| In vitro hepatic maturation of human embryonic stem cells by using a mesenchymal cell line derived from murine fetal livers.
December 31, 2009 at 8:10 am
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| | In vitro hepatic maturation of human embryonic stem cells by using a mesenchymal cell line derived from murine fetal livers. Cell Tissue Res. 2009 Dec 30; Authors: Ishii T, Yasuchika K, Fukumitsu K, Kawamoto T, Kawamura-Saitoh M, Amagai Y, Ikai I, Uemoto S, Kawase E, Suemori H, Nakatsuji N Hepatocytes derived from human embryonic stem cells (hESCs) are an attractive cell source for regenerative medicine. We previously reported the differentiation of hESCs into alpha-fetoprotein (AFP)-producing endodermal cells by using extracellular matrix and growth factors. We also reported the establishment of the MLSgt20 cell line, which was derived from mesenchymal cells residing in murine fetal livers and accelerated the hepatic maturation of both murine hepatic progenitor cells and murine ESCs. In this study, hESC-derived AFP-producing cells were isolated by using a flow cytometer and co-cultured with MLSgt20 cells. The co-cultured hESC-derived AFP-producing cells had the immunocytological characteristics of hepatocytes, expressed mature hepatocyte markers (as indicated by reverse transcription and the polymerase chain reaction), and displayed higher hepatocyte functions including ammonia removal, cytochrome P450 3A4/7 activity, and the ability to produce and store glycogen. However, the MLSgt20 cells did not directly cause undifferentiated hESCs to mature into hepatocyte-like cells. The co-culture method was thus successfully shown to induce the differentiation of hESC-derived endodermal cells into functional hepatocyte-like cells. PMID: 20041263 [PubMed - as supplied by publisher] |
| Activation of Wnt/beta-Catenin Signaling Increases Insulin Sensitivity through a Reciprocal Regulation of Wnt10b and SREBP-1c in Skeletal Muscle Cells.
December 31, 2009 at 8:10 am
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| | Activation of Wnt/beta-Catenin Signaling Increases Insulin Sensitivity through a Reciprocal Regulation of Wnt10b and SREBP-1c in Skeletal Muscle Cells. PLoS One. 2009;4(12):e8509 Authors: Abiola M, Favier M, Christodoulou-Vafeiadou E, Pichard AL, Martelly I, Guillet-Deniau I BACKGROUND: Intramyocellular lipid accumulation is strongly related to insulin resistance in humans, and we have shown that high glucose concentration induced de novo lipogenesis and insulin resistance in murin muscle cells. Alterations in Wnt signaling impact the balance between myogenic and adipogenic programs in myoblasts, partly due to the decrease of Wnt10b protein. As recent studies point towards a role for Wnt signaling in the pathogenesis of type 2 diabetes, we hypothesized that activation of Wnt signaling could play a crucial role in muscle insulin sensitivity. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that SREBP-1c and Wnt10b display inverse expression patterns during muscle ontogenesis and regeneration, as well as during satellite cells differentiation. The Wnt/beta-catenin pathway was reactivated in contracting myotubes using siRNA mediated SREBP-1 knockdown, Wnt10b over-expression or inhibition of GSK-3beta, whereas Wnt signaling was inhibited in myoblasts through silencing of Wnt10b. SREBP-1 knockdown was sufficient to induce Wnt10b protein expression in contracting myotubes and to activate the Wnt/beta-catenin pathway. Conversely, silencing Wnt10b in myoblasts induced SREBP-1c protein expression, suggesting a reciprocal regulation. Stimulation of the Wnt/beta-catenin pathway i) drastically decreased SREBP-1c protein and intramyocellular lipid deposition in myotubes; ii) increased basal glucose transport in both insulin-sensitive and insulin-resistant myotubes through a differential activation of Akt and AMPK pathways; iii) restored insulin sensitivity in insulin-resistant myotubes. CONCLUSIONS/SIGNIFICANCE: We conclude that activation of Wnt/beta-catenin signaling in skeletal muscle cells improved insulin sensitivity by i) decreasing intramyocellular lipid deposition through downregulation of SREBP-1c; ii) increasing insulin effects through a differential activation of the Akt/PKB and AMPK pathways; iii) inhibiting the MAPK pathway. A crosstalk between these pathways and Wnt/beta-catenin signaling in skeletal muscle opens the exciting possibility that organ-selective modulation of Wnt signaling might become an attractive therapeutic target in regenerative medicine and to treat obese and diabetic populations. PMID: 20041157 [PubMed - in process] |
| Current concepts in platelet transfusion.
December 31, 2009 at 8:10 am
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| | Current concepts in platelet transfusion. Asian J Transfus Sci. 2009 Jan;3(1):18-21 Authors: Mohanty D This is the era of component therapy. Therefore there is a need for rational use of platelet concentrate. Lot of knowledge has been added recently in the field of platelet specially about the platelet rich plasma and its application in clinical practice. The current review focuses on improvement in preparation of platelet rich plasma, the procedure to make the same more safe and its rational use. Furthermore newer aspects of platelet concentrate use in surgical practice and for regenerative medicine has also been discussed. It also covers some progress and hurdles in preparation of platelet substitutes. PMID: 20041092 [PubMed - in process] |
| Effect of cyclic loading on in vitro degradation of poly(L-lactide-co-glycolide) scaffolds.
December 31, 2009 at 8:10 am
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| | Effect of cyclic loading on in vitro degradation of poly(L-lactide-co-glycolide) scaffolds. J Biomater Sci Polym Ed. 2010;21(1):53-66 Authors: Yang Y, Tang G, Zhao Y, Yuan X, Fan Y The in vitro degradation performance of porous scaffolds is very important in tissue engineering, especially the scaffold implanted in the environment imitating the repaired tissue. In this paper, the effect of cyclic loading on in vitro degradation of porous poly(L-lactide-co-glycolide) (PLGA) scaffolds was studied by incubating the samples in phosphate-buffered saline at 37 degrees C and pH 7.4 under dynamic conditions (cyclic loading) and static conditions (shaking water bath) for 12 weeks. The results showed earlier morphological variations and faster reduction in mass, dimensions and relative molecular mass of the scaffolds under dynamic conditions. Mechanical properties (the compressive modulus and the compressive strength) of the PLGA scaffolds under both conditions tended to increase in the first 3 weeks, but showed a decrease tendency afterward. The scaffolds under dynamic conditions were too brittle to be further characterized after degradation for 6 weeks, while those under static conditions endured degradation until week 8. The degradation mechanism of the PLGA scaffolds under cyclic loading was clearly explained and a three-stage degradation model based on the degradation behaviors of the scaffolds under two conditions was presented. PMID: 20040153 [PubMed - in process] |
| Controlled release of stromal-cell-derived factor-1 from gelatin hydrogels enhances angiogenesis.
December 31, 2009 at 8:10 am
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| | Controlled release of stromal-cell-derived factor-1 from gelatin hydrogels enhances angiogenesis. J Biomater Sci Polym Ed. 2010;21(1):37-51 Authors: Kimura Y, Tabata Y Controlled release of a chemokine, stromal-cell-derived factor-1 (SDF-1), could be achieved with gelatin hydrogels of release carrier. Gelatin was chemically derivatized to give it different electric charge and hydrophobicity. Among the derivatives, succinylated gelatin (Succ) of an anionic charge was the most suitable for preparation of the hydrogel in terms of SDF-1 release. The time profile of SDF-1 release from the hydrogel of succinylated gelatin could be controlled by changing the water content of hydrogel which could be modified by changing the conditions of hydrogel preparation. When evaluated after the subcutaneous implantation of Succ hydrogels incorporating SDF-1 or injection of SDF-1 solution, significantly stronger angiogenesis by the hydrogel was observed. The hydrogel implantation also enhanced the mRNA level of SDF-1 receptor at the site implanted. It is possible that the gelatin hydrogel enabled SDF-1 to be released locally, resulting in an enhanced angiogenesis at the site implanted. PMID: 20040152 [PubMed - in process] |
| Effects of the surface characteristics of polyhydroxyalkanoates on the metabolic activities and morphology of human mesenchymal stem cells.
December 31, 2009 at 8:10 am
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| | Effects of the surface characteristics of polyhydroxyalkanoates on the metabolic activities and morphology of human mesenchymal stem cells. J Biomater Sci Polym Ed. 2010;21(1):17-36 Authors: Yu BY, Chen PY, Sun YM, Lee YT, Young TH Polyhydroxyalkanoates (PHAs) are a newer family of biomaterials for tissue-engineering applications. The objective of this study is to investigate the behavior of human mesenchymal stem cells (hMSCs) grown on various PHA films. The surface characteristics of PHA co-polymer films were varied by the content of 3-hydroxyvalerate (HV) or 3-hydroxyhexanoate (HHx) and by the film preparation methods such as compression-molding and solvent-casting. Hyaluronic acid (HA) was further applied to modify the surface properties of PHA membranes. As HV content increased, the crystallinity and the hydrophobicity of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) membranes decreased and the metabolic activity of hMSCs raised, although the distribution and morphology of hMSCs did not show significant variation. Hyaluronic acid (HA) coating on PHA membranes could improve the metabolic activity and reduce the death rate of hMSCs. Aggregates and spheroidal clusters of hMSCs were found on the surface of cast poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) membranes. The growth of hMSCs was remarkably influenced by various surface characteristics of the PHA films. PMID: 20040151 [PubMed - in process] |
| Germ plasm-like Dot cells maintain the wound regenerative function after in vitro expansion.
December 31, 2009 at 8:10 am
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| | Germ plasm-like Dot cells maintain the wound regenerative function after in vitro expansion. Clin Exp Pharmacol Physiol. 2009 Dec 19; Authors: Kong W, Li S, Peter Lorenz H Summary 1. Wounds in fetal skin heal without scar, however the mechanism is unknown. We have identified a novel group of protein and nucleotides-positive particles in fetal and adult mouse blood and in human blood, and termed them "Dot cells". Freshly isolated Dot cells regenerate wounds with less scarring and can be cultured without feeder layers. 2. Because the morphology of Dot cells has never been described, in this study we describe the specific characterizations of Dot cells, including their growth pattern in vitro, and their expressions of stem cell markers using FACS analyses and immunofluorescent histology. Our data indicates that cultured Dot cells express stem cell surface markers and embryonic stem cell transcription markers, such as Oct4, Nanog and Sox-2. In addition, Dot cells express VASA, the germ plasm specific marker. 3. To confirm if Dot cells maintain their wound regenerative activity after in vitro expansion, in vitro cultured Dot cells were transplanted to wounded mice. Dot cells from albino mice maintain their wound regenerative activities after intravenous transplantation to black-background diabetic mice. Also, Dot cells regenerate both the epithelial and dermal cells in the wounds of wild-type mice. The regenerated hair follicles, smooth muscle, and dermal tissues express transiently to VASA. 4. Our data demonstrate that Dot cells are newly identified organisms located in the blood and bone marrow of mammals. They express germ cell, embryonic stem cell and adult stem cell markers. Dot cells maintain their regenerative function after in vitro expansion. PMID: 20039903 [PubMed - as supplied by publisher] |
| Calcium Phosphate Coated Electrospun Fiber Matrices as Scaffolds for Bone Tissue Engineering.
December 31, 2009 at 8:10 am
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| | Calcium Phosphate Coated Electrospun Fiber Matrices as Scaffolds for Bone Tissue Engineering. Langmuir. 2009 Dec 29; Authors: Nandakumar A, Yang L, Habibovic P, van Blitterswijk C Electrospun polymeric scaffolds are used for various tissue engineering applications. In this study, we applied a biomimetic coating method to provide electrospun scaffolds from a block copolymer-poly(ethylene oxide terephthalate)-poly(buthylene terephthalate), with a calcium phosphate layer to improve their bioactivity in bone tissue engineering. The in vitro studies with human mesenchymal stem cells demonstrated cell proliferation on both uncoated and coated samples. No significant effect of calcium phosphate coating was observed on the expression of alkaline phosphatase in vitro. Implantation of scaffold-goat mesenchymal stem cells constructs subcutaneously in nude mice resulted in bone formation in the calcium phosphate coated samples, in contrast to the uncoated ones, where no new bone formation was observed. The results of this study showed that the biomimetic method can successfully be used to coat electrospun scaffolds with a calcium phosphate layer, which improved the in vivo bioactivity of the polymer. PMID: 20039599 [PubMed - as supplied by publisher] |
| Reconstruction of a human hemicornea through natural scaffolds compatible with the growth of corneal epithelial stem cells and stromal keratocytes.
December 31, 2009 at 8:10 am
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| | Reconstruction of a human hemicornea through natural scaffolds compatible with the growth of corneal epithelial stem cells and stromal keratocytes. Mol Vis. 2009;15:2084-93 Authors: Barbaro V, Ferrari S, Fasolo A, Ponzin D, Di Iorio E PURPOSE: To reconstruct a human hemicornea in vitro by means of limbal stem cells cultured onto human keratoplasty lenticules (HKLs) and to obtain a natural corneal graft for clinical applications. METHODS: Limbal stem cells were seeded onto HKLs with or without the presence of feeder layers of lethally irradiated 3T3-J2 cells and compared with the current "gold standard" scaffold, i.e., the fibrin glue. The effects of the scaffold on the preservation of stemness and/or induction of differentiation pathways were investigated through analysis of a variety of markers, including p63 and DeltaNp63alpha for stemness, 14-3-3sigma for early differentiation, keratins 3, 14, 12, and 19 to determine cell phenotype, and alpha6, beta1, and beta4 integrins to evaluate interactions with the stroma. Integrity of the stroma was assessed through analysis of keratan sulfate, CD-34 and aldehyde dehydrogenase 3A1 (ALDH3A1) (for keratocytes), visual system homeobox 1 (VSX1), and alpha-smooth muscle actin (alpha-SMA) (for fibroblasts and myofibroblasts). The structural properties of the reconstructed "hemicornea" were investigated through scanning electron microscopy. To evaluate the preservation of the stemness potential, cells were trypsinized from each scaffold and clonogenic/proliferative characteristics analyzed. RESULTS: Limbal stem cells expanded onto HKLs gave rise to a stratified squamous keratinized epithelium morphologically similar to that of normal corneas. The resulting corneal epithelium was characterized by basal expression of p63 and DeltaNp63alpha, while expression of 14-3-3sigma, keratin 3, and keratin 12 was found in the upper cell layers. The basal cuboidal epithelial cells were anchored to the basement membrane and expressed keratin 14 and alpha6, beta1, and beta4 integrins. In the stroma of HKLs, keratocytes maintained the biosynthetic and phenotypic appearances typical of resting/quiescent cells and expressed keratan sulfate, CD-34, and ALDH3A1. Fibroblastic transformation was observed with the appearance of VSX1 and alpha-SMA. Scanning electron microscopy analysis showed that HKLs maintained their native conformation with collagen fibrils interconnected to the network and parallel to the corneal surface. HKLs did not alter the clonogenic/proliferative capacity of limbal stem cells. No differences were seen when HKL was compared to fibrin glue, one of the scaffolds currently used for limbal stem cell transplantation. CONCLUSIONS: Our findings demonstrate that HKL could be a suitable scaffold for corneal epithelial stem cells as they were shown to proliferate, express differentiation markers, and bind to the underlying stroma with no alterations in clonogenic potential. HKLs have some advantages over currently used scaffolds, such as the possibility to allow cell growth with no feeder layers, to be freeze dried, and to preserve the integrity and viability of stromal keratocytes. The development of a tissue-engineered "hemicornea" might offer new therapeutic perspectives to patients affected by total limbal stem cell deficiency with stromal scarring. PMID: 19862337 [PubMed - indexed for MEDLINE] |
| In vivo evaluation of a novel scaffold for artificial corneas prepared by using ultrahigh hydrostatic pressure to decellularize porcine corneas.
December 31, 2009 at 8:10 am
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| | In vivo evaluation of a novel scaffold for artificial corneas prepared by using ultrahigh hydrostatic pressure to decellularize porcine corneas. Mol Vis. 2009;15:2022-8 Authors: Sasaki S, Funamoto S, Hashimoto Y, Kimura T, Honda T, Hattori S, Kobayashi H, Kishida A, Mochizuki M PURPOSE: To evaluate the stability and biocompatibility of artificial corneal stroma that was prepared by using ultrahigh hydrostatic pressurization treatment to decellularize corneas. METHODS: The porcine cornea was decellularized by two methods, a detergent method and an ultrahigh hydrostatic pressure (UHP) method. Either 1% w/v Triton X-100 or sodium dodecyl sulfate (SDS) was used for the detergent method, and 10,000 atmospheres (atm; 7.6x10(6) mmHg) was applied to the cornea for 10 min at 10 degrees C by a high-pressure machine for the UHP method. Hematoxylin-eosin staining was performed to confirm the removal of the corneal cells, and then decellularized porcine corneal stroma was implanted into rabbit corneal pockets. After eight weeks, the rabbit eyes were enucleated to examine the tissue compatibility of the implanted stroma. RESULTS: Complete decellularization was confirmed only in corneas treated by the UHP method, and little inflammation was seen when they were implanted into the rabbit corneal pockets. CONCLUSIONS: Porcine corneal stroma completely decellularized by the UHP method has extremely high biocompatibility and is a possible corneal scaffold for an artificial cornea. PMID: 19844587 [PubMed - indexed for MEDLINE] |
| Fabrication and characterization of prosurvival growth factor releasing, anisotropic scaffolds for enhanced mesenchymal stem cell survival/growth and orientation.
December 31, 2009 at 8:10 am
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| | Fabrication and characterization of prosurvival growth factor releasing, anisotropic scaffolds for enhanced mesenchymal stem cell survival/growth and orientation. Biomacromolecules. 2009 Sep 14;10(9):2609-18 Authors: Wang F, Li Z, Tamama K, Sen CK, Guan J Scaffolds that not only mimic the mechanical and structural properties of the target tissue but also support cell survival/growth are likely necessary for the development of mechanically functional cardiovascular tissues. To reach these goals, we have generated scaffolds that are elastic to approximate soft tissue mechanical properties, are nanofibrous to mimic fibrous nature of extracellular matrix (ECM), have aligned structure to guide cellular alignment, and are capable of releasing insulin-like growth factor (IGF-1) to administrate cellular growth and survival. We have developed a technique that can quickly fabricate (<3 h) such scaffolds by simultaneously electrospinning elastase-sensitive polyurethaneurea nanofibers, encapsulating IGF-1 into poly(lactide-co-glycolide) (PLGA) microspheres and assembling them into scaffolds. Scaffold morphology, mechanical properties, degradation with or without elastase, and bioactivity of the released IGF-1 were assessed. The scaffolds had degree of alignment approximately 70%. They were flexible and relatively strong, with tensile strengths of 3.4-11.1 MPa, elongations at break of 71-88%, and moduli of 2.3-7.9 MPa at the alignment direction. IGF-1 release profile and bioactivity were dependent on PLGA content and molecular weight and IGF-1 loading. The released IGF-1 remained bioactive for 4 weeks. The fabricated nanofibers were elastase-sensitive with weight remaining <59% after a 4-week degradation in the presence of elastase. Mesenchymal stem cells (MSCs) were seeded on the scaffolds and cultured either under normal culture conditions (21% O(2), 5% CO(2), and 20% fetal bovine serum (FBS)) or hypoxia/nutrient starvation conditions (5% O(2), 5% CO(2), and 1% FBS) to evaluate the effect of IGF-1 loading on cell growth and survival. Under normal culture conditions, MSCs were found to align on the scaffolds with a degree of alignment matching that of the scaffold. The IGF-1 loaded scaffolds enhanced MSC growth during a 7-day culture period, with higher IGF-1 content showing better stimulus effect. Under hypoxia/nutrient starvation conditions, the IGF-1 loaded scaffolds were found to significantly improve MSC survival. PMID: 19689108 [PubMed - indexed for MEDLINE] | | | 
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