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| Engineering Musculoskeletal Tissues with Human Embryonic Germ Cell Derivatives.
February 24, 2010 at 9:40 AM
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| | Engineering Musculoskeletal Tissues with Human Embryonic Germ Cell Derivatives. Stem Cells. 2010 Feb 22; Authors: Varghese S, Hwang N, Ferran A, Hillel A, Theprungsirikul P, Canver AC, Zhang Z, Gearhart J, Elisseeff J The cells derived from differentiating embryoid bodies of human embryonic germ (hEG) cells express a broad spectrum of gene markers and have been induced towards ecto- and endodermal lineages. We describe here in vitro and in vivo differentiation of hEG-derived cells (LVEC line) towards mesenchymal tissues. The LVEC cells express many surface marker proteins characteristic of mesenchymal stem cells and differentiated into cartilage, bone, and fat. Homogenous hyaline cartilage was generated from cells after 63 population-doublings. In vivo results demonstrate cell survival, differentiation, and tissue formation. The high proliferative capacity of hEG-derived cells and their ability to differentiate and form 3D mesenchymal tissues without teratoma formation underscores their significant potential for regenerative medicine. The adopted coculture system also provides new insights into how a microenvironment comprising of extracellular and cellular components may be ha! rnessed to generate hierarchically complex tissues from pluripotent cells. PMID: 20178108 [PubMed - as supplied by publisher] | | |
| In vivo cyclic loading as a potent stimulatory signal for bone formation inside tissue engineering scaffold.
February 24, 2010 at 9:40 AM
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| | In vivo cyclic loading as a potent stimulatory signal for bone formation inside tissue engineering scaffold. Eur Cell Mater. 2010;19:41-9 Authors: Roshan-Ghias A, Terrier A, Bourban PE, Pioletti DP In clinical situations, bone defects are often located at load bearing sites. Tissue engineering scaffolds are future bone substitutes and hence they will be subjected to mechanical stimulation. The goal of this study was to test if cyclic loading can be used as stimulatory signal for bone formation in a bone scaffold. Poly(L-lactic acid) (PLA)/ 5% beta-tricalcium phosphate (beta-TCP) scaffolds were implanted in both distal femoral epiphyses of eight rats. Right knees were stimulated (10N, 4Hz, 5 min) five times, every two days, starting from the third day after surgery while left knees served as control. Finite element study of the in vivo model showed that the strain applied to the scaffold is similar to physiological strains. Using micro-computed tomography (CT), all knees were scanned five times after the surgery and the related bone parameters of the newly formed bone were quantified. Statistical modeling was used to estimate the evolution of these parameters! as a function of time and loading. The results showed that mechanical stimulation had two effects on bone volume (BV): an initial decrease in BV at week 2, and a long-term increase in the rate of bone formation by 28%. At week 13, the BV was then significantly higher in the loaded scaffolds. PMID: 20178097 [PubMed - in process] | | |
| Derivation of the King's College London human embryonic stem cell lines.
February 24, 2010 at 9:40 AM
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| | Derivation of the King's College London human embryonic stem cell lines. In Vitro Cell Dev Biol Anim. 2010 Feb 23; Authors: Stephenson EL, Braude PR Since the derivation of the first human embryonic stem cell (hESC) line in 1998, there has been substantial interest in the potential of these cells for regenerative medicine and cell therapy and in the use of hESCs carrying clinically relevant genetic mutations as models for disease research and therapeutic target identification. There is still a need to improve derivation efficiency and further the understanding of the basic biology of these cells and to develop clinical grade culture systems with the aim of producing cell lines suitable for subsequent manipulation for therapy. The derivation of initial hESC lines at King's College London is discussed here, with focus on derivation methodology. Each of the derivations was distinctive. Although the stage and morphology of each blastocyst were generally similar in each attempt, the behaviour of the colonies was unpredictable; colony morphology and development was different with each attempt. Days 5, 6 and 7 blasto! cysts were used successfully, and the number of days until appearance of stem-like cells varied from 4 to 14 d. Routine characterisation analyses were performed on three lines, all of which displayed appropriate marker expression and survived cryopreservation-thaw cycles. From the lines discussed, four are at various stages of the deposition process with the UKSCB, one is pending submission and two are unsuitable for banking. Continued open and transparent reporting of results and collaborations will maximise the efficiency of derivation and facilitate the development of standardised protocols for the derivation and early culture of hESC lines. PMID: 20178004 [PubMed - as supplied by publisher] | | |
| Derivation of a new human embryonic stem cell line, Endeavour-2, and its characterization.
February 24, 2010 at 9:40 AM
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| | Derivation of a new human embryonic stem cell line, Endeavour-2, and its characterization. In Vitro Cell Dev Biol Anim. 2010 Feb 23; Authors: Sidhu KS, Ryan JP, Lees JG, Tuch BE Here, we describe the derivation of a novel human embryonic stem cell (hESC) line, Endeavour-2 (E-2), propagated on human fetal fibroblasts (HFF) in a serum-replacement media. The inner cell mass (ICM) was manually dissected from the blastocyst without using immunodissection and, therefore, antibodies from animal sources. A total of 20 embryos were thawed and cultured, eight embryos were hatched, and five ICMs were obtained. They were transferred onto HFF used as feeder layer, and one colony representing the initial cell proliferation of a new hESC line, E-2, was obtained. The newly emerged hESC colony was passaged first by physical dissection and subsequently by enzymatic dissociation. E-2 has been in culture for over 6 months and has been shown to possess typical features of a pluripotent hESC line including expression of stem cell surface markers (SSEA4, TRA-160, and integrin alpha-6), intracellular alkaline phosphatase, and pluripotency gene markers, OCT4 and ! NANOG. This hESC line shows lineage-specific differentiation into various representative cell types expressing markers characteristic of the three somatic germ layers under both in vitro and in vivo conditions. E-2 line shows a normal karyotype (46 XX) and has been successfully cryopreserved and thawed several times using slow-freezing procedures. E-2 adds to the repertoire of existing hESC lines for research and development purposes in the field of regenerative medicine. PMID: 20178001 [PubMed - as supplied by publisher] | | |
| Derivation and characterization of three new human embryonic stem cell lines in Finland.
February 24, 2010 at 9:40 AM
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| | Derivation and characterization of three new human embryonic stem cell lines in Finland. In Vitro Cell Dev Biol Anim. 2010 Feb 23; Authors: Skottman H Human embryonic stem cell (hESC) lines can be established from the preimplantation embryos. Due to their ability to differentiate into all three embryonic layers, hESC are of significant interest as a renewable source of cell material for different applications, especially for cell replacement therapy. Since the establishment of the first hESC lines in 1998, several studies have described the derivation and culture of new hESC lines using various derivation methods and culture conditions. Our group has currently established eight new hESC lines of which three of the latest ones are described in a more detailed way in this report. The described lines have been established using mechanical derivation methods for surplus bad quality embryos and culture conditions containing human foreskin fibroblast feeder cells and serum-free culture medium. All the new lines have a normal karyotype and typical hESC characteristics analyzed in vitro. The described hESC lines are ava! ilable for research purposes upon request ( www.regea.fi ). PMID: 20177999 [PubMed - as supplied by publisher] | | |
| Derivation of human embryonic stem cells at the Center of Regenerative Medicine in Barcelona.
February 24, 2010 at 9:40 AM
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| | Derivation of human embryonic stem cells at the Center of Regenerative Medicine in Barcelona. In Vitro Cell Dev Biol Anim. 2010 Feb 23; Authors: Aran B, Rodríguez-Pizà I, Raya A, Consiglio A, Muñoz Y, Barri PN, Izpisúa JC, Veiga A We report here the legislative issues related to embryo research and human embryonic stem cell (hESC) research in Spain and the derivation of nine hESC lines at the Center of Regenerative Medicine in Barcelona. You can find the information for obtaining our lines for research purposes at blc@cmrb.eu . PMID: 20177997 [PubMed - as supplied by publisher] | | |
| Characterization and differentiation potential of rat ventral mesencephalic neuronal progenitor cells immortalized with SV40 large T antigen.
February 24, 2010 at 9:40 AM
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| | Characterization and differentiation potential of rat ventral mesencephalic neuronal progenitor cells immortalized with SV40 large T antigen. Cell Tissue Res. 2010 Feb 23; Authors: Nobre A, Kalve I, Cesnulevicius K, Rangancokova D, Ratzka A, Halfer N, Wesemann M, Krampfl K, Claus P, Grothe C Neuronal progenitor cells (NPCs) possess high potential for use in regenerative medicine. To overcome their limited mitotic competence, various immortalization strategies have been applied that allow their prolonged maintenance and expansion in vitro. Such immortalized cells can be used for the design and discovery of new cell-based therapies for neurodegenerative diseases, such as Parkinson's disease. We immortalized rat ventral mesencephalic NPCs by using SV40 large T antigen (SV40Tag). All cell clones displayed a two- to three-fold higher proliferation rate compared with the primary cells. In order to induce dopaminergic differentiation of generated cell clones, both glial-derived neurotrophic factor and di-butyryl cyclic adenosine monophosphate were applied. Treated cells were then characterized regarding the expression of dopaminergic lineage markers, differentiation of various cell populations, calcium imaging in the presence of kainate, and immunohistochemi! stry after intrastriatal transplantation. Treated cells displayed morphological maturation, and calcium imaging revealed neuronal properties in the presence of kainate. These cells also expressed low mRNA levels of the dopamine transporter and tyrosine hydroxylase (TH), although no TH-immunopositive neurons were found. Intrastriatal transplantation into the neurotoxin-lesioned rats did not induce further differentiation. As an alternative approach, we silenced SV40Tag with short interfering RNA, but this was not sufficient to trigger differentiation into dopaminergic neurons. Nevertheless, neuronal and glial cells were detected as shown by beta-tubulin type III and glial fibrillary acidic protein staining, respectively. SV40Tag cells are suitable for carrying out controlled genetic modifications as shown by overexpression of enhanced green fluorescence protein after efficient non-viral transfection. PMID: 20177706 [PubMed - as supplied by publisher] | | |
| Histone Deacetylase Inhibitors Attenuate Acute Lung Injury During Cecal Ligation and Puncture-Induced Polymicrobial Sepsis.
February 24, 2010 at 9:40 AM
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| | Histone Deacetylase Inhibitors Attenuate Acute Lung Injury During Cecal Ligation and Puncture-Induced Polymicrobial Sepsis. World J Surg. 2010 Feb 23; Authors: Zhang L, Jin S, Wang C, Jiang R, Wan J BACKGROUND: The histone deacetylase (HDAC) inhibitors have emerged as the useful reagents that epigenetically modulate the expression of various genes. In the present study, the effects of HDAC inhibitors on the expression of inflammation-related genes and lung injury during sepsis were investigated. METHODS: Mice were pretreated with two structurally unrelated HDAC inhibitors, Trichostatin A (TSA) and sodium butyrate (SB). Thirty minutes later, mice underwent cecal ligation and puncture (CLP)-induced sepsis. Lung injury and the expression of inflammation-related molecules were determined. In addition, survival was assessed post-CLP. RESULTS: Our results indicated that administration of TSA or SB alleviated sepsis-induced lung injury. This was accompanied by reduced neutrophil infiltration, decreased intercellular adhesion molecule-1 (ICAM-1) and E-selectin expression in lung tissue, and lower interleukin-6 (IL-6) level in plasma. In addition, treatment with HDAC ! inhibitors significantly prolonged the survival time of CLP mice. CONCLUSIONS: These data indicated that the HDAC inhibitors, based on modulating the key enzymes linked to acetylation modification, effectively attenuate intrapulmonary inflammatory response, thus significantly alleviating lung injury during sepsis. PMID: 20177680 [PubMed - as supplied by publisher] | | |
| Follistatin-like-1, a diffusible mesenchymal factor determines the fate of epithelium.
February 24, 2010 at 9:40 AM
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| | Follistatin-like-1, a diffusible mesenchymal factor determines the fate of epithelium. Proc Natl Acad Sci U S A. 2010 Feb 22; Authors: Umezu T, Yamanouchi H, Iida Y, Miura M, Tomooka Y Mesenchyme is generally believed to play critical roles in "secondary induction" during organogenesis. Because of the complexity of tissue interactions in secondary inductions, however, little is known about the precise mechanisms at the cellular and molecular levels. We have demonstrated that, in mouse oviductal development, the mesenchyme determines the fate of undetermined epithelial cells to become secretory or cilial cells. We have established a model for studying secondary induction by establishing clonal epithelial and mesenchymal cell lines from perinatal p53(-/-) mouse oviducts. The signal sequence trap method collected candidate molecules secreted from mesenchymal cell lines. Naive epithelial cells exposed to Follistatin-like-1 (Fstl1), one of the candidates, became irreversibly committed to expressing a cilial epithelial marker and differentiated into ciliated cells. We concluded that Fstl1 is one of the mesenchymal factors determining oviductal epithel! ial cell fate. This is a unique demonstration that the determination of epithelial cell fate is induced by a single diffusible factor. PMID: 20176958 [PubMed - as supplied by publisher] | | |
| The effect of BMP-2 on micro and macroscale osteointegration of biphasic calcium phosphate scaffolds with multiscale porosity.
February 24, 2010 at 9:40 AM
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| | The effect of BMP-2 on micro and macroscale osteointegration of biphasic calcium phosphate scaffolds with multiscale porosity. Acta Biomater. 2010 Feb 19; Authors: Lan Levengood SK, Polak SJ, Poellmann MJ, Hoelzle DJ, Maki AJ, Clark SG, Wheeler MB, Wagoner Johnson AJ It is well established that scaffolds for applications in bone tissue engineering require interconnected pores on the order of 100um for bone growth, and nutrient and waste transport. As a result, most studies of have focused on macroporosity (>100um). More recently, researchers have also investigated the role of microporosity in calcium phosphaste (CaP) based scaffolds. Osteointegration into macropores improves when scaffold rods or struts contain micropores, typically defined as pores less than approximately 50um. We recently demonstrated multiscale osteointegration, or growth into both macropores and micropores (<10um), of rods in biphasic CaP (BCP) scaffolds. The combined effect of BMP-2, a potent osteoinductive growth factor, and multiscale porosity has yet to be investigated. In this study, we implanted BCP scaffolds into porcine mandibles for 3, 6, 12, and 24 weeks and evaluated the effect of BMP-2 on multiscale osteointegration. Results showed, for t! his in vivo model, that BMP-2 influences osteointegration at the microscale, but not at the macroscale. Cell density was higher in the rod micropores for scaffolds containing BMP-2 compared to controls at all time points, but BMP-2 was not required for bone formation in micropores. In contrast, there was essentially no difference in fraction of bone in macropores for scaffolds with BMP-2 compared to controls. Additionally, bone in macropores seemed to have reached steady state by 3 weeks. Multiscale osteointegration results in composites that are fully osteointegrated, with no "dead space," and are likely to have not only a continuous cell network, but also the potential for enhanced load transfer and improved mechanical properties. PMID: 20176148 [PubMed - as supplied by publisher] | | |
| Fibroblasts facilitate the engraftment of embryonic stem cell-derived cardiomyocytes on three-dimensional collagen matrices and aggregation in hanging drops.
February 24, 2010 at 9:40 AM
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| | Fibroblasts facilitate the engraftment of embryonic stem cell-derived cardiomyocytes on three-dimensional collagen matrices and aggregation in hanging drops. Stem Cells Dev. 2010 Feb 22; Authors: Pfannkuche K, Neuss S, Pillekamp F, Frenzel L, Attia W, Hannes T, Salber J, Hoss M, Zenke M, Fleischmann B, Hescheler J, Saric T There is growing interest in use of cardiomyocytes purified from embryonic stem (ES) cells for tissue engineering and cardiomyoplasty. However, most of transplanted cells are lost shortly after transplantation due to the lack of integration into the host tissue and subsequent apoptosis. Here we examine whether murine embryonic fibroblasts (MEFs) can support the integration of purified murine ES cell-derived cardiomyocytes in a three-dimensional tissue culture model based on a freezed-dryed collagen matrix with tubular structure. Collagen matrix was seeded either with cardiomyocytes alone or in combination with MEFs. The collagen sponges that were transplanted with cardiomyocytes alone did neither show morphological nor functional integration of viable cells. Cardiomyocytes also did not appear to be capable of attaching quantitatively to any of 16 different two-dimensional biomaterials. However, cardiomyocytes co-cultured with MEFs formed fibre-like structures of r! od-shaped cells with organized sarcomeric structure that contracted spontaneously. Electrical coupling between cardiomyocytes was suggested by strong expression of connexin 43. In addition, MEFs as well as cardiac fibroblasts supported re-aggregation of dissociated cardiomyocytes in hanging drops in the absence of collagen matrix. We conclude that fibroblasts promote cardiomyocyte engraftment and formation of functional three-dimensional tissue in vitro. Elucidation of the mechanism of this phenomenon may help improving the integration of cardiomyocytes in vivo. PMID: 20175666 [PubMed - as supplied by publisher] | | |
| Invited commentary.
February 24, 2010 at 9:40 AM
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| | Invited commentary. J Vasc Surg. 2010 Jan;51(1):183 Authors: Golden MA PMID: 20117501 [PubMed - indexed for MEDLINE] | | |
| The fate of an endothelium layer after preconditioning.
February 24, 2010 at 9:40 AM
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| | The fate of an endothelium layer after preconditioning. J Vasc Surg. 2010 Jan;51(1):174-83 Authors: Yazdani SK, Tillman BW, Berry JL, Soker S, Geary RL BACKGROUND: A strategy in minimizing thrombotic events of vascular constructs is to seed the luminal surface with autologous endothelial cells (ECs). The task of seeding ECs can be achieved via bioreactors, which induce mechanical forces (shear stress, strain, pressure) onto the ECs. Although bioreactors can achieve a confluent layer of ECs in vitro, their acute response to blood remains unclear. Moreover, the necessary mechanical conditions that will increase EC adhesion and function remain unclear. We hypothesize that preconditioning seeded endothelium under physiological flow will enhance their retention and function. OBJECTIVE: To determine the role of varying preconditioning protocols on seeded ECs in vitro and in vivo. METHODS: Scaffolds derived from decelluarized arteries seeded with autologous ECs were preconditioned for 9 days. Three specific protocols, low steady shear stress (SS), high SS, and cyclic SS were investigated. After preconditioning, the seed! ed grafts were exposed to 15 minutes of blood via an ex vivo arteriovenous shunt model or alternately an in vivo arteriovenous bypass graft model. RESULTS: The shunt model demonstrated ECs remained intact for all conditions. In the arteriovenous bypass model, only the cyclic preconditioned grafts remained intact, maintained morphology, and resisted the attachment of circulating blood elements such as platelets, red blood cells, and leukocytes. Western blotting analysis demonstrated an increase in the protein expression of eNOS and prostaglandin I synthase for the cyclic high shear stress-conditioned cells relative to cells conditioned with high shear stress alone. CONCLUSION: Cyclic preconditioning has been shown here to increase the ECs ability to resist blood flow-induced shear stress and the attachment of circulating blood elements, key attributes in minimizing thrombotic events. These studies may ultimately establish protocols for the formation of a more durable endothe! lial monolayer that may be useful in the context of small vess! el arter ial reconstruction. PMID: 20117500 [PubMed - indexed for MEDLINE] | | |
| Bladder dysfunction in a new mutant mouse model with increased superoxide--lack of nitric oxide?
February 24, 2010 at 9:40 AM
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| | Bladder dysfunction in a new mutant mouse model with increased superoxide--lack of nitric oxide? J Urol. 2010 Feb;183(2):780-5 Authors: Soler R, Füllhase C, Lu B, Bishop CE, Andersson KE PURPOSE: Nitric oxide mediates urethral smooth muscle relaxation and may also be involved in detrusor activity control. Mice with mutation in the Immp2l gene have high superoxide ion levels and a consequent decrease in the bioavailable amount of nitric oxide. We studied bladder function in this mouse model. MATERIAL AND METHODS: Young male mutants at ages 4 to 6 months, old female mutants at age 18 months and healthy WT age matched controls were used. The detrusor contractile response to carbachol and electrical field stimulation was tested in isolated detrusor strips in organ baths. In vivo bladder function was evaluated by cystometry in conscious animals. RESULTS: Young male mutants had significantly lower micturition and higher post-void residual volume than WT controls. They had pronounced voiding difficulty and strained when initiating micturition. Detrusor contractile responses to carbachol and electrical field stimulation were similar in mutant and WT mice.! Old female mutant mice had lower bladder capacity and micturition volume, and higher micturition frequency and bladder-to-body weight ratio than WT controls. In the in vitro study detrusor strips from mutants showed a lower maximum response to carbachol. CONCLUSIONS: Mice with mutation in the Immp2l gene have bladder dysfunction, mainly characterized by emptying abnormalities in young males and increased detrusor activity in old females. Detrusor function was preserved in young males and impaired in old females. These animals are a natural model of oxidative stress with low bioavailable nitric oxide. Thus, they are interesting tools in which to evaluate the role of these conditions on bladder dysfunction. PMID: 20022053 [PubMed - indexed for MEDLINE] | | |
| Alginate-encapsulated human hepatoma C3A cells for use in a bioartificial liver device - the hybrid-MDS.
February 24, 2010 at 9:40 AM
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| | Alginate-encapsulated human hepatoma C3A cells for use in a bioartificial liver device - the hybrid-MDS. Int J Artif Organs. 2009 Nov;32(11):769-78 Authors: Harm S, Stroble K, Hartmann J, Falkenhagen D PURPOSE: The aim of this study was to encapsulate C3A cells into alginate microcapsules with an average diameter of < or =100 microm, thus enabling them to be recirculated in a bioartificial liver device based on MDS (Microsphere-based Detoxification System) technology. The microcapsules have to be permeable for essential proteins such as albumin. METHODS: C3A cells were encapsulated using alginate. The resulting alginate beads were coated with poly(diallyldimethylammoniumchloride) (pDADMAC) and poly(sodium-p-styrenesulfonate) (pSS). Their mechanical stability was tested by recirculation of the microcapsule suspension, while their permeability was determined by reverse-size exclusion chromatography and by the use of a confocal laser microscope. The metabolic activities of encapsulated C3A cells were compared to freely growing adherent C3A cells in static cultivation models. The metabolic functionality of encapsulated C3A cells in static conditions was compared ! to encapsulated C3A cells in a dynamic model. RESULTS: The mean diameter of the resulting microcapsules was 86 mum. Our experiments show that these microcapsules were permeable for albumin and the high flow rate of 600 ml/min in a dynamic model has no influence on the survival and the metabolic activities of the encapsulated cells during the tested time of 24 hours. CONCLUSIONS: Alginate microcapsules containing C3A cells can be used to produce albumin and growth factors in a bioartificial or hybrid liver support system. Thanks to their small diameter, the microcapsules in suspension can be recirculated in the MDS. PMID: 20020408 [PubMed - indexed for MEDLINE] | | |
| Innovation in basic science: stem cells and their role in the treatment of paediatric cardiac failure--opportunities and challenges.
February 24, 2010 at 9:40 AM
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| | Innovation in basic science: stem cells and their role in the treatment of paediatric cardiac failure--opportunities and challenges. Cardiol Young. 2009 Nov;19 Suppl 2:74-84 Authors: Kaushal S, Jacobs JP, Gossett JG, Steele A, Steele P, Davis CR, Pahl E, Vijayan K, Asante-Korang A, Boucek RJ, Backer CL, Wold LE Heart failure is a leading cause of death worldwide. Current therapies only delay progression of the cardiac disease or replace the diseased heart with cardiac transplantation. Stem cells represent a recently discovered novel approach to the treatment of cardiac failure that may facilitate the replacement of diseased cardiac tissue and subsequently lead to improved cardiac function and cardiac regeneration. A stem cell is defined as a cell with the properties of being clonogenic, self-renewing, and multipotent. In response to intercellular signalling or environmental stimuli, stem cells differentiate into cells derived from any of the three primary germ layers: ectoderm, endoderm, and mesoderm, a powerful advantage for regenerative therapies. Meanwhile, a cardiac progenitor cell is a multipotent cell that can differentiate into cells of any of the cardiac lineages, including endothelial cells and cardiomyocytes. Stem cells can be classified into three categories: ! (1) adult stem cells, (2) embryonic stem cells, and (3) induced pluripotential cells. Adult stem cells have been identified in numerous organs and tissues in adults, including bone-marrow, skeletal muscle, adipose tissue, and, as was recently discovered, the heart. Embryonic stem cells are derived from the inner cell mass of the blastocyst stage of the developing embryo. Finally through transcriptional reprogramming, somatic cells, such as fibroblasts, can be converted into induced pluripotential cells that resemble embryonic stem cells. Four classes of stem cells that may lead to cardiac regeneration are: (1) Embryonic stem cells, (2) Bone Marrow derived stem cells, (3) Skeletal myoblasts, and (4) Cardiac stem cells and cardiac progenitor cells. Embryonic stem cells are problematic because of several reasons: (1) the formation of teratomas, (2) potential immunologic cellular rejection, (3) low efficiency of their differentiation into cardiomyocytes, typically 1% in culture! , and (4) ethical and political issues. As of now, bone marrow! derived stem cells have not been proven to differentiate reproducibly and reliably into cardiomyocytes. Skeletal myoblasts have created in vivo myotubes but have not electrically integrated with the myocardium. Cardiac stem cells and cardiac progenitor cells represent one of the most promising types of cellular therapy for children with cardiac failure. PMID: 19857353 [PubMed - indexed for MEDLINE] | | |
| Endothelial progenitor cells as a sole source for ex vivo seeding of tissue-engineered heart valves.
February 24, 2010 at 9:40 AM
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| | Endothelial progenitor cells as a sole source for ex vivo seeding of tissue-engineered heart valves. Tissue Eng Part A. 2010 Jan;16(1):257-67 Authors: Sales VL, Mettler BA, Engelmayr GC, Aikawa E, Bischoff J, Martin DP, Exarhopoulos A, Moses MA, Schoen FJ, Sacks MS, Mayer JE PURPOSES: We investigated whether circulating endothelial progenitor cells (EPCs) can be used as a cell source for the creation of a tissue-engineered heart valve (TEHV). METHODS: Trileaflet valved conduits were fabricated using nonwoven polyglycolic acid/poly-4-hydroxybutyrate polymer. Ovine peripheral blood EPCs were dynamically seeded onto a valved conduit and incubated for 7, 14, and 21 days. RESULTS: Before seeding, EPCs were shown to express CD31(+), eNOS(+), and VE-Cadherin(+) but not alpha-smooth muscle actin. Histological analysis demonstrated relatively homogenous cellular ingrowth throughout the valved conduit. TEHV constructs revealed the presence of endothelial cell (EC) markers and alpha-smooth muscle actin(+) cells comparable with native valves. Protein levels were comparable with native valves and exceeded those in unseeded controls. EPC-TEHV demonstrated a temporal pattern of matrix metalloproteinases-2/9 expression and tissue inhibitors of metall! oproteinase activities comparable to that of native valves. Mechanical properties of EPC-TEHV demonstrated significantly greater stiffness than that of the unseeded scaffolds and native valves. CONCLUSIONS: Circulating EPC appears to have the potential to provide both interstitial and endothelial functions and could potentially serve as a single-cell source for construction of autologous heart valves. PMID: 19698056 [PubMed - indexed for MEDLINE] | | |
| Human embryonic stem cell-derived keratinocytes exhibit an epidermal transcription program and undergo epithelial morphogenesis in engineered tissue constructs.
February 24, 2010 at 9:40 AM
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| | Human embryonic stem cell-derived keratinocytes exhibit an epidermal transcription program and undergo epithelial morphogenesis in engineered tissue constructs. Tissue Eng Part A. 2010 Jan;16(1):213-23 Authors: Metallo CM, Azarin SM, Moses LE, Ji L, de Pablo JJ, Palecek SP Human embryonic stem (hES) cells are an attractive source of cellular material for scientific, diagnostic, and potential therapeutic applications. Protocols are now available to direct hES cell differentiation to specific lineages at high purity under relatively defined conditions; however, researchers must establish the functional similarity of hES cell derivatives and associated primary cell types to validate their utility. Using retinoic acid to initiate differentiation, we generated high-purity populations of keratin 14+ (K14) hES cell-derived keratinocyte (hEK) progenitors and performed microarray analysis to compare the global transcriptional program of hEKs and primary foreskin keratinocytes. Transcriptional patterns were largely similar, though gene ontology analysis identified that genes associated with signal transduction and extracellular matrix were upregulated in hEKs. In addition, we evaluated the ability of hEKs to detect and respond to environmenta! l stimuli such as Ca(2+), serum, and culture at the air-liquid interface. When cultivated on dermal constructs formed with collagen gels and human dermal fibroblasts, hEKs survived and proliferated for 3 weeks in engineered tissue constructs. Maintenance at the air-liquid interface induced stratification of surface epithelium, and immunohistochemistry results indicated that markers of differentiation (e.g., keratin 10, involucrin, and filaggrin) were localized to suprabasal layers. Although the overall tissue morphology was significantly different compared with human skin samples, organotypic cultures generated with hEKs and primary foreskin keratinocytes were quite similar, suggesting these cell types respond to this microenvironment in a similar manner. These results represent an important step in characterizing the functional similarity of hEKs to primary epithelia. PMID: 19686061 [PubMed - indexed for MEDLINE] | | |
| Controlled dynamization to enhance reconstruction capacity of tissue-engineered bone in healing critically sized bone defects: an in vivo study in goats.
February 24, 2010 at 9:40 AM
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| | Controlled dynamization to enhance reconstruction capacity of tissue-engineered bone in healing critically sized bone defects: an in vivo study in goats. Tissue Eng Part A. 2010 Jan;16(1):201-12 Authors: Hou T, Li Q, Luo F, Xu J, Xie Z, Wu X, Zhu C Tissue-engineered bone (TEB) has shown to be an effective alternative to conventional gold-standard autogenous bone for the repair of critically sized bone defects (CSBD). Moderate axial interfragmentary movement (IFM) has been shown to promote bone healing in conventional models. This study explored the use of IFM to enhance the capacity of TEB in the repair of CSBD using a goat model. CSBD were created in a goat model. Dynamic intramedullary rods designed to supply axial IFMs within 10% of the interfragmentary strain were used to stabilize CSBD goat femur models, whose bone defects were filled with TEB. Bone regeneration was evaluated using radionuclide bone imaging, roentgenographic analysis, periosteal callus area, computed tomography value score, biomechanical analysis, and histological observation. Compared with the static intramedullary rods, the dynamic intramedullary rod group showed an increase in early-stage callus formation and blood supply to the call! us tissue, better differentiation of fibrous and cartilaginous tissue into bone tissue, improved strength and stiffness of callus tissue in late-stage healing, and overall better functional recovery of the goat femur. This showed that moderate axial IFM could promote the osteogenesis and reconstruction of TEB in vivo. PMID: 19678758 [PubMed - indexed for MEDLINE] | | |
| Transplantation of a tissue-engineered human vascularized cardiac muscle.
February 24, 2010 at 9:40 AM
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| | Transplantation of a tissue-engineered human vascularized cardiac muscle. Tissue Eng Part A. 2010 Jan;16(1):115-25 Authors: Lesman A, Habib M, Caspi O, Gepstein A, Arbel G, Levenberg S, Gepstein L Myocardial regeneration strategies have been hampered by the lack of sources for human cardiomyocytes (CMs) and by the significant donor cell loss following transplantation. We assessed the ability of a three-dimensional tissue-engineered human vascularized cardiac muscle to engraft in the in vivo rat heart and to promote functional vascularization. Human embryonic stem cell-derived CMs alone or with human endothelial cells (human umbilical vein endothelial cells) and embryonic fibroblasts (triculture constructs) were seeded onto biodegradable porous scaffolds. The resulting tissue constructs were transplanted to the in vivo rat heart and formed cardiac tissue grafts. Immunostaining studies for human-specific CD31 and alpha-smooth muscle actin demonstrated the formation of both donor (human) and host (rat)-derived vasculature within the engrafted triculture tissue constructs. Intraventricular injection of fluorescent microspheres or lectin resulted in their incorp! oration by human-derived vessels, confirming their functional integration with host coronary vasculature. Finally, the number of blood vessels was significantly greater in the triculture tissue constructs (60.3 +/- 8/mm(3), p < 0.05) when compared with scaffolds containing only CMs (39.0 +/- 14.4/mm(3)). In conclusion, a tissue-engineered human vascularized cardiac muscle can be established ex vivo and transplanted in vivo to form stable grafts. By utilizing a multicellular preparation we were able to increase biograft vascularization and to show that the preexisting human vessels can become functional and contribute to tissue perfusion. PMID: 19642856 [PubMed - indexed for MEDLINE] | | |
| Hypoxic preconditioning of human mesenchymal stem cells overcomes hypoxia-induced inhibition of osteogenic differentiation.
February 24, 2010 at 9:40 AM
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| | Hypoxic preconditioning of human mesenchymal stem cells overcomes hypoxia-induced inhibition of osteogenic differentiation. Tissue Eng Part A. 2010 Jan;16(1):153-64 Authors: Volkmer E, Kallukalam BC, Maertz J, Otto S, Drosse I, Polzer H, Bocker W, Stengele M, Docheva D, Mutschler W, Schieker M Osteogenic differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts is a prerequisite for subsequent bone formation. Numerous studies have explored osteogenic differentiation under standard tissue culture conditions, which usually employ 21% of oxygen. However, bone precursor cells such as hMSCs reside in stem cell niches of low-oxygen atmospheres. Furthermore, they are subjected to low oxygen concentrations when cultured on three-dimensional scaffolds in vitro, and even more so after transplantation when vascularization has yet to be established. Similarly, hMSCs are exposed to low oxygen in the fracture microenvironment following bony injury. Recent studies revealed that hypoxic preconditioning improves cellular engraftment and survival in low-oxygen atmospheres. In our study we investigated the osteogenic differentiation potential of hMSCs under 2% O(2) (hypoxia) in comparison to a standard tissue culture oxygen atmosphere of 21% (normoxia). We ! assessed the osteogenic differentiation of hMSCs following hypoxic preconditioning to address whether this pretreatment is beneficial for subsequent differentiation processes as well. To validate our findings we carefully characterized the extent of hypoxia exerted and its effect on cell survival and proliferation. We found that hMSCs proliferate better if cultured under 2% of oxygen. We confirmed that osteogenic differentiation of hMSCs is indeed inhibited if osteogenic induction is carried out under constant hypoxia. Finally, we showed for the first time that hypoxic preconditioning of hMSCs prior to osteogenic induction restores osteogenic differentiation of hMSCs under hypoxic conditions. Collectively, our results indicate that maintaining constant levels of oxygen improves the osteogenic potential of hMSCs and suggest that low oxygen concentrations may preserve the stemness of hMSCs. In addition, our data support the hypothesis that if low-oxygen atmospheres are expect! ed at the site of implantation, hypoxic pretreatment may be be! neficial for the cells' subsequent in vivo performance. PMID: 19642854 [PubMed - indexed for MEDLINE] | | |
| Correlation of donor age and telomerase activity with in vitro cell growth and replicative potential for dermal fibroblasts and keratinocytes.
February 24, 2010 at 9:40 AM
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| | Correlation of donor age and telomerase activity with in vitro cell growth and replicative potential for dermal fibroblasts and keratinocytes. J Tissue Viability. 2009 Nov;18(4):109-16 Authors: Ng MH, Aminuddin BS, Hamizah S, Lynette C, Mazlyzam AL, Ruszymah BH Previous studies suggested telomerase activity as a determinant of cell replicative capacity by delaying cell senescence. This study aimed to evaluate the feasibility of adopting telomerase activity as a selection criterion for in vitro expanded skin cells before autologous transplantation. Fibroblasts and keratinoctyes were derived from the same consenting patients aged 9-69 years, and cultured separately in serum-supplemented and serum-free media, respectively. Telomerase activity of fresh and cultured cells were measured and correlated with cell growth rate, donor age and passage number. The results showed that telomerase activity and cell growth were independent of donor age for both cell types. Telomerase was expressed in freshly digested epidermis and dermis and continued expressing in vitro. Keratinocytes consistently showed 3-12 folds greater telomerase activity than fibroblast both in vivo and in vitro. Conversely, growth rate for fibroblast exceeded that! of keratinocyte. Telomerase activity decreased markedly at Passage 6 for keratinocytes and ceased by Passage 3 for fibroblasts. The decrease or cessation of telomerase activity coincided with senescence for keratinocyte but not for fibroblast, implying a telomerase-regulated cell senescence for the former and hence a predictor of replicative capacity for this cell type. Relative telomerase activity for fibroblasts from the younger age group was significantly higher than that from the older age group; 69.7% higher for fresh isolates and 31.1% higher at P0 (p<0.05). No detectable telomerase activity was to be found at later subcultures for both age groups. Similarly for keratinocytes, telomerase activity in the younger age group was significantly higher (p<0.05) compared to that in the older age group; 507.7% at P0, 36.8% at P3 and the difference was no longer significant at P6. In conclusion, the study provided evidence that telomerase sustained the proliferation of k! eratinocytes but not fibroblasts. Telomerase activity is an im! portant criterion for continued survival and replication of keratinocytes, hence its positive detection before transplantation is desirable. Inferring from our results, the use of keratinocytes from Passage 3 or lesser for construction of skin substitute or cell-based therapy is recommended owing to their sustained telomerase expression. PMID: 19632116 [PubMed - indexed for MEDLINE] | | |
| Collagen type II enhances chondrogenesis in adipose tissue-derived stem cells by affecting cell shape.
February 24, 2010 at 9:40 AM
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| | Collagen type II enhances chondrogenesis in adipose tissue-derived stem cells by affecting cell shape. Tissue Eng Part A. 2010 Jan;16(1):81-90 Authors: Lu Z, Doulabi BZ, Huang C, Bank RA, Helder MN Ideally, biomaterials have inductive properties, favoring specific lineage differentiation. For chondrogenic induction, these properties have been attributed to collagen type II. However, the underlying mechanisms are largely unknown. This study aimed to investigate whether collagen type II favors chondrogenic induction by affecting cell shape through beta1 integrins and Rho A/Rock signaling. For this purpose, adipose tissue-derived stem cells (ASCs) were encapsulated in collagen type I or II gels and cultured in plain and chondrogenic medium. It was demonstrated that (i) ASCs showed more efficient chondrogenic induction (higher collagen X, aggrecan, sox6, sox9, and collagen II gene expression) in both plain and chondrogenic media in collagen type II versus collagen type I gels; (ii) ASCs showed lower Rock 2 gene expression and a more rounded cell shape in collagen type II versus type I gels when grown in plain medium; (iii) Rock inhibitor (Y27632) more effectivel! y enhanced chondrogenic gene expression of ASCs in collagen type I than in collagen type II gels, and diminished differences in chondrogenic gene expression and cell shape of ASCs between the two gel types; and (iv) beta1 integrins blocking not only reduced the differences of chondrogenic gene expression but also eliminated the differences of Rock 1 and Rock 2 gene expressions and cell shape when comparing ASCs embedded in collagen type I and II gels. We conclude that collagen type II provides the inductive signaling for chondrogenic differentiation in ASCs by evoking a round cell shape through beta1 integrin-mediated Rho A/Rock signaling pathway. PMID: 19624244 [PubMed - indexed for MEDLINE] | | |
| Promoted adipogenesis of rat mesenchymal stem cells by transfection of small interfering RNA complexed with a cationized dextran.
February 24, 2010 at 9:40 AM
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| | Promoted adipogenesis of rat mesenchymal stem cells by transfection of small interfering RNA complexed with a cationized dextran. Tissue Eng Part A. 2010 Jan;16(1):21-31 Authors: Nagane K, Jo J, Tabata Y The objective of this study is to investigate the possibility of small interfering RNA (siRNA) complexed with a cationized dextran of nonviral carrier to biologically modify the adipogenesis extent of bone marrow-derived mesenchymal stem cells (MSC). Spermine was chemically introduced to the hydroxyl groups of dextran to prepare the cationized dextran (spermine-dextran). The spermine-dextran could form a complex with siRNA, and the physicochemical properties were changed by the molecular weight of dextran, the molar percentage of spermine introduced to dextran, and the molar ratio of nitrogen molecule of spermine-dextran to the phosphorous ones of siRNA (N/P ratio). The gene expression level of luciferase or green fluorescence protein was significantly suppressed by transfection with the complex of spermine-dextran and siRNA. The gene expression level by the complex decreased with an increase in the extent of complex internalized. Biochemical experiments revealed ! that culture in an adipogenic differentiation medium allowed MSC to differentiate into adipogenic cells. However, upon culturing with siRNA of anti-transcription coactivator containing PDZ-binding motif (TAZ) for osteogenic differentiation complexed with the spermine-dextran, the adipogenesis of MSC was further promoted. It is concluded that the spemine-dextran was a promising nonviral carrier to suppress the expression level of differentiation gene, resulting in the modification of cell differentiation direction. PMID: 19604070 [PubMed - indexed for MEDLINE] | | | | 
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