| Stem Cell Platforms for Regenerative Medicine.
September 26, 2009 at 6:34 am
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| | Stem Cell Platforms for Regenerative Medicine. Clin Transl Sci. 2009 Jun 1;2(3):222-227 Authors: Nelson TJ, Behfar A, Yamada S, Martinez-Fernandez A, Terzic A The pandemic of chronic degenerative diseases associated with aging demographics mandates development of effective approaches for tissue repair. As diverse stem cells directly contribute to innate healing, the capacity for de novo tissue reconstruction harbors a promising role for regenerative medicine. Indeed, a spectrum of natural stem cell sources ranging from embryonic to adult progenitors has been recently identified with unique characteristics for regeneration. The accessibility and applicability of the regenerative armamentarium has been further expanded with stem cells engineered by nuclear reprogramming. Through strategies of replacement to implant functional tissues, regeneration to transplant progenitor cells or rejuvenation to activate endogenous self-repair mechanisms, the overarching goal of regenerative medicine is to translate stem cell platforms into practice and achieve cures for diseases limited to palliative interventions. Harnessing the full potential of each platform will optimize matching stem cell-based biologics with the disease-specific niche environment of individual patients to maximize the quality of long-term management, while minimizing the needs for adjunctive therapy. Emerging discovery science with feedback from clinical translation is therefore poised to transform medicine offering safe and effective stem cell biotherapeutics to enable personalized solutions for incurable diseases. PMID: 19779576 [PubMed - as supplied by publisher] |
| Conserved expression patterns predict microRNA targets.
September 26, 2009 at 6:34 am
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| | Conserved expression patterns predict microRNA targets. PLoS Comput Biol. 2009 Sep;5(9):e1000513 Authors: Ritchie W, Rajasekhar M, Flamant S, Rasko JE microRNAs (miRNAs) are major regulators of gene expression and thereby modulate many biological processes. Computational methods have been instrumental in understanding how miRNAs bind to mRNAs to induce their repression but have proven inaccurate. Here we describe a novel method that combines expression data from human and mouse to discover conserved patterns of expression between orthologous miRNAs and mRNA genes. This method allowed us to predict thousands of putative miRNA targets. Using the luciferase reporter assay, we confirmed 4 out of 6 of our predictions. In addition, this method predicted many miRNAs that act as expression enhancers. We show that many miRNA enhancer effects are mediated through the repression of negative transcriptional regulators and that this effect could be as common as the widely reported repression activity of miRNAs. Our findings suggest that the indirect enhancement of gene expression by miRNAs could be an important component of miRNA regulation that has been widely neglected to date. PMID: 19779543 [PubMed - in process] |
| The effects of low oxygen on self-renewal and differentiation of embryonic stem cells.
September 26, 2009 at 6:34 am
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| | The effects of low oxygen on self-renewal and differentiation of embryonic stem cells. Curr Opin Organ Transplant. 2009 Sep 24; Authors: Millman JR, Tan JH, Colton CK PURPOSE OF REVIEW: To summarize recent reports on the effects of low oxygen on the undifferentiated phenotype and differentiation of embryonic stem cells (ESCs). RECENT FINDINGS: The oxygen level to which ESCs are exposed is an important environmental parameter. Under conditions maintaining the undifferentiated phenotype, low oxygen reduces spontaneous differentiation of human ESCs but reduces pluripotency gene expression in mouse ESCs, although reports are conflicting. Differentiation under low oxygen increases generation of neurons, cardiomyocytes, hematopoietic progenitors, endothelial cells, and chondrocytes. Many of the effects of low oxygen have been attributed to action by hypoxia inducible factor-1alpha (HIF-1alpha). The oxygen level in the gas phase (pO2gas) is often different than that experienced by the cells (pO2cell) and is unrecognized by investigators, which makes interpretation of the literature difficult. This difference increases with high cell densities, high cellular oxygen consumption rates, and large medium heights. The problem can be addressed by use of oxygen-permeable culture dishes and by estimation of pO2cell with mathematical models. SUMMARY: Low oxygen influences aspects of ESC pluripotency and differentiation. A better understanding of its effects and mechanism along with better estimation and control of pO2cell is important for applying low oxygen culture to regenerative medicine applications. PMID: 19779343 [PubMed - as supplied by publisher] |
| Evaluation of a Respiratory Assist Catheter that Uses an Impeller Within a Hollow Fiber Membrane Bundle.
September 26, 2009 at 6:34 am
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| | Evaluation of a Respiratory Assist Catheter that Uses an Impeller Within a Hollow Fiber Membrane Bundle. ASAIO J. 2009 Sep 22; Authors: Mihelc KM, Frankowski BJ, Lieber SC, Moore ND, Hattler BG, Federspiel WJ Respiratory assist using an intravenous catheter may be a potential treatment for patients suffering from acute or acute-on-chronic lung failure. The objective of this study was to evaluate a novel respiratory catheter that uses an impeller within the fiber bundle to enhance gas exchange efficiency, thus requiring a smaller fiber bundle and insertional size (25 Fr) and permitting simple percutaneous insertion. Bench testing of gas exchange in deionized water was used to evaluate eight impeller designs. The three best performing impeller designs were evaluated in acute studies in four calves (122 +/- 10 kg). Gas exchange increased significantly with increasing impeller rotation rate. The degree of enhancement varied with impeller geometry. The maximum gas exchange efficiency (exchange per unit surface area) for the catheter with the best performing impeller was 529 +/- 20 ml CO2/min/m and 513 +/- 21 ml CO2/min/m for bench and animal studies, respectively, at a rotation rate of 20,000 rpm. Absolute CO2 exchange was 37 and 36 ml CO2/min, respectively. Active mixing by rotating impellers produced 70% higher gas exchange efficiency than pulsating balloon catheters. The sensitivity of gas exchange to impeller design suggests that further improvements can be made by computational fluid dynamics-based optimization of the impeller. PMID: 19779302 [PubMed - as supplied by publisher] |
| Biological evaluation of collagen-chitosan scaffolds for dermis tissue engineering.
September 26, 2009 at 6:34 am
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| | Biological evaluation of collagen-chitosan scaffolds for dermis tissue engineering. Biomed Mater. 2009 Sep 25;4(5):55008 Authors: Sun LP, Wang S, Zhang ZW, Wang XY, Zhang QQ Three-dimensional collagen-chitosan scaffolds were fabricated with type I collagen and chitosan through freeze drying and glutaraldehyde cross-linking. Dermal fibroblasts were isolated from neonatal Sprague-Dawley rat skin by dispase II/collagenase I digestion. The fibroblasts were then seeded into the scaffolds to construct tissue-engineered dermis. The microstructure of the scaffolds as well as the fibroblasts' proliferation, cytokine secretion and cell cycle were investigated. Flow cytometry analysis indicated that cells in the scaffolds proliferated steadily. IL-6 concentration measurement by the ELISA test suggested that the scaffolds could promote secretion of the fibroblasts' cytokine. These results show that the fibroblasts and the scaffolds interact well with each other, and the fibroblasts have better proliferation ability and biological activity in the scaffolds than in monolayer culture. The scaffolds are a promising candidate for tissue repair and regeneration with enhanced biostability and good cytocompatibility. PMID: 19779250 [PubMed - as supplied by publisher] |
| AN IN VITRO ASSESSMENT OF A CELL-CONTAINING COLLAGENOUS EXTRACELLULAR MATRIX-LIKE SCAFFOLD FOR BONE TISSUE ENGINEERING.
September 26, 2009 at 6:34 am
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| | AN IN VITRO ASSESSMENT OF A CELL-CONTAINING COLLAGENOUS EXTRACELLULAR MATRIX-LIKE SCAFFOLD FOR BONE TISSUE ENGINEERING. Tissue Eng Part A. 2009 Sep 24; Authors: Pedraza CE, Marelli B, Chicatun F, McKee MD, Nazhat SN Extracellular matrix (ECM) consists of a complex mixture of macromolecules such as collagens, proteoglycans, glycoproteins and elastic fibres. ECM is essential to preserving tissue architecture, signaling to cells and regulating calcification in mineralized tissues. Osteoblasts in culture secrete and assemble an extensive ECM rich in type I collagen, and other noncollagenous proteins, that can be mineralized. Three-dimensional (3D) matrix models can be used in vitro to most appropriately resemble the geometry and biochemistry of natural ECMs. In the present study, MC3T3-E1 mouse calvarial pre-osteoblasts were cultured within a dense, 3D collagenous ECM-like scaffold produced through the method of plastic compression (PC). PC rapidly produces scaffolds of collagen density approaching native tissue levels with enhanced biomechanical properties while maintaining the viability of resident cells. The proliferation, morphology, and gene expression of seeded MC3T3s, as well as collagen production and matrix mineralization, were investigated for up to 7 weeks in culture. Soluble collagen secretion ranged in concentration from 5 to 30 g/ml over a 24-h period, concomitant with a steady rate of collagen mRNA expression. Expression of osteogenic markers such as tissue-nonspecific alkaline phosphatase (Alpl), bone sialoprotein (Bsp) and osteopontin (Opn) examined by biochemical assay and RT-PCR demonstrated cell differentiation. Pericellular voids of ECM around cells, together with evidence of MMP13 mRNA expression, suggested matrix remodelling. Ultrastructural analyses, x-ray microanalysis, micro-computed tomography as well as FTIR and imaging all confirmed the formation of a calcium-phosphate mineral phase within the fibrillar collagen matrix. In conclusion, pre-osteoblastic MC3T3 cells seeded within an ECM-like dense collagen scaffold secrete matrix proteins and induce scaffold mineralization in a manner potentially appropriate for bone tissue engineering uses. PMID: 19778181 [PubMed - as supplied by publisher] |
| Carbodiimide Conjugation of Fibronectin on Collagen Basal Lamina Analogs Enhances Cellular Binding Domains and Epithelialization.
September 26, 2009 at 6:34 am
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| | Carbodiimide Conjugation of Fibronectin on Collagen Basal Lamina Analogs Enhances Cellular Binding Domains and Epithelialization. Tissue Eng Part A. 2009 Sep 24; Authors: Bush KA, Pins GD In order to improve the regenerative potential of biomaterials used as bioengineered scaffolds, it is necessary to strategically incorporate biologically active molecules that promote in vivo cellular processes that lead to a fully functional tissue. This work evaluates the effects of strategically binding FN to collagen basal lamina analogs to enhance keratinocyte functions necessary for complete skin regeneration. We found that FN passively adsorbed to collagen-GAG basal lamina analogs enhanced epithelial thickness and keratinocyte proliferation when compared to non-treated basal lamina analogs at 3 days of air/liquid (A/L) interface culture. Additionally we evaluated the availability of FN cellular binding site domains when FN was either passively adsorbed or (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)) conjugated to basal lamina analogs fabricated from collagen-GAG coprecipiate or self-assembled type I collagen. It was found that EDC conjugation of FN significantly enhanced FN binding site presentation as well as epithelial thickness. Overall the results gained from this study will be used to improve the regenerative capacity of basal lamina analogs for bioengineered skin substitutes as well as the development of bioengineered scaffolds for other tissue engineering applications. PMID: 19778179 [PubMed - as supplied by publisher] |
| Finite Element Analysis of Fluid Flow Conditions in Cell Culture.
September 26, 2009 at 6:34 am
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| | Finite Element Analysis of Fluid Flow Conditions in Cell Culture. Tissue Eng Part C Methods. 2009 Sep 24; Authors: Salvi JD, Lim JY, Donahue HJ Numerous studies in tissue engineering and biomechanics utilize fluid flow stimulation, both unidirectional and oscillatory, in order to analyze the effects of shear stresses on cell behavior. However, it has typically been assumed that these shear stresses are uniform and that cell and substrate properties do not adversely affect these assumptions. With the increasing utilization of fluid flow in cell biology, it would be beneficial to determine the validity of various experimental protocols. Since it is difficult to determine the velocity profiles and shear stresses empirically, we utilized the finite element method (FEM). By using FEM, we determined the effects of cell confluence on fluid flow, the effects of cell height on the uniformity of shear stresses, apparent shear stresses exhibited by cells cultured on various substrates, and the effects of oscillatory fluid flow relative to unidirectional flow. FEM analyses could successfully analyze flow patterns over cells for various cell confluence and shape and substrate characteristics. Our data suggest the benefits of the utilization of oscillatory fluid flow and the use of substrates that stimulate cell spreading in the distribution of more uniform shear stresses across the surface of cells. Also, we demonstrated that cells cultured on nanotopographies are exposed to greater apparent shear stresses than cells on flat controls when using the same fluid flow conditions. FEM thus provides an excellent tool for development of experimental protocols and the design of bioreactor systems. PMID: 19778171 [PubMed - as supplied by publisher] |
| In vivo generation of thick, vascularized hepatic tissue from collagen hydrogel-based hepatic units.
September 26, 2009 at 6:34 am
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| | In vivo generation of thick, vascularized hepatic tissue from collagen hydrogel-based hepatic units. Tissue Eng Part C Methods. 2009 Sep 24; Authors: Zhao Y, Xu Y, Zhang B, Wu X, Xu F, Liang W, Li R In vivo engineering of hepatic tissue based on primary hepatocytes offers new perspectives for the treatment of liver diseases. However, generation of thick, three-dimensional liver tissue has been limited by the lack of vasculature in the tissue-engineered constructs. Here, we used collagen hydrogel as a matrix to generate engineered hepatic units to reconstitute 3-dimensional, vascularized hepatic tissue in vivo. Hepatocytes harvested from Sprague-Dawley rats were mixed with liquid type collagen, concentrated Dulbecco's Modified Eagle's Medium (DMEM, 2x) and hepatocyte maintenance medium to create hepatocyte/collagen hydrogel constructs. The constructs were then dissociated into cylindrical hepatic units (diameter/height:2000-4000mum/500-1000mum). Stacking of hepatic units under the subcutaneous space resulted in significant cell engraftment, with the formation of large fused hepatic system (more than 0.5cm thick) containing blood vessels. In contrast, only less cell engraftment could be achieved when hepatocytes were transplanted in a manner of whole constructs. Functional maintenance of the engineered hepatic tissue was confirmed by the expression of liver-specific mRNA and proteins. The engineered hepatic tissue has the ability to respond to the regenerative stimulus. In conclusion, large hepatic tissue containing blood vessels could be engineered in vivo by merging small hepatic units. This approach for tissue engineering is simple, and represents an efficient way to engineer hepatic tissue in vivo. PMID: 19778169 [PubMed - as supplied by publisher] |
| Tissue-engineered small intestine and stomach form from autologous tissue in a preclinical large animal model.
September 26, 2009 at 6:34 am
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| | Tissue-engineered small intestine and stomach form from autologous tissue in a preclinical large animal model. J Surg Res. 2009 Oct;156(2):205-12 Authors: Sala FG, Kunisaki SM, Ochoa ER, Vacanti J, Grikscheit TC BACKGROUND: Tissue-engineered small intestine, stomach, large intestine, esophagus, and gastroesophageal (GE) junction have been successfully formed from syngeneic cells, and employed as a rescue therapy in a small animal model. The purpose of this study is to determine if engineered intestine and stomach could be generated in an autologous, preclinical large animal model, and to identify if the tissue-engineered intestine retained features of an intact stem cell niche. METHODS: A short segment of jejunum or stomach was resected from 6-wk-old Yorkshire swine. Organoid units, multicellular clusters with predominantly epithelial content, were generated and loaded onto biodegradable scaffold tubes. The constructs were then implanted intraperitoneally in the autologous host. Seven wk later, all implants were harvested and analyzed using histology and immunohistochemistry techniques. RESULTS: Autologous engineered small intestine and stomach formed. Tissue-engineered intestinal architecture replicated that of native intestine. Histology revealed tissue-engineered small intestinal mucosa composed of a columnar epithelium with all differentiated intestinal cell types adjacent to an innervated muscularis mucosae. Intestinal subepithelial myofibroblasts, specialized cells that participate in the stem cell niche formation, were identified. Moreover, cells positive for the putative intestinal stem cell marker, doublecortin and CaM kinase-like-1 (DCAMKL-1) expression were identified at the base of the crypts. Finally, tissue-engineered stomach also formed with antral-type mucosa (mucus cells and surface foveolar cells) and a muscularis. CONCLUSION: We successfully generated tissue-engineered intestine with correct architecture, including features of an intact stem cell niche, in the pig model. To our knowledge, this is the first demonstration in which tissue-engineered intestine was successfully generated in an autologous manner in an animal model, which may better emulate a human host and the intended therapeutic pathway for humans. PMID: 19665143 [PubMed - indexed for MEDLINE] |
| Control of myotube contraction using electrical pulse stimulation for bio-actuator.
September 26, 2009 at 6:34 am
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| | Control of myotube contraction using electrical pulse stimulation for bio-actuator. J Artif Organs. 2009;12(2):131-7 Authors: Yamasaki K, Hayashi H, Nishiyama K, Kobayashi H, Uto S, Kondo H, Hashimoto S, Fujisato T The contractility of tissue-engineered muscle on the application of electrical signals is required for the development of bio-actuators and for muscle tissue regeneration. Investigations have already reported on the contraction of myotubes differentiated from myoblasts and the construction of tissue-engineered skeletal muscle using electrical pulses. However, the relationship between myotube contraction and electrical pulses has not been quantitatively evaluated. We quantitatively investigated the effect of electrical pulse frequency on the excitability of myotubes and developed bio-actuators made of tissue-engineered skeletal muscle. C2C12 cells were seeded on a collagen-coated dish and in collagen gel and were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and antibiotics. When the cells reached confluence or after 2 days in culture, the medium was shifted to DMEM containing 7% horse serum to allow them to differentiate to C2C12 myotubes. We electrically stimulated the myotubes and tissue-engineered skeletal muscle, and contractions were observed under a microscope. The myotubes contracted synchronously with electrical pulses between 0.5 and 5 Hz and unfused tetanus was generated at 10 Hz. The contractile performance of tissue-engineered skeletal muscle made of collagen gel and C2C12 was similar to that of the myotubes. Both the rheobase and chronaxie of the myotubes were lowest when the electric field was applied parallel to the myotube axis, and the values were 8.33 +/- 2.78 mA and 1.19 +/- 0.38 ms, respectively. The motion of C2C12 myotube contraction depended on the pulse frequency and showed anisotropy in the electric field. These results suggest that a tissue-engineered bio-actuator may be controlled using electrical signals. PMID: 19536631 [PubMed - indexed for MEDLINE] |
| Integrative analysis of genome-wide RNA interference screens.
September 26, 2009 at 6:34 am
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| | Integrative analysis of genome-wide RNA interference screens. Sci Signal. 2009;2(70):pt4 Authors: Berndt JD, Biechele TL, Moon RT, Major MB High-throughput genetic screens have exponentially increased the functional annotation of the genome over the past 10 years. Likewise, genome-scale efforts to map DNA methylation, chromatin state and occupancy, messenger RNA expression patterns, and disease-associated genetic polymorphisms, and proteome-wide efforts to map protein-protein interactions, have also created vast resources of data. An emerging trend involves combining multiple types of data, referred to as integrative screening. Examples include papers that report integrated data generated from large-scale RNA interference screens on the Wnt/beta-catenin pathway with either genotypic or proteomic data in colorectal cancer. These studies demonstrate the power of data integration to generate focused, validated data sets and to identify high-confidence candidate genes for follow-up experiments. We present the ongoing evolution and new strategies for the integrative screening approach with respect to understanding and treating human disease. PMID: 19436058 [PubMed - indexed for MEDLINE] |
| A novel method for the generation of reaggregated organotypic cultures that permits juxtaposition of defined cell populations.
September 26, 2009 at 1:34 am
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| | A novel method for the generation of reaggregated organotypic cultures that permits juxtaposition of defined cell populations. Genesis. 2009 May;47(5):346-51 Authors: Sheridan JM, Taoudi S, Medvinsky A, Blackburn CC Cellular reaggregation methods have been used to generate in vitro organotypic cultures as a means to elucidate the cellular and molecular requirements of organogenesis. However, reproducibility from experiment to experiment has remained problematic and furthermore, current protocols do not support reaggregation of many important tissues. Here, using the thymus as a model organ, we present a novel reaggregation method termed "compaction reaggregation" that offers improved kinetics of reaggregation and greatly improved efficiency. Using compaction reaggregation we have been able to reaggregate the aorta-gonad- mesonephros region, a tissue that previously proved refractory to commonly used reaggregation methods, enabling the study of hematopoietic stem cell emergence and expansion. Additionally, compaction reaggregation permits the juxtaposition of different cell layers within the aggregated structure thus providing the means to study inductive interactions between different cell populations in vitro. PMID: 19370754 [PubMed - indexed for MEDLINE] | | | 
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