| Generation of functional human hepatic endoderm from human induced pluripotent stem cells.
October 31, 2009 at 6:01 am
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| | Generation of functional human hepatic endoderm from human induced pluripotent stem cells. Hepatology. 2009 Sep 29; Authors: Sullivan GJ, Hay DC, Park IH, Fletcher J, Hannoun Z, Payne CM, Dalgetty D, Black JR, Ross JA, Samuel K, Wang G, Daley GQ, Lee JH, Church GM, Forbes SJ, Iredale JP, Wilmut I With the advent of induced pluripotent stem cell (iPSC) technology, it is now feasible to generate iPSCs with a defined genotype or disease state. When coupled with direct differentiation to a defined lineage, such as hepatic endoderm (HE), iPSCs would revolutionize the way we study human liver biology and generate efficient "off the shelf" models of human liver disease. Here, we show the "proof of concept" that iPSC lines representing both male and female sexes and two ethnic origins can be differentiated to HE at efficiencies of between 70%-90%, using a method mimicking physiological relevant condition. The iPSC-derived HE exhibited hepatic morphology and expressed the hepatic markers albumin and E-cadherin, as assessed by immunohistochemistry. They also expressed alpha-fetoprotein, hepatocyte nuclear factor-4a, and a metabolic marker, cytochrome P450 7A1 (Cyp7A1), demonstrating a definitive endodermal lineage differentiation. Furthermore, iPSC-derived hepatocytes produced and secreted the plasma proteins, fibrinogen, fibronectin, transthyretin, and alpha-fetoprotein, an essential feature for functional HE. Additionally iPSC-derived HE supported both CYP1A2 and CYP3A4 metabolism, which is essential for drug and toxicology testing. Conclusion: This work is first to demonstrate the efficient generation of hepatic endodermal lineage from human iPSCs that exhibits key attributes of hepatocytes, and the potential application of iPSC-derived HE in studying human liver biology. In particular, iPSCs from individuals representing highly polymorphic variants in metabolic genes and different ethnic groups will provide pharmaceutical development and toxicology studies a unique opportunity to revolutionize predictive drug toxicology assays and allow the creation of in vitro hepatic disease models. (HEPATOLOGY 2009.). PMID: 19877180 [PubMed - as supplied by publisher] |
| Genome modification in human embryonic stem cells.
October 31, 2009 at 6:01 am
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| | Genome modification in human embryonic stem cells. J Cell Physiol. 2009 Oct 28; Authors: Tenzen T, Zembowicz F, Cowan CA Induced pluripotent stem cell (iPSC) technology has emerged as the most promising method for generating patient-specific human embryonic stem (ES) cells and adult stem cells (Takahashi et al., 2007, Cell 131:861-872; Wernig et al., 2007, Nature 448:318-324; Park et al., 2008, Nature 451:141-146). So far, most studies of direct reprogramming have been done by using lentiviruses/retroviruses encoding the reprogramming factors. This represents a major limitation to therapeutic applications since viral integration in the host genome increases the risk of tumorigenicity, and low-level residual expression of reprogramming factors may alter the differentiation potential of the human iPSCs (hiPSCs). As a result, more attention has been paid to developing new techniques to manipulate the human genome, with the goal of making safer hiPSCs that have fewer or no lesions or alterations in the genome. Additionally, the efficiency of reprogramming and of homologous recombination in gene therapy must be improved, if iPSC technology is to be a viable tool in regenerative medicine. Here, we summarize the recent developments in human genome manipulation for generating hiPSCs and advances in homologous recombination for gene targeting. J. Cell. Physiol. (c) 2009 Wiley-Liss, Inc. PMID: 19877154 [PubMed - as supplied by publisher] |
| Inpp5f Is a Polyphosphoinositide Phosphatase That Regulates Cardiac Hypertrophic Responsiveness.
October 31, 2009 at 6:01 am
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| | Inpp5f Is a Polyphosphoinositide Phosphatase That Regulates Cardiac Hypertrophic Responsiveness. Circ Res. 2009 Oct 29; Authors: Zhu W, Trivedi CM, Zhou D, Yuan L, Lu MM, Epstein JA Rationale: Cardiac hypertrophy occurs in response to a variety of extrinsic and intrinsic stimuli that impose increased biomechanical stress. The phosphatidylinositol 3-kinase/Akt pathway has previously been strongly associated with hypertrophic signaling in the heart, and with the control of cell size in multiple contexts. This pathway is tightly regulated by many factors, including a host of kinases and phosphatases that function at multiple steps in the signaling cascade. For example, the PTEN (phosphatase and tensin homolog) tumor suppressor protein is a phosphoinositide 3-phosphatase that, by metabolizing PtdIns(3,4,5)P3, acts in direct antagonism to growth factor-stimulated phosphatidylinositol 3-kinase. Inhibition of PTEN leads to cardiomyocyte hypertrophy. Another polyphoinositide phosphatase, inositol polyphosphate-5-phosphatase F (Inpp5f) has recently been implicated in regulation of cardiac hypertrophy. Like PTEN, this phosphatase can degrade PtdIns(3,4,5)P3 and thus modulates the phosphatidylinositol 3-kinase/Akt pathway. Objective: To characterize the role of Inpp5f in regulating cardiac hypertrophy. Methods and Results: We generated homozygous Inpp5f knockout mice and cardiac specific Inpp5f overexpression transgenic mice. We evaluated their hearts for biochemical, structural and functional changes. Inpp5f knockout mice have augmented hypertrophy and reactivation of the fetal gene program in response to stress when compared to wild-type littermates. Furthermore, cardiac overexpression of Inpp5f in transgenic mice reduces hypertrophic responsiveness. Conclusions: Our results suggest that Inpp5f is a functionally important endogenous modulator of cardiac myocyte size and of the cardiac response to stress. PMID: 19875726 [PubMed - as supplied by publisher] |
| VEGF Induces Differentiation of Functional Endothelium From Human Embryonic Stem Cells. Implications for Tissue Engineering.
October 31, 2009 at 6:01 am
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| | VEGF Induces Differentiation of Functional Endothelium From Human Embryonic Stem Cells. Implications for Tissue Engineering. Arterioscler Thromb Vasc Biol. 2009 Oct 29; Authors: Nourse MB, Halpin DE, Scatena M, Mortisen DJ, Tulloch NL, Hauch KD, Torok-Storb B, Ratner BD, Pabon L, Murry CE OBJECTIVE: Human embryonic stem cells (hESCs) offer a sustainable source of endothelial cells for therapeutic vascularization and tissue engineering, but current techniques for generating these cells remain inefficient. We endeavored to induce and isolate functional endothelial cells from differentiating hESCs. METHODS AND RESULTS: To enhance endothelial cell differentiation above a baseline of approximately 2% in embryoid body (EB) spontaneous differentiation, 3 alternate culture conditions were compared. Vascular endothelial growth factor (VEGF) treatment of EBs showed the best induction, with markedly increased expression of endothelial cell proteins CD31, VE-Cadherin, and von Willebrand Factor, but not the hematopoietic cell marker CD45. CD31 expression peaked around days 10 to 14. Continuous VEGF treatment resulted in a 4- to 5-fold enrichment of CD31(+) cells but did not increase endothelial proliferation rates, suggesting a primary effect on differentiation. CD31(+) cells purified from differentiating EBs upregulated ICAM-1 and VCAM-1 in response to TNFalpha, confirming their ability to function as endothelial cells. These cells also expressed multiple endothelial genes and formed lumenized vessels when seeded onto porous poly(2-hydroxyethyl methacrylate) scaffolds and implanted in vivo subcutaneously in athymic rats. Collagen gel constructs containing hESC-derived endothelial cells and implanted into infarcted nude rat hearts formed robust networks of patent vessels filled with host blood cells. CONCLUSIONS: VEGF induces functional endothelial cells from hESCs independent of endothelial cell proliferation. These enrichment methods increase endothelial cell yield, enabling applications for revascularization as well as basic studies of human endothelial biology. We demonstrate the ability of hESC-derived endothelial cells to facilitate vascularization of tissue-engineered implants. PMID: 19875721 [PubMed - as supplied by publisher] |
| Targeting aldehyde dehydrogenase: a potential approach for cell labeling.
October 31, 2009 at 6:01 am
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| | Targeting aldehyde dehydrogenase: a potential approach for cell labeling. Nucl Med Biol. 2009 Nov;36(8):919-29 Authors: Vaidyanathan G, Song H, Affleck D, McDougald DL, Storms RW, Zalutsky MR, Chin BB INTRODUCTION: To advance the science and clinical application of stem cell therapy, the availability of a highly sensitive, quantitative and translational method for tracking stem cells would be invaluable. Because hematopoetic stem cells express high levels of the cytosolic enzyme aldehyde dehydrogenase-1A1 (ALDH1), we sought to develop an agent that is specific to ALDH1 and thus to cells expressing the enzyme. Such an agent might be also helpful in identifying tumors that are resistant to cyclophosphomide chemotherapy because ALDH1 is known to be responsible for this resistance. METHODS: We developed schemes for the synthesis of two radioiodinated aldehdyes - N-formylmethyl-5-[*I]iodopyridine-3-carboxamide ([*I]FMIC) and 4-diethylamino-3-[*I]iodobenzaldehyde ([*I]DEIBA)-at no-carrier-added levels from their respective tin precursors. These agents were evaluated using pure ALDH1 and tumor cells that expressed the enzyme. RESULTS: The average radiochemical yields for the synthesis of [(125)I]FMIC and [(125)I]DEIBA were 70+/-5% and 47+/-14%, respectively. ALDH1 converted both compounds to respective acids suggesting their suitability as ALDH1 imaging agents. Although ability of ALDH1 within the cells to oxidize one of these substrates was shown, specific uptake in ALDH-expressing tumor cells could not be demonstrated. CONCLUSION: To pursue this approach for ALDH1 imaging, radiolabeled aldehydes need to be designed such that, in addition to being good substrates for ALDH1, the cognate products should be sufficiently polar so as to be retained within the cells. PMID: 19875048 [PubMed - in process] |
| Biocompatibility, alignment degree and mechanical properties of an electrospun chitosan-P(LLA-CL) fibrous scaffold.
October 31, 2009 at 6:01 am
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| | Biocompatibility, alignment degree and mechanical properties of an electrospun chitosan-P(LLA-CL) fibrous scaffold. J Biomater Sci Polym Ed. 2009;20(14):2117-28 Authors: Chen F, Su Y, Mo X, He C, Wang H, Ikada Y Chitosan-poly(L-lactide-co-epsilon-caprolactone) (P(LLA-CL)) complex fibers, fibrous mats and a tubular scaffold have been obtained through electrospinning. Due to their high porosity, there were more porcine iliac artery endothelial cells (PIECs) attached to fiber mats than to tissue-culture plate (TCP) and coverslips. The cells could grow and spread well on nanofiber mats. There were many of native extracellular matrix (ECM)-like colloids above and under the surface of fibrous mats after cell culturing. The two-dimensional fast Fourier transform (2-D FFT) approach was used to analysis alignment degree of fibers collected on a rotary mandrel. The relations among mechanical properties, alignment degree, fiber diameter and rotary speed are discussed. Aligned fibers with various alignment degrees could be found through adjusting rotary speed. Fiber alignment was the variable most closely associated with the regulation of stress and strain. In this study, we show a feasible approach for producing scaffold with controllable mechanical property for soft tissue engineering through electrospinning. PMID: 19874681 [PubMed - in process] |
| Fabrication of nano-fibrous PLLA scaffold reinforced with chitosan fibers.
October 31, 2009 at 6:01 am
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| | Fabrication of nano-fibrous PLLA scaffold reinforced with chitosan fibers. J Biomater Sci Polym Ed. 2009;20(14):1995-2002 Authors: Wang X, Song G, Lou T, Peng W In this study, a nano-fibrous PLLA scaffold reinforced by micro-scale chitosan fibers was fabricated using thermally-induced phase separation (TIPS). The morphology, porosity, mechanical performance and pH changes in in vitro degradation of the scaffold were also investigated. Results showed that the mechanical properties of the scaffold increased with the amount of chitosan fibers embedded, and the pH in in vitro degradation of the scaffold changed more slowly than that of the pure nano-fibrous PLLA scaffold without chitosan fibers. The new composite scaffold might be a very promising scaffold for tissue engineering. PMID: 19874673 [PubMed - in process] |
| Comparison of bone marrow stromal cell behaviors on poly(caprolactone) with or without surface modification: studies on cell adhesion, survival and proliferation.
October 31, 2009 at 6:01 am
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| | Comparison of bone marrow stromal cell behaviors on poly(caprolactone) with or without surface modification: studies on cell adhesion, survival and proliferation. J Biomater Sci Polym Ed. 2009;20(14):1975-93 Authors: Zhang H, Hollister S Poly(caprolactone) (PCL) is a promising biodegradable polymer for tissue engineering. However, intrinsically poor cell-adhesive properties of PCL may limit its application. In this study, the PCL film surface was modified with RGDC peptide by a chemical immobilization procedure. Furthermore, bone marrow stromal cell (BMSC) behaviors including attachment, spreading, focal adhesion formation, focal adhesion kinase (FAK) activation, apoptosis and proliferation when cultured on the modified PCL films were investigated. Our results demonstrated that PCL with RGD modification promoted initial BMSC attachment, spreading and focal adhesion formation. At a later time point (12 h), BMSC attachment on both RGD peptide-modified PCL and PCL-NH(2) films significantly increased compared to untreated PCL films. Importantly, FAK phosphorylation was significantly increased only on the films with RGD-modified films, not on the PCL-NH(2) films, demonstrating that PCL with RGD modification had an advantage in initiating the specific integrin-mediated signal transduction and might play an important role in the subsequent retardation in cell death and enhancement in cell proliferation. The present results provide more evidence that functionalizing PCL with RGD peptides may be a feasible way to improve the interaction between BMSC and PCL substrate, which is important in tissue engineering. PMID: 19874672 [PubMed - in process] |
| Not a process of simple vicariousness, the differentiation of human adipose-derived mesenchymal stem cells to renal tubular epithelial cells plays an important role in acute kidney injury repairing.
October 31, 2009 at 6:01 am
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| | Not a process of simple vicariousness, the differentiation of human adipose-derived mesenchymal stem cells to renal tubular epithelial cells plays an important role in acute kidney injury repairing. Stem Cells Dev. 2009 Oct 29; Authors: Li K, Han Q, Yan X, Liao L, Zhao RC The recent findings indicate that under conditions of severe tubular injuries, transplantation of mesenchymal stem cells (MSCs) may comprise a promising treatment in acute kidney diseases; nevertheless, the underling mechanism is still under debate. To investigate the differentiation characteristics and the role of MSCs in renal tubular injury, human adipose-derived MSCs (hAD-MSCs) were transplanted into ischemia-reperfusion (I/R) kidneys in C57BL/6 mouse model. Results showed that hAD-MSCs were able to differentiate toward renal tubular epithelium at early stage of injuries. The differentiated donor cells replaced the vacant space left over by the dead cells, contributed to maintenance of structural integrity and proceeding to subsequent tissue repair process. Furthermore, MSCs as supportive cells may promote repair via secreting cytokines. The differentiation and replacement of MSCs at extremely early stage play important roles for the subsequent self-repair and renewal of functional cells. Direct differentiation of MSCs, as an important mechanism of injured kidney repair, warrants further investigation. PMID: 19874085 [PubMed - as supplied by publisher] |
| Restoration of Runx1 expression in the Tie2 cell compartment rescues definitive hematopoietic stem cells and extends life of Runx1 knockout animals until birth.
October 31, 2009 at 6:01 am
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| | Restoration of Runx1 expression in the Tie2 cell compartment rescues definitive hematopoietic stem cells and extends life of Runx1 knockout animals until birth. Stem Cells. 2009 Jul;27(7):1616-24 Authors: Liakhovitskaia A, Gribi R, Stamateris E, Villain G, Jaffredo T, Wilkie R, Gilchrist D, Yang J, Ure J, Medvinsky A Mice deficient in the runt homology domain transcription factor Runx1/AML1 fail to generate functional clonogenic hematopoietic cells and die in utero by embryonic day 12.5. We previously generated Runx1 reversible knockout mice, in which the Runx1 locus can be restored by Cre-mediated recombination. We show here that selective restoration of the Runx1 locus in the Tie2 cell compartment rescues clonogenic hematopoietic progenitors in early Runx1-null embryos and rescues lymphoid and myeloid lineages during fetal development. Furthermore, fetal liver cells isolated from reactivated Runx1 embryos are capable of long-term multilineage lymphomyeloid reconstitution of adult irradiated recipients, demonstrating the rescue of definitive hematopoietic stem cells. However, this rescue of the definitive hematopoietic hierarchy is not sufficient to rescue the viability of animals beyond birth, pointing to an essential role for Runx1 in other vital developmental processes. PMID: 19544462 [PubMed - indexed for MEDLINE] | | | 
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