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Immortalized Cell Lines (immortalized + cell_line)
Selected AbstractsImmortalized cell lines from mouse xiphisternum preserve chondrocyte phenotypeJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2006Manas K. Majumdar Chondrocytes are unique to cartilage and the study of these cells in vitro is important for advancing our understanding of the role of these cells in normal homeostasis and disease including osteoarthritis (OA). As there are limitations to the culture of primary chondrocytes, cell lines have been developed to overcome some of these obstacles. In this study, we developed a procedure to immortalize and characterize chondrocyte cell lines from mouse xiphisternum. The cells displayed a polygonal to fibroblastic morphology in monolayer culture. Gene expression studies using quantitative PCR showed that the cell lines responded to bone morphogenetic protein 2 (BMP-2) by increased expression of matrix molecules, aggrecan, and type II collagen together with transcriptional factor, Sox9. Stimulation by IL-1 results in the increased expression of catabolic effectors including MMP-13, nitric oxide synthase, ADAMTS4, and ADAMTS5. Cells cultured in alginate responded to BMP-2 by increased synthesis of proteoglycan (PG), a major matrix molecule of cartilage. IL-1 treatment of cells in alginate results in increased release of PG into the conditioned media. Further analysis of the media showed the presence of Aggrecanase-cleaved aggrecan fragments, a signature of matrix degradation. These results show that the xiphisternum chondrocyte cell lines preserve their chondrocyte phenotype cultured in either monolayer or 3-dimensional alginate bead culture systems. In summary, this study describes the establishment of chondrocyte cell lines from the mouse xiphisternum that may be useful as a surrogate model system to understand chondrocyte biology and to shed light on the underlying mechanism of pathogenesis in OA. J. Cell. Physiol. 209: 551,559, 2006. © 2006 Wiley-Liss, Inc. [source] Proceedings of the Australian Physiological and Pharmacological Society Symposium: New Frontiers in Muscle Research Gene transfer: manipulating and monitoring function in cells and tissuesCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 8 2001Rekha G Panchal SUMMARY 1. The ectopic expression of genes has proven to be an extremely valuable tool for biologists. The most widely used systems involve electrically or chemically mediated transfer of genes to immortalized cell lines and, at the other end of the spectrum, transgenic animal models. As would be expected, there are compromises to be made when using either of these broad approaches. Immortalized cell lines have limited ,physiological relevance' and transgenic approaches are costly and out of the reach of many laboratories. There is also significant time required for the de novo generation of a transgenic animal. 2. As a viable alternative to these approaches, we describe the use of recombinant adenovirus and Sindbis virus to deliver genes to cells and tissues. 3. We exemplify this approach with studies from our laboratories: (i) an investigation of Ca2+ handling deficits in cardiac myocytes of hypertrophied hearts using infection with recombinant adenovirus encoding either green fluorescent protein (GFP) or the sarcoplasmic/endoplasmic reticulum calcium-ATPase (Serca2a); (ii) a study of the mechanism of macrophage/microglial migration by infection of embryonic phagocytes with a GFP-encoding virus and coculture with brain slices to then track the movement of labelled cells; and (iii) we are also exploiting the natural tropism of the Sindbis virus to label neurons in hippocampal brain slices in culture to resolve high-resolution structure and to map neuronal connectivity. 4. Further development of these approaches should open new avenues of investigation for the study of physiology in a range of cells and tissues. [source] Characterization of a chemokine receptor CCR5-negative T cell line and its use in determining human immunodeficiency virus type 1 phenotypeJOURNAL OF MEDICAL VIROLOGY, Issue 2 2008Dorothea Binninger-Schinzel Abstract A human CD4-positive T cell line from a donor homozygous negative for the chemokine receptor CCR5 was established, characterized, and used for determining the coreceptor usage of human immunodeficiency virus type 1 (HIV-1) isolates. Clones of this IL-2 dependent human T-cell lymphotropic virus type 1 (HTLV-I) immortalized cell line, named IsnoR5 clones 1 and 2, are susceptible to infection by HIV-1 isolates that use CXCR4 as a coreceptor but resistant to infection by CCR5 tropic HIV-1 viruses. HIV-1 isolates whose replication is inhibited in IsnoR5 cells in the presence of the bicyclam AMD 3100, a CXCR4 specific inhibitor, utilize a coreceptor distinct from CCR5 and CXCR4. Using a panel of primary HIV-1 isolates we have shown that a single T cell line is sufficient to discriminate between use of CCR5, CXCR4 or an alternative coreceptor. As IsnoR5 clone 1 cells revealed the existence of even minor populations of CXCR4-using virus variants, they could be useful for the early identification of changes in coreceptor usage in HIV infected individuals facilitating the timely introduction of appropriate clinical treatments. J. Med. Virol. 80:192,200, 2008. © 2007 Wiley-Liss, Inc. [source] Selective induction of human beta-defensin mRNAs by Actinobacillus actinomycetemcomitans in primary and immortalized oral epithelial cellsMOLECULAR ORAL MICROBIOLOGY, Issue 6 2003E. C. Feucht Human beta-defensin-2, and -3 (hBD-2, -3) are small inducible antimicrobial peptides involved in host defense. Actinobacillus actinomycetemcomitans, a gram-negative facultative anaerobe, is frequently associated with oral disease in humans. A. actinomycetemcomitans, strain JP2, was examined for its ability to modulate hBD-2 and -3 gene expression in normal human oral epithelial cells (NHOECs) and in OKF6/Tert cells, an immortalized cell line derived from human oral epithelial cells. Stimulation of both cell types by live bacteria, at a minimal bacteria/cell ratio of 500 : 1, resulted in increased hBD-3 gene expression. This was not evinced for hBD-2 in either cell type with live bacteria, even at bacteria/cell ratios exceeding 500 : 1. The increased hBD-3 gene expression was dependent upon viable bacteria, and not their lipopolysaccharides (LPS), since heat-killed A. actinomycetemcomitans did not induce hBD-3 transcript expression. The overall similarity between results obtained in OKF6/Tert cells and NHOECs suggest that the OKF6/Tert cell line may be a useful tool in the study of beta-defensin expression in oral epithelium. [source] Molecular characterization of conditionally immortalized cell lines derived from mouse early embryonic inner earDEVELOPMENTAL DYNAMICS, Issue 4 2004John A. Germiller Abstract Inner ear sensory hair cells (HCs), supporting cells (SCs), and sensory neurons (SNs) are hypothesized to develop from common progenitors in the early embryonic otocyst. Because little is known about the molecular signals that control this lineage specification, we derived a model system of early otic development: conditionally immortalized otocyst (IMO) cell lines from the embryonic day 9.5 Immortomouse. This age is the earliest stage at which the otocyst can easily be separated from surrounding mesenchymal, nervous system, and epithelial cells. At 9.5 days post coitum, there are still pluripotent cells in the otocyst, allowing for the eventual identification of both SN and HC precursors,and possibly an elusive inner ear stem cell. Cell lines derived from primitive precursor cells can also be used as blank canvases for transfections of genes that can affect lineage decisions as the cells differentiate. It is important, therefore, to characterize the "baseline state" of these cell lines in as much detail as possible. We characterized seven representative "precursor-like" IMO cell populations and the uncloned IMO cells, before cell sorting, at the molecular level by polymerase chain reaction (PCR) and immunocytochemistry (IHC), and one line (IMO-2B1) in detail by real-time quantitative PCR and IHC. Many of the phenotypic markers characteristic of differentiated HCs or SCs were detected in IMO-2B1 proliferating cells, as well as during differentiation for up to 30 days in culture. These IMO cell lines represent a unique model system for studying early stages of inner ear development and determining the consequences of affecting key molecular events in their differentiation. Developmental Dynamics 231:815,827, 2004. © 2004 Wiley-Liss, Inc. [source] Genotype-dependent priming to self- and xeno-cannibalism in heterozygous and homozygous lymphoblasts from patients with Huntington's diseaseJOURNAL OF NEUROCHEMISTRY, Issue 4 2006Elisabetta Mormone Abstract In the present work, we studied the mitochondrial function and cell death pathway(s) in heterozygous and homozygous immortalized cell lines from patients with Huntington's disease (HD). Heterozygosis was characterized by specific alterations in mitochondrial membrane potential, a constitutive hyperpolarization state of mitochondria, and was correlated with an increased susceptibility to apoptosis. Lymphoblasts from homozygous patients, on the other hand, were characterized by a significant percentage of cells displaying autophagic vacuoles. These cells also demonstrated a striking attitude towards significant cannibalistic activity. Considering the pathogenic role of cell death in HD, our study provides new and useful insights into the role of mitochondrial dysfunction, i.e. hyperpolarization, in hijacking HD heterozygous cells towards apoptosis and HD homozygous cells towards a peculiar phenotype characterized by both self- and xeno-cannibalism. These events can, however, be viewed as an ultimate attempt to survive rather than a way to die. The present work underlines the possibility that HD-associated mitochondrial defects could tentatively be by-passed by the cells by activating cellular ,phagic' activities, including so-called ,mitophagy' and ,cannibalism', that only finally lead to cell death. [source] Differential expression of periodontal ligament-specific markers and osteogenic differentiation in human papilloma virus 16-immortalized human gingival fibroblasts and periodontal ligament cellsJOURNAL OF PERIODONTAL RESEARCH, Issue 2 2007S.-H. Pi Background and Objective:, Periodontal ligament cells and gingival fibroblasts are important in the remodeling of periodontal tissue, but human papilloma virus (HPV)16-immortalized cell lines derived from human periodontal ligament cells and gingival fibroblasts has not been characterized. The purpose of this study was to establish and differentially characterize the immortalized cell lines from gingival fibroblasts and periodontal ligament by HPV16 transfection. Material and Methods:, Cell growth, cell cycle analysis, western blot for cell cycle regulatory proteins and osteogenic differentiation markers, and reverse transcription,polymerase chain reaction for periodontal ligament-specific markers were performed. Results:, Both immortalized cell lines (immortalized gingival fibroblasts and immortalized periodontal ligament cells) grew faster than primary cultured gingival fibroblasts or periodontal ligament cells. Immortalized gingival fibroblasts and immortalized periodontal ligament cells overexpressed proteins p16 and p21, and exhibited degradation of proteins pRb and p53, which normally cause cell cycle arrest in G2/M-phase. Western blotting and reverse transcription,polymerase chain reaction for periodontal ligament-specific and osteogenic differentiation marker studies demonstrated that a cell line, designated IPDL, mimicked periodontal ligament gene expression for alkaline phosphatase, osteonectin, osteopontin, bone sialoprotein, bone morphogenic protein-2, periostin, S-100A4 and PDLs17. Conclusion:, These results indicate that IPDL and immortalized gingival fibroblast cell lines consistently retain normal periodontal ligament and gingival fibroblast phenotypes, respectively, and periodontal ligament markers and osteogenic differentiation in IPDL are distinct from immortalized gingival fibroblast cells. [source] Human neural stem cells genetically modified for brain repair in neurological disordersNEUROPATHOLOGY, Issue 3 2004Seung U. Kim Existence of multipotent neural stem cells (NSC) has been known in developing or adult mammalian CNS, including humans. NSC have the capacity to grow indefinitely and have multipotent potential to differentiate into three major cell types of CNS, neurons, astrocytes and oligodendrocytes. Stable clonal lines of human NSC have recently been generated from the human fetal telencephalon using a retroviral vector encoding v-myc. One of the NSC lines, HB1.F3, carries normal human karyotype of 46XX and has the ability to self-renew, differentiate into cells of neuronal and glial lineages, and integrate into the damaged CNS loci upon transplantation into the brain of animal models of Parkinson disease, HD, stroke and mucopolysaccharidosis. F3 human NSC were genetically engineered to produce L-dihydroxyphenylalanine (L-DOPA) by double transfection with cDNA for tyrosine hydroxylase and guanosine triphosphate cylohydrolase-1, and transplantation of these cells in the brain of Parkinson disease model rats led to L-DOPA production and functional recovery. Proactively transplanted F3 human NSC in rat striatum, supported the survival of host striatal neurons against neuronal injury caused by 3-nitropro-pionic acid in rat model of HD. Intravenously introduced through the tail vein, F3 human NSC were found to migrate into ischemic lesion sites, differentiate into neurons and glial cells, and improve functional deficits in rat stroke models. These results indicate that human NSC should be an ideal vehicle for cell replacement and gene transfer therapy for patients with neurological diseases. In addition to immortalized human NSC, immortalized human bone marrow mesenchymal stem cell lines have been generated from human embryonic bone marrow tissues with retroviral vectors encording v-myc or teromerase gene. These immortalized cell lines of human bone marrow mesenchymal stem cells differentiated into neurons/glial cells, bone, cartilage and adipose tissue when they were grown in selective inducing media. There is further need for investigation into the neurogenic potential of the human bone marrow stem cell lines and their utility in animal models of neurological diseases. [source] Gene expression changes in human cells after exposure to mobile phone microwavesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 17 2006Daniel Remondini Abstract Possible biological effects of mobile phone microwaves were investigated in,vitro. In this study, which was part of the 5FP EU project REFLEX (Risk Evaluation of Potential Environmental Hazards From Low-Energy Electromagnetic Field Exposure Using Sensitive in,vitro Methods), six human cell types, immortalized cell lines and primary cells, were exposed to 900 and 1800,MHz. RNA was isolated from exposed and sham-exposed cells and labeled for transcriptome analysis on whole-genome cDNA arrays. The results were evaluated statistically using bioinformatics techniques and examined for biological relevance with the help of different databases. NB69 neuroblastoma cells, T,lymphocytes, and CHME5 microglial cells did not show significant changes in gene expression. In EA.hy926 endothelial cells, U937,lymphoblastoma cells, and HL-60 leukemia cells we found between 12 and 34,up- or down-regulated genes. Analysis of the affected gene families does not point towards a stress response. However, following microwave exposure, some but not all human cells might react with an increase in expression of genes encoding ribosomal proteins and therefore up-regulating the cellular metabolism. [source] | |||||||||||||