| Human Induced Pluripotent Stem Cell Lines Show Similar Stress Defence Mechanisms and Mitochondrial Regulation to Human Embryonic Stem Cells.
January 15, 2010 at 10:17 AM
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| | Human Induced Pluripotent Stem Cell Lines Show Similar Stress Defence Mechanisms and Mitochondrial Regulation to Human Embryonic Stem Cells. Stem Cells. 2010 Jan 13; Authors: Armstrong L, Tilgner K, Saretzki G, Atkinson SP, Stojkovic M, Moreno R, Przyborski S, Lako M The generation of induced pluripotent stem cells (iPSC) has enormous potential for the development of patient specific regenerative medicine. Human embryonic stem cells (hESC) are able to defend their genomic integrity by maintaining low levels of reactive oxygen species (ROS) through a combination of enhanced removal capacity and limited production of these molecules. Such limited ROS production stems partly from the small numbers of mitochondria present in hESC, thus it was important to determine that human iPSC (hiPSC) generation is able to eliminate the extra mitochondria present in the parental fibroblasts (reminiscent of "bottleneck" situation after fertilisation) and to show that hiPSC have similar antioxidant defences to hESC. We were able to generate seven hiPSC lines from adult human dermal fibroblasts and have fully characterised two of those clones. Both hiPSC clones express pluripotency markers and are able to differentiate in vitro into cells belongi! ng to all three germ layers. One of these clones is able to produce fully differentiated teratoma, whilst the other hiPSC clone is unable to silence the viral expression of OCT4 and c-MYC, produce fully differentiated teratoma and unable to downregulate the expression of some of the pluripotency genes during the differentiation process. In spite of these differences, both clones show similar ROS stress defence mechanisms and mitochondrial biogenesis to hESC. Together our data suggest that during the reprogramming process, certain cellular mechanisms are in place to ensure that hiPSC are provided with the same defence mechanisms against accumulation of ROS as the hESC. PMID: 20073085 [PubMed - as supplied by publisher] | | |
| Regulation of valvular interstitial cell calcification by adhesive peptide sequences.
January 15, 2010 at 10:17 AM
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| | Regulation of valvular interstitial cell calcification by adhesive peptide sequences. J Biomed Mater Res A. 2010 Jan 13; Authors: Gu X, Masters KS Knowledge of how valvular interstitial cells (VICs) interact with the extracellular matrix (ECM) would aid in not only better understanding the etiology of valvular disease but also constructing appropriate environments for valve tissue engineering. In this work, the calcification of VICs cultured on ECM coatings (fibronectin, fibrin, collagen, and laminin) or ECM-derived peptide sequences (RGDS, YIGSR, and DGEA) was quantified via several techniques. Neutralizing antibodies to specific adhesion receptors were also applied, followed by quantification of phenotypic markers related to valve calcification. The calcification of VICs varied with the ECM component or peptide that was presented on the culture substrate. VICs calcified the most on RGDS and least on YIGSR and DGEA, while blocking specific receptors revealed that disruption of VIC binding via the alpha(5)beta(1) integrin or the 67-kDa laminin receptor had a dramatic calcification-stimulating effect. Binding! via the alpha(2)beta(1) integrin did not alter calcification or VIC phenotype. These findings were translated to 3D peptide-modified scaffold environments that demonstrated varying levels of disease expression by VICs. Thus, specific adhesion receptors play a significant role in mediating the interactions between VICs and ECM that lead to calcification, which provides important information regarding the mechanisms of valvular disease and scaffold design for valve tissue engineering. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res 2010. PMID: 20073077 [PubMed - as supplied by publisher] | | |
| HPLC purification of adenoviral vectors.
January 15, 2010 at 10:17 AM
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| | HPLC purification of adenoviral vectors. Methods Mol Biol. 2010;594:395-408 Authors: Eglon M, McGrath B, O'Brien T Adenoviruses are attractive vectors for gene therapy where short-term transgene expression is required. In order to meet the clinical requirements of adenovirus for use beyond the laboratory, advanced methods are required for the purification and quantitation of recombinant adenoviral vectors (rAd). Chromatographic systems offer the advantages of linear scalability and reproducibility, and this method describes a laboratory-scale process based on liquid chromatography, which can be technically transferred and readily scaled-up according to the demands of the laboratory or clinic in which it will be used. PMID: 20072933 [PubMed - in process] | | |
| Generation of Antioxidant Adenovirus Gene Transfer Vectors Encoding CuZnSOD, MnSOD, and Catalase.
January 15, 2010 at 10:17 AM
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| | Generation of Antioxidant Adenovirus Gene Transfer Vectors Encoding CuZnSOD, MnSOD, and Catalase. Methods Mol Biol. 2010;594:381-93 Authors: Duffy AM, O'Brien T, McMahon JM Replication-deficient adenovirus gene transfer vectors are very useful for the experimental delivery of genes into cells and are widely used both in vitro and in vivo to determine the effects of transgene expression. Having a broad cell tropism, these vectors allow efficient transduction of many cell types and permit transfer of large amounts of DNA with resulting high expression levels within the target cell. Manganese superoxide dismutase (MnSOD), copper zinc superoxide dismutase (CuZnSOD) and catalase are all known antioxidants whose over-expression can result in amelioration of pathology brought about by an excess of reactive oxygen species within a cell. Their use has been suggested as therapies for many conditions, including cardiovascular disease, arthritis, diabetes, cancer, and damage to central nervous system cells. This chapter describes the methodology commonly used for production of replication-deficient adenovirus vectors encoding MnSOD, CuZnSOD, and! catalase. PMID: 20072932 [PubMed - in process] | | |
| Equine embryos and embryonic stem cells: Defining reliable markers of pluripotency.
January 15, 2010 at 10:17 AM
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| | Equine embryos and embryonic stem cells: Defining reliable markers of pluripotency. Theriogenology. 2010 Jan 11; Authors: Paris DB, Stout TA Cartilage and tendon injuries are a significant source of animal wastage and financial loss within the horse-racing industry. Moreover, both cartilage and tendon have limited intrinsic capacity for self-repair, and the functionally inferior tissue produced within a lesion may reduce performance and increase the risk of reinjury. Stem cells offer tremendous potential for accelerating and improving tissue healing, and adult mesenchymal stem cells (MSCs) are already used to treat cartilage and tendon injuries in horses. However, MSCs are scarce in the bone marrow isolates used, have limited potential for proliferation and differentiation in vitro, and do not appear to noticeably improve long-term functional repair. Embryonic stem cells (ESCs) or induced pluripotent stem (iPS) cells could overcome many of the limitations and be used to generate tissues of value for equine regenerative medicine. To date, six lines of putative ESCs have been described in the horse. All ! expressed stem cell-associated markers and exhibited longevity and pluripotency in vitro, but none have been proven to exhibit pluripotency in vivo. Moreover, it is becoming clear that the markers used to characterize the putative ESCs were inadequate, primarily because studies in domestic species have revealed that they are not specific to ESCs or the pluripotent inner cell mass, but also because the function of most in the maintenance of pluripotency is not known. Future derivation and validation of equine embryonic or other pluripotent stem cells would benefit greatly from a reliable panel of molecular markers specific to pluripotent cells of the developing horse embryo. PMID: 20071015 [PubMed - as supplied by publisher] | | |
| Student perspectives and opinions on their experience at an undergraduate outreach dental teaching centre at Cardiff: a 5-year study.
January 15, 2010 at 10:17 AM
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| | Student perspectives and opinions on their experience at an undergraduate outreach dental teaching centre at Cardiff: a 5-year study. Eur J Dent Educ. 2010 Feb;14(1):12-6 Authors: Lynch CD, Ash PJ, Chadwick BL AIM: Outreach teaching is now regarded as a desirable component of undergraduate dental teaching programmes in the UK. A purpose-built undergraduate dental outreach-training centre was opened in Cardiff in 2002. The aim of this paper is to report student perspectives and opinions on their experience at this unit over a 5-year period. METHODS: Final year dental students at Cardiff University were invited to report their comments on the St David's Primary Care Unit at various times during their placement there. Information was recorded for undergraduate students who commenced final year in 2003, 2004, 2005, 2006 and 2007 (n = 257). RESULTS: After 1 year, the most common favourable aspects reported by students included the availability of a suitably trained nurse for all procedures (n = 191), ready access to helpful/approachable teaching staff (n = 145), and closeness of learning experience to subsequent practice (n = 122). Many students commented on their growing co! nfidence in their own abilities whilst in the unit. CONCLUSION: Overwhelmingly, students reported their enthusiasm for training in an outreach teaching unit, preferring it to traditional dental school environments. Inherent in the comments recorded for each student was a sense of growing confidence in their abilities and development of reflective practice. Further work is needed to identify the impact of this form of dental student training on subsequent practices in Vocational Training and independent clinical careers. PMID: 20070794 [PubMed - in process] | | |
| Differentiation of human adipose-derived stem cells into beating cardiomyocytes.
January 15, 2010 at 10:17 AM
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| | Differentiation of human adipose-derived stem cells into beating cardiomyocytes. J Cell Mol Med. 2010 Jan 11; Authors: Choi YS, Dusting GJ, Stubbs S, Arunothayaraj S, Han XL, Collas P, Morrison WA, Dilley RJ ABSTRACT Background Human adipose-derived stem cells (ASCs) may differentiate into cardiomyocytes and this provides a source of donor cells for tissue engineering. Methods In this study, we evaluated cardiomyogenic differentiation protocols using a DNA demethylating agent 5-azacytidine (5-aza), a modified cardiomyogenic medium (MCM), a histone deacetylase inhibitor trichostatinA (TSA), and co-culture with neonatal rat cardiomyocytes. Results 5-aza treatment reduced both cardiac actin and TropT mRNA expression. Incubation in MCM only slightly increased gene expression (1.5 to 1.9 fold) and the number of cells co-expressing nkx2.5/sarcomeric alpha-actin (27.2% vs. 0.2% in control). TSA treatment increased cardiac actin mRNA expression 11-fold after 1 week, which could be sustained for 2 weeks by culturing cells in cardiomyocyte culture medium. TSA-treated cells also stained positively for cardiac myosin heavy chain, alpha-actin, TropI and connexin43, however none of! these treatments produced beating cells. ASCs in non-contact co-culture showed no cardiac differentiation, however ASCs co-cultured in direct contact co-culture exhibited a time-dependent increase in cardiac actin mRNA expression (up to 33 fold) between days 3 and 14. Immunocytochemistry revealed co-expression of GATA4 and Nkx2.5, alpha-actin, TropI, and cMHC in CM-DiI labeled ASCs. Most importantly, many of these cells showed spontaneous contractions accompanied by calcium transients in culture. Human ASC showed synchronous Ca(2+) transient and contraction synchronous with surrounding rat cardiomyocytes (106.08 beats/min). Gap junctions also formed between them as observed by dye transfer. Conclusion In conclusion, cell-to-cell interaction was identified as a key inducer for cardiomyogenic differentiation of hASCs. This method was optimized by co-culture with contracting cardiomyocytes and provides a potential cardiac differentiation system to progress applications for ca! rdiac cell therapy or tissue engineering. PMID: 20070436 [PubMed - as supplied by publisher] | | |
| The anatomical basis for a novel classification of osteoarthritis and allied disorders.
January 15, 2010 at 10:17 AM
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| | The anatomical basis for a novel classification of osteoarthritis and allied disorders. J Anat. 2010 Jan 7; Authors: McGonagle D, Tan AL, Carey J, Benjamin M Abstract Osteoarthritis (OA) has historically been classified as 'primary' where no discernible cause was evident and 'secondary' where a triggering factor was apparent. Irrespective of the triggering events, late-stage OA is usually characterized by articular cartilage attrition and consequently the anatomical basis for disease has been viewed in terms of cartilage. However, the widespread application of magnetic resonance imaging in early OA has confirmed several different anatomical abnormalities within diseased joints. A key observation has been that several types of primary or idiopathic OA show ligament-related pathology at the time of clinical presentation, so these categories of disease are no longer idiopathic - at least from the anatomical perspective. There is also ample evidence for OA initiation in other structures including menisci and bones in addition to articular cartilage. Therefore, a new classification for OA is proposed, which is based on the ! anatomical sites of earliest discernible joint structural involvement. The major proposed subgroups within this classification are ligament-, cartilage-, bone-, meniscal- and synovial-related, in addition to disease that is mixed pattern or multifocal in origin. We show how such a structural classification for OA provides a useful reference framework for staging the magnitude of disease. For late-stage or end-stage/whole organ disease, the final common pathway of these different scenarios, joint replacement strategies are likely to remain the only viable option. However, for younger subjects in particular, near the time of clinical disease onset, this scheme has implications for therapy targeted to specific anatomical locations. Thus, in the same way that tumours can be classified and staged according to their tissue of origin and extent of involvement, OA can likewise be anatomically classified and staged. This has implications for therapeutic strategies including regenera! tive medicine therapy development. PMID: 20070426 [PubMed - as supplied by publisher] | | |
| Osteogenic differentiation of human umbilical cord mesenchymal stromal cells in polyglycolic acid scaffolds.
January 15, 2010 at 10:17 AM
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| | Osteogenic differentiation of human umbilical cord mesenchymal stromal cells in polyglycolic acid scaffolds. Tissue Eng Part A. 2010 Jan 13; Authors: Wang L, Dormer NH, Bonewald L, Detamore MS Although human umbilical cord mesenchymal stromal cells (hUCMSCs) have been shown to differentiate along an osteogenic lineage in monolayer culture, the potential of these cells has seldom before been investigated in three-dimensional (3D) scaffolds for bone tissue engineering applications. In this 6-week study, we observed osteogenic differentiation of hUCMSCs on polyglycolic acid (PGA) non-woven mesh scaffolds, and compared seeding densities for potential use in bone tissue engineering. Cells were seeded into PGA meshes with densities of 5, 25 or 50 million cells/ml scaffold and then cultured in osteogenic medium. Cell proliferation, osteogenic differentiation, and matrix formation were evaluated at weeks 0, 3, and 6. Osteogenic differentiation was observed based on positive alkaline phosphatase activity (ALP) and an increase of collagen production and calcium incorporation into the extracellular matrix (ECM), which increased with higher cell density. During dif! ferentiation, runt-related transcription factor (RUNX2), type I collagen (CI) and osteocalcin (OCN) gene expression were also increased. In conclusion, exposed to osteogenic signals, hUCMSCs differentiated along an osteogenic lineage as determined by the expression of osteogenic markers and matrix formation, and increasing the density of hUCMSCs seeded onto 3D PGA scaffolds led to better osteogenic differentiation. PMID: 20070186 [PubMed - as supplied by publisher] | | |
| The N-terminal RASSF family: a new group of Ras-association-domain-containing proteins, with emerging links to cancer formation.
January 15, 2010 at 10:17 AM
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| | The N-terminal RASSF family: a new group of Ras-association-domain-containing proteins, with emerging links to cancer formation. Biochem J. 2010 Jan 15;425(2):303-11 Authors: Sherwood V, Recino A, Jeffries A, Ward A, Chalmers AD The RASSF (Ras-association domain family) has recently gained several new members and now contains ten proteins (RASSF1-10), several of which are potential tumour suppressors. The family can be split into two groups, the classical RASSF proteins (RASSF1-6) and the four recently added N-terminal RASSF proteins (RASSF7-10). The N-terminal RASSF proteins have a number of differences from the classical RASSF members and represent a newly defined set of potential Ras effectors. They have been linked to key biological processes, including cell death, proliferation, microtubule stability, promoter methylation, vesicle trafficking and response to hypoxia. Two members of the N-terminal RASSF family have also been highlighted as potential tumour suppressors. The present review will summarize what is known about the N-terminal RASSF proteins, addressing their function and possible links to cancer formation. It will also compare the N-terminal RASSF proteins with the classica! l RASSF proteins and ask whether the N-terminal RASSF proteins should be considered as genuine members or imposters in the RASSF family. PMID: 20025613 [PubMed - indexed for MEDLINE] | | |
| Cell-seeded polyurethane-fibrin structures--a possible system for intervertebral disc regeneration.
January 15, 2010 at 10:17 AM
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| | Cell-seeded polyurethane-fibrin structures--a possible system for intervertebral disc regeneration. Eur Cell Mater. 2009;18:27-38; discussion 38-9 Authors: Mauth C, Bono E, Haas S, Paesold G, Wiese H, Maier G, Boos N, Graf-Hausner U Nowadays, intervertebral disc (IVD) degeneration is one of the principal causes of low back pain involving high expense within the health care system. The long-term goal is the development of a medical treatment modality focused on a more biological regeneration of the inner nucleus pulposus (NP). Hence, interest in the endoscopic implantation of an injectable material took center stage in the recent past. We report on the development of a novel polyurethane (PU) scaffold as a mechanically stable carrier system for the reimplantation of expanded autologous IVD-derived cells (disc cells) to stimulate regenerative processes and restore the chondrocyte-like tissue within the NP. Primary human disc cells were seeded into newly developed PU spheroids which were subsequently encapsulated in fibrin hydrogel. The study aims to analyze adhesion properties, proliferation capacity and phenotypic characterization of these cells. Polymerase chain reaction was carried out to de! tect the expression of genes specifically expressed by native IVD cells. Biochemical analyses showed an increased DNA content, and a progressive enhancement of total collagen and glycosaminoglycans (GAG) was observed during cell culture. The results suggest the synthesis of an appropriate extracellular matrix as well as a stable mRNA expression of chondrogenic and/or NP specific markers. In conclusion, the data presented indicate an alternative medical approach to current treatment options of degenerated IVD tissue. PMID: 19802794 [PubMed - indexed for MEDLINE] | | |
| Conserved expression patterns predict microRNA targets.
January 15, 2010 at 10:17 AM
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| | Conserved expression patterns predict microRNA targets. PLoS Comput Biol. 2009 Sep;5(9):e1000513 Authors: Ritchie W, Rajasekhar M, Flamant S, Rasko JE microRNAs (miRNAs) are major regulators of gene expression and thereby modulate many biological processes. Computational methods have been instrumental in understanding how miRNAs bind to mRNAs to induce their repression but have proven inaccurate. Here we describe a novel method that combines expression data from human and mouse to discover conserved patterns of expression between orthologous miRNAs and mRNA genes. This method allowed us to predict thousands of putative miRNA targets. Using the luciferase reporter assay, we confirmed 4 out of 6 of our predictions. In addition, this method predicted many miRNAs that act as expression enhancers. We show that many miRNA enhancer effects are mediated through the repression of negative transcriptional regulators and that this effect could be as common as the widely reported repression activity of miRNAs. Our findings suggest that the indirect enhancement of gene expression by miRNAs could be an important component of ! miRNA regulation that has been widely neglected to date. PMID: 19779543 [PubMed - indexed for MEDLINE] | | | |
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