii, Prem P. Dwivedi, Randall H. Grose, Barry C. Powell, Jorge Filmus, Peter J. Anderson, Cory J. Xian, Dwivedi, Prem P, Grose, Randall H, Filmus, Jorge, Hii, Charles ST, Xian, Cory J, Anderson, Peter J, and Powell, Barry C
- Subjects
animal structures ,Histology ,Glypican ,Physiology ,Endocrinology, Diabetes and Metabolism ,BMP2 ,Fluorescent Antibody Technique ,Biology ,Bone morphogenetic protein ,Transfection ,bone ,glypican ,Bone morphogenetic protein 2 ,osteogenesis ,Craniosynostosis ,Mesoderm ,Glypicans ,Osteogenesis ,medicine ,Humans ,Immunoprecipitation ,Bone growth ,Fibrous joint ,proteoglycan ,Microscopy, Confocal ,Mesenchymal stem cell ,Anatomy ,Cranial Sutures ,medicine.disease ,Flow Cytometry ,Cell biology ,Bone morphogenetic protein 7 ,craniosynostosis ,medicine.anatomical_structure ,embryonic structures ,Bone Morphogenetic Proteins ,Signal Transduction - Abstract
From birth, the vault of the skull grows at a prodigious rate, driven by the activity of osteoblastic cells at the fibrous joints (sutures) that separate the bony calvarial plates. One in 2500 children is born with a medical condition known as craniosynostosis because of premature bony fusion of the calvarial plates and a cessation of bone growth at the sutures. Bone morphogenetic proteins (BMPs) are potent growth factors that promote bone formation. Previously, we found that Glypican-1 (GPC1) and Glypican-3 (GPC3) are expressed in cranial sutures and are decreased during premature suture fusion in children. Although glypicans are known to regulate BMP signalling, a mechanistic link between GPC1, GPC3 and BMPs and osteogenesis has not yet been investigated. We now report that human primary suture mesenchymal cells coexpress GPC1 and GPC3 on the cell surface and release them into the media. We show that they inhibit BMP2, BMP4 and BMP7 activities,which both physically interact with BMP2 and that immunoblockade of endogenous GPC1 and GPC3 potentiates BMP2 activity. In contrast, increased levels of GPC1 and GPC3 as a result of over expression or the addition of recombinant protein, inhibit BMP2 signalling and BMP2-mediated osteogenesis. We demonstrate that BMP signalling in suture mesenchymal cells is mediated by both SMAD-dependent and SMAD-independent pathways and that GPC1 and GPC3 inhibit both pathways. GPC3 inhibition of BMP2 activity is independent of attachment of the glypican on the cell surface and post-translational glycanation, and thus appears to be mediated by the core glypican protein. The discovery that GPC1 and GPC3 regulate BMP2-mediated osteogenesis,and that inhibition of endogenous GPC1 and GPC3 potentiates BMP2 responsiveness of human suture mesenchymal cells, indicates how down regulation of glypican expression could lead to the bony suture fusion that characterizes craniosynostosis.
- Published
- 2013
6. Boning up on glypicans-opportunities for new insights into bone biology
- Author
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Dwivedi, Prem, Lam, N, and Powell, Barry C
- Subjects
WNT ,hedgehog ,receptor ,chondrocyte ,endochondral ,BMP ,glypican ,bone - Abstract
Bone formation is remarkable for the convergence in the activity of four major signalling pathways, the bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (HH) and wingless-integrated (WNT) pathways. These pathways cooperate in morphogenetic, proliferative and differentiative processes that underpin the development, growth and repair of skeletal structures. They are regulated by pathway-specific modulators and by another class of molecules, the glypicans. Glypicans are proteoglycans located on the cell surface, where they act as coreceptors to promote or inhibit signalling by ligands of the BMP, FGF, HH and WNT pathways, through protein–protein and protein–carbohydrate interactions. In this review, we discuss glypican structure, expression and function in the context of bone development and growth, with emphasis on the long bone growth plate where five of the six glypicans are expressed in overlapping patterns in the chondrogenic zone. Analyses of gene knockout models and the human conditions of Simpson–Golabi–Behmel syndrome and omodysplasia, which arise from mutations in glypican 3 (GPC3) and GPC6, respectively, highlight both subtle and striking effects of glypicans on bone growth. We draw attention to challenges and areas of opportunity, where the actions of glypicans on BMP, FGF, HH and WNT signalling might be profitably studied to help illuminate the complex interplay of signalling that drives bone growth. Refereed/Peer-reviewed
- Published
- 2013
7. Development of an efficient, non-viral transfection method for studying gene function and bone growth in human primary cranial suture mesenchymal cells reveals that the cells respond to BMP2 and BMP3
- Author
-
Barry C. Powell, Peter J. Anderson, Prem P. Dwivedi, Dwivedi, Prem P, Anderson, Peter J, and Powell, Barry C
- Subjects
skull ,Mesenchymal ,Cell Survival ,lcsh:Biotechnology ,Green Fluorescent Proteins ,BMP2 ,Bone Morphogenetic Protein 2 ,Bone Morphogenetic Protein 3 ,Nucleofection ,mesenchymal ,Biology ,Bone morphogenetic protein ,Transfection ,bone ,Bone morphogenetic protein 2 ,Mice ,Glypicans ,Genes, Reporter ,lcsh:TP248.13-248.65 ,Animals ,Humans ,Primary cell culture ,nucleofection ,Bone ,Cells, Cultured ,Bone growth ,Methodology Article ,Electroporation ,Mesenchymal stem cell ,Skull ,Mesenchymal Stem Cells ,Cranial Sutures ,luciferase ,Molecular biology ,Cell biology ,transfection ,Liposomes ,Stem cell ,primary cell culture ,Plasmids ,Biotechnology - Abstract
Background Achieving efficient introduction of plasmid DNA into primary cultures of mammalian cells is a common problem in biomedical research. Human primary cranial suture cells are derived from the connective mesenchymal tissue between the bone forming regions at the edges of the calvarial plates of the skull. Typically they are referred to as suture mesenchymal cells and are a heterogeneous population responsible for driving the rapid skull growth that occurs in utero and postnatally. To better understand the molecular mechanisms involved in skull growth, and in abnormal growth conditions, such as craniosynostosis, caused by premature bony fusion, it is essential to be able to easily introduce genes into primary bone forming cells to study their function. Results A comparison of several lipid-based techniques with two electroporation-based techniques demonstrated that the electroporation method known as nucleofection produced the best transfection efficiency. The parameters of nucleofection, including cell number, amount of DNA and nucleofection program, were optimized for transfection efficiency and cell survival. Two different genes and two promoter reporter vectors were used to validate the nucleofection method and the responses of human primary suture mesenchymal cells by fluorescence microscopy, RT-PCR and the dual luciferase assay. Quantification of bone morphogenetic protein (BMP) signalling using luciferase reporters demonstrated robust responses of the cells to both osteogenic BMP2 and to the anti-osteogenic BMP3. Conclusions A nucleofection protocol has been developed that provides a simple and efficient, non-viral alternative method for in vitro studies of gene and protein function in human skull growth. Human primary suture mesenchymal cells exhibit robust responses to BMP2 and BMP3, and thus nucleofection can be a valuable method for studying the potential competing action of these two bone growth factors in a model system of cranial bone growth.
- Full Text
- View/download PDF
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