|  |  |  | | | | | TE-RegenMed-StemCell feed | | | | | | | | | | | | | | | | | | | Small-molecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly efficient neural induction from human pluripotent stem cells. J Neurosci Res. 2011 Feb;89(2):117-26 Authors: Morizane A, Doi D, Kikuchi T, Nishimura K, Takahashi J The balance of bone morphogenic protein (BMP), transforming growth factor-β (TGFβ)/activin/nodal, and Wnt signals regulates the early lineage segregation of human embryonic stem cells (ESCs). Here we demonstrate that a combination of small-molecule inhibitors of BMP (Dorsomorphin) and TGFβ/activin/nodal (SB431542) signals promotes highly efficient neural induction from both human ESCs and induced pluripotent stem cells (iPSCs). The combination of small molecules had effects on both cell survival and purity of neural differentiation, under conditions of stromal (PA6) cell coculture and feeder-free floating aggregation culture, for all seven pluripotent stem cell lines that we studied, including three ESC and four iPSC lines. Small molecule compounds are stable and cost effective, so our findings provide a promising strategy for controlled production of neurons in regenerative medicine. © 2010 Wiley-Liss, Inc. PMID: 21162120 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Expression of doublecortin reveals articular chondrocyte lineage in mouse embryonic limbs. Genesis. 2010 Dec 15; Authors: Zhang Q, Cigan AD, Marrero L, Lopreore C, Liu S, Ge D, Savoie FH, You Z The doublecortin (Dcx) gene encodes a microtubule-binding protein that was originally found in immature neurons. In this study, we used two mouse strains that express reporter genes (LacZ and enhanced green fluorescence protein, respectively) driven by the endogenous Dcx promoter. We found that Dcx was expressed in the mesenchymal cells in the mouse embryonic limb buds. A population of the mesenchymal cells continued Dcx expression after they differentiated into joint interzone cells and then articular chondrocytes. In contrast, the endochondral chondrocytes lost Dcx expression when the mesenchymal cells differentiated into endochondral chondrocytes. These data support a concept that the articular and endochondral chondrocytes originate from the same mesenchymal cells that express Dcx. In contrast to the notion that articular chondrocytes are derived from de-differentiated endochondral chondrocytes, our findings demonstrate that the lineages of articular and endochondral chondrocytes bifurcate at the stage of endochondral chondrogenesis. © 2010 Wiley-Liss, Inc. PMID: 21162077 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Theoretical and Experimental Analysis of Anti-A Antibody Capture in Novel Integrated Bead and Hollow Fiber Modules. Ann Biomed Eng. 2010 Dec 16; Authors: Alikhani A, Federspiel WJ Anti-A/B antibody removal from blood reduces the hyperacute rejection risk following ABO-incompatible transplantation. We are developing an integrated bead and fiber module (BSAF) that selectively removes anti-A from blood. In BSAF blood flows through the inner lumen of microfiltration fibers. Starling flow carries plasma from the inner fiber lumen to the beads in the shell compartment where antibodies bind to covalently attached antigens on the beads. In this study, we developed a mathematical model to guide the choice of key design and operational parameters for a clinical BSAF device. The model demonstrated that for a given flow rate and reservoir volume, antibody removal rate was dependent on the magnitude of a lumped parameter, k (L) m (B)/Q (s), that characterizes the ratio of antibody uptake rate by the beads to the Starling flow rate in the device. The highest antibody removal rate was predicted for the perfusion limited regime, when k (L) m (B)/Q (s) → 10; Once this maximum limit was obtained, any further increase in the antibody removal rate was only possible by increasing the flow rate. Key model predictions were validated in a series of experiments. The model was then used to conceptually design a BSAF capable of a clinically relevant rate of anti-A removal. PMID: 21161682 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | β Cell Protection by Inhibition of iNOS Through Lentiviral Vector-Based Strategies. Methods Mol Biol. 2011;704:153-68 Authors: Hynes SO, McCabe C, O'Brien T Cytoprotective gene transfer to pancreatic islet β cellβ cell s may prove useful in preventing their destruction and prolonging islet graft survival after transplantation in patients with type 1 diabetes mellitus. A host of therapeutically relevant transgenes may potentially be incorporated into an appropriate gene delivery vehicle and used for islet modification. To examine this, we utilised a robust model of cytokine-induced β cell pathophysiology. Using this model, it is clear that antioxidant gene transfer confers no cytoprotective benefit. In contrast, we demonstrated that gene-based approaches to inhibit the activation of NF-κBNF-κB following cytokine exposure harbours therapeutic utility in preserving islet β cell viability in the face of cytokine toxicity. We identified that NF-κB-dependent induction of iNOSiNOS is a critical determinant of β cell fate following cytokine exposure. Having identified the pivotal role of iNOS activation in cytokine-induced β cell pathophysiology, lentiviral vectors may be used to efficiently deliver small interfering RNARNA molecules to confer efficient iNOS gene silencing. We have shown that lentiviral vector-based shRNA delivery holds significant promise in preserving β cell viability following cytotoxic cytokine exposure. PMID: 21161636 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | MicroRNAs in embryonic stem cell function and fate. Genes Dev. 2010 Dec 15;24(24):2732-41 Authors: Tiscornia G, Izpisúa Belmonte JC Since their discovery in the early 1990s, microRNAs (miRs) have gone from initially being considered an oddity to being recognized as a level of gene expression regulation that is integral to the normal function of cells and organisms. They are implicated in many if not all biological processes in animals, from apoptosis and cell signaling to organogenesis and development. Our understanding of cell regulatory states, as determined primarily by transcription factor (TF) profiles, is incomplete without consideration of the corresponding miR profile. The miR complement of a cell provides robust and redundant control over the output of hundreds of possible targets for each miR. miRs are common components of regulatory pathways, and in some cases can constitute on-off switches that regulate crucial fate decisions. In this review, we summarize our current knowledge about the biogenesis and regulation of miRs and describe their involvement in the pathways that regulate cell division, pluripotency, and reprogramming to the pluripotent state. PMID: 21159814 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | The Gamma Catenin/CBP Complex Maintains Survivin Transcription in β-catenin Deficient/Depleted Cancer Cells. Curr Cancer Drug Targets. 2010 Dec 15; Authors: Kim YM, Ma H, Oehler VG, Gang EJ, Nguyen C, Masiello D, Liu H, Zhao Y, Radich J, Kahn M Previously, we demonstrated that survivin expression is CBP/β-catenin/TCF-dependent. Now, using NCI-H28 cells, which harbor a homozygous deletion of β-catenin, we demonstrate that survivin transcription can similarly be mediated by nuclear γ-catenin. ICG-001, a specific inhibitor of binding to the N-terminus of CBP, effectively attenuates survivin expression. We demonstrate that γ-catenin by binding to TCF family members and specifically recruiting the coactivator CBP drives survivin transcription particularly in β-catenin-deficient cells. We also examined the relative expression of γ-catenin and β-catenin in 90 cases of chronic myeloid leukemia (CML) in a published gene expression microarray data base. A statistically significant negative correlation between γ-catenin and β-catenin was found in AP/BC cases (-0.389, P = 0.006). Furthermore, in subsequent independent validation studies by qPCR in 28 CP and BC patients increased γ-catenin expression predominated in BC cases and was associated with concomitantly increased survivin expression. Gene expression was 3- and 6-fold greater in BC patients as compared to CP patients, for γ-catenin and survivin, respectively. Consistent with this observation, nuclear γ-catenin accumulation was evident in this population consistent with a potential transcriptional role. Combined treatment with imatinib mesylate (IM) and ICG-001 significantly inhibited colony formation in sorted CD34(+) CML progenitors (survivin(+)/γ-catenin(high)/β-catenin(low)) isolated from one BC and one AP patient resistant to IM. Therefore, we believe that the ability of ICG-001 to block both the CBP/γ-catenin interaction and the CBP/β-catenin interaction may have clinical significance in cancers in which γ-catenin plays a significant transcriptional role. PMID: 21158719 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Tissue engineering: a modular, hydroxyapatite-binding version of vascular endothelial growth factor (adv. Mater. 48/2010). Adv Mater. 2010 Dec 21;22(48):5436 Authors: Lee JS, Wagoner Johnson AJ, Murphy WL PMID: 21161993 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Tissue Engineering in Plastic and Reconstructive Surgery. Handchir Mikrochir Plast Chir. 2010 Dec;42(6):327-328 Authors: Horch RE PMID: 21161857 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Resazurin dye as a reliable tool for determination of cell number and viability in mesenchymal stem cell culture. Bull Exp Biol Med. 2010 Jul;150(1):155-7 Authors: Dienstknecht T, Ehehalt K, Jenei-Lanzl Z, Zellner J, Mueller M, Berner A, Nerlich M, Angele P Human mesenchymal stem cells are a valuable cell source for tissue engineering. Determination of cell number and viability is crucial. However, this can be tested only at the end of cell culture. This study shows that Resazurin dye staining is a reliable tool for evaluation of cell number and viability in culture without cell perturbation. PMID: 21161076 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Evaluation of collagen gel microstructure by scanning electron microscopy. Bull Exp Biol Med. 2010 Jul;150(1):151-4 Authors: Pogorelov AG, Selezneva II We performed qualitative comparison of freeze drying and chemical drying as methods of preparing 3D wet specimens for scanning electron microscopy. Human fibroblasts immobilized in collagen gel were used as a model system. Specimens fixed with glutaraldehyde were frozen in liquid nitrogen and freeze-dried at low temperature in high vacuum. In parallel experiments, glutaraldehyde-fixed samples were dehydrated in ascending ethanol solutions, absolute ethanol, and 100% hexamethyldisilazane and then dried at room temperature. Scanning electron microscopy microphotographs of collagen fibers and cells were characterized by high resolution and the absence of collapsed or deformed structures even at high magnification (×50,000) for both chemical drying and high-vacuum freeze drying. However, high-vacuum freeze drying is superior to chemical drying for the investigation of the internal space of 3D scaffolds, because sample fracture can be prepared directly in liquid nitrogen. These techniques are a part of the sample preparation process for scanning electron microscopy and can also be used for studies of cell adhesion, morphology, and arrangement in wet specimens (3D gels and flexible tissue engineering scaffolds). PMID: 21161075 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | The evolution of extracellular matrix. Mol Biol Cell. 2010 Dec;21(24):4300-5 Authors: Ozbek S, Balasubramanian PG, Chiquet-Ehrismann R, Tucker RP, Adams JC We present a perspective on the molecular evolution of the extracellular matrix (ECM) in metazoa that draws on research publications and data from sequenced genomes and expressed sequence tag libraries. ECM components do not function in isolation, and the biological ECM system or "adhesome" also depends on posttranslational processing enzymes, cell surface receptors, and extracellular proteases. We focus principally on the adhesome of internal tissues and discuss its origins at the dawn of the metazoa and the expansion of complexity that occurred in the chordate lineage. The analyses demonstrate very high conservation of a core adhesome that apparently evolved in a major wave of innovation in conjunction with the origin of metazoa. Integrin, CD36, and certain domains predate the metazoa, and some ECM-related proteins are identified in choanoflagellates as predicted sequences. Modern deuterostomes and vertebrates have many novelties and elaborations of ECM as a result of domain shuffling, domain innovations and gene family expansions. Knowledge of the evolution of metazoan ECM is important for understanding how it is built as a system, its roles in normal tissues and disease processes, and has relevance for tissue engineering, the development of artificial organs, and the goals of synthetic biology. PMID: 21160071 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Bioactivation of dermal scaffolds with a non-viral copolymer-protected gene vector. Biomaterials. 2010 Dec 13; Authors: Reckhenrich AK, Hopfner U, Krötz F, Zhang Z, Koch C, Kremer M, Machens HG, Plank C, Egaña JT The use of scaffolds in skin tissue engineering is accompanied with low regeneration rates and high risk of infection. In this study, we activated an FDA-approved collagen scaffold for dermal regeneration by incorporation of copolymer-protected gene vectors (COPROGs) to induce a temporary release of VEGF. In vitro results show that the presence of COPROGs did not affect the distribution, attachment, proliferation and viability of cells in the scaffold. A transient release of VEGF was observed for up to 3 weeks. Moreover a high amount of VEGF was also found in the cells and associated with the scaffold. In a full skin defect model in nude mice, VEGF levels were significantly increased compared to controls in VEGF gene activated scaffolds 14 d after implantation, but not in skin from the wound edge. Results showed an increased amount of non-adherent cells, especially erythrocytes, and von Willebrandt factor (vWF) and a yellow red appearance of gene activated scaffolds in relation to controls. This suggests the presence of leaky vessels. In this work we show that the bioactivation of collagen scaffolds with COPROGs presents a new technology that allows a local release of therapeutic proteins thus enhancing the regenerative potential in vivo. PMID: 21159378 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Candida glabrata and Candida albicans co-infection of an in vitro oral epithelium. J Oral Pathol Med. 2010 Dec 15; Authors: Silva S, Henriques M, Hayes A, Oliveira R, Azeredo J, Williams DW J Oral Pathol Med (2010) Background: Candida albicans is regarded as the leading of candidosis. However, Candida glabrata has emerged as an important pathogen of oral mucosa, occurring both singly or in mixed species infections, often with C. albicans. Compared with C. albicans, little is known about the role of C. glabrata in oral infection. The aim of this study was to examine single and mixed species infection of oral epithelium involving C. glabrata and establish its ability to invade and damage tissue. Methods: A reconstituted human oral epithelium (RHOE) was infected only with C. glabrata, or simultaneously with C. glabrata and C. albicans. The ability of both species to invade the tissue was examined using species specific peptide nucleic acid (PNA) probe hybridization and confocal laser scanning microscopy. Epithelial damage was assessed by measuring lactate dehydrogenase (LDH) activity. Results: Candida glabrata strains were able to colonize the RHOE, in a strain dependent manner. Candida glabrata single infection after 12 h, generally revealed no invasion of the RHOE, which contrasted with extensive tissue invasion demonstrated by C. albicans. Mixed infection showed that C. albicans enhanced the invasiveness of C. glabrata, and led to increased LDH release by the RHOE, which paralleled the observed histological damage. Conclusions: The results obtained demonstrating enhanced invasion and increased tissue damage caused by mixed C. glabrata and C. albicans infections have important clinical significance and highlight the need to identify Candida species involved in oral candidosis. PMID: 21158929 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Pediatric cardiopulmonary bypass adaptations for long-term survival of baboons undergoing pulmonary artery replacement. J Extra Corpor Technol. 2010 Sep;42(3):223-31 Authors: Whittaker C, Grist G, Bert A, Brasky K, Neighbors S, McFall C, Hilbert SL, Drake WB, Cromwell M, Mueller B, Lofland GK, Hopkins RA Cardiopulmonary bypass (CPB) protocols of the baboon (Papio cynocephalus anubis) are limited to obtaining experimental data without concern for long-term survival. In the evaluation of pulmonary artery tissue engineered heart valves (TEHVs), pediatric CPB methods are adapted to accommodate the animals' unique physiology enabling survival up to 6 months until elective sacrifice. Aortic access was by a 14F arterial cannula and atrial access by a single 24F venous cannula.The CPB circuit includes a 3.3 L/min flow rated oxygenator, 1/4" x %" arterial-venous loop, 3/8" raceway, and bubble trap. The prime contains 700 mL Plasma-Lyte, 700 units heparin, 5 mL of 50% dextrose, and 20 mg amiodarone. Heparinization (200 u/kg) targets an activated clotting time of 350 seconds. Normothermic CPB was initiated at a 2.5 L/m2/min cardiac index with a mean arterial pressure of 55-80 mmHg. Weaning was monitored with transesophageal echocardiogram. Post-CPB circuit blood was re-infused. Chest tubes were removed with cessation of bleeding. Extubation was performed upon spontaneous breathing. The animals were conscious and upright 3 hours post-CPB. Bioprosthetic valves or TEHVs were implanted as pulmonary replacements in 20 baboons: weight = 27.5 +/- 5.6 kg, height = 73 +/- 7 cm, body surface area = 0.77 m2 +/- 0.08, mean blood flow = 1.973 +/- .254 L/min, core temperature = 37.1 +/- .1 degree C, and CPB time = 60 +/- 40 minutes. No acidosis accompanied CPB. Sixteen animals survived, four expired. Three died of right ventricular failure and one of an anaphylactoid reaction. Surviving animals had normally functioning replacement valves and ventricles. Baboon CPB requires modifications to include high systemic blood pressure for adequate perfusion into small coronary arteries, careful CPB weaning to prevent ventricular distention, and drug and fluid interventions to abate variable venous return related to a muscularized spleno-splanchnic venous capacity. PMID: 21114226 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Endogenous patterns of mechanical stress are required for branching morphogenesis. Integr Biol (Camb). 2010 Sep;2(9):424-34 Authors: Gjorevski N, Nelson CM Spatial patterning of cell behaviors establishes the regional differences within tissues that collectively develop branched organs into their characteristic treelike shapes. Here we show that the pattern of branching morphogenesis of three-dimensional (3D) engineered epithelial tissues is controlled in part by gradients of endogenous mechanical stress. We used microfabrication to build model mammary epithelial tissues of defined geometry that branched in a stereotyped pattern when induced with growth factors. Branches initiated from sites of high mechanical stress within the tissues, as predicted numerically and measured directly using 3D traction force microscopy. Branch sites were defined by activation of focal adhesion kinase (FAK), inhibition of which disrupted morphogenesis. Stress, FAK activation, and branching were all altered by manipulating cellular contractility, matrix stiffness, intercellular cohesion and tissue geometry. These data suggest that the pattern and magnitude of mechanical stress across epithelial tissues cooperate with biochemical signals to specify branching pattern. PMID: 20717570 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | [Reconstruction of rabbit corneal stroma with skin fibroblasts]. Zhonghua Yan Ke Za Zhi. 2009 Sep;45(9):827-33 Authors: Zhang YQ, Zhang WJ, Liu W, Hu XJ, Zhou GD, Cui L, Cao YL To explore whether skin fibroblasts could be used as a cell source for reconstruction of the corneal stroma. PMID: 20137290 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Autologous chondrocyte implantation versus ACI using 3D-bioresorbable graft for the treatment of large full-thickness cartilage lesions of the knee. Arch Orthop Trauma Surg. 2010 Aug;130(8):957-64 Authors: Erggelet C, Kreuz PC, Mrosek EH, Schagemann JC, Lahm A, Ducommun PP, Ossendorf C In autologous chondrocyte implantation (ACI), the periosteum patch which is sutured over the cartilage defect has been identified as a major source of complications such as periosteal hypertrophy. In the present retrospective study, we compared midterm results of first-generation ACI with a periosteal patch to second generation ACI using a biodegradable collagen fleece (BioSeed-C) in 82 patients suffering from chronic posttraumatic and degenerative cartilage lesions of the knee. PMID: 19711090 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Amniocytes can serve a dual function as a source of iPS cells and feeder layers. Hum Mol Genet. 2010 Dec 14; Authors: Anchan RM, Quaas P, Gerami-Naini B, Bartake H, Griffin A, Zhou Y, Day D, Eaton J, George LL, Naber C, Turbe-Doan A, Park PJ, Hornstein MD, Maas RL Clinical barriers to stem cell therapy include the need for efficient derivation of histocompatible stem cells and the zoonotic risk inherent to human stem cell xenoculture on mouse feeder cells. We describe a system for efficiently deriving induced pluripotent stem (iPS) cells from human and mouse amniocytes, and for maintaining the pluripotency of these iPS cells on mitotically inactivated feeder layers prepared from the same amniocytes. Both cellular components of this system are thus autologous to a single donor. Moreover, the use of human feeder cells reduces the risk of zoonosis. Generation of iPS cells using retroviral vectors from short or long-term cultured human and mouse amniocytes using four factors, or two factors in mouse, occurs in 5 to 7 days with 0.5% efficiency. This efficiency is greater than reported for mouse and human fibroblasts using similar viral infection approaches, and does not appear to result from selective reprogramming of Oct4(+) or c-Kit(+) amniocyte subpopulations. Derivation of amniocyte-derived iPS (AdiPS) cell colonies, which express pluripotency markers and exhibit appropriate microarray expression and DNA methylation properties, was facilitated by live immunostaining. AdiPS cells also generate embryoid bodies in vitro and teratomas in vivo. Furthermore, mouse and human amniocytes can serve as feeder layers for iPS cells and for mouse and human ES cells. Thus, human amniocytes provide an efficient source of autologous iPS cells and, as feeder cells, can also maintain iPS and ES cell pluripotency without the safety concerns associated with xenoculture. PMID: 21156717 [PubMed - as supplied by publisher] | | | | | | | | | |  | |  | |  |
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