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Vitro Culture (vitro + culture)
Terms modified by Vitro Culture Selected AbstractsIn Vitro Culture and Differentiation of Buffalo (Bubalus bubalis) SpermatogoniaREPRODUCTION IN DOMESTIC ANIMALS, Issue 2 2010B Xie Contents The objective of this study was to develop a culture system which could support buffalo spermatogonia differentiation into spermatids in vitro. Testes from 3- to 5-month-old buffaloes were decapsulated and seminiferous tubules were enzymatically dissociated to recover spermatogonia and sertoli cells. The cells were cultured in modified Dulbecco modified Eagle medium supplemented with different concentrations of foetal bovine serum, retinol, testosterone for 2 months at 37°C. Spermatogonia and sertoli cells were identified with an antibody against c-kit or GATA4, respectively. The viability of spermatogonia in the media supplemented with different concentrations of serum was all significantly higher (p < 0.05) compared with that in the medium without serum. A-paired or A-aligned spermatogonia and spermatogonial colonies (AP-positive) were observed after 7,10 days of culture and spermatid-like cells with a flagellum (6,8 ,m) appeared after 30 days of culture. For cultured conditions, retinol could not significantly promote the formation of spermatid-like cells (p > 0.05), whereas supplementation of testosterone could significantly promote (p < 0.05) the formation of spermatid-like cells after 41 days of culture. The expression of the spermatid-specific marker gene (PRM2) was identified after 30 days of culture by RT-PCR. Yet, the transition protein 1 (TP1, a haploid makers) was not detected. Meanwhile, spermatids developed in vitro were also confirmed by Raman spectroscopy. These results suggest that buffalo spermatogonia could differentiate into spermatids in vitro based on the analysis of their morphology, PRM2 expression and Raman spectroscopy. Yet, the normality of the spermatid-like cells was not supported by TP1 expression. [source] In Vitro Culture of the Obligate Parasite Spongospora subterranea (Cercozoa; Plasmodiophorida) Associated with Root-Inducing Transferred-DNA Transformed Potato Hairy RootsTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 6 2007XINSHUN QU ABSTRACT. Spongospora subterranea is a soil-borne, obligate parasitic protist that causes powdery scab of potatoes. In this study, an in vitro culture system was developed for the maintenance and proliferation of the protist in potato hairy roots. The hairy roots of potato were induced in vitro with Agrobacterium rhizogenes. Cystosori of S. subterranea from potato scab lesions were surface disinfested and used to inoculate potato hairy roots. Plasmodia, zoosporangia, and cystosori were observed microscopically in the hairy roots within 6 wk after inoculation, indicating the completion of the life cycle of S. subterranea in vitro. This is the first in vitro culture system for S. subterranea, and will be a valuable tool to study fundamental and practical aspects of the biology of the parasite. [source] Myosins of Babesia bovis: Molecular characterisation, erythrocyte invasion, and phylogenyCYTOSKELETON, Issue 4 2002A.E. Lew Abstract Using degenerate primers, three putative myosin sequences were amplified from Australian isolates of Babesa bovis and confirmed as myosins (termed Bbmyo-A, Bbmyo-B, and Bbmyo-C) from in vitro cultures of the W strain of B. bovis. Comprehensive analysis of 15 apicomplexan myosins suggests that members of Class XIV be defined as those with greater than 35% myosin head sequence identity and that these be further subclassed into groups bearing above 50,60% identity. Bbmyo-A protein bears a strong similarity with other apicomplexan myosin-A type proteins (subclass XIVa), the Bbmyo-B myosin head protein sequence exhibits low identity (35,39%) with all members of Class XIV, and 5,-sequence of Bbmyo-C shows strong identity (60%) with P. falciparum myosin-C protein. Domain analysis revealed five divergent IQ domains within the neck of Pfmyo-C, and a myosin-N terminal domain as well as a classical IQ sequence unusually located within the head converter domain of Bbmyo-B. A cross-reacting antibody directed against P. falciparum myosin-A (Pfmyo-A) revealed a zone of approximately 85 kDa in immunoblots prepared with B. bovis total protein, and immunofluorescence inferred stage-specific myosin-A expression since only 25% of infected erythrocytes with mostly paired B. bovis were immuno-positive. Multiplication of B. bovis in in vitro culture was inhibited by myosin- and actin-binding drugs at concentrations lower than those that inhibit P. falciparum. This study identifies and classifies three myosin genes and an actin gene in B. bovis, and provides the first evidence for the participation of an actomyosin-based motor in erythrocyte invasion in this species of apicomplexan parasite. Cell Motil. Cytoskeleton 52:202,220, 2002. © 2002 Wiley-Liss, Inc. [source] Contribution of mesothelium-derived cells to liver sinusoids in avian embryosDEVELOPMENTAL DYNAMICS, Issue 3 2004J.M. Pérez-Pomares Abstract The developing liver is vascularized through a complex process of vasculogenesis that leads to the differentiation of the sinusoids. The main structural elements of the sinusoidal wall are endothelial and stellate (Ito) cells. We have studied the differentiation of the hepatic sinusoids in avian embryos through confocal colocalization of differentiation markers, in ovo direct labeling of the liver mesothelium, induced invasion of the developing chick liver by quail proepicardial cells, and in vitro culture of chimeric aggregates. Our results show that liver mesothelial cells give rise to mesenchymal cells which intermingle between the growing hepatoblast cords and become incorporated to the sinusoidal wall, contributing to both endothelial and stellate cell populations. We have also shown that the proepicardium, a mesothelial tissue anatomically continuous with liver mesothelium, is able to form sinusoid-like vessels into the hepatic primordium as well as in cultured aggregates of hepatoblasts. Thus, both intrinsic or extrinsic mesothelium-derived cells have the developmental potential to contribute to the establishment of liver sinusoids. Developmental Dynamics 229:465,474, 2004. © 2004 Wiley-Liss, Inc. [source] T-box gene products are required for mesenchymal induction of epithelial branching in the embryonic mouse lungDEVELOPMENTAL DYNAMICS, Issue 1 2003Judith A. Cebra-Thomas Abstract The regulation of signaling pathways is a prerequisite for coordinating the induction between mesenchymal and epithelial tissues during morphogenesis. Mesenchymal FGF10 is known to be an important paracrine factor regulating the branching morphogenesis of the bronchial epithelium. By using antisense oligonucleotides (AS ODNs) and in vitro culture of embryonic lungs, we demonstrate that the transcription factors Tbx4 and Tbx5 are critical for the expression of mesenchymal FGF10. Treatment of embryonic lung cultures with AS ODNs to Tbx4 and Tbx5 reduces the level of these transcripts, suppresses Fgf10 expression in the mesenchyme, and completely eliminates the formation of new lung branches. If FGF10 is locally replaced in these AS ODN-treated lungs, epithelial branching is restored. These studies provide evidence that the production of branching signals by the lung mesenchyme is mediated by T-box genes. © 2002 Wiley-Liss, Inc. [source] Induction of chondrogenesis in neural crest cells by mutant fibroblast growth factor receptorsDEVELOPMENTAL DYNAMICS, Issue 2 2002Anita Petiot Abstract Activating mutations in human fibroblast growth factor receptors (FGFR) result in a range of skeletal disorders, including craniosynostosis. Because the cranial bones are largely neural crest derived, the possibility arises that increased FGF signalling may predispose to premature/excessive skeletogenic differentiation in neural crest cells. To test this hypothesis, we expressed wild-type and mutant FGFRs in quail embryonic neural crest cells. Chondrogenesis was consistently induced when mutant FGFR1-K656E or FGFR2-C278F were electroporated in ovo into stage 8 quail premigratory neural crest, followed by in vitro culture without FGF2. Neural crest cells electroporated with wild-type FGFR1 or FGFR2 cDNAs exhibited no chondrogenic differentiation in culture. Cartilage differentiation was accompanied by expression of Sox9, Col2a1, and osteopontin. This closely resembled the response of nonelectroporated neural crest cells to FGF2 in vitro: 10 ng/ml induces chondrogenesis, Sox9, Col2a1, and osteopontin expression, whereas 1 ng/ml FGF2 enhances cell survival and Sox9 and Col2a1 expression, but never induces chondrogenesis or osteopontin expression. Transfection of neural crest cells with mutant FGFRs in vitro, after their emergence from the neural tube, in contrast, produced chondrogenesis at a very low frequency. Hence, mutant FGFRs can induce cartilage differentiation when electroporated into premigratory neural crest cells but this effect is drastically reduced if transfection is carried out after the onset of neural crest migration. © 2002 Wiley-Liss, Inc. [source] Characteristics of strawberry plants propagated by in vitro bioreactor culture and ex vitro propagation methodENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 3 2009Samir C. Debnath Abstract Reproducible protocol for regeneration of complete plantlets from ,Bounty' strawberry (Fragaria ananassa Duch.), using a combination of gelled medium and bioreactor system, has been standardized. Sepals, leaf discs, and petiole halves produced multiple buds and shoots when cultured on semi solid-gelled medium containing 4 ,M thidiazuron (TDZ) for 4 wk followed by transferring in liquid medium containing 2,,M TDZ in a bioreactor system and cultured for another 4 wk. TDZ induced shoot proliferation at 0.1,,M in the bioreactor system but inhibited shoot elongation. TDZ-induced shoots were elongated and rooted in vitro on gelled medium containing 2,,M zeatin. Such bioreactor-derived tissue culture (BC) plantlets obtained from sepal explants were grown ex vitro and compared with those propagated by tissue culture on gelled medium (GC) and by conventional runner cuttings (RC), for growth, morphology, anthocyanin content, and antioxidant activity after three growth seasons. The BC and GC plants produced more crowns, runners, leaves, and berries than the RC plants although berry weight per plant did not differ significantly. BC and GC plants produced berries with more anthocyanin contents and antioxidant activities than those produced by the RC plants. However, intersimple sequence repeat (ISSR) marker assay produced a homogenous amplification profile in the tissue culture and donor control plants confirming the clonal fidelity of micropropagated plants. In vitro culture on TDZ and zeatin-containing nutrient media apparently induced the juvenile branching characteristics that favored enhanced vegetative growth with more crown, runners, leaf, and berry production. [source] Production of Taxol fromPhyllosticta spinarum, an endophytic fungus ofCupressus sp.ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2008R. Senthil Kumaran Abstract Taxol production during the cultivation on a modified liquid and potato dextrose broth medium was indicated for the first time to occur in Phyllosticta spinarum, an endophytic fungus isolated from the needles of Cupressus sp. The presence of taxol in the fungal culture filtrate was confirmed by chromatographic and spectroscopic methods of analysis. The amount of taxol produced by this fungus was quantified by high performance liquid chromatography. The maximum amount of taxol production was obtained in this fungus when grown on M1D medium (235,,g/L) followed by PDB medium (125,,g/L). The results indicate that P.,spinarum is an excellent candidate for taxol production. The production rate was 4.7,×,103 -fold higher than that found in the culture broth of an earlier reported fungus, Taxomyces andreanae. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of human cancer cells tested in an apoptotic assay. [source] Disruption of nasopharyngeal epithelium by pneumococci is density-linkedEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 4 2003K. Lagrou Abstract Background The aim of this project was to study the influence of pneumococci on nasopharyngeal epithelial integrity as a function of time and pneumococcal density. Materials and methods Cell layers of an in vitro model of human nasopharyngeal epithelium were inoculated with different pneumococcal strains. The transepithelial electrical resistance (TEER), a measure of the integrity of the cell layers, and the pneumococcal concentration in the apical fluid on the epithelial cells were measured at different times after inoculation. Results Pneumococci caused a decrease in the TEER when a density of 1 × 107 CFU mL,1 was reached. The growth rate of pneumococci in our in vitro model differed between the strains tested and, for the same strain, between in vitro culture on the epithelial cells and broth culture. Differences in timing of the onset of decrease in the TEER between strains were the result of differences in growth rate on the epithelial cells. Antibiotic-induced lysis of pneumococci caused an immediate decrease in the TEER of the cell layers. Conclusion Pneumococci cause a decrease in the TEER at a density of 1 × 107 CFU mL,1. Our hypothesis is that this decrease in the TEER is the result of quorum-induced lysis of the pneumococci. [source] Platelet-derived growth factor (PDGF) in human acute myelogenous leukemia: PDGF receptor expression, endogenous PDGF release and responsiveness to exogenous PDGF isoforms by in vitro cultured acute myelogenous leukemia blastsEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 6 2001Brynjar Foss Abstract: We investigated effects of Platelet-derived growth factor (PDGF) and Platelet factor 4 (PF-4) on the functional characteristics of native, human acute myelogenous leukemia (AML) blasts. AML blast expression of the PDGF-receptor ,-chain was detected for a subset of patients (45%), whereas PDGF-receptor ,-chain expression was detected for most patients (90%). Constitutive AML blast release of the PDGF-AB isoform (the major form also derived from normal platelets) was detected for 43% of patients, whereas PDGF-BB release was not detected for any patient. The PDGF isoforms AA, AB and BB had dose-dependent and divergent effects on spontaneous and cytokine-dependent AML blast proliferation, whereas for constitutive cytokine secretion (IL-1,, IL-6, TNF-,) inhibitory effects were rare and all three isoforms usually had no effect or enhanced the constitutive secretion. The PDGF effects were caused by a direct effect on the AML blasts and were not dependent on the presence of serum. The PDGF effects could also be detected after in vitro culture of AML cells in the presence of IL-4+granulocyte-macrophage colony stimulating factor. PF-4 had divergent effects on proliferation and cytokine secretion by native AML blasts. Our results suggest that exogenous (e.g. platelet-secreted) PDGF and PF-4 can function as regulators of leukemic hematopoiesis and possibly also modulate the function of residual AML cells in peripheral blood stem cell grafts. On the other hand, endogenous release of PDGF-AB by native blasts may modulate the function of normal cells in the bone marrow microenvironment (e.g. bone marrow stromal cells). [source] In vitro culture of skin-homing T lymphocytes from inflammatory skin diseasesEXPERIMENTAL DERMATOLOGY, Issue 5 2005Karen Bang Abstract:, We, in this study, describe how T lymphocytes in a skin biopsy can proliferate in vitro for up to 3 months by using T-cell growth factors , interleukin-2 (IL-2) and IL-4 yielding approximately 100,160 million T lymphocytes within 1 month. We established cell lines from three tuberculin skin tests, four positive patch tests, 15 of 16 biopsies from atopic dermatitis (AD), 15 of 19 biopsies from mycosis fungoides (MF), 12 of 24 biopsies from psoriasis vulgaris, which was significantly less than AD (P < 0.05), and with a reduced cumulative number of lymphocytes (P < 0.05). Omitting IL-2 and IL-4 led to immediate halt of proliferation. Blood mononuclear cells from patients and biopsies from healthy persons never gave cell lines. All cells were T lymphocytes expressing CD45RO+, HLA-DR+ and CD150. The CD7 expression was significantly increased in cell lines from AD (P < 0.05). T-cell receptor ,-chain studies by using reverse transcription-polymerase chain reaction showed that all T lymphocytes had access to the skin compartment. Single-stranded conformational analysis showed clonally expanded T cells numbering between 40 and 60 clones. After approximately 2 months of growth, the mean CD4+ : CD8+ ratio was for AD 1.20, MF 0.65 and psoriasis 0.85. Patients with AD treated with cyclosporin-A had almost no growth of CD8+ cells in vitro. Our findings indicate a changed homeostasis among skin-homing lymphocytes for in vitro culture. Our culture system of skin-homing T lymphocytes leads to a prominent cellular expansion allowing for a range of studies of in vivo activated skin T lymphocytes. [source] Correlation of psoriasis activity with abundance of CD25+CD8+ T cells: conditions for cloning T cells from psoriatic plaquesEXPERIMENTAL DERMATOLOGY, Issue 10 2004Wayan M. Kohlmann Abstract:, The role of T cells in the pathogenesis of psoriasis is widely acknowledged. However, key aspects of their precise function in the disease as well as the relative pathogenic contribution of T-cell subsets are still unknown. T-cell clones have been isolated from psoriatic plaques but a study of conditions affecting the isolation and expansion of T-cell clones from psoriatic skin has not been reported to date. Here, we observe a correlation of disease activity with the frequency of the CD3+CD8+CD25+ subset. We show that prolonged in vitro culture changes the phenotypic subset distribution of T-cell lines derived from psoriatic skin and that T-cell clones can be isolated by sorting of CD25+ cells emigrated from skin fragments after 7 days. We evaluate various conditions affecting expansion of psoriatic T-cell clones in vitro and show that blocking apoptosis can facilitate proliferation of activated T-cell clones in vitro. Our results indicate a prominent role of the CD8+CD25+ T-cell subset in disease pathogenesis and should be useful in the design of experiments aiming at a systematic analysis of the specificity of clones present in psoriatic plaques. [source] Association of immunological disorders in lethal side effect of NSAIDs on ,-glucan-administered miceFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1 2001Hideaki Takahashi Abstract (1,3)-,- d -Glucan (,-glucan) is a biological response modifier that regulates host immune response. We have found that the combination of a ,-glucan and a non-steroidal anti-inflammatory drug (NSAID), indomethacin (IND), induced lethal toxicity in mice [Yoshioka et al. (1998) FEMS Immunol. Med. Microbiol., 21, 171,179]. This study was undertaken to analyze the mechanism of the lethal side effect. Combination of a ,-glucan and IND increased the number of leukocytes, especially macrophages and neutrophils, in various organs and these cells were activated. The activated state of these cells was supported by the enhanced production of interferon-, in the presence of IND in vitro culture of the peritoneal exudate cells. Intestinal bacterial flora was translocated into the peritoneal cavity in these mice to cause peritonitis. Comparing the toxicity of various NSAIDs, nabumetone, a partially cyclooxygenase-2-selective NSAID with weaker toxicity to the gastrointestinal tract, did not exhibit a lethal side effect. These facts strongly suggested that gastrointestinal damage by NSAIDs was more severe in ,-glucan-administered mice, resulting in peritonitis by enteric bacteria and leading to death. [source] Evidence for Antinociceptive Activity of Botulinum Toxin Type A in Pain ManagementHEADACHE, Issue 2003K. Roger Aoki PhD The neurotoxin, botulinum toxin type A, has been used successfully, in some patients, as an analgesic for myofascial pain syndromes, migraine, and other headache types. The toxin inhibits the release of the neurotransmitter, acetylcholine, at the neuromuscular junction thereby inhibiting striated muscle contractions. In the majority of pain syndromes where botulinum toxin type A is effective, inhibiting muscle spasms is an important component of its activity. Even so, the reduction of pain often occurs before the decrease in muscle contractions suggesting that botulinum toxin type A has a more complex mechanism of action than initially hypothesized. Current data points to an antinociceptive effect of botulinum toxin type A that is separate from its neuromuscular activity. The common biochemical mechanism, however, remains the same between botulinum toxin type A's effect on the motor nerve or the sensory nerve: enzymatic blockade of neurotransmitter release. The antinociceptive effect of the toxin was reported to block substance P release using in vitro culture systems.1 The current investigation evaluated the in vivo mechanism of action for the antinociceptive action of botulinum toxin type A. In these studies, botulinum toxin type A was found to block the release of glutamate. Furthermore, Fos, a product of the immediate early gene, c- fos, expressed with neuronal stimuli was prevented upon peripheral exposure to the toxin. These findings suggest that botulinum toxin type A blocks peripheral sensitization and, indirectly, reduces central sensitization. The recent hypothesis that migraine involves both peripheral and central sensitization may help explain how botulinum toxin type A inhibits migraine pain by acting on these two pathways. Further research is needed to determine whether the antinociceptive mechanism mediated by botulinum toxin type A affects the neuronal signaling pathways that are activated during migraine. [source] Liver endothelial cells promote LDL-R expression and the uptake of HCV-like particles in primary rat and human hepatocytes,HEPATOLOGY, Issue 2 2006Yaakov Nahmias Low-density lipoprotein (LDL) is an important carrier of plasma cholesterol and triglycerides whose concentration is regulated by the liver parenchymal cells. Abnormal LDL regulation is thought to cause atherosclerosis, while viral binding to LDL has been suggested to facilitate hepatitis C infection. Primary hepatocytes quickly lose the ability to clear LDL during in vitro culture. Here we show that the coculture of hepatocytes with liver sinusoidal endothelial cells (LSEC) significantly increases the ability of hepatocytes to uptake LDL in vitro. LDL uptake does not increase when hepatocytes are cocultured with other cell types such as fibroblasts or umbilical vein endothelial cells. We find that LSECs induce the hepatic expression of the LDL receptor and the epidermal growth factor receptor. In addition, while hepatocytes in single culture did not take up hepatitis C virus (HCV)-like particles, the hepatocytes cocultured with LSECs showed a high level of HCV-like particle uptake. We suggest that coculture with LSECs induces the emergence of a sinusoidal surface in primary hepatocytes conducive to the uptake of HCV-like particles. In conclusion, our findings describe a novel model of polarized hepatocytes in vitro that can be used for the study of LDL metabolism and hepatitis C infection. (HEPATOLOGY 2006;43:257,265.) [source] Gene expression characteristics of CD28null memory phenotype CD8+ T cells and its implication in T-cell agingIMMUNOLOGICAL REVIEWS, Issue 1 2005Monchou Fann Summary:, Accumulation of CD28nullCD8+ T cells is considered as one of the hallmarks of aging in the human immune system. However, the precise changes of CD28nullCD8+ T cells, compared to those of the precursor CD28+CD8+ memory T cells, have not been determined. In this study, we present an analysis of the global gene expression profiles of CD28+ and CD28null memory phenotype CD8+ T cells. These two CD8+ T subsets exhibited an overall similar gene expression profile with only a few dozen genes that were differentially expressed. A wide range of functions, including co-stimulation, effector activity, signaling, and transcription, were possessed by these differentially expressed genes, reflecting significant functional changes of CD28null memory phenotype CD8+ T cells from their CD28+ counterparts. In addition, CD28null memory CD8+ T cells expressed several natural killer cell receptors and high levels of granzymes, perforin, and FasL, indicating an increasing capacity for cytotoxicity during memory CD8+ T-cell aging. Interestingly, in vitro culture of these two subsets with interleukin-15 showed that similar gene expression changes occurred in both subsets. Our analysis provides the gene expression portraits of CD28null memory phenotype CD8+ T cells and alteration from their CD28+ counterparts and suggests potential mechanisms of T-cell aging. [source] Trehalose and trehalose-hydrolyzing enzyme in the haemolymph of Locusta migratoria infected with Metarhizium anisopliae strain CQMa102INSECT SCIENCE, Issue 4 2007HUA ZHAO Abstract Topical application of the Metarhizium anisopliae var. acridum specialist strain CQMa102 to the locust Locusta migratoria manilensis results in changes of the concentrations of trehalose and glucose in the haemolymph. Micrographs of the locust haemolymph shows Metarhizium anisopliae can effectivly penetrate the external skeleton of locust and after 2 days infection, the hyphae body will appear in the haemolymph of infected insects. The time in decrease of trehalose concentration coincided with that in increase of trehalose-hydrolysing enzyme activity in the haemolymph of the fungus-infected insects. Overlay gel analysis indicated there was considerably more trehalose-hydrolysing activity in the haemolymph of locusts infected by fungus than in controls. A comparable isoform was identified in in vitro culture of the fungus, suggesting a fungal origin for the in vivo enzyme. Haemolymph trehalose decreased significantly during mycosis of locusts by M. anisopliae. All these results suggested that this fungus may take advantage of competing nutrient utilization against the insect by its trehalose-hydrolyzing enzyme secretion. It may provide fundamental knowledge for fungal pathogenesis. [source] Photoinitiating polymerization to prepare biocompatible chitosan hydrogelsJOURNAL OF APPLIED POLYMER SCIENCE, Issue 2 2008Xiaohong Hu Abstract Chitosan hydrogels were prepared from water soluble chitosan derivatives (chitosan-MA-LA, CML) by photoinitiating polymerization under the existence of Irgacure2959 and the irradiation of UV light. The CML was obtained by amidation of the amine groups of chitosan with lactic acid and methacrylic acid. Gelation time of the hydrogel could be adjusted within a range of 5,50 min, and controlled by factors such as the degree of MA substitution, initiator concentration, existence of oxygen, and salt. The dry hydrogel adsorbed tens to hundred times of water, forming a highly hydrated gel. The swelling ratio was smaller at the higher degree of MA substitution, higher pH, and higher salt concentration. Rheological test showed that the hydrogel is elastomeric in the measuring frequency range, with a storage modulus and loss modulus of 0.8,7 kPa and 10,100 Pa, respectively. In vitro culture of chondrocytes demonstrated that the cells could normally proliferate in the extractant of the hydrogels, showing no cytotoxicity at lower initiator concentration. By contrast, the extractant of the hydrogel made by the redox initiating system, i.e., ammonium persulfate (APS) and N,N,N,,N,-tetramethylethylenediamine (TEMED), showed apparent cytotoxicity. Thus, the chitosan hydrogels initiated by the Irgacure2959 have better comprehensive properties, in particular better biocompatibility, and are more suitable for biomedical applications. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source] Human islet-derived precursor cells can cycle between epithelial clusters and mesenchymal phenotypesJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 8b 2009Behrous Davani Abstract We showed previously that undifferentiated, proliferating human islet-derived precursor cells (hIPCs) are a type of mesenchymal stem/stromal cell (MSC) that can be induced by serum deprivation to form clusters and ultimately differentiate in vitro to endocrine cells. We also demonstrated that partially differentiated hIPC clusters, when implanted under the kidney capsules of mice, continued to differentiate in vivo into hormone-producing cells. However, we noted that not all hIPC preparations yielded insulin-secreting cells in vivo and that in some animals no hormone-expressing cells were found. This suggested that the implanted cells were not always irreversibly committed to further differentiation and may even de-differentiate to a mesenchymal phenotype. In this study, we show that human cells with a mesenchymal phenotype are indeed found in the grafts of mice implanted with hIPCs in epithelial cell clusters (ECCs), which are obtained after 4-day in vitro culture of hIPCs in serum-free medium (SFM); mesenchymal cells were predominant in some grafts. We could mimic the transition of ECCs to de-differentiated mesenchymal cells in vitro by exposure to foetal bovine serum (FBS) or mouse serums, and to a significantly lesser extent to human serum. In a complementary series of experiments, we show that mouse serum and FBS are more effective stimulants of mesenchymal hIPC migration than is human serum. We found that proliferation was not needed for the transition from ECCs to de-differentiated cells because mitomycin-treated hIPCs that could not proliferate underwent a similar transition. Lastly, we show that cells exhibiting a mesenchymal phenotype can be found in grafts of adult human islets in mice. We conclude that epithelial-to-mesenchymal transition (EMT) of cells in hIPC ECCs can occur following implantation in mice. This potential for EMT of human islets or differentiated precursor cells must be considered in strategies for cell replacement therapy for diabetes. [source] Ex vivo organ culture of adipose tissue for in situ mobilization of adipose-derived stem cells and defining the stem cell nicheJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2010Young-Il Yang In spite of the advances in the knowledge of adipose-derived stem cells (ASCs), in situ location of ASCs and the niche component of adipose tissue (AT) remain controversial due to the lack of an appropriate culture system. Here we describe a fibrin matrix-supported three-dimensional (3D) organ culture system for AT which sustains the ASC niche and allows for in situ mobilization and expansion of ASCs in vitro. AT fragments were completely encapsulated within the fibrin matrix and cultured under dynamic condition. The use of organ culture of AT resulted in a robust outgrowth and proliferation in the fibrin matrix. The outgrown cells were successfully recovered from fibrin by urokinase treatment. These outgrown cells fulfilled the criteria of mesenchymal stem cells, adherence to plastic, multilineage differentiation, and cell surface molecule expression. In vitro label retaining assay revealed that newly divided cells during the culture resided in interstitium between adipocytes and capillary endothelial cells. These interstitial stromal cells proliferated and outgrew into the fibrin matrix. Both in situ mobilized and outgrown cells expressed CD146 and ,-smooth muscle actin (SMA), but no endothelial cell markers (CD31 and CD34). The structural integrity and spatial approximation of CD31,/CD34,/CD146+/SMA+ interstitial stromal cells, adipocytes, and capillary endothelial cells were well preserved during in vitro culture. Our results suggest that ASCs are natively associated with the capillary wall and more specifically, belong to a subset of pericytes. Furthermore, organ culture of AT within a fibrin matrix-supported 3D environment can recapitulate the ASC niche in vitro. J. Cell. Physiol. 224: 807,816, 2010. © 2010 Wiley-Liss, Inc. [source] Control of human articular chondrocyte differentiation by reduced oxygen tensionJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Christopher L. Murphy Cell number is often a limiting factor in studies of chondrocyte physiology, particularly for human investigations. Chondrocytes can be readily proliferated in monolayer culture, however, differentiated phenotype is soon lost. We therefore endeavored to restore normal phenotype to human chondrocytes after serial passage in monolayer culture by manipulating cell morphology and oxygen tension towards the in vivo state. Third passage cells were encapsulated in alginate and exposed to either 20% or more physiologic 5% oxygen tensions. To assess cell phenotype, gene expression was measured using TaqMan real-time PCR. Encapsulated, primary chondrocytes cultured in 20% oxygen were used as a positive reference. Passaged human chondrocytes were fibroblastic in appearance and had lost normal phenotype as evidenced by a decrease in expression of collagen II, aggrecan, and sox9 genes of 66, 6, and 14 fold, respectively; with concomitant high expression of type I collagen (22 fold increase). A partial regaining of the differentiated phenotype was observed by encapsulation in 20% oxygen; however, even after 4 weeks, collagen II gene expression was not fully restored. Collagen II and aggrecan expression were increased, on average, 3 fold, in 5% oxygen tension compared to 20% cultures. Furthermore, matrix glycosaminoglycan (GAG) levels were significantly increased in reduced oxygen. In fact, after 4 weeks in 5% oxygen, encapsulated third passage cells had collagen II expression fully regained and aggrecan and sox9 levels actually exceeding primary cell levels in 20% oxygen. Our results show that the phenotype of serially passaged human articular chondrocytes is more fully restored by combining encapsulation with culture in more physiological levels of oxygen. Sox9, an essential transcription factor for chondrocyte differentiation is strongly implicated in this process since its expression was upregulated almost 27 fold. These findings have implications for the optimal conditions for the in vitro culture of chondrocytes. © 2004 Wiley-Liss, Inc. [source] Studies on BrdU labeling of hematopoietic cells: Stem cells and cell linesJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2003Lizhen Pang Studies using chronic in vivo BrdU exposure, isolating primitive stem cells, and determining BrdU labeling, indicate that stem cells cycle. BrdU is also incorporated into DNA during damage/repair. DNA, which has incorporated BrdU due to cycle transit is heavier than normal, while the density of DNA with damage/repair incorporation is intermediate. DNA density of purified lineage,rhodamine low (rholow) Hoechst low (Holow) stem cells or FDC-P1 cell line cells,was assessed in vitro, after exposure to cytokines and BrdU (cycling model) or cytokines and BrdU with bleomycin to induce strand breaks and hydroxyurea to halt cycle progression (damage/repair model). We determined DNA density using cesium chloride (CsCl) gradients and either fluorometry or dot blot chemiluminesence. DNA from BrdU labeled cycling Lin-rholoHolo or FDC-P1 cells was heavier than normal DNA, while damage repair DNA had an intermediate density. We then assessed BrdU labeling of Lin-rholoHolo cells in vivo. We found that 70.9% of lin-rholoHolo cells labeled at 5 weeks. DNA density of these cells was low, in the damage/repair range, but similar results were obtained with stem cells, which had proliferated in vivo. Dilution of BrdU in in vitro culture of proliferating FDC-P1 cells also resulted in damage/repair density. We conclude that in vitro BrdU labeling models can distinguish between proliferation and damage/repair, but that we cannot obtain high enough in vivo levels to address this issue. All together, while we cannot absolutely exclude damage/repair as contributing to stem cell BrdU labeling, the data indicate that primitive bone marrow stem cells are probably a cycling population. J. Cell. Physiol. 197: 251,260, 2003© 2003 Wiley-Liss, Inc. [source] Generation of pluripotent stem cells from eggs of aging miceAGING CELL, Issue 2 2010Junjiu Huang Summary Oocytes can reprogram genomes to form embryonic stem (ES) cells. Although ES cells largely escape senescence, oocytes themselves do senesce in the ovaries of most mammals. It remains to be determined whether ES cells can be established using eggs from old females, which exhibit reproductive senescence. We attempted to produce pluripotent stem cell lines from artificial activation of eggs (also called pES) from reproductive aged mice, to determine whether maternal aging affects pES cell production and pluripotency. We show that pES cell lines were generated with high efficiency from reproductive aged (old) mice, although parthenogenetic embryos from these mice produced fewer ES clones by initial two passages. Further, pES cell lines generated from old mice showed telomere length, expression of pluripotency molecular markers (Oct4, Nanog, SSEA1), alkaline phosphatase activity, teratoma formation and chimera production similar to young mice. Notably, DNA damage was reduced in pES cells from old mice compared to their progenitor parthenogenetic blastocysts, and did not differ from that of pES cells from young mice. Also, global gene expression differed only minimally between pES cells from young and old mice, in contrast to marked differences in gene expression in eggs from young and old mice. These data demonstrate that eggs from old mice can generate pluripotent stem cells, and suggest that the isolation and in vitro culture of ES cells must select cells with high levels of DNA and telomere integrity, and/or with capacity to repair DNA and telomeres. [source] Guidelines for species descriptions of diplomonad flagellates from fishJOURNAL OF FISH DISEASES, Issue 1 2002S L Poynton Diplomonad flagellates are common commensals of the digestive tract, and less common pathogenic parasites occurring in the digestive tract and systemically in numerous fish species. Many aspects of infections are poorly understood, including host-flagellate specificity, geographic ranges, and pathogenicity of different species. Much confusion is attributable to inadequate determination of genus and species. Although older literature reports Hexamita, Octomitus and Spironucleus from fish, recent studies confirm only Spironucleus. To address this problem, we describe ultrastructural features of trophozoites permitting reliable identification to genus and species, and techniques for their elucidation. Pioneering work by Brugerolle and colleagues established that genera can be distinguished by transmission electron microscopy (TEM). We now demonstrate that at the species level, surface ornamentations (especially at the posterior end of the body), and the pattern of bands of microtubules accompanying the flagellar pocket (in transverse section through the middle of the body), are of particular taxonomic value. Both scanning and TEM are essential for robust species descriptions and type material must be deposited in a recognized reference collection. Taxonomic studies are enhanced by in vitro culture, with tolerance and optimum for different conditions providing important supplementary information. Molecular characterization of fish diplomonads is in its infancy. [source] Investigation of hatching and early post-embryonic life of freshwater crayfish by in vitro culture, behavioral analysis, and light and electron microscopyJOURNAL OF MORPHOLOGY, Issue 7 2008Günter Vogt Abstract The late embryonic and early post-embryonic life period of freshwater crayfish, which is the main time period of organogenesis, is poorly investigated because of the protective brooding behavior of crayfish mothers. A combination of in vitro culture, behavioral observations, and microscopic investigations of organs involved in hatching, attachment, exploration of the environment, and searching and processing of food yielded deeper insights in this important period of life. Experiments were performed with the robust parthenogenetic marbled crayfish. The following results were obtained: (1) Marbled crayfish can be raised in simple in vitro systems from 80% embryonic development to juvenile Stage 4 with up to 100% survival; (2) Hatching is prepared by chemical weakening of the egg shell and completed by levering actions of the hatchling's appendages; (3) The telson thread, a safety line that keeps the hatchling secured to the mother, is formed by secretions from the telson and the detaching inner layer of the egg case; (4) Molting Stage-1 juveniles are secured by an anal thread that results from delayed molting of the hindgut; (5) Active attachment of the hatchlings to the maternal pleopods with their 1st pereiopods is achieved by an innate fixed action pattern; (6) In vitro, juveniles are motile from Stage 2 despite incomplete development of their balance controlling statocysts. Movement pattern and social behavior vary greatly among individuals; and (7) Feeding starts in Stage 3, when the mouthparts and the gastric mill are fully developed. Onset of feeding is innate and does not require maternal contributions. In vitro culture of the isogenic marbled crayfish is recommended for broader use in research because it enables not only time and stage-specific sampling but also precisely timed experimental manipulations. J. Morphol., 2008. © 2008 Wiley-Liss, Inc. [source] MRG15, a component of HAT and HDAC complexes, is essential for proliferation and differentiation of neural precursor cellsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 7 2009Meizhen Chen Abstract Neurogenesis during development depends on the coordinated regulation of self-renewal and differentiation of neural precursor cells (NPCs). Chromatin regulation is a key step in self-renewal activity and fate decision of NPCs. However, the molecular mechanism or mechanisms of this regulation is not fully understood. Here, we demonstrate for the first time that MRG15, a chromatin regulator, is important for proliferation and neural fate decision of NPCs. Neuroepithelia from Mrg15 -deficient embryonic brain are much thinner than those from control, and apoptotic cells increase in this region. We isolated NPCs from Mrg15 -deficient and wild-type embryonic whole brains and produced neurospheres to measure the self-renewal and differentiation abilities of these cells in vitro. Neurospheres culture from Mrg15 -deficient embryo grew less efficiently than those from wild type. Measurement of proliferation by means of BrdU (bromodeoxyuridine) incorporation revealed that Mrg15 -deficient NPCs have reduced proliferation ability and apoptotic cells do not increase during in vitro culture. The reduced proliferation of Mrg15 -deficient NPCs most likely accounts for the thinner neuroepithelia in Mrg15 -deficient embryonic brain. Moreover, we also demonstrate Mrg15 -deficient NPCs are defective in differentiation into neurons in vitro. Our results demonstrate that MRG15 has more than one function in neurogenesis and defines a novel role for this chromatin regulator that integrates proliferation and cell-fate determination in neurogenesis during development. © 2008 Wiley-Liss, Inc. [source] Adhesion of perichondrial cells to a polylactic acid scaffoldJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 4 2003Alexander Giurea Abstract The number of chondrogenic cells available locally is an important factor in the repair process for cartilage defects. Previous studies demonstrated that the number of transplanted rabbit perichondrial cells (PC) remaining in a cartilage defect in vivo, after being carried into the site in a polylactic acid (PLA) scaffold, declined markedly within two days. This study examined the ability of in vitro culture of PC/PLA constructs to enhance subsequent biomechanical stability of the cells and the matrix content in an in vitro screening assay. PC/PLA constructs were analyzed after 1 h, 1 and 2 weeks of culture. The biomechanical adherence of PC to the PLA scaffold was tested by subjecting the PC/PLA constructs to a range of flow velocities (0.25,25 mm/s), spanning the range estimated to occur under conditions of construct insertion in vivo. The adhesion of PC to the PLA carrier was increased significantly by 1 and 2 weeks of incubation, with 25 mm/s flow causing a 57% detachment of cells after 1 h of seeding, but only 7% and 16% after 1 and 2 weeks of culture, respectively (p > 0.001). This adherence was associated with marked deposition of glycosaminoglycan and collagen. These findings suggest that pre-incubation of PC-laden PLA scaffolds markedly enhances the stability of the indwelling cells. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Behaviors of ATP-dependent chromatin remodeling factors during maturation of bovine oocytes in vitroMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2010Gabbine Wee The mammalian oocyte undergoes dynamic changes in chromatin structure to reach complete maturation. However, little known is about behaviors of ATP-dependent chromatin remodeling factors (ACRFs) during meiosis. Here, we found that respective ACRFs may differently behave in the process of oocyte maturation in the bovine. All ACRFs interacted with oocytic chromatin at the germinal vesicle (GV) stage. Mi-2 and hSNF2H disappeared from GV-chromatin within 1,hr of in vitro culture whereas Brg-1 and BAF-170 were retained throughout germinal vesicle break down (GVBD). Brg-1 was localized on the condensed chromatin outside, whereas BAF-170 was entirely excluded from condensed chromatin. Thereafter, Brg-1 and BAF-170 interacted with metaphase I and metaphase II chromosomes. These results imply that Mi-2 and hSNF2H may initiate the meiotic resumption, and Brg-1 and BAF-170 may support chromatin condensation during meiosis. In addition, DNA methylation and methylation of histone H3 at lysine 9 (H3K9) seem to be constantly retained in the oocyte chromatin throughout in vitro maturation. Inhibition of ACRF activity by treatment with the inhibitor apyrase led to retarded chromatin remodeling in bovine oocytes, thereby resulting in poor development of fertilized embryos. Therefore, these results indicate that precise behaviors of ACRFs during meiosis are critical for nuclear maturation and subsequent embryonic development in the bovine. Mol. Reprod. Dev. 77: 126,135, 2010. © 2009 Wiley-Liss, Inc. [source] Molecular Reproduction & Development: Volume 76, Issue 2MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2009Article first published online: 29 DEC 200 Reconstructed rat embryos. Cloned rat embryos were created by cumulus cell nuclear transfer followed by electrofusion. These individuals were imaged after overnight in vitro culture. See the accompanying article by Popova et al. in this issue. [source] Determination of sex and scrapie resistance genotype in preimplantation ovine embryosMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2009Florence Guignot Abstract The aim of this study was to test the accuracy of genotype diagnosis after pre-amplification of DNA extracted from biopsies obtained by microblade cutting of ovine embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer to recipients. Sex and PrP genotypes were determined. Sex diagnosis was done by PCR amplification of ZFX/ZFY and SRY sequences after PEP-PCR while PrP genotype determination was performed after specific pre-amplification of specific target including codons 136, 154 and 171. Embryos were collected at Day 7 after oestrus. Blastocysts and expanded blastocysts were biopsied immediately after collection whereas compacted morulae were biopsied after 24 hr of in vitro culture. Eighty-nine biopsied embryos were frozen by vitrification. Fresh and vitrified whole embryos were kept as control. DNA of biopsies was extracted and pre-amplified. Sex diagnosis was efficient for 96.6% of biopsies and PrP genotyping was determined in 95.8% of codons. After embryo transfer, no significant difference was observed in lambing rate between biopsied, vitrified control and fresh embryos (54.5%, 60% and 66.6%, respectively). Embryo survival rate was not different between biopsied and whole vitrified embryos (P,=,0.38). At birth, 96.7% of diagnosed sex and 95.4% of predetermined codons were correct. Lamb PrP profiles were in agreement with parental genotype. PEP-PCR coupled with sex diagnosis and nested PCR coupled with PrP genotype predetermination are very accurate techniques to genotype ovine embryo before transfer. These original results allow planning of selection of resistant genotype to scrapie and sex of offspring before transfer of cryopreserved embryo. Mol. Reprod. Dev. 76: 183,190, 2009. © 2008 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||||||||