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| KATP Ahannel-Deficient Dilated Cardiomyopathy Proteome Remodeled by Embryonic Stem Cell Therapy.
June 24, 2010 at 6:54 AM
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| | KATP Ahannel-Deficient Dilated Cardiomyopathy Proteome Remodeled by Embryonic Stem Cell Therapy. Stem Cells. 2010 Jun 22; Authors: Lindor JZ, Arrell DK, Yamada S, Nelson TJ, Terzic A Transplantation of pluripotent stem cells has proven beneficial in heart failure, yet the proteomic landscape underlying repair remains largely uncharacterized. In a genetic model of dilated cardiomyopathy elicited by pressure overload in the KCNJ11 null mutant, proteome-wide profiles were here resolved by means of a systems approach prior to and following disease manifestation in the absence or presence of embryonic stem cell treatment. Comparative two-dimensional gel electrophoresis revealed a unique cardiomyopathic proteome in the absence of therapy, remodeled in response to stem cell treatment. Specifically, LTQ-Orbitrap mass spectrometry determined the identities of 93 and 109 differentially expressed proteins from treated and untreated cardiomyopathic hearts, respectively. Mapped protein-protein relationships and corresponding neighborhoods incorporated the stem cell-dependent subproteome into a non-stochastic network with divergent composition from the stem cell-independent counterpart. Stem cell intervention produced a distinct proteome signature across a spectrum of biological processes ranging from energetic metabolism, oxidoreductases, and stress-related chaperones to processes supporting protein synthesis/degradation, signaling and transport regulation, cell structure and scaffolding. In the absence of treatment, bioinformatic interrogation of the disease-only proteome network prioritized adverse cardiac outcomes, ablated or ameliorated following stem cell transplantation. Functional and structural measurements validated improved myocardial contractile performance, reduced ventricular size and decreased cardiac damage in the treated cohort. Unbiased systems assessment unmasked "Cardiovascular Development" as a prioritized biological function in stem cell-reconstructed cardiomyopathic hearts. Thus, embryonic stem cell treatment transformed the cardiomyopathic proteome to demote disease-associated adverse effects and sustain a pro-cardiogenic developmental response, supplying a regenerative substrate for heart failure repair. PMID: 20572010 [PubMed - as supplied by publisher] | | |
| Reprogramming adult hematopoietic cells.
June 24, 2010 at 6:54 AM
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| | Reprogramming adult hematopoietic cells. Curr Opin Hematol. 2010 Jul;17(4):271-5 Authors: Kaneko S, Otsu M, Nakauchi H PURPOSE OF REVIEW: Recent advances in molecular biology research have culminated in development of technologies to generate pluripotent stem cells from somatic cells. In addition to skin fibroblasts, hematopoietic cells also have been shown to be amenable to reprogramming to pluripotency. The present review discusses the relevance of these findings to basic researches and regenerative medicine, and how researchers can take advantage of hematopoietic cell reprogramming technologies. RECENT FINDINGS: In 2006, Yamanaka and his colleagues published their amazing observation that murine somatic cells can be reprogrammed to the embryonic stem cell-like state simply by retroviral-mediated introduction of three or four defined factors. Soon after, human cells also were shown to be amenable to similar reprogramming. Generation of induced pluripotent cells from several types of hematopoietic cells of both murine and human origins now has been reported. SUMMARY: Reprogramming adult hematopoietic cells will provide opportunities to obtain valuable materials with minimum risk and burden to patients. Reprogrammed cells can be used in research to elucidate disease mechanisms and in drug or toxicity screening. In clinical settings, patient-derived induced pluripotent stem cells may be used to generate mature functional cells for various therapies. PMID: 20571391 [PubMed - in process] | | |
| Regeneration of intervertebral discs in a rat disc degeneration model by implanted adipose-tissue-derived stromal cells.
June 24, 2010 at 6:32 AM
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| | Regeneration of intervertebral discs in a rat disc degeneration model by implanted adipose-tissue-derived stromal cells. Acta Neurochir (Wien). 2010 Jun 24; Authors: Jeong JH, Lee JH, Jin ES, Min JK, Jeon SR, Choi KH BACKGROUND: Because adipose-tissue-derived stromal cell (ADSC) is readily accessible and abundant in stem cell, ADSC may be a better candidate for cell therapy and tissue engineering. This study investigated the potential of ADSC implantation to restore disc in a rat IVD model. METHODS: The first coccygeal disc segments of a Sprague-Dawley rat was left undamaged as a control (NC) group, and other two segments were damaged by needle injection. Two weeks later, ADSCs (TS) group or saline (IN) group was transplanted into each of the two damaged segments. RESULTS: At 6 weeks after transplantation, the TS group showed a significantly smaller reduction in disc height than the IN group and exhibited a restoration of MRI signal intensity. Hematoxylin and eosin staining revealed a greater restoration of the inner annulus structure in the TS group. Anti-Human Nucleic Antibody, collagen type II, and aggrecan, staining showed positive findings at 2 weeks after transplantation in TS group. CONCLUSIONS: ADSCs show potential for restoring degenerative discs and may prove effective in the treatment of IVD. PMID: 20571835 [PubMed - as supplied by publisher] | | |
| Nanoporous protein matrix made of amyloid fibrils of beta(2)-microglobulin.
June 24, 2010 at 6:23 AM
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| | Nanoporous protein matrix made of amyloid fibrils of beta(2)-microglobulin. Biotechnol Prog. 2010 Jun 1; Authors: Ahn M, Kang S, Koo HJ, Lee JH, Lee YS, Paik SR Amyloid fibrils are considered as novel nanomaterials because of their nanoscale width, a regular constituting structure of cross beta-sheet conformation, and considerable mechanical strength. By using an amyloidogenic protein of beta(2)-microglobulin (beta(2)M) related to dialysis-related amyloidosis, nanoporous protein matrix has been prepared. The beta(2)M granules made of around 15 monomers showed an average size of 23.1 nm. They formed worm-like fibrils at pH 7.4 in 20 mM sodium phosphate containing 0.15 M NaCl following vigorous nondirectional shaking incubation, in which they became laterally associated and interwound to generate the porous amyloid fibrillar matrix with an average pore size of 30-50 nm. This nanoporous protein matrix was demonstrated to be selectively disintegrated by reducing agents, such as tris-(2-carboxyethyl) phosphine. High surface area with nanopores on the surface has been suggested to make the matrix of beta(2)M amyloid fibrils particularly suitable for applications in the area of nanobiotechnology including drug delivery and tissue engineering. (c) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010. PMID: 20572297 [PubMed - as supplied by publisher] | | |
| Cryopreservation of human bone marrow-derived mesenchymal stem cells with reduced dimethylsulfoxide and well-defined freezing solutions.
June 24, 2010 at 6:23 AM
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| | Cryopreservation of human bone marrow-derived mesenchymal stem cells with reduced dimethylsulfoxide and well-defined freezing solutions. Biotechnol Prog. 2010 Jun 1; Authors: Liu Y, Xu X, Ma X, Martin-Rendon E, Watt S, Cui Z The aim of this study is to investigate the feasibility of using well defined, serum-free freezing solutions with a reduced level of dimethylsulfoxide (DMSO) of 7.5, 5, and 2.5% (v/v) in the combination with polyethylene glycol (PEG) or trehalose to cryopreserve human bone marrow-derived mesenchymal stem cells (hBMSCs), a main source of stem cells for cell therapy and tissue engineering. The standard laboratory freezing protocol of around 1 degrees C/min was used in the experiments. The efficiency of 1,2-propandiol on cryopreservation of hBMSCs was explored. We measured the post-thawing cell viability and early apoptotic behaviors, cell metabolic activities, and growth dynamics. Cell morphology and osteogenic, adipogenic and chondrogenic differentiation capability were also tested after cryopreservation. The results showed that post-thawing viability of hBMSCs in 7.5% DMSO (v/v), 2.5% PEG (w/v), and 2% bovine serum albumin (BSA) (w/v) was comparable with that obtained in conventional 10% DMSO, that is, 82.9 +/- 4.3% and 82.7 +/- 3.7%, respectively. In addition, 5% DMSO (v/v) with 5% PEG (w/v) and 7.5% 1,2-propandiol (v/v) with 2.5% PEG (w/v) can provide good protection to hBMSCs when 2% albumin (w/v) is present. Enhanced cell viability was observed with the addition of albumin to all tested freezing solutions. (c) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010. PMID: 20572296 [PubMed - as supplied by publisher] | | |
| A Simple Method for Cell Sheet Fabrication Using Mica Surfaces Grafted with Peptide Detergent A(6)K.
June 24, 2010 at 6:23 AM
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| | A Simple Method for Cell Sheet Fabrication Using Mica Surfaces Grafted with Peptide Detergent A(6)K. Macromol Biosci. 2010 Jun 22; Authors: Qiu F, Chen Y, Cheng J, Wang C, Xu H, Zhao X Cell sheet technology is a very important strategy for scaffold-free tissue engineering. In order to fabricate cell sheets by a simple method, peptide detergent A(6)K was grafted on mica surfaces by dropping its aqueous solution at different concentrations on the surface. As revealed by surface topographical observation and water contact angle measurement, the most hydrophobic surface was obtained using peptide solution at the concentration of 0.2 mg . mL(-1). The peptide-grafted mica surface was used to culture mouse preosteoblast cell MC3T3-E1. After the cells reached confluence and the peptide was biodegraded, an intact cell sheet was peeled from the mica. This simple method does not need any non-biological reagents or complicated procedures, and may have great potential in tissue engineering based on cell sheet technology. PMID: 20572268 [PubMed - as supplied by publisher] | | |
| Tissue engineering and the use of stem/progenitor cells for airway epithelium repair.
June 24, 2010 at 6:23 AM
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| | Tissue engineering and the use of stem/progenitor cells for airway epithelium repair. Eur Cell Mater. 2010;19:284-99 Authors: Roomans GM Stem/progenitor cells can be used to repair defects in the airway wall, resulting from e.g., tumors, trauma, tissue reactions following long-time intubations, or diseases that are associated with epithelial damage. Several potential sources of cells for airway epithelium have been identified. These can be divided into two groups. The first group consists of endogenous progenitor cells present in the respiratory tract. This group can be subdivided according to location into (a) a ductal cell type in the submucosal glands of the proximal trachea, (b) basal cells in the intercartilaginous zones of the lower trachea and bronchi, (c) variant Clara cells (Clarav-cells) in the bronchioles and (d) at the junctions between the bronchioles and the alveolar ducts, and (e) alveolar type II cells. This classification of progenitor cell niches is, however, controversial. The second group consists of exogenous stem cells derived from other tissues in the body. This second group can be subdivided into: (a) embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, or amniotic fluid stem cells, (b) side-population cells from bone marrow or epithelial stem cells present in bone marrow or circulation and (c) fat-derived mesenchymal cells. Airway epithelial cells can be co-cultured in a system that includes a basal lamina equivalent, extracellular factors from mesenchymal fibroblasts, and in an air-liquid interface system. Recently, spheroid-based culture systems have been developed. Several clinical applications have been suggested: cystic fibrosis, acute respiratory distress syndrome, chronic obstructive lung disease, pulmonary fibrosis, pulmonary edema, and pulmonary hypertension. Clinical applications so far are few, but include subglottic stenosis, tracheomalacia, bronchiomalacia, and emphysema. PMID: 20571996 [PubMed - in process] | | |
| Precise cell patterning using cytophobic self-assembled monolayer deposited on top of semi-transparent gold.
June 24, 2010 at 6:23 AM
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| | Precise cell patterning using cytophobic self-assembled monolayer deposited on top of semi-transparent gold. Biomed Microdevices. 2010 Jun 23; Authors: Jing G, Perry SF, Tatic-Lucic S This paper reports a simple and effective method for cell patterning by using a self-assembled monolayer (SAM)-treated glass surface which is surrounded by semi-transparent gold coated with another type of SAM. Specifically, a hydrophobic SAM, derived from 1-hexadecanethiol (HDT), was coated on the gold surface to prevent cell growth, and a hydrophilic SAM, derived from 3-trimethoxysilyl propyl-diethylenetriamine (DETA), was coated on the exposed glass surface to promote cell growth. The capabilities of this technique are as follows: 1) single-cell resolution, 2) easy alignment of the cell patterns to the structures already existing on the substrate, 3) visualization and verification of the predefined cytophobic/cytophilic pattern prior to cell growth, and 4) convenient monitoring cell growth at the same location for an extended long term period of time. Whereas a number of earlier techniques have demonstrated the single cell resolution, or visualization and verification of the cytophobic/cytophilic patterns prior to cell growth, we believe that our technique is unique in possessing all of these beneficial qualities at the same time. The distinguishing characteristic of our technique is, however, that the use of semi-transparent Cr/Au film allows for convenient brightfield pattern visualization and offers an advantage over previously developed methods which require fluorescent imaging. We have successfully demonstrated the patterning of four different kinds of cells using this technique: immortalized mouse hypothalamic neurons (GT1-7), mouse osteoblast cells (MC3T3), mouse fibroblast cells (NIH3T3) and primary rat hippocampal neurons. This study was performed with a specific ultimate application-the creation of a multi electrode array (MEA) with predefined localization of cell bodies on top of the electrodes, as well as predefined patterns for cell extensions to grow in between the electrodes. With that goal in mind, we have also determined critical parameters for patterning of each of these cell types, such as the minimum size of a cell-adherent island for exclusively anchoring one cell or two cells, as well as the width of the cytophilic pathway between two islands that enables cell extensions to grow, while preventing the anchoring of the cell bodies. Additionally, we have provided statistical analysis of the occupancy for various sizes and shape of cell-anchoring islands. As demonstrated here, we have developed a novel and reliable cell patterning technique, which can be utilized in various applications, such as biosensors or tissue engineering. PMID: 20571865 [PubMed - as supplied by publisher] | | |
| Regeneration of intervertebral discs in a rat disc degeneration model by implanted adipose-tissue-derived stromal cells.
June 24, 2010 at 6:23 AM
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| | Regeneration of intervertebral discs in a rat disc degeneration model by implanted adipose-tissue-derived stromal cells. Acta Neurochir (Wien). 2010 Jun 24; Authors: Jeong JH, Lee JH, Jin ES, Min JK, Jeon SR, Choi KH BACKGROUND: Because adipose-tissue-derived stromal cell (ADSC) is readily accessible and abundant in stem cell, ADSC may be a better candidate for cell therapy and tissue engineering. This study investigated the potential of ADSC implantation to restore disc in a rat IVD model. METHODS: The first coccygeal disc segments of a Sprague-Dawley rat was left undamaged as a control (NC) group, and other two segments were damaged by needle injection. Two weeks later, ADSCs (TS) group or saline (IN) group was transplanted into each of the two damaged segments. RESULTS: At 6 weeks after transplantation, the TS group showed a significantly smaller reduction in disc height than the IN group and exhibited a restoration of MRI signal intensity. Hematoxylin and eosin staining revealed a greater restoration of the inner annulus structure in the TS group. Anti-Human Nucleic Antibody, collagen type II, and aggrecan, staining showed positive findings at 2 weeks after transplantation in TS group. CONCLUSIONS: ADSCs show potential for restoring degenerative discs and may prove effective in the treatment of IVD. PMID: 20571835 [PubMed - as supplied by publisher] | | |
| Novel Antibacterial Nanofibrous PLLA Scaffolds.
June 24, 2010 at 6:23 AM
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| | Novel Antibacterial Nanofibrous PLLA Scaffolds. J Control Release. 2010 Jun 3; Authors: Feng K, Sun H, Bradley MA, Dupler EJ, Giannobile WV, Ma PX In order to achieve high local bioactivity and low systemic side effects of antibiotics in the treatment of dental, periodontal and bone infections, a localized and temporally controlled delivery system is crucial. In this study, a three-dimensional (3D) porous tissue engineering scaffold was developed with the ability to release antibiotics in a controlled fashion for long-term inhibition of bacterial growth. The highly soluble antibiotic drug, Doxycycline (DOXY), was successfully incorporated into PLGA nanospheres using a modified water-in-oil-in-oil (w/o/o) emulsion method. The PLGA nanospheres (NS) were then incorporated into prefabricated nanofibrous PLLA scaffolds with a well interconnected macroporous structure. The release kinetics of DOXY from four different PLGA NS formulations on a PLLA scaffold was investigated. DOXY could be released from the NS-scaffolds in a locally and temporally controlled manner. The DOXY release is controlled by DOXY diffusion out of the NS and is strongly dependent upon the physical and chemical properties of the PLGA. While PLGA50-6.5K, PLGA50-64K, and PLGA75-113K NS-scaffolds discharge DOXY rapidly with a high initial burst release, PLGA85-142K NS-scaffold can extend the release of DOXY to longer than 6 weeks with a low initial burst release. Compared to NS alone, the NS incorporated on a 3-D scaffold had significantly reduced the initial burst release. In vitro antibacterial tests of PLGA85 NS-scaffold demonstrated its ability to inhibit common bacterial growth (S.aureus and E.coli) for a prolonged duration. The successful incorporation of DOXY onto 3-D scaffolds and its controlled release from scaffolds extends the usage of nano-fibrous scaffolds from the delivery of large molecules such as growth factors to the delivery of small hydrophilic drugs, allowing for a broader application and a more complex tissue engineering strategy. PMID: 20570700 [PubMed - as supplied by publisher] | | |
| An injectable calcium phosphate-alginate hydrogel-umbilical cord mesenchymal stem cell paste for bone tissue engineering.
June 24, 2010 at 6:23 AM
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| | An injectable calcium phosphate-alginate hydrogel-umbilical cord mesenchymal stem cell paste for bone tissue engineering. Biomaterials. 2010 Jun 5; Authors: Zhao L, Weir MD, Xu HH The need for bone repair has increased as the population ages. Stem cell-scaffold approaches hold immense promise for bone tissue engineering. However, currently, preformed scaffolds for cell delivery have drawbacks including the difficulty to seed cells deep into the scaffold, and inability for injection in minimally-invasive surgeries. Current injectable polymeric carriers and hydrogels are too weak for load-bearing orthopedic applications. The objective of this study was to develop an injectable and mechanically-strong stem cell construct for bone tissue engineering. Calcium phosphate cement (CPC) paste was combined with hydrogel microbeads encapsulating human umbilical cord mesenchymal stem cells (hUCMSCs). The hUCMSC-encapsulating composite paste was fully injectable under small injection forces. Cell viability after injection matched that in hydrogel without CPC and without injection. Mechanical properties of the construct matched the reported values of cancellous bone, and were much higher than previous injectable polymeric and hydrogel carriers. hUCMSCs in the injectable constructs osteodifferentiated, yielding high alkaline phosphatase, osteocalcin, collagen type I, and osterix gene expressions at 7 d, which were 50-70 fold higher than those at 1 d. Mineralization by the hUCMSCs at 14 d was 100-fold that at 1 d. In conclusion, a fully injectable, mechanically-strong, stem cell-CPC scaffold construct was developed. The encapsulated hUCMSCs remained viable, osteodifferentiated, and synthesized bone minerals. The new injectable stem cell construct with load-bearing capability may enhance bone regeneration in minimally-invasive and other orthopedic surgeries. PMID: 20570346 [PubMed - as supplied by publisher] | | |
| Differential regulation of immature articular cartilage compressive moduli and Poisson's ratios by in vitro stimulation with IGF-1 and TGF-beta1.
June 24, 2010 at 6:23 AM
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| | Differential regulation of immature articular cartilage compressive moduli and Poisson's ratios by in vitro stimulation with IGF-1 and TGF-beta1. J Biomech. 2010 Jun 4; Authors: Williams GM, Dills KJ, Flores CR, Stender ME, Stewart KM, Nelson LM, Chen AC, Masuda K, Hazelwood SJ, Klisch SM, Sah RL Mechanisms of articular cartilage growth and maturation have been elucidated by studying composition-function dynamics during in vivo development and in vitro culture with stimuli such as insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta 1 (TGF-beta1). This study tested the hypothesis that IGF-1 and TGF-beta1 regulate immature cartilage compressive moduli and Poisson's ratios in a manner consistent with known effects on tensile properties. Bovine calf articular cartilage from superficial-articular (S) and middle-growth (M) regions were analyzed fresh or following culture in medium with IGF-1 or TGF-beta1. Mechanical properties in confined (CC) and unconfined (UCC) compression, cartilage matrix composition, and explant size were assessed. Culture with IGF-1 resulted in softening in CC and UCC, increased Poisson's ratios, substantially increased tissue volume, and accumulation of glycosaminoglycan (GAG) and collagen (COL). Culture with TGF-beta1 promoted maturational changes in the S layer, including stiffening in CC and UCC and increased concentrations of GAG, COL, and pyridinoline crosslinks (PYR), but little growth. Culture of M layer explants with TGF-beta1 was nearly homeostatic. Across treatment groups, compressive moduli in CC and UCC were positively related to GAG, COL, and PYR concentrations, while Poisson's ratios were negatively related to concentrations of these matrix components. Thus, IGF-1 and TGF-beta1 differentially regulate the compressive mechanical properties and size of immature articular cartilage in vitro. Prescribing tissue growth, maturation, or homeostasis by controlling the in vitro biochemical environment with such growth factors may have applications in cartilage repair and tissue engineering. PMID: 20570267 [PubMed - as supplied by publisher] | | |
| Bioluminescence imaging of dual gene expression at the single-cell level.
June 24, 2010 at 6:23 AM
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| | Bioluminescence imaging of dual gene expression at the single-cell level. Biotechniques. 2010 Jun;48(6):460-2 Authors: Kwon H, Enomoto T, Shimogawara M, Yasuda K, Nakajima Y, Ohmiya Y Bioluminescence imaging reveals the long-term dynamics of individual gene expression in a single cell. However, methods for simultaneously imaging multiple gene expression patterns have been unknown to date. Here, we constructed a dual-path optical luminescence imaging system using a two-color reporter system and could simultaneously track two gene expression patterns for several days in a single cell. Address correspondence to Yoshihiro Ohmiya, Regenerative Medicine/Tissue Engineering Division, Research Center for Cooperative Projects, Hokkaido University Graduate School of Medicine, Kita-15, Nishi-7, Kita-Ku, Sapporo 060-8638, Japan. e-mail: PMID: 20569221 [PubMed - in process] | | |
| Effect of Parathyroid Hormone on Type X and Type II Collagen Expression in Mesenchymal Stem Cells from Osteoarthritis Patients.
June 24, 2010 at 6:23 AM
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| | Effect of Parathyroid Hormone on Type X and Type II Collagen Expression in Mesenchymal Stem Cells from Osteoarthritis Patients. Tissue Eng Part A. 2010 Jun 22; Authors: Mwale F, Yao G, Ouellet JA, Petit A, Antoniou J A major drawback of current cartilage and intervertebral disc tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients express type X collagen (COL10), a marker of late-stage chondrocyte hypertrophy (associated with endochondral ossification). Parathyroid hormone (PTH)) regulate endochondral ossification by inhibiting chondrocyte differentiation toward hypertrophy. In the present study, we investigated the effect of PTH on the expression of COL10 in MSCs from OA patients and analyzed the potential mechanisms related to its effect. MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA. Expanded cells were then incubated for 0-48h without (Control) or with 100 nM PTH (1-34). Protein expression and phosphorylation were measured by Western blot. Results showed that PTH (1-34) inhibited in a time-dependent manner the expression of COL10 in MSCs from OA patients. In parallel, PTH (1-34) stimulated the expression of COL2, a marker of chondrogenic differentiation. Results also showed that PTH (1-34) inhibited in a sustained manner the phosphorylation of p38 and AKT protein kinase signaling pathways. Interestingly, the modulation of COL2 and COL10 gene expression was significant as rapidly as after 1h in the presence of PTH (1-34), changes in the phosphorylation of p38 and AKT was significant only after 6h. This suggests that while p38 and AKT protein kinase signaling pathways may not be required to initiate the regulation of the expression of COL2 and COL10 by PTH (1-34), these pathways may modulate later events necessary for preventing precocious MSC hypertrophy. PMID: 20569194 [PubMed - as supplied by publisher] | | |
| Hydrogels Based on Dual Curable Chitosan-graft-Polyethylene Glycol-graft-Methacrylate: Application to Layer-by-Layer Cell Encapsulation.
June 24, 2010 at 6:23 AM
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| | Hydrogels Based on Dual Curable Chitosan-graft-Polyethylene Glycol-graft-Methacrylate: Application to Layer-by-Layer Cell Encapsulation. ACS Appl Mater Interfaces. 2010 Jun 22; Authors: Poon YF, Cao Y, Liu Y, Chan V, Chan-Park MB Ultraviolet (UV) photo-cross-linkable hydrogels have been commonly used for three-dimensional (3D) encapsulation of cells. Previous UV cross-linkable hydrogels have employed one-shot hardening of mixtures of hydrogels and cells. Here we propose an alternative method of making hydrogel-encapsulated cell constructs through layer by layer (LBL) buildup of alternating layers of cells and hydrogel. The LBL method potentially permits better spatial control of different cell types and control of cell orientation. Each hydrogel layer must be hardened before deposition of the next layer of cells. A UV-curable gel precursor that can also be gelled at physiological temperature is desirable to avoid repeated UV exposure of cells after deposition of each successive hydrogel layer. We designed, synthesized, and applied such a precursor, dual-curable-both thermoresponsive and UV-curable-chitosan-graft-polyethylene glycol-graft-methacrylate (CEGx-MA) copolymer (x is the PEG molecular weight in Daltons). We found that CEG350-MA copolymer solutions (5 wt % polymer) formed physical gels at approximately 37 degrees C and could be further photopolymerized to form thermally stable dual-cured hydrogels. This material was applied to the creation of a two-layer LBL smooth muscle cell (SMC)/hydrogel construct using temperature elevation to approximately 37 degrees C to gel each hydrogel layer. The physically gelled two-layered hydrogel/cell construct was finally exposed to a single UV shot to improve its mechanical properties and render it thermally stable. CEG350-MA solution and gel are nontoxic to SMCs. Cells remained mostly viable when they were encapsulated inside both physically gelled and dual-cured CEG350-MA and suffered little damage from the single brief UV exposure. The combination of LBL tissue engineering with a dual curable hydrogel precursor such as CEG350-MA permits the buildup of viable thick and complex tissues in a stable, biocompatible, and biodegradable matrix. PMID: 20568698 [PubMed - as supplied by publisher] | | |
| Synthetic surfaces for human embryonic stem cell culture.
June 24, 2010 at 6:23 AM
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| | Synthetic surfaces for human embryonic stem cell culture. J Biotechnol. 2010 Apr 1;146(3):143-6 Authors: Kolhar P, Kotamraju VR, Hikita ST, Clegg DO, Ruoslahti E Human embryonic stem cells (hESCs) have numerous potential biomedical applications owing to their unique abilities for self-renewal and pluripotency. Successful clinical application of hESCs and derivatives necessitates the culture of these cells in a fully defined environment. We have developed a novel peptide-based surface that uses a high-affinity cyclic RGD peptide for culture of hESCs under chemically defined conditions. PMID: 20132848 [PubMed - indexed for MEDLINE] | | |
| Comparison of Reprogramming Efficiency Between Transduction of Reprogramming Factors, Cell-Cell Fusion, and Cytoplast Fusion.
June 24, 2010 at 6:05 AM
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| | Comparison of Reprogramming Efficiency Between Transduction of Reprogramming Factors, Cell-Cell Fusion, and Cytoplast Fusion. Stem Cells. 2010 Jun 22; Authors: Hasegawa K, Zhang P, Wei Z, Pomeroy JE, Lu W, Pera MF Reprogramming human somatic cells into pluripotent cells opens up new possibilities for transplantation therapy, the study of disease, and drug screening. In addition to somatic cell nuclear transfer, several approaches to reprogramming human cells have been reported: transduction of defined transcription factors to generate induced pluripotent stem cell (iPSC), human embryonic stem cell (hESC)-somatic cell fusion, and hESC cytoplast-somatic cell fusion or exposure to extracts of hESC. Here we optimized techniques for hESC-human fibroblast fusion, and enucleation and cytoplast fusion, and then compared the reprogramming efficiency between iPSC generation, cell-fusion and cytoplast-fusion. Compared to iPSC, hESC-fusion provided for much faster and efficient reprogramming of somatic cells. The reprogramming required more than 4 weeks and the efficiency was less than 0.001% in iPSC generation, and it was less than 10 days and more than 0.005% in hESC-fusion. In addition, fusion yielded almost no partially reprogrammed cell colonies. However, the fused cells were tetraploid or aneuploid. hESC cytoplast fusion could initiate reprogramming but was never able to complete reprogramming. These data indicate that in cell fusion, as in nuclear transfer, reprogramming through direct introduction of a somatic nucleus into the environment of a pluripotent cell provides for relatively efficient reprogramming. The findings also suggest that the nucleus of the host pluripotent cell may contain components that accelerate the reprogramming process. PMID: 20572011 [PubMed - as supplied by publisher] | | |
| Efficient KRT14 Targeting and Functional Characterization of Transplanted Human Keratinocytes for the Treatment of Epidermolysis Bullosa Simplex.
June 24, 2010 at 6:05 AM
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| | Efficient KRT14 Targeting and Functional Characterization of Transplanted Human Keratinocytes for the Treatment of Epidermolysis Bullosa Simplex. Mol Ther. 2010 Jun 22; Authors: Petek LM, Fleckman P, Miller DG Inherited skin blistering conditions collectively named epidermolysis bullosa (EB) cause significant morbidity and mortality due to the compromise of the skin's barrier function, the pain of blisters, inflammation, and in some cases scaring and cancer. The simplex form of EB is usually caused by dominantly inherited mutations in KRT5 or KRT14. These mutations result in the production of proteins with dominant-negative activity that disrupt polymerization of intermediate filaments in the basal keratinocyte layer and result in a weak epidermal-dermal junction. The genome of adeno-associated virus (AAV) vectors can recombine with chromosomal sequence so that mutations can be corrected, or production of proteins with dominant-negative activity can be disrupted. We demonstrate a clinically feasible strategy for efficient targeting of the KRT14 gene in normal and EB-affected human keratinocytes. Using a gene-targeting vector with promoter trap design, targeted alteration of one allele of KRT14 occurred in 100% of transduced cells and transduction frequencies ranged from 0.1 to 0.6% of total cells. EBS patient keratinocytes with precise modifications of the mutant allele are preferentially recovered from targeted cell populations. Single epidermal stem cell clones produced histologically normal skin grafts after transplantation to athymic mice and could generate a sufficient number of cells to transplant the entire skin surface of an individual. PMID: 20571545 [PubMed - as supplied by publisher] | | |
| Dissecting spatio-temporal protein networks driving human heart development and related disorders.
June 24, 2010 at 6:05 AM
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| | Dissecting spatio-temporal protein networks driving human heart development and related disorders. Mol Syst Biol. 2010 Jun 22;6:381 Authors: Lage K, Møllgård K, Greenway S, Wakimoto H, Gorham JM, Workman CT, Bendsen E, Hansen NT, Rigina O, Roque FS, Wiese C, Christoffels VM, Roberts AE, Smoot LB, Pu WT, Donahoe PK, Tommerup N, Brunak S, Seidman CE, Seidman JG, Larsen LA Aberrant organ development is associated with a wide spectrum of disorders, from schizophrenia to congenital heart disease, but systems-level insight into the underlying processes is very limited. Using heart morphogenesis as general model for dissecting the functional architecture of organ development, we combined detailed phenotype information from deleterious mutations in 255 genes with high-confidence experimental interactome data, and coupled the results to thorough experimental validation. Hereby, we made the first systematic analysis of spatio-temporal protein networks driving many stages of a developing organ identifying several novel signaling modules. Our results show that organ development relies on surprisingly few, extensively recycled, protein modules that integrate into complex higher-order networks. This design allows the formation of a complicated organ using simple building blocks, and suggests how mutations in the same genes can lead to diverse phenotypes. We observe a striking temporal correlation between organ complexity and the number of discrete functional modules coordinating morphogenesis. Our analysis elucidates the organization and composition of spatio-temporal protein networks that drive the formation of organs, which in the future may lay the foundation of novel approaches in treatments, diagnostics, and regenerative medicine. PMID: 20571530 [PubMed - in process] | | | | 
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