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| In this issue: Biotechnology Journal 2/2010.
February 13, 2010 at 6:24 AM
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| | In this issue: Biotechnology Journal 2/2010. Biotechnol J. 2010 Feb 11;5(2):136 Authors: BARLEY, A GREEN LIGAND FACTORY: Erlendsson et al., Biotechnol. J. 2009, 5, 163-171Growth factors are commonly used as cell culture supplements and might gain even more importance in stem cell research, tissue engineering and regenerative medicine or as human therapeutics. Recombinant growth factors are commercially produced in bacteria, yeast, insect and mammalian cells. While these systems are efficient, there is still a need for serum and endotoxin free production systems. Therefore, researchers from Island and co-workers produced biologically active recombinant human Flt3 ligand in transgenic barley seeds. This ligand can then be purified with a tag using immobilized metal ion affinity chromatography (IMAC). The Flt3 is posttranslationally modified and exhibits comparable biological activity to commercial Flt3 ligand. This study demonstrates that plant molecular farming is a viable approach for the bioproduction of human-derived growth factors. TRANSGENIC CROP ! SAFETY: Herman et al., Biotechnol. J. 2009, 5, 172-182To evalutate the safety of transgenic crops one can analyse the composition of the transgenic crop and compare it to the parent plant or seed. If the composition of nutrients is found to be equivalent to that of non-transgenic variant that is considered safe, then further safety assessment of the transgenic crop can focus solely on the intended modification, like the expression of a transgenic protein. Statistical methods that are used in clinical medicine to compare new generic drugs with brand-name drugs can then be applied to evaluate the equivalence. However, commonly used equivalence limits are shown to be a poor model for comparing transgenic crops with an array of reference crop varieties. Researchers from Dow AgroSciences LLC (USA) suggest an alternate model applied to corn, cotton and soybean seed samples. ADVANCED BIOFUELS FROM MICROBES: Peralta-Yahya and Keasling, Biotechnol. J. 2009, 5, 147-162The climate cha! nge and energy security are the major challenges of current ti! mes. Bio fuels produced from renewable resourses are a cost-effective alternative. Ethanol produced by microorganisms is currently the major biofuel in the transportation sector. However, ethanol's corrosivity and hygroscopicity make it incompatible with existing fuel storage and distribution infrastructure and limits its economic use. Advanced biofuels, such as long chain alcohols and isoprenoid- and fatty acid-based biofuels, have physical properties that more closely resemble petroleum-derived fuels. Therefore, they are attractive candidates for the replacement of petroleum-derived fuels. Authors from the University of California, Berkeley, review recent developments in the engineering of metabolic pathways for the production of advanced biofuels by microorganisms, most importantly Escherichia coli and Saccharomyces cerevisiae. PMID: 20151449 [PubMed - as supplied by publisher] | | |
| Fusion of uniluminal vascular spheroids: A model for assembly of blood vessels.
February 13, 2010 at 6:24 AM
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| | Fusion of uniluminal vascular spheroids: A model for assembly of blood vessels. Dev Dyn. 2010 Feb 11;239(3):spcone Authors: Fleming PA, Argraves WS, Gentile C, Neagu A, Forgacs G, Drake CJ Blood vessel formation via vascular fusion. When placed into hanging drop culture, five uniluminal vascular spheroids fuse to form a single, larger diameter spheroid with an outer layer of smooth muscle alpha actin positive cells (red) and an inner PECAM-1 positive endothelium (green) surrounding a large central lumen. From Fleming et al., Developmental Dynamics 239:398-406, 2010. PMID: 20151401 [PubMed - as supplied by publisher] | | |
| Rescue of Developmental Defects by Blastocyst Stem Cell Injection: Towards Elucidation of Neomorphic Corrective Pathways.
February 13, 2010 at 6:24 AM
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| | Rescue of Developmental Defects by Blastocyst Stem Cell Injection: Towards Elucidation of Neomorphic Corrective Pathways. J Cardiovasc Transl Res. 2009 Oct 14;3(1):66 Authors: Zhao Q, Beck A, Vitale JM, Schneider JS, Terzic A, Fraidenraich D Stem cell-based therapy is an exciting area of high potential for regenerative medicine. To study disease prevention, we inject mouse embryonic stem cells (ESCs) into a variety of mouse blastocysts, most of which harbor mutations. Mice derived from these mutant blastocysts develop human-like diseases, either at developmental stages or in the adult, but blastocyst injection of ESCs prevents disease from occurring. Rather than entirely repopulating the affected organs, with just 20% of chimerism, the ESCs replenish protein levels that are absent in mutant mice, and induce novel or "neomorphic" signals that help circumvent the requirements for the mutations. We also show data indicating that the "neomorphic" mechanisms arise as a result of blastocyst injection of ESCs, regardless of the nature of the host blastocyst (mutant or wild-type). Thus, blastocyst injection of ESCs not only allows the study of disease prevention, but also unveils novel pathways whose activati! on may aid in the correction of congenital or acquired disease. PMID: 20151025 [PubMed - as supplied by publisher] | | |
| Reconstitution of Experimental Neurogenic Bladder Dysfunction Using Skeletal Muscle-Derived Multipotent Stem Cells.
February 13, 2010 at 6:24 AM
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| | Reconstitution of Experimental Neurogenic Bladder Dysfunction Using Skeletal Muscle-Derived Multipotent Stem Cells. Transplantation. 2010 Feb 10; Authors: Nitta M, Tamaki T, Tono K, Okada Y, Masuda M, Akatsuka A, Hoshi A, Usui Y, Terachi T BACKGROUND.: Postoperative neurogenic bladder dysfunction is a major complication of radical hysterectomy for cervical cancer and is mainly caused by unavoidable damage to the bladder branch of the pelvic plexus (BBPP) associated with colateral blood vessels. Thus, we attempted to reconstitute disrupted BBPP and blood vessels using skeletal muscle-derived multipotent stem cells that show synchronized reconstitution capacity of vascular, muscular, and peripheral nervous systems. METHODS.: Under pentobarbital anesthesia, intravesical pressure by electrical stimulation of BBPP was measured as bladder function. The distal portion of BBPP with blood vessels was then cut unilaterally (experimental neurogenic bladder model). Measurements were performed before, immediately after, and at 4 weeks after transplantation as functional recovery. Stem cells were obtained from the right soleus and gastrocnemius muscles after enzymatic digestion and cell sorting as CD34/45 (Sk-34)! and CD34/45 (Sk-DN). Suspended cells were autografted around the damaged region, whereas medium alone and CD45 cells were transplanted as control groups. To determine the morphological contribution of the transplanted cells, stem cells obtained from green fluorescent protein transgenic mouse muscles were transplanted into a nude rat model and were examined by immunohistochemistry and immunoelectron microscopy. RESULTS.: At 4 weeks after surgery, the transplantation group showed significantly higher functional recovery ( approximately 80%) than the two controls ( approximately 28% and 24%). The transplanted cells showed an incorporation into the damaged peripheral nerves and blood vessels after differentiation into Schwann cells, perineurial cells, vascular smooth muscle cells, pericytes, and fibroblasts around the bladder. CONCLUSION.: Transplantation of multipotent Sk-34 and Sk-DN cells is potentially useful for the reconstitution of damaged BBPP. PMID: 20150836 [PubMed - as supplied by publisher] | | |
| Quantitative Structure-Cytotoxicity Relationship of Newly Synthesized Tropolones Determined by a Semiempirical Molecular-orbital Method (PM5).
February 13, 2010 at 6:24 AM
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| | Quantitative Structure-Cytotoxicity Relationship of Newly Synthesized Tropolones Determined by a Semiempirical Molecular-orbital Method (PM5). Anticancer Res. 2010 Jan;30(1):129-33 Authors: Ishihara M, Wakabayashi H, Motohashi N, Sakagami H A semiempirical molecular-orbital method (CONFLEX/PM5) was applied to delineate the relationship between the cytotoxicity (evaluated by 50% cytotoxic concentration, CC(50)) of twenty-four tropolone-related compounds and their molecular weight or one of the following eleven chemical descriptors: the heat of formation (COSMO, non-COSMO; kcal/mole), stability of hydration (=COSMO-nonCOSMO (DeltaH); kcal/mole), dipole moment (D), hydrophobicity (log P), highest occupied molecular orbital energy (E(HOMO); eV), lowest unoccupied molecular orbital energy (E(LUMO); eV), absolute hardness [eta=(E(LUMO)-E(HOMO))/2; eV)], absolute electron negativity [chi=-(E(LUMO)+E(HOMO))/2; eV], reactivity index (omega=chi(2)/2eta; eV), surface area (A(2)) and volume (A(3)) of the molecule. No good correlation was found with the unseparated twenty-four compounds all together, but modest to high correlation was found after separation into three different groups of compounds, depending on t! he structural similarity. Particular descriptors could be used to evaluate the biological activity of newly synthesized tropolones. PMID: 20150627 [PubMed - in process] | | |
| Pharmaceutical modulation of canonical Wnt signaling in multipotent stromal cells for improved osteoinductive therapy.
February 13, 2010 at 6:24 AM
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| | Pharmaceutical modulation of canonical Wnt signaling in multipotent stromal cells for improved osteoinductive therapy. Proc Natl Acad Sci U S A. 2010 Feb 11; Authors: Krause U, Harris S, Green A, Ylostalo J, Zeitouni S, Lee N, Gregory CA Human mesenchymal stem cells (hMSCs) from bone marrow are regarded as putative osteoblast progenitors in vivo and differentiate into osteoblasts in vitro. Positive signaling by the canonical wingless (Wnt) pathway is critical for the differentiation of MSCs into osteoblasts. In contrast, activation of the peroxisome proliferator-activated receptor-gamma (PPARgamma)-mediated pathway results in adipogenesis. We therefore compared the effect of glycogen-synthetase-kinase-3beta (GSK3beta) inhibitors and PPARgamma inhibitors on osteogenesis by hMSCs. Both compounds altered the intracellular distribution of beta-catenin and GSK3beta in a manner consistent with activation of Wnt signaling. With osteogenic supplements, the GSK3beta inhibitor 6-bromo-indirubin-3'-oxime (BIO) and the PPARgamma inhibitor GW9662 (GW) enhanced early osteogenic markers, alkaline phosphatase (ALP), and osteoprotegerin (OPG) by hMSCs and transcriptome analysis demonstrated up-regulation of genes ! encoding bone-related structural proteins. At higher doses of the inhibitors, ALP levels were attenuated, but dexamethasone-induced biomineralization was accelerated. When hMSCs were pretreated with BIO or GW and implanted into experimentally induced nonself healing calvarial defects, GW treatment substantially increased the capacity of the cells to repair the bone lesion, whereas BIO treatment had no significant effect. Further investigation indicated that unlike GW, BIO induced cell cycle inhibition in vitro. Furthermore, we found that GW treatment significantly reduced expression of chemokines that may exacerbate neutrophil- and macrophage-mediated cell rejection. These data suggest that use of PPARgamma inhibitors during the preparation of hMSCs may enhance the capacity of the cells for osteogenic cytotherapy, whereas adenine analogs such as BIO can adversely affect the viability of hMSC preparations in vitro and in vivo. PMID: 20150512 [PubMed - as supplied by publisher] | | |
| Direct Myocardial Implantation of Human Fetal Stem Cells in Heart Failure Patients: Long-term Results.
February 13, 2010 at 6:24 AM
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| | Direct Myocardial Implantation of Human Fetal Stem Cells in Heart Failure Patients: Long-term Results. Heart Surg Forum. 2010 Feb 1;13(1):E31-5 Authors: Benetti F, Peñherrera E, Maldonado T, Vera YD, Subramanian V, Geffner L Background: End-stage heart failure (HF) is refractory to current standard medical therapy, and the number of donor hearts is insufficient to meet the demand for transplantation. Recent studies suggest autologous stem cell therapy may regenerate cardiomyocytes, stimulate neovascularization, and improve cardiac function and clinical status. Although human fetal-derived stem cells (HFDSCs) have been studied for the treatment of a variety of conditions, no clinical studies have been reported to date on their use in treating HF. We sought to determine the efficacy and safety of HFDSC treatment in HF patients.Methods and Results: Direct myocardial transplantation of HFDSCs by open-chest surgical procedure was performed in 10 patients with HF due to nonischemic, nonchagasic dilated cardiomyopathy. Before and after the procedure, and with no changes in their preoperative doses of medications (digoxin, furosemide, spironolactone, angiotensin-converting enzyme inhibitors, ! angiotensin receptor blockers, betablockers), patients were assessed for New York Heart Association (NYHA) class, performance in the exercise tolerance test (ETT), ejection fraction (EF), left ventricular end-diastolic dimension (LVEDD) via transthoracic echocardiography, performance in the 6-minute walk test, and performance in the Minnesota congestive HF test. All 10 patients survived the operation. One patient had a stroke 3 days after the procedure, and although she later recovered, she was unable to perform the follow-up tests. Another male patient experienced pericardial effusion 3 weeks after the procedure. Although it resolved spontaneously, the patient abandoned his control tests and died 5 months after the procedure. An autopsy of the myocardium suggested that new young cells were present in the cardiomyocyte mix. At 40 months, the mean (+/-SD) NYHA class decreased from 3.4 +/- 0.5 to 1.33 +/- 0.5 (P = .001); the mean EF increased 31%, from 26.6% +/- 4% to 34.8% +! /- 7.2% (P = .005); and the mean ETT increased 291.3%, from 4.! 25 minut es to 16.63 minutes (128.9% increase in metabolic equivalents, from 2.46 to 5.63) (P < .0001); the mean LVEDD decreased 15%, from 6.85 +/- 0.6 cm to 5.80 +/- 0.58 cm (P < .001); mean performance in the 6-minute walk test increased by 43.2%, from 251 +/- 113.1 seconds to 360 +/- 0 seconds (P = .01); the mean distance increased 64.4%, from 284.4 +/- 144.9 m to 468.2 +/- 89.8 m (P = .004); and the mean result in the Minnesota test decreased from 71 +/- 27.3 to 6 +/- 5.9 (P < .001).Conclusion: Although these initial findings suggest direct myocardial implantation of HFDSCs is feasible and improves cardiac function in HF patients at 40 months, more clinical research is required to confirm these observations. PMID: 20150037 [PubMed - in process] | | |
| Switching cell fate: the remarkable rise of induced pluripotent stem cells and lineage reprogramming technologies.
February 13, 2010 at 6:24 AM
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| | Switching cell fate: the remarkable rise of induced pluripotent stem cells and lineage reprogramming technologies. Trends Biotechnol. 2010 Feb 8; Authors: Selvaraj V, Plane JM, Williams AJ, Deng W Cell reprogramming, in which a differentiated cell is made to switch its fate, is an emerging field with revolutionary prospects in biotechnology and medicine. The recent discovery of induced pluripotency by means of in vitro reprogramming has made way for unprecedented approaches for regenerative medicine, understanding human disease and drug discovery. Moreover, recent studies on regeneration and repair by direct lineage reprogramming in vivo offer an attractive novel alternative to cell therapy. Although we continue to push the limits of current knowledge in the field of cell reprogramming, the mechanistic elements that underlie these processes remain largely elusive. This article reviews landmark developments in cell reprogramming, current knowledge, and technological developments now on the horizon with significant promise for biomedical applications. PMID: 20149468 [PubMed - as supplied by publisher] | | |
| Extracellular matrix-mediated osteogenic differentiation of murine embryonic stem cells.
February 13, 2010 at 6:24 AM
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| | Extracellular matrix-mediated osteogenic differentiation of murine embryonic stem cells. Biomaterials. 2010 Feb 8; Authors: Evans ND, Gentleman E, Chen X, Roberts CJ, Polak JM, Stevens MM Embryonic stem cells (ESCs) are pluripotent and have the ability to differentiate into mineralising cells in vitro. The use of pluripotent cells in engineered bone substitutes will benefit from the development of bioactive scaffolds which encourage cell differentiation and tissue development. Extracellular matrix (ECM) may be a suitable candidate for use in such scaffolds since it plays an active role in cellular differentiation. Here, we test the hypothesis that tissue-specific ECM influences the differentiation of murine ESCs. We induced murine ESCs to differentiate by embryoid body formation, followed by dissociation and culture on ECM prepared by decellularisation of either osteogenic cell (MC3T3-E1) or non-osteogenic cell (A549) cultures, or on defined collagen type I matrix. We assessed osteogenic differentiation by formation of mineralised tissue and osteogenic gene expression, and found it to be significantly greater on MC3T3-E1 matrices than on any other ! matrix. The osteogenic effect of MC3T3-E1 matrix was reduced by heat treatment and abolished by trypsin, suggesting a bioactive proteinaceous component. These results demonstrate that decellularised bone-specific ECM promotes the osteogenic differentiation of ESCs. Our results are of fundamental interest and may help in tailoring scaffolds for tissue engineering applications which both incorporate tissue-specific ECM signals and stimulate stem-cell differentiation. PMID: 20149448 [PubMed - as supplied by publisher] | | |
| Rat hepatocyte aggregate formation on discrete aligned nanofibers of type-I collagen-coated poly(l-lactic acid).
February 13, 2010 at 6:24 AM
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| | Rat hepatocyte aggregate formation on discrete aligned nanofibers of type-I collagen-coated poly(l-lactic acid). Biomaterials. 2010 Feb 8; Authors: Feng ZQ, Chu XH, Huang NP, Leach MK, Wang G, Wang YC, Ding YT, Gu ZZ Primary hepatocytes cultured in three dimensional tissue constructs composed of multicellular aggregates maintain normal differentiated cellular function in vitro while cultured monolayers do not. Here, we report a technique to induce hepatocyte aggregate formation using type-I collagen-coated poly(l-lactic acid) (PLLA) discrete aligned nanofibers (disAFs) by providing limited cell-substrate adhesion strength and restricting cell migration to uniaxial movement. Kinetics of aggregate formation, morphology and biochemical activities of rat hepatocyte aggregates were tested over a 15 day culture period. Evidence was provided that physical cues from disAFs quickly induced the formation of aggregates. After 3 days in culture, 88.3% of free hepatocytes on disAFs were incorporated into aggregates with an average diameter of 61 +/- 18 mum. Hepatocyte aggregates formed on disAFs displayed excellent cell retention, cell activity and stable functional expression in terms of ! albumin secretion, urea synthesis and phase I and II (CYP1A and UGT) metabolic enzyme activity compared to monolayer culture of hepatocytes on tissue culture plastic (TCP) with type-I collagen as well as on meshes of type-I collagen-coated PLLA random nanofibers (meshRFs). These results suggest that disAFs may be a suitable method to maintain large-scale hepatic cultures with high activity for tissue engineering research and potential therapeutic applications, such as bioartificial liver devices. PMID: 20149442 [PubMed - as supplied by publisher] | | |
| Template synthesized poly(varepsilon-caprolactone) nanowire surfaces for neural tissue engineering.
February 13, 2010 at 6:24 AM
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| | Template synthesized poly(varepsilon-caprolactone) nanowire surfaces for neural tissue engineering. Biomaterials. 2010 Feb 8; Authors: Bechara SL, Judson A, Popat KC Tissue engineering therapies targeted at nerve regeneration in spinal cord injuries (SCI) have broad social and economic benefits to the American population. Due to the complicated pathophysiology of SCI, there are very few options available for functional regeneration of the spinal column. Nanotechnology offers interesting avenues to explore tissue engineering in SCI. In this study, we have developed a novel solvent free nanotemplating technique for fabricating poly(varepsilon-caprolactone) (PCL) surfaces with controlled arrays of high aspect ratio substrate-bound nanowires for the growth and maintenance of differentiated states of neuronal cells. PC12 cells were used to evaluate the ability of nanowire surfaces to promote neuronal phenotypic behavior. Cell adhesion, proliferation and viability were investigated for up to 4 days of culture using fluorescence microscopy, scanning electron microscopy (SEM) and MTT activity. Our results indicate significantly higher! cell adhesion and subsequent proliferation and viability of PC12 cells cultured on nanowire surfaces as compared to control surfaces without any nanoarchitecture. Further, the adhered cells were maintained in a differentiated state for 7 days and neuronal network formation and expression of neuronal markers were investigated using fluorescence microscopy, SEM and immunofluorescence. Cells on nanowire surfaces expressed key neuronal markers and demonstrated neuronal phenotypic behavior as compared to the cells on control surfaces. PMID: 20149440 [PubMed - as supplied by publisher] | | |
| An anisotropic nanofiber/microsphere composite with controlled release of biomolecules for fibrous tissue engineering.
February 13, 2010 at 6:24 AM
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| | An anisotropic nanofiber/microsphere composite with controlled release of biomolecules for fibrous tissue engineering. Biomaterials. 2010 Feb 9; Authors: Ionescu LC, Lee GC, Sennett BJ, Burdick JA, Mauck RL Aligned nanofibrous scaffolds can recapitulate the structural hierarchy of fiber-reinforced tissues of the musculoskeletal system. While these electrospun fibrous scaffolds provide physical cues that can direct tissue formation when seeded with cells, the ability to chemically guide a population of cells, without disrupting scaffold mechanical properties, would improve the maturation of such constructs and add additional functionality to the system both in vitro and in vivo. In this study, we developed a fabrication technique to entrap drug-delivering microspheres within nanofibrous scaffolds. We hypothesized that entrapping microspheres between fibers would have a less adverse impact on mechanical properties than placing microspheres within the fibers themselves, and that the composite would exhibit sustained release of multiple model compounds. Our results show that microspheres ranging from 10 approximately 20 microns in diameter could be electrospun in a dose-! dependent manner to form nanofibrous composites. When delivered in a sacrificial PEO fiber population, microspheres remained securely entrapped between slow-degrading PCL fibers after removal of the sacrificial delivery component. Stiffness and modulus of the composite decreased with increasing microsphere density for composites in which microspheres were entrapped within each fiber, while stiffness did not change when microspheres were entrapped between fibers. The release profiles of the composite structures were similar to free microspheres, with an initial burst release followed by a sustained release of the model molecules over 4 weeks. Further, multiple model molecules were released from a single scaffold composite, demonstrating the capacity for multi-factor controlled release ideal for complex growth factor delivery from these structures. PMID: 20149432 [PubMed - as supplied by publisher] | | |
| Effects of collagen membranes enriched with in vitro-differentiated N1E-115 cells on rat sciatic nerve regeneration after end-to-end repair.
February 13, 2010 at 6:24 AM
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| | Effects of collagen membranes enriched with in vitro-differentiated N1E-115 cells on rat sciatic nerve regeneration after end-to-end repair. J Neuroeng Rehabil. 2010 Feb 11;7(1):7 Authors: Amado S, Rodrigues JM, Luis AL, Armada-da-Silva PA, Vieira M, Gartner A, Simoes MJ, Veloso AP, Fornaro M, Raimondo S, Varejao AS, Geuna S, Mauricio AC ABSTRACT: Peripheral nerves possess the capacity of self-regeneration after traumatic injury but the extent of regeneration is often poor and may benefit from exogenous factors that enhance growth. The use of cellular systems is a rational approach for delivering neurotrophic factors at the nerve lesion site, and in the present study we investigated the effects of enwrapping the site of end-to-end rat sciatic nerve repair with an equine type III collagen membrane enriched or not with N1E-115 pre-differentiated neural cells. After neurotmesis, the sciatic nerve was repaired by end-to-end suture (End-to-End group), end-to-end suture enwrapped with an equine collagen type III membrane (End-to-EndMemb group); and end-to-end suture enwrapped with an equine collagen type III membrane previously covered with neural cells pre-differentiated in vitro from N1E-115 cells (End-to-EndMembCell group). Along the postoperative, motor and sensory functional recovery was evaluated ! using extensor postural thrust (EPT), withdrawal reflex latency (WRL) and ankle kinematics. After 20 weeks animals were sacrificed and the repaired sciatic nerves were processed for histological and stereological analysis. Results showed that enwrapment of the rapair site with a collagen membrane, with or without neural cell enrichment, did not lead to any significant improvement in most of functional and stereological predictors of nerve regeneration that we have assessed, with the exception of EPT which recovered significantly better after neural cell enriched membrane employment. It can thus be concluded that this particular type of nerve tissue engineering approach has very limited effects on nerve regeneration after sciatic end-to-end nerve reconstruction in the rat. PMID: 20149260 [PubMed - as supplied by publisher] | | |
| Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes.
February 13, 2010 at 6:24 AM
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| | Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes. Arthritis Res Ther. 2010 Feb 11;12(1):R22 Authors: Minogue BM, Richardson SM, Zeef LA, Freemont AJ, Hoyland JA ABSTRACT: INTRODUCTION: Nucleus pulposus (NP) cells have a phenotype similar to articular cartilage (AC) cells. However, the matrix of the NP is clearly different to that of AC suggesting that specific cell phenotypes exist. The aim of this study was to identify novel genes that could be used to distinguish bovine NP cells from AC and annulus fibrosus (AF) cells and to further determine their expression in normal and degenerate human intervertebral disc (IVD) cells. METHODS: Microarrays were conducted on bovine AC, AF and NP cells, using Affymetrix Genechip(R) Bovine Genome Arrays. Differential expression levels for a number of genes were confirmed by quantitative real time polymerase chain reaction (qRT-PCR) on bovine, AC, AF and NP cells, as well as separated bovine NP and notochordal (NC) cells. Expression of these novel markers were further tested on normal human AC, AF and NP cells, and degenerate AF and NP cells. RESULTS: Microarray comparisons between NP/AC! &AF and NP/AC identified 34 NP-specific and 49 IVD-specific genes respectively that were differentially expressed [greater than or equal to]100 fold. A subset of these were verified by qRT-PCR and shown to be expressed in bovine NC cells. Eleven genes (SNAP25, KRT8, KRT18, KRT19, CDH2, IBSP, VCAN, TNMD, BASP1, FOXF1 & FBLN1) were also differentially expressed in normal human NP cells although to a lesser degree. Four genes (SNAP25, KRT8, KRT18 and CDH2) were significantly decreased in degenerate human NP cells, while three genes (VCAN, TNMD and BASP1) were significantly increased in degenerate human AF cells. The IVD negative marker FBLN1 was significantly increased in both degenerate human NP and AF cells. CONCLUSIONS: This study has identified a number of novel genes that characterise the bovine and human NP and IVD transcriptional profiles and allows for discrimination between AC, AF and NP cells. Furthermore, the similarity in expression profiles of the separate! d NP and NC cell populations suggests that these two cell type! s may be derived from a common lineage. Although interspecies variation, together with changes with IVD degeneration were noted, use of this gene expression signature will benefit tissue engineering studies where defining the NP phenotype is paramount. PMID: 20149220 [PubMed - as supplied by publisher] | | |
| Generation of genetically modified animals using spermatogonial stem cells.
February 13, 2010 at 6:24 AM
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| | Generation of genetically modified animals using spermatogonial stem cells. Dev Growth Differ. 2010 Feb 10; Authors: Takehashi M, Kanatsu-Shinohara M, Shinohara T Spermatogonial stem cells (SSCs) provide the foundation for spermatogenesis, and are unique tissue-specific stem cells because of their ability to transmit genetic information to offspring. Generation of knockout mice using mouse SSCs became feasible after the successful establishment of protocols for the transplantation and long-term culture of these cells, called germline stem (GS) cells. Furthermore, SSCs can acquire pluripotentiality similar to that of embryonic stem (ES) cells, in addition to their highly differentiated spermatogenic potential. These ES-like cells, called multipotent GS (mGS) cells, are capable of generating knockout mice in a manner similar to that of ES cells. The use of GS and mGS cells for animal transgenesis has added a new dimension to gene-targeting technology using ES cells and somatic cell nuclear transfer, which has limited application. Furthermore, for regenerative medicine purposes, the use of mGS will settle problems such as ethi! cs issues and immunological rejection associated with ES cells, as well as risks of insertional mutagenesis associated with integrated genes into induced pluripotent stem cells. PMID: 20148923 [PubMed - as supplied by publisher] | | |
| DNA-incorporating nanomaterials in biotechnological applications.
February 13, 2010 at 6:24 AM
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| | DNA-incorporating nanomaterials in biotechnological applications. Nanomedicine (Lond). 2010 Feb;5(2):319-34 Authors: Stadler A, Chi C, van der Lelie D, Gang O The recently developed ability to controllably connect biological and inorganic objects on a molecular scale opens a new page in biomimetic methods with potential applications in biodetection, tissue engineering, targeted therapeutics and drug/gene delivery. Particularly in the biodetection arena, a rapid development of new platforms has largely been stimulated by a spectrum of novel nanomaterials with physical properties that offer efficient, sensitive and inexpensive molecular sensing. Recently, DNA-functionalized nano-objects have emerged as a new class of nanomaterials that can be controllably assembled in predesigned structures. Such DNA-based nanoscale structures might provide a new detection paradigm due to their regulated optical, electrical and magnetic responses, chemical heterogeneity and high local biomolecular concentration. The specific biorecognition DNA and its physical-chemical characteristics allows for an exploitation of DNA-functionalized nanom! aterials for sensing of nucleic acids, while a broad tunability of DNA interactions permits extending their use for detection of proteins, small molecules and ions. We discuss the progress that was achieved in the last decade in the exploration of new detection methods based on DNA-incorporating nanomaterials as well as their applications to gene delivery. The comparison between various detection platforms, their sensitivity and selectivity, and specific applications are reviewed. PMID: 20148641 [PubMed - in process] | | |
| Conference Scene: NanoBiotech Montreux 2009.
February 13, 2010 at 6:24 AM
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| | Conference Scene: NanoBiotech Montreux 2009. Nanomedicine (Lond). 2010 Feb;5(2):177-9 Authors: Hollis V, Morgan H The 13th Annual European Conference on Micro & Nanoscale Technologies for the Biosciences (NanoBioTech) was held on the picturesque shores of Lake Geneva in Montreux, Switzerland from 16-18th November, 2009. The Eden Palace au Lac Hotel, with its stunning views of the Alps across the Lake, once again provided an exquisite venue. The NanoBioTech conference focuses on the biological, chemical and medical applications of nano- and micro-technologies, with topics ranging from the development of nanomaterials for cell and tissue engineering to the use of integrated microfluidic systems for single biomolecule analysis. With over 130 delegates from more than 25 countries worldwide, NanoBioTech 2009 provided a diverse and invigorating forum for discussion. PMID: 20148630 [PubMed - in process] | | |
| Gene therapy in transplantation: Toward clinical trials.
February 13, 2010 at 6:24 AM
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| | Gene therapy in transplantation: Toward clinical trials. Curr Opin Mol Ther. 2009 Oct;11(5):504-12 Authors: Ritter T, Nosov M, Griffin MD The genetic modification of organs or cells is an attractive approach to protect allogeneic transplants from acute rejection and other complications. The transplant setting offers a unique opportunity to utilize ex vivo gene therapy for the modification of allogeneic organs and tissues prior to implantation. However, significant challenges exist in the application of this concept to human organ transplantation, including the large number of potential molecular targets, the diversity and safety profile of available vector delivery systems and the merging of gene-based therapies with existing immunosuppressive regimens. Accordingly, many different therapeutic concepts and vector systems have been investigated in preclinical studies with the aim of prolonging allograft survival. However, the translation of promising gene therapy strategies to transplant clinical trials has lagged behind the progress made in other medical fields. This review describes the recent precl! inical applications of gene transfer to transplantation, and critically evaluates the degree to which gene therapy has been tested clinically in organ transplant recipients. PMID: 19806498 [PubMed - indexed for MEDLINE] | | |
| alpha-Fetoprotein as a modulator of the pro-inflammatory response of human keratinocytes.
February 13, 2010 at 6:24 AM
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| | alpha-Fetoprotein as a modulator of the pro-inflammatory response of human keratinocytes. Br J Pharmacol. 2009 Nov;158(5):1236-47 Authors: Potapovich AI, Pastore S, Kostyuk VA, Lulli D, Mariani V, De Luca C, Dudich EI, Korkina LG BACKGROUND AND PURPOSE: The immunomodulatory effects of alpha-fetoprotein (AFP) on lymphocytes and macrophages have been described in vitro and in vivo. Recombinant forms of human AFP have been proposed as potential therapeutic entities for the treatment of autoimmune diseases. We examined the effects of embryonic and recombinant human AFP on the spontaneous, UVA- and cytokine-induced pro-inflammatory responses of human keratinocytes. EXPERIMENTAL APPROACH: Cultures of primary and immortalized human keratinocytes (HaCaT) and human blood T lymphocytes were used. The effects of AFP on cytokine expression were studied by bioplexed elisa and quantitative reverse transcriptase polymerase chain reaction assay. Kinase and nuclear factor kappa B (NFkappaB) phosphorylation were quantified by intracellular elisa. Nuclear activator protein 1 and NFkappaB DNA binding activity was measured by specific assays. Nitric oxide and H(2)O(2) production and redox status were assessed ! by fluorescent probe and biochemical methods. KEY RESULTS: All forms of AFP enhanced baseline expression of cytokines, chemokines and growth factors. AFP dose-dependently increased tumour necrosis factor alpha-stimulated granulocyte macrophage colony stimulating factor and interleukin 8 expression and decreased tumour necrosis factor alpha-induced monocyte chemotactic protein 1 and IP-10 (interferon gamma-produced protein of 10 kDa) expression. AFP induced a marked activator protein 1 activation in human keratinocytes. AFP also increased H(2)O(2) and modulated nitrite/nitrate levels in non-stimulated keratinocytes whereas it did not affect these parameters or cytokine release from UVA-stimulated cells. Phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Akt1 but not NFkappaB was activated by AFP alone or by its combination with UVA. CONCLUSIONS AND IMPLICATIONS: Exogenous AFP induces activation of human keratinocytes, with de novo expression of a number of! pro-inflammatory mediators and modulation of their pro-inflam! matory r esponse to cytokines or UVA. AFP may modulate inflammatory events in human skin. PMID: 19785658 [PubMed - indexed for MEDLINE] | | |
| Tgfbeta signal inhibition cooperates in the induction of iPSCs and replaces Sox2 and cMyc.
February 13, 2010 at 6:24 AM
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| | Tgfbeta signal inhibition cooperates in the induction of iPSCs and replaces Sox2 and cMyc. Curr Biol. 2009 Nov 3;19(20):1718-23 Authors: Maherali N, Hochedlinger K Ectopic expression of Oct4, Sox2, cMyc, and Klf4 confers a pluripotent state upon several differentiated cell types, generating induced pluripotent stem cells (iPSCs) [1-8]. iPSC derivation is highly inefficient, and the underlying mechanisms are largely unknown. This low efficiency suggests the existence of additional cooperative factors whose identification is critical for understanding reprogramming. In addition, the therapeutic use of iPSCs relies on the development of efficient nongenetic means of factor delivery, and although a handful of replacement molecules have been identified, their use yields a further reduction to the already low reprogramming efficiency [9-11]. Thus, the identification of compounds that enhance rather than solely replace the function of the reprogramming factors will be of great use. Here, we demonstrate that inhibition of Tgfbbeta signaling cooperates in the reprogramming of murine fibroblasts by enabling faster, more efficient indu! ction of iPSCs, whereas activation of Tgfbeta signaling blocks reprogramming. In addition to exhibiting a strong cooperative effect, the Tgfbeta receptor inhibitor bypasses the requirement for exogenous cMyc or Sox2, highlighting its dual role as a cooperative and replacement factor. The identification of a highly characterized pathway operating in iPSC induction will open new avenues for mechanistic dissection of the reprogramming process. PMID: 19765992 [PubMed - indexed for MEDLINE] | | | | 
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