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Long-term Culture (long-term + culture)
Selected AbstractsLong-term culture of Xenopus presumptive ectoderm in a nutrient-supplemented culture mediumDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5-6 2003Yasuto Fukui Animal cap assay is a useful experimental model for investigating the activity of inducers in amphibian development. This assay has revealed that activin A is a potent mesoderm-inducing factor. However, it has been very difficult to induce highly differentiated tissues such as cartilage in a 3,4 day culture period. It was recently reported that jaw cartilage was induced in vitro in an animal cap that had been cultured for 14 days in Steinberg's solution using the sandwich culture method and activin A. Under these conditions, necrosis was occasionally observed in the explants. In this study, we have achieved long-term animal cap cultures in a nutrient-supplemented culture medium designated RDX. This medium was made by modifying the saline concentration of the RD medium previously developed as a basal medium for the serum-free culture of various kinds of mammalian cells. The explants cultured in RDX grew more vigorously compared with those in Steinberg's solution. RDX medium promoted a wider variety of tissue induction and gene expression in the animal caps than Steinberg's solution, and also increased the frequency of cartilage induction. Therefore, the supplemental nutrients may support and promote the differentiation of cartilage. This long-term culture method using RDX medium is useful for studying the differentiation of tissues or organs such as cartilage in vitro. [source] Lentivirus-mediated bifunctional cell labeling for in vivo melanoma studyPIGMENT CELL & MELANOMA RESEARCH, Issue 3 2009Chi-Ping Day Summary Lentiviral vectors (LVs) are capable of labeling a broad spectrum of cell types, achieving stable expression of transgenes. However, for in vivo studies, the duration of marker gene expression has been highly variable. We have developed a series of LVs harboring different promoters for expressing reporter gene in mouse cells. Long-term culture and colony formation of several LV-labeled mouse melanoma cells showed that promoters derived from mammalian house-keeping genes, especially those encoding RNA polymerase II (Pol2) and ferritin (FerH), provided the highest consistency for reporter expression. For in vivo studies, primary B16BL6 mouse melanoma were infected with LVs whose luciferase,green fluorescence protein fusion gene (Luc/GFP) was driven by either Pol2 or FerH promoters. When transplanted into syngeneic C57BL/6 mice, Luc/GFP-labeled B16BL6 mouse melanoma cells can be monitored by bioluminescence imaging in vivo, and GFP-positive cells can be isolated from the tumors by fluorescence-activated cell sorter. Pol2-Luc/GFP labeling, while lower in activity, was more sustainable than FerH-Luc/GFP labeling in B16BL6 over consecutive passages into mice. We conclude that Pol-2-Luc/GFP labeling allows long-term in vivo monitoring and tumor cell isolation in immunocompetent mouse melanoma models. [source] The influence of sex on the chondrogenic potential of muscle-derived stem cells: Implications for cartilage regeneration and repairARTHRITIS & RHEUMATISM, Issue 12 2008Tomoyuki Matsumoto Objective To explore possible differences in muscle-derived stem cell (MDSC) chondrogenic differentiation in vitro and articular cartilage regeneration in vivo between murine male MDSCs (M-MDSCs) and female MDSCs (F-MDSCs). Methods Three different populations of M- and F-MDSCs (n = 3 of each sex) obtained via preplate technique, which separates cells based on their variable adhesion characteristics, were compared for their in vitro chondrogenic potential using pellet culture. Cells were assayed with and without retroviral transduction to express bone morphogenetic protein 4 (BMP-4). The influence of both expression of stem cell marker Sca1 and in vitro expansion on the chondrogenic potential of M- and F-MDSCs was also determined. Additionally, BMP-4,transduced M- and F-MDSCs were applied to a full-thickness articular cartilage defect (n = 5 each) on the femur of a nude rat, and the quality of the repaired tissue was evaluated by macroscopic and histologic examination. Results With and without BMP-4 gene transduction, M-MDSCs produced significantly larger pellets with a richer extracellular matrix, compared with F-MDSCs. Sca1 purification influenced the chondrogenic potential of MDSCs, especially M-MDSCs. Long-term culture did not affect the chondrogenic potential of M-MDSCs but did influence F-MDSCs. M-MDSCs repaired articular cartilage defects more effectively than did F-MDSCs at all time points tested, as assessed both macroscopically and histologically. Conclusion Our findings demonstrate that sex influences the chondrogenic differentiation and articular cartilage regeneration potential of MDSCs. Compared with female MDSCs, male MDSCs display more chondrogenic differentiation and better cartilage regeneration potential. [source] Lep d 2 polymorphisms in wild and cultured Lepidoglyphus destructor mitesFEBS JOURNAL, Issue 4 2003Liselotte Kaiser We have previously cloned, expressed and characterized two variants of the major allergen Lep d 2 from cultured Lepidoglyphus destructor mites. These variants, Lep d 2.0101 and Lep d 2.0201, differ at 13 amino acid positions. In this study we investigated Lep d 2 sequence diversity between wild and cultured mites. PCR, Southern blot and DNA sequence analysis revealed the presence of two different Lep d 2 genes, one with and one without an intron. In addition, two new variants of Lep d 2, Lep d 2.0102 and Lep d 2.0202, were found at different frequencies in wild and cultured mites. When we expressed the Lep d 2 variants and compared their IgE binding properties by ELISA inhibition, we found that Lep d 2.0102 was a more potent inhibitor than Lep d 2.0101, and to a lesser extent Lep d 2.0202 was more potent than Lep d 2.0201. Long-term cultures of peripheral blood mononuclear cells were used to assess the ability of the expressed Lep d 2 variants to induce cytokine release. Although cells from different individuals released different amounts of interferon-, and interleukin-5, no consistent cytokine release pattern could be linked to any specific Lep d 2 variant. In conclusion, we show that both cultured and wild Lepidoglyphus destructor mites contain the same pattern of polymorphism. Furthermore, this Lep d 2 sequence diversity seems not to have any significant impact on the allergens IgE binding or its ability to induce T cell cytokine release. [source] Proliferative responses to growth factors decline rapidly during postnatal maturation of mammalian hair cell epitheliaEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2007Rende Gu Abstract Millions of lives are affected by hearing and balance deficits that arise as a consequence of sensory hair cell loss. Those deficits affect mammals permanently, but hearing and balance recover in nonmammals after epithelial supporting cells divide and produce replacement hair cells. Hair cells are not effectively replaced in mammals, but balance epithelia cultured from the ears of rodents and adult humans can respond to hair cell loss with low levels of supporting cell proliferation. We have sought to stimulate vestibular proliferation; and we report here that treatment with glial growth factor 2 (rhGGF2) yields a 20-fold increase in cell proliferation within sheets of pure utricular hair cell epithelium explanted from adult rats into long-term culture. In epithelia from neonates, substantially greater proliferation responses are evoked by rhGGF2 alone, insulin alone and to a lesser degree by serum even during short-term cultures, but all these responses progressively decline during the first 2 weeks of postnatal maturation. Thus, sheets of utricular epithelium from newborn rats average >,40% labelling when cultured for 72 h with bromo-deoxyuridine (BrdU) and either rhGGF2 or insulin. Those from 5- and 6-day-olds average 8,15%, 12-day-olds average <,1% and after 72 h there is little or no labelling in epithelia from 27- and 35-day-olds. These cells are the mammalian counterparts of the progenitors that produce replacement hair cells in nonmammals, so the postnatal quiescence described here is likely to be responsible for at least part of the mammalian ear's unique vulnerability to permanent sensory deficits. [source] Binding partners L1 cell adhesion molecule and the ezrin-radixin-moesin (ERM) proteins are involved in development and the regenerative response to injury of hippocampal and cortical neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2004Matilda A. Haas Abstract Regeneration of the adult central nervous system may require recapitulation of developmental events and therefore involve the re-expression of developmentally significant proteins. We have investigated whether the L1 cell adhesion molecule, and its binding partner, the ezrin-radixin-moesin (ERM) proteins are involved in the neuronal regenerative response to injury. Hippocampal and cortical neurons were cultured in vitro on either an L1 substrate or poly-L-lysine, and ERM and other neuronal proteins were localized immunocytochemically both developmentally and following neurite transection of neurons maintained in long-term culture. Activated ERM was localized to growth cones up to 7 days in vitro but relatively mature cultures (21 days in vitro) were devoid of active ERM proteins. However, ERM proteins were localized to the growth cones of sprouting neuronal processes that formed several hours after neurite transection. In addition, the L1 substrate, relative to poly-L-lysine, resulted in significantly longer regenerative neurites, as well as larger growth cones with more filopodia. Furthermore, neurons derived from the cortex formed significantly longer post-injury neurite sprouts at 6 h post-injury than hippocampal derived neurons grown on both substrates. We have demonstrated that L1 and the ERM proteins are involved in the neuronal response to injury, and that neurons derived from the hippocampus and cortex may have different post-injury regenerative neurite sprouting abilities. [source] Long-term establishment, characterization and manipulation of cell lines from mouse basal cell carcinoma tumorsEXPERIMENTAL DERMATOLOGY, Issue 9 2006Po-Lin So Abstract:, There have been few reports of successful long-term culture of cells established from cutaneous basal cell carcinoma (BCC) tumors. Here, we describe techniques that have enabled us to establish three long-term cultures of BCC cells isolated from BCC tumors that arose in irradiated Patched 1 (Ptch1)+/, mice. All three cell lines showed cellular morphology similar to that of BCC tumors and could be propagated for at least 20 passages. In addition, similar to BCC tumors, all cell lines had lost the wildtype Ptch1 allele, expressed BCC molecular markers, and responded similarly to cyclopamine, a small molecule inhibitor of Hedgehog signaling. Finally, we describe an efficient electroporation technique for DNA transfection into the BCC cell lines and show that they have activated Hedgehog signaling activity, albeit at a level lower than that of murine BCCs in vivo. These data indicate that the cell lines are bona fide long-term cultures of BCC cells and that DNA plasmids can be introduced into the BCC cell lines with relatively high transfection efficiency using a modified electroporation technique. [source] Gap junction-mediated intercellular communication in a long-term primary mouse hepatocyte culture systemHEPATOLOGY, Issue 5 2003Stephanie A. Stoehr Gap junction-mediated intercellular communication (GJIC) is critical for maintaining integral cellular processes including differentiation and growth control. The disruption of GJIC has been correlated with aberrant function in many cell types, including hepatocytes in vivo; therefore it is imperative that cellular model systems support intercellular communication to simulate normal cellular functions. Functional GJIC has been shown in long-term primary rat hepatocyte cultures, which have been implemented widely to study various aspects of hepatocellular function; however, the onset of transgenic technology in murine species has necessitated the development of a primary mouse hepatocyte system. In this report, we analyze GJIC in a dimethylsulfoxide (DMSO)-containing long-term primary mouse hepatocyte culture system. The cells retain morphologic and biochemical characteristics of differentiated hepatocytes through day 30 post plating, including liver-specific gene expression. We further show that connexin32 and connexin26 expression and gap junction plaque formation increase over time in culture concomitant with an increase in GJIC between adjoining primary mouse hepatocytes. In conclusion, the findings described in this study make it possible to maintain differentiated primary mouse hepatocytes that also show GJIC in long-term culture for 30 days. In addition, this system has the potential to be extended to study primary mouse hepatocytes isolated from genetically engineered mice. [source] Steroid hormone receptors and coregulators in endocrine-resistant and estrogen-independent breast cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 4 2006Nanna Sarvilinna Abstract Resistance to hormonal therapy is often a problem in the treatment of breast cancer patients. It has been suggested that resistance could be explained by altered nuclear hormone receptor or coregulator levels or inappropriately increased agonist activity of selective estrogen receptor modulator (SERM). To test these hypotheses, we have established novel MCF-7 cell line-derived in vitro models of anti-estrogen- and progestin-resistant and estrogen-independent breast cancer by long-term culture in the presence of toremifene and medroxyprogesterone acetate (MPA) and in the absence of estradiol, respectively. Using cell growth and multiprobe ribonuclease protection assays, the expression of 5 nuclear hormone receptors and 9 coregulators as well as the alterations in the cell proliferation and target gene transcription in response to hormonal treatments were studied. Progesterone receptor (PR) expression was decreased and silencing mediator for retinoid acid and thyroid hormone receptors (SMRT) and amplified in breast cancer-1 (AIB1) expression increased in anti-estrogen-resistant cells. Estrogen caused PR and ER, upregulation in all cell lines, but we did not observe increased agonist activity of anti-estrogen measured by regulation of these estrogen target genes. Basal ER, levels and estrogenic growth response were decreased and p300/CBP-associated factor (pCAF) and AIB1 upregulated by estrogen in progestin-resistant cells, but coregulator levels were unchanged. Estrogen-independent cells were still estrogen-responsive and PR, nuclear receptor corepressor (N-CoR) and SMRT expression was increased whereas steroid receptor coactivator-1 (SRC-1a) and CBP-related protein p300 (p300) expression decreased. Their growth was inhibited by toremifene, but estradiol was able to abrogate this effect, which might have interesting clinical implications concerning the use of postmenopausal hormone replacement therapy. © 2005 Wiley-Liss, Inc. [source] Biomolecular characterization of human glioblastoma cells in primary cultures: Differentiating and antiangiogenic effects of natural and synthetic PPAR, agonistsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2008E. Benedetti Gliomas are the most commonly diagnosed malignant brain primary tumors. Prognosis of patients with high-grade gliomas is poor and scarcely affected by radiotherapy and chemotherapy. Several studies have reported antiproliferative and/or differentiating activities of some lipophylic molecules on glioblastoma cells. Some of these activities in cell signaling are mediated by a class of transcriptional factors referred to as peroxisome proliferator-activated receptors (PPARs). PPAR, has been identified in transformed neural cells of human origin and it has been demonstrated that PPAR, agonists decrease cell proliferation, stimulate apoptosis and induce morphological changes and expression of markers typical of a more differentiated phenotype in glioblastoma and astrocytoma cell lines. These findings arise from studies mainly performed on long-term cultured transformed cell lines. Such experimental models do not exactly reproduce the in vivo environment since long-term culture often results in the accumulation of further molecular alterations in the cells. To be as close as possible to the in vivo condition, in the present work we investigated the effects of PPAR, natural and synthetic ligands on the biomolecular features of primary cultures of human glioblastoma cells derived from surgical specimens. We provide evidence that PPAR, agonists may interfere with glioblastoma growth and malignancy and might be taken in account as novel antitumoral drugs. J. Cell. Physiol. 217: 93,102, 2008. © 2008 Wiley-Liss, Inc. [source] Prostate carcinoma cells selected by long-term exposure to reduced oxygen tension show remarkable biochemical plasticity via modulation of superoxide, HIF-1, levels, and energy metabolismJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Jeanne Bourdeau-Heller Cancer cells are able to tolerate levels of O2 that are damaging or lethal to normal cells; we hypothesize that this tolerance is the result of biochemical plasticity which maintains cellular homeostasis of both energy levels and oxidation state. In order to examine this hypothesis, we used different O2 levels as a selective agent during long-term culture of DU145 prostate cancer cells to develop three isogenic cell lines that grow in normoxic (4%), hyperoxic (21%), or hypoxic (1%) O2 conditions. Growth characteristics and O2 consumption differed significantly between these cell lines without changes in ATP levels or altered sensitivity to 2-deoxy- D -glucose, an inhibitor of glycolysis. O2 consumption was significantly higher in the hyperoxic line as was the level of endogenous superoxide. The hypoxic cell line regulated the chemical gradient of the proton motive force (PMF) independent of the electrical component without O2 -dependent changes in Hif-1, levels. In contrast, the normoxic line regulated Hif-1, without tight regulation of the chemical component of the PMF noted in the hypoxic cell line. From these studies, we conclude that selection of prostate cancer cells by long-term exposure to low ambient levels of O2 resulted in cells with unique biochemical properties in which energy metabolism, reactive oxygen species (ROS), and HIF-1, levels are modulated to allow cell survival and growth. Thus, cancer cells exhibit remarkable biochemical plasticity in response to various O2 levels. J. Cell. Physiol. 212:744,752, 2007. © 2007 Wiley-Liss, Inc. [source] DNA methylation pattern changes upon long-term culture and aging of human mesenchymal stromal cellsAGING CELL, Issue 1 2010Simone Bork Summary Within 2,3 months of in vitro culture-expansion, mesenchymal stromal cells (MSC) undergo replicative senescence characterized by cell enlargement, loss of differentiation potential and ultimate growth arrest. In this study, we have analyzed DNA methylation changes upon long-term culture of MSC by using the HumanMethylation27 BeadChip microarray assessing 27 578 unique CpG sites. Furthermore, we have compared MSC from young and elderly donors. Overall, methylation patterns were maintained throughout both long-term culture and aging but highly significant differences were observed at specific CpG sites. Many of these differences were observed in homeobox genes and genes involved in cell differentiation. Methylation changes were verified by pyrosequencing after bisulfite conversion and compared to gene expression data. Notably, methylation changes in MSC were overlapping in long-term culture and aging in vivo. This supports the notion that replicative senescence and aging represent developmental processes that are regulated by specific epigenetic modifications. [source] Telomere uncapping during in vitro T-lymphocyte senescenceAGING CELL, Issue 1 2009Amel Chebel Summary Normal lymphocytes represent examples of somatic cells that are able to induce telomerase activity when stimulated. As previously reported, we showed that, during lymphocyte long-term culture and repeated stimulations, the appearance of senescent cells is associated with telomere shortening and a progressive drop in telomerase activity. We further showed that this shortening preferentially occured at long telomeres and was interrupted at each stimulation by a transitory increase in telomere length. In agreement with the fact that telomere uncapping triggers lymphocyte senescence, we observed an increase in ,-H2AX and 53BP1 foci as well as in the percentage of cells exhibiting DNA damage foci in telomeres. Such a DNA damage response may be related to the continuous increase of p16ink4a upon cell stimulation and cell aging. Remarkably, at each stimulation, the expression of shelterin genes, such as hTRF1, hTANK1, hTIN2, hPOT1 and hRAP1, was decreased. We propose that telomere dysfunction during lymphocyte senescence caused by iterative stimulations does not only result from an excessive telomere shortening, but also from a decrease in shelterin content. These observations may be relevant for T-cell biology and aging. [source] Growth hormone regulates osteogenic marker mRNA expression in human periodontal fibroblasts and alveolar bone-derived cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 4 2003H. R. Haase Background:, Growth hormone (GH) is a potent regulator of bone formation. The proposed mechanism of GH action is through the stimulation of osteogenic precursor cell proliferation and, following clonal expansion of these cells, promotion of differentiation along the osteogenic lineage. Objectives:, We tested this hypothesis by studying the effects of GH on primary cell populations of human periodontal ligament cells (PLC) and alveolar bone cells (ABC), which contain a spectrum of osteogenic precursors. Methods:, The cell populations were assessed for mineralization potential after long-term culture in media containing ,-glycerophosphate and ascorbic acid, by the demonstration of mineral deposition by Von Kossa staining. The proliferative response of the cells to GH was determined over a 48-h period using a crystal violet dye-binding assay. The profile of the cells in terms of osteogenic marker expression was established using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for alkaline phosphatase (ALP), osteopontin, osteocalcin, bone sialoprotein (BSP), as well as the bone morphogenetic proteins BMP-2, BMP-4 and BMP-7. Results:, As expected, a variety of responses were observed ranging from no mineralization in the PLC populations to dense mineralized deposition observed in one GH-treated ABC population. Over a 48-h period GH was found to be non-mitogenic for all cell populations. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) BSP mRNA expression correlated well with mineralizing potential of the cells. The change in the mRNA expression of the osteogenic markers was determined following GH treatment of the cells over a 48-h period. GH caused an increase in ALP in most cell populations, and also in BMP expression in some cell populations. However a decrease in BSP, osteocalcin and osteopontin expression in the more highly differentiated cell populations was observed in response to GH. Conclusion:, The response of the cells indicates that while long-term treatment with GH may promote mineralization, short-term treatment does not promote proliferation of osteoblast precursors nor induce expression of late osteogenic markers. [source] Cholangiocytes as immune modulators in rotavirus-induced murine biliary atresiaLIVER INTERNATIONAL, Issue 8 2009Barrett H. Barnes Abstract Background/Aims: Biliary atresia (BA) is a progressive disease characterized by bile duct inflammation and fibrosis. The aetiology is unknown and may be due to a virus-induced, autoimmune-mediated injury of cholangiocytes. Cholangiocytes are not only targets of injury but may also modulate hepatic inflammation. The aim of this study was to determine the immune profile of murine cholangiocytes and the ability to function as antigen-presenting cells (APCs) in culture with Rhesus rotavirus (RRV), poly I:C (viral mimic) or interferon-,/tumour necrosis factor-,. Methods/Results: Both the cholangiocyte cell line (long-term culture) and fresh, ex vivo cholangiocytes expressed APC surface markers major histocompatibility complex (MHC)-class I and II and CD40, while only the cultured cell line expressed costimulatory molecules B7-1 and B7-2. Despite APC expression, cultured cholangiocytes were unable to function as competent APCs in T-cell proliferation assays. Furthermore, both cultured and ex vivo cholangiocytes expressed RNA transcripts for many pro-inflammatory cytokines and chemokines. Conclusions: Although cholangiocytes contain APC molecules, they are incompetent at antigen presentation and cannot elicit effective T-cell activation. Upregulation of MHC-class I and II found in BA mice may serve to prime the cholangiocyte as a target for immune-mediated injury. Cholangiocytes produced many pro-inflammatory cytokines and chemokines in the setting of RRV infection and T-helper type 1 cytokine milieu, suggesting a role of cholangiocytes as immune modulators promoting the ongoing inflammation that exists in RRV-induced BA. [source] Rapid prenatal diagnosis of common trisomies: discordant results between QF-PCR analysis and karyotype analysis on long-term culture for a case of trisomy 18 detected in CVSPRENATAL DIAGNOSIS, Issue 12 2006S. K. Allen Abstract Objectives QF-PCR analysis can be used as a rapid test to diagnose primary trisomy in prenatal samples. Mosaicism in CVS detected by QF-PCR has previously been reported; however, no case has so far been reported in which the QF-PCR result was completely discrepant to that of the karyotype analysis from a long-term culture. Methods A CVS, referred because of a high serum screening risk of 1:10 for Down Syndrome and 1:110 for Edwards Syndrome, was tested by QF-PCR analysis and chromosome analysis of cultured cells. Subsequent analyses were carried out on a follow-up amniotic fluid sample and foetal tissue samples. Results Conflicting results were obtained between QF-PCR analysis on two independent fronds from the chorionic villi and chromosome analysis on cultured CVS. Cytogenetic and molecular analysis on a subsequent amniotic fluid sample indicated trisomy 18 with no evidence of mosaicism. Analysis of follow-up tissue confirmed trisomy in a foetal skin sample and mosaicism for trisomy 18 in four placental sites tested. Conclusion We report here an apparently normal CVS QF-PCR result that was completely discrepant with the trisomy 18 positive karyotype result on long-term culture. This has important implications regarding our current testing protocol. Copyright © 2006 John Wiley & Sons, Ltd. [source] Proteomic and transcriptomic analysis of human CD8+ T lymphocytes over-expressing telomerasePROTEOMICS - CLINICAL APPLICATIONS, Issue 3 2007Lynne Thadikkaran Abstract Human T lymphocytes have a finite life span resulting from progressive telomere shortening that occurs at each cell division, eventually leading to chromosomal instability. It has been shown that ectopic expression of the human telomerase reverse transcriptase (hTERT) gene into various human cells results in the extension of their replicative life span, without inducing changes associated with transformation. However, it is still unclear whether cells that over-express telomerase are physiologically and biochemically indistinguishable from normal cells. To address this question, we compared the proteome of young and aged human CD8+ T lymphocytes with that of T cells transduced with hTERT. Interestingly, we found no global changes in the protein pattern in young T cells, irrespective of telomerase expression. In contrast, several relevant proteins with differential expression patterns were observed in hTERT-transduced T cells with extended life span upon long-term culture. Altogether, our data revealed that T lymphocytes over-expressing telomerase displayed an intermediate protein pattern, sharing a similar protein expression not only with young T cells, but also with aged T cells. Finally, the results obtained from this global proteomic approach are in agreement with the overall gene transcription profiling performed on the same T-cell derived clones. [source] The selective use of rapid aneuploidy screening in prenatal diagnosisAUSTRALIAN AND NEW ZEALAND JOURNAL OF OBSTETRICS AND GYNAECOLOGY, Issue 1 2009Jan E. DICKINSON Aims: To evaluate the diagnostic utility and costing of the selective use of rapid aneuploidy screening (RAS) for chorion villus sampling (CVS) and amniocentesis specimens. Methods: CVS and amniocenteses performed between 2000 and 2006 were identified. Cases were subdivided into two groups: (i) RAS in addition to long-term culture and (ii) long-term chromosome culture alone. The frequency of RAS, the proportion of abnormal results and the cytogenetic costings were reviewed. Results: A total of 3315 procedures were performed: 730 CVS and 2585 amniocenteses. An abnormal karyotype culture was present in 366 of 3315 (11%). For CVS an abnormal culture was present in 164 (22.5%). RAS (short-term culture/direct preparation) was selectively used in 399 cases (54.6%) with an abnormal result in 128 (32% of RAS). For amniocentesis, 206 chromosome abnormalities were present (8.0% of specimens). RAS (interphase FISH) was selectively used in 580 amniocenteses (22.4%). FISH was requested in 95 (66.4%) of the 143 abnormal cases potentially detectable with standard probes. There was a progressive increase in utilisation of RAS for amniocentesis (8.9% in 2000 to 43.3% of cases in 2006, P < 0.001). CVS RAS was stable. This liberalisation resulted in a fourfold increase in expenditure for FISH and cost/abnormality detected ($A970 per abnormal result in 2000 to $A4015 per abnormal result in 2006). Conclusion: The selective use of prenatal RAS results in a reasonably high detection rate for chromosomal anomalies. Liberalisation of RAS, however, is an expensive cytogenetic model. An approach based on some predictive level of risk combined with resource funding levels may be a more pragmatic approach. [source] Long,term culture of multibacillary leprosy macrophages isolated from skin lesions: a new model to study Mycobacterium leprae,human cell interactionBRITISH JOURNAL OF DERMATOLOGY, Issue 2 2007D.F. Moura Summary Background, Leprosy is characterized by a disease spectrum having two polar clinical forms dependent on the presence or not of cell-mediated immunity. In the tuberculoid forms, granuloma-activated macrophages kill Mycobacterium leprae in conjunction with a Th1 response while, in multibacillary (MB) lesions, M. leprae nonactivated macrophages infiltrate the nerves and internal organs together with a Th2 response. The functional properties and activation pathways of macrophages isolated from patients with MB leprosy remain only partially understood. Objectives, To establish an ex vivo methodology capable of evaluating the activation pathways, grade and fate of cultured macrophages isolated from MB lesions. Methods, Skin biopsies from patients with borderline tuberculoid, bordeline lepromatous and lepromatous leprosy (LL) were characterized by immunohistochemistry and transcriptional analysis. To isolate inflammatory cells, a portion of the samples was submitted to enzymatic digestion. These same cells, maintained in culture for a minimum 7-day period, were characterized morphologically and via flow cytometry at different culture time points. Cytokine [interferon (IFN)-,, tumour necrosis factor (TNF)-, and interleukin (IL)-10] mRNA levels were quantified by real-time polymerase chain reaction and protein secretion in the culture supernatants was measured by enzyme-linked immunosorbent assay and the nitric oxide levels by Griess reagent. Results, RNA expression in tuberculoid and MB lesions showed the profile expected of characteristic Th1 and Th2 responses, respectively. The inflammatory cells in all biopsies were successfully isolated. Although the number of cells varied between biopsies, it was highest in LL biopsies. The frequency of isolated CD14+ and CD3+ cells measured by flow cytometry correlated with the percentages of macrophages and lymphocytes in the lesions. Throughout the culture period, CD68+ macrophages showed morphological changes. A progressive increase in cell number and reduction of infected cells were perceptible in the cultures. In contrast to the biopsies, TNF-,, IFN-, and IL-10 expression in the tuberculoid and MB leprosy cells in 24-h culture and the cytokine levels in the supernatants did not differ significantly. During the culture period, cytokine expression in the MB cells progressively declined, whereas, from days 1 to 7, nitrite levels progressively increased. After day 40, the remaining macrophages were able to ingest fluorescein isothiocyanate-labelled M. leprae. These data need to be confirmed. Conclusions, This study confirmed the feasibility of obtaining ex vivo macrophages from leprosy lesions and keeping them in long-term culture. This procedure may open new pathways to studying the interaction between M. leprae and human macrophages, which might, in turn, lead to the development of therapeutic tools capable of overcoming the specific anergy found in patients with MB leprosy. [source] Bone morphogenetic protein 4 modulates c-Kit expression and differentiation potential in murine embryonic aorta-gonad-mesonephros haematopoiesis in vitroBRITISH JOURNAL OF HAEMATOLOGY, Issue 2 2007Caroline J. Marshall Summary The transforming growth factor- , -related factor bone morphogenetic protein 4 (BMP4) is expressed in the human embryonic aorta-gonad-mesonephros (AGM) coincident with the emergence of haematopoietic cells and influences postnatal mammalian haematopoietic stem cells in vitro. To investigate the role of BMP4 in mammalian embryonic haematopoiesis, cells were isolated from murine AGM and two populations of CD34+ cells with different levels of c-Kit expression and multipotency were identified. CD34+/c-Kithigh cells express CD45 and are haematopoietic-restricted progenitors. In contrast, CD34+/c-Kitlow cells are Flk1+/CD45neg and generate adherent colonies in ex vivo culture that resemble haemangioblast colonies identified in other systems. The addition of BMP4 to AGM cells resulted in expansion of the CD34+/c-Kitlow cell pool within 48 h, via a combination of down modulation of the c-Kit receptor in CD34+/c-Kithigh cells and proliferation. In long-term culture, BMP4 increased the growth/survival of CD34+/c-Kithigh haematopoietic progenitors, effects that were blocked by BMP inhibitors. CD34+/c-Kithigh progenitors cultured with BMP4 also generated adherent colonies typical of c-Kitlow cells. These results suggest that BMP4 regulates c-Kit expression and differentiation potential in CD34+ AGM cells and supports a role for BMP signalling in the maintenance of multipotency during embryonic haematopoiesis, providing an insight into stem cell homeostasis within the mammalian haematopoietic niche. [source] Intergenerational acclimation in aphid overwinteringECOLOGICAL ENTOMOLOGY, Issue 1 2008S. J. POWELL Abstract 1.,When first instar nymphs and adults of the grain aphid Sitobion avenae (Fabricius) (Hemiptera: Aphidiae) were maintained in long-term cultures (>6 months) at 20 °C and 10 °C, the LT50 decreased from ,8 and ,8.8 °C to ,16.0 and ,13.5 °C, respectively. 2.,When aphids from the 20 °C culture were transferred to 10 °C, there was a progressive increase in cold tolerance through three successive generations. Transfer of newly moulted pre-reproductive adults reared at 10 °C for three generations back to 20 °C resulted in a rapid loss of cold hardiness in their nymphal offspring. 3.,In all generations reared at 10 °C, first born nymphs were more cold hardy than those born later in the birth sequence. The LT50 of nymphs produced on the first day of reproduction in the first, second and third generations maintained at 10 °C were ,14.8, ,17.0 and ,16.6 °C, respectively. Thereafter, nymphal cold hardiness decreased over the subsequent 14 days of reproduction in each generation at 10 °C with mean LT50 values of ,10.3, ,12.6 and ,14.8 °C, respectively. By contrast, the cold tolerance of first born nymphs of aphids reared continuously at 20 °C did not differ in comparison with later born siblings. The LT50 of adult aphids was also unaffected by ageing. 4.,The ecological relevance of these findings is discussed in relation to the overwintering survival of aphids such as S. avenae. [source] Folate deficiency in human peripheral blood lymphocytes induces chromosome 8 aneuploidy but this effect is not modified by riboflavinENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2010Juan Ni Abstract Chromosome 8 aneuploidy is a common event in certain cancers but whether folate (F) deficiency induces chromosome 8 aneuploidy is not known. Furthermore the impact of riboflavin (R) deficiency, which may alter activity of a key enzyme in folate metabolism, on these events is unknown. Therefore, the aim of our research was to test the following hypotheses: (a) F deficiency induces chromosome 8 aneuploidy; (b) chromosome 8 aneuploidy is affected by F deficiency to a similar degree as chromosome 17 and (c) R deficiency aggravates the risk of aneuploidy caused by F deficiency. These hypotheses were tested in long-term cultures of lymphocytes from twenty female healthy volunteers (aged 30,48 years). Lymphocytes were cultured in each of the four possible combinations of low (L) and high (H) F (LF, 20 nmol/L, HF 200 nmol/L, respectively) and L and H R (LR 1 nmol/L, HR 500 nmol/L, respectively) media (LFLR, LFHR, HFLR, HFHR) for 9 days. Chromosomes 8 and 17 aneuploidy was measured in mononucleated (MONO) and cytokinesis-blocked binucleated (BN) cells using dual-color fluorescence in situ hybridization (FISH) with fluorescent centromeric probes specific for chromosomes 8 and 17. Culture in LF media (LFLR or LFHR) induced significant and similar increases in frequencies of aneuploidy of chromosomes 8 and 17 (P < 0.001) relative to culture in HF media (HFLR or HFHR). There was no significant effect of R concentration on aneuploidy frequency for either chromosome. We conclude that F deficiency is a possible cause of chromosome 8 aneuploidy. Environ. Mol. Mutagen. 2010. © 2009 Wiley-Liss, Inc. [source] Exploration of the functional hierarchy of the basal layer of human epidermis at the single-cell level using parallel clonal microcultures of keratinocytesEXPERIMENTAL DERMATOLOGY, Issue 4 2010Nicolas O. Fortunel Please cite this paper as: Exploration of the functional hierarchy of the basal layer of human epidermis at the single-cell level using parallel clonal microcultures of keratinocytes. Experimental Dermatology 2010. Abstract:, The basal layer of human epidermis contains both stem cells and keratinocyte progenitors. Because of this cellular heterogeneity, the development of methods suitable for investigations at a clonal level is dramatically needed. Here, we describe a new method that allows multi-parallel clonal cultures of basal keratinocytes. Immediately after extraction from tissue samples, cells are sorted by flow cytometry based on their high integrin-,6 expression and plated individually in microculture wells. This automated cell deposition process enables large-scale characterization of primary clonogenic capacities. The resulting clonal growth profile provided a precise assessment of basal keratinocyte hierarchy, as the size distribution of 14-day-old clones ranged from abortive to highly proliferative clones containing 1.7 × 105 keratinocytes (17.4 cell doublings). Importantly, these 14-day-old primary clones could be used to generate three-dimensional reconstructed epidermis with the progeny of a single cell. In long-term cultures, a fraction of highly proliferative clones could sustain extensive expansion of >100 population doublings over 14 weeks and exhibited long-term epidermis reconstruction potency, thus fulfilling candidate stem cell functional criteria. In summary, parallel clonal microcultures provide a relevant model for single-cell studies on interfollicular keratinocytes, which could be also used in other epithelial models, including hair follicle and cornea. The data obtained using this system support the hierarchical model of basal keratinocyte organization in human interfollicular epidermis. [source] Long-term establishment, characterization and manipulation of cell lines from mouse basal cell carcinoma tumorsEXPERIMENTAL DERMATOLOGY, Issue 9 2006Po-Lin So Abstract:, There have been few reports of successful long-term culture of cells established from cutaneous basal cell carcinoma (BCC) tumors. Here, we describe techniques that have enabled us to establish three long-term cultures of BCC cells isolated from BCC tumors that arose in irradiated Patched 1 (Ptch1)+/, mice. All three cell lines showed cellular morphology similar to that of BCC tumors and could be propagated for at least 20 passages. In addition, similar to BCC tumors, all cell lines had lost the wildtype Ptch1 allele, expressed BCC molecular markers, and responded similarly to cyclopamine, a small molecule inhibitor of Hedgehog signaling. Finally, we describe an efficient electroporation technique for DNA transfection into the BCC cell lines and show that they have activated Hedgehog signaling activity, albeit at a level lower than that of murine BCCs in vivo. These data indicate that the cell lines are bona fide long-term cultures of BCC cells and that DNA plasmids can be introduced into the BCC cell lines with relatively high transfection efficiency using a modified electroporation technique. [source] Primary hepatocyte culture supports hepatitis C virus replication: A model for infection-associated hepatocarcinogenesis,HEPATOLOGY, Issue 6 2010Krishna Banaudha Analysis of progressive changes in hepatic gene expression that underlie hepatocarcinogenesis following hepatitis C virus (HCV) infection require examination of long-term cultures of normally differentiating primary human hepatocytes. We report a culture system of primary hepatocytes that support productive replication of infectious HCV. Hepatic functions were analyzed by reverse-transcription polymerase chain reaction amplification of total cell RNA from cultures maintained in serum-free defined medium for up to 190 days. Sustained hepatic function was assessed by expression of albumin, alpha-fetoprotein, cytochrome P4502E1, cytokeratin-18, type-1 collagen, transforming growth factor-beta 1, matrix metalloproteinase-2 (MMP-2), MMP-13, and interferon alpha-receptors 1 and 2. Normally differentiated human primary hepatocytes supported productive replication of infectious clones of HCV genotypes 1a, 1b, and 2a; virus infection was inhibited by antibodies against CD81 virus entry factor. Virus released into the culture media of HCV-infected primary hepatocytes repeatedly passage to naïve hepatocytes. Replication of the three HCV genotypes shows interferon sensitivity observed in natural infections. Conclusion: Sustained cultures of physiologic host cells for the propagation of infectious HCV strains should accelerate studies of host response to HCV infection and progressive liver disease. Hepatology 2010;51:1922,1932 [source] Prenatal diagnosis and postnatal follow-up of a child with mosaic trisomy 22 with several levels of mosaicism in different tissuesJOURNAL OF OBSTETRICS AND GYNAECOLOGY RESEARCH (ELECTRONIC), Issue 5 2010Vincenzo Mazza Abstract We report on the case of a patient with mosaic trisomy 22, who was diagnosed prenatally by amniocentesis during the 16th week of pregnancy. In the foetus, three trisomic clones were found out of the nine that were analyzed (the other six clones had a 46,XY karyotype). Cytogenetic analysis of cord blood during the 20th week of pregnancy showed a normal male karyotype; however, a placental biopsy that was performed at the same time showed 100% and 95% trisomic cells in the chromosomal analysis of direct and long-term cultures, respectively. A follow-up ultrasonographic examination excluded major congenital malformations and the abdominal and cranial circumferences were normal until the 24th week of pregnancy. At this point, a deflection of the growth curve occurred and the values were persistently below the 3rd centile until birth. After birth, karyotypic and fluorescent in situ hybridisation analyses performed on the fibroblasts of the neonate showed that 3,4% of the cell lines were trisomic, and studies using microsatellite markers showed normal allelic segregation, which excluded uniparental disomy. The period of postnatal follow-up was characterised by a significant growth deficit (height and head circumference were less than the 3rd centile) and by mental retardation. The present case is compatible with other earlier reports that showed that the levels of trisomy 22 are tissue-specific and are of little help in establishing the prognosis of the chromosomal abnormality. [source] Tissue culture methods to study neurological disorders: Establishment of immortalized Schwann cells from murine disease modelsNEUROPATHOLOGY, Issue 1 2003Kazuhiko Watabe Previously, the authors have established spontaneously immortalized cell lines from long-term cultures of normal adult mouse Schwann cells. Establishment of such Schwann cell lines derived from murine disease models may greatly facilitate studies of the cellular mechanisms of their peripheral nervous system lesions in the relevant diseases. Recently, the authors have established immortalized Schwann cell lines derived from Niemann,Pick disease type C mice (NPC; spm/spm) and globoid cell leukodystrophy mice (twitcher). In the present study, long-term cultures were maintained of Schwann cells derived from dorsal root ganglia and consecutive peripheral nerves of another NPC mouse (npcnih/npcnih, npcnih/+), myelin P0 protein-deficient mice (P0,/,, P0+/,) with their wild-type littermates (P0+/+), and neurofibromatosis type 1 gene (NF1)-deficient mice (Nf1Fcr/+) for 8,10 months, and immortalized cell lines from all these animals established spontaneously. These cell lines had spindle-shaped Schwann cell morphology and distinct Schwann cell phenotypes and retained genomic and biochemical abnormalities, sufficiently representing the in vivo pathological features of the mutant mice. These immortalized Schwann cell lines can be useful in studies of nervous system lesions in these mutant mice and relevant human disorders. [source] Improved gene transfer and normalized enzyme levels in primitive hematopoietic progenitors from patients with mucopolysaccharidosis type I using a bioreactorTHE JOURNAL OF GENE MEDICINE, Issue 12 2004Dao Pan Abstract Background One of the major barriers to the clinical application of hematopoietic stem cell (HSC) gene therapy has been relatively low gene transfer efficiency. Other inadequacies of current transduction protocols are related to their multi-step procedures, e.g., using tissue-culture flasks, roller bottles or gas-permeable bags for clinical application. Methods In comparison with a conventional bag transduction protocol, a ,closed' hollow-fiber bioreactor system (HBS) was exploited to culture and transduce human peripheral blood CD34+ progenitor cells (PBPCMPS) from patients with mucopolysaccharidosis type I (MPS I) using an amphotropic retroviral vector based on a murine Moloney leukemia virus LN prototype. Both short-term colony-forming cell (CFC) and long-term culture initiating cell (LTCIC) assays were employed to determine transduction frequency and transgene expression in committed progenitor cells and primitive progenitors with multi-lineage potentials. Results A novel ultrafiltration-transduction method was established to culture and transduce enzyme-deficient PBPCMPS over a 5-day period without loss in viability and CD34 identity (n = 5). Significantly higher transduction efficiencies were achieved in primary CFC that derived from the HBS (5.8,14.2%) in comparison with those from gas-permeable bags (undetectable to 1.7%; p < 0.01). Up to 15-fold higher-than-normal enzyme activity was found in selected PBPCMPS -LP1CD transductants. Moreover, higher gene transfer (4.4-fold) and expression in very primitive progenitors were observed in products from the HBS compared with bag experiments as indicated by CFC derived from primitive LTCIC. Remarkably, with relatively modest gene transfer levels in LTCIC from HBS experiments, the expression of the IDUA transgene corrected the enzyme-deficiency in 5-week long-term cultures (LTC). Conclusions MPS I progenitor cells achieved normalized enzyme levels in LTC after transduction in a HBS system. These studies demonstrate the advantages of a bioreactor-transduction system for viral-mediated stem cell gene transfer. Copyright © 2004 John Wiley & Sons, Ltd. [source] Spermatogonial stem cells: characteristics and experimental possibilities,APMIS, Issue 11-12 2005PEDRO M. APONTE The continuation of the spermatogenic process throughout life relies on a proper regulation of self-renewal and differentiation of the spermatogonial stem cells. These are single cells situated on the basal membrane of the seminiferous epithelium. Only 0.03% of all germ cells are spermatogonial stem cells. They are the only cell type that can repopulate and restore fertility to congenitally infertile recipient mice following transplantation. Although numerous expression markers have been helpful in isolating and enriching spermatogonial stem cells, such as expression of THY-1 and GFR,-1 and absence of c-kit, no specific marker for this cell type has yet been identified. Much effort has been put into developing a protocol for the maintenance of spermatogonial cells in vitro. Recently, coculture systems of testicular cells on various feeder cells have made it possible to culture spermatogonial stem cells for a long period of time, as was demonstrated by the transplantation assay. Even expansion of testicular cells, including the spermatogonial stem cells, has been achieved. In these culture systems, hormones and growth factors are investigated for their role in the process of proliferation of spermatogonial stem cells. At the moment the best culture system known still consists of a mixture of testicular cells with about 1.33% spermatogonial stem cells. Recently pure SV40 large T immortalized spermatogonial stem cell lines have been established. These c-kit-negative cell lines did not show any differentiation in vitro or in vivo. A telomerase immortalized c-kit-positive spermatogonial cell line has been established that was able to differentiate in vitro. Spermatocytes and even spermatids were formed. However, spermatogonial stem cell activity by means of the transplantation assay was not tested for this cell line. Both the primary long-term cultures and immortalized cell lines have represented a major step forward in investigating the regulation of spermatogonial self-renewal and differentiation, and will be useful for identifying specific molecular markers. [source] CD56-expressing T cells that have features of senescence are expanded in rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 1 2007Joshua J. Michel Objective T cells deficient in CD28 expression have been implicated in the pathogenesis of rheumatoid arthritis (RA). Given that CD28-null T cells are functionally heterogeneous, we undertook this study to screen for novel receptors on these cells. Methods Seventy-two patients with RA (ages 35,84 years) and 53 healthy persons (32 young controls ages 19,34 years, 21 older controls ages 39,86 years) were recruited. Phenotypes and proliferative capacity of T cells from fresh leukocytes and of long-term cultures were monitored by flow cytometry. Lung biopsy specimens from patients with RA-associated interstitial pneumonitis (IP) were examined by immunohistochemistry. Receptor functionality was assessed by crosslinking bioassays. Results Chronic stimulation of CD28+ T cells in vitro yielded progenies that lacked CD28 but that gained CD56. Ex vivo analysis of leukocytes from patients with extraarticular RA showed a higher frequency of CD56+,CD28-null T cells than in patients with disease confined to the joints or in healthy controls. CD56+,CD28-null T cells had nil capacity for proliferation, consistent with cellular senescence. CD56+ T cells had skewed T cell receptor (TCR) ,/,-chain usage and restricted TCR third complementarity-determining region spectra. Histologic studies showed that CD56+ T cells were components of cellular infiltrates in RA-associated IP. CD56 crosslinking on T cells sufficiently induced cytokine production, although CD56/TCR coligation induced higher production levels. Conclusion Chronic activation of T cells induces counterregulation of CD28 and CD56 expression. The loss of CD28 is accompanied by the gain of CD56 that confers TCR-independent and TCR-dependent activation pathways. We propose that accumulation of CD56+ T cells in RA contributes to maladaptive immune responses and that CD56+ T cells are potential targets for therapy. [source] | ||||||||||||||||||||||||||||