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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Transduced cells gave rise to cystic bodies with similar CD34 cell content and profiles upon development.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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The transduced hES-CD34 cells also gave rise to apparently normal macrophages that expressed the transgene as shown by GFP expression.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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These results are consistent with those of others that showed normal differentiation of hES cells to other cell types following lentiviral transduction .
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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A requirement for successful cellular and HIV-1 gene therapy is that mature end stage cells derived from CD34 progenitor cells be phenotypically and functionally normal to maintain and restore the body's immunological function.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Accordingly, hES cell derived macrophages were evaluated to determine if they met these criteria.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Macrophages display distinct cell surface markers upon end stage differentiation.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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To determine whether hES cell derived macrophages display these surface markers, FACS analysis was performed to detect the presence of CD14, HLA-DR (MHCII), CD4, CCR5, and CXCR4.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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As observed in Fig 2A, both nontransduced and transduced hES cell derived macrophages expressed all of these markers with some differences in their levels of expression.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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HLA-DR, CD4, and CCR5 expression profiles were comparable between all cell types analyzed.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Even though all cell types analyzed stained positive for CD14, relative expression of CD14 was slightly lower on hES cell derived macrophages compared to fetal liver CD34 cell derived macrophages.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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On the contrary, the levels of CXCR4, a chemokine receptor involved in cellular homing, were found to be higher on hES-CD34 cell derived macrophages.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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This may be due to inherent differences in the cell types and/or due to their physiological state at the time of harvest .
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Additional hES cell lines need to be evaluated in the future to establish if these differences are consistent.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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A major functional role of macrophages in vivo is their ability to serve as professional antigen presenting cells.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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During this process macrophages present antigen peptide fragments complexed with both classes of MHC molecules and deliver a costimulatory signal through the expression of B7 molecules.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Upon stimulation with LPS, hES-CD34 cell derived macrophages had shown upregulation of the costimulatory molecule B7.1 similar to cells derived from fetal liver.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Furthermore, the hES-CD34 cell derived macrophages also showed a normal capacity to ingest foreign particles in phagocytosis assays using E.coli Bioparticles.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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In addition to antigen presentation and phagocytosis, macrophages also play a critical role in inflammation and secrete cytokines in response to external stimuli.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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When exposed to LPS, the hES-CD34 cell derived macrophages secreted two important cytokines IL-1 and TNF-α similar to that of fetal liver derived cells.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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The above data has established that phenotypically and functionally normal macrophages could be derived from hES-CD34 cells.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Macrophages in addition to playing important physiological roles are also major cell targets for certain viral infections, particularly HIV-1.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Here we evaluated the susceptibility of hES-CD34 cell derived macrophages to be productively infected with HIV-1.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Similar to that of fetal liver CD34 cell derived cells, the hES-CD34 macrophages also supported HIV-1 infection although the levels of viral yield differed somewhat.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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However this should not be a major concern for testing anti-HIV genes in these cells.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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In all the above experiments, the vector transduced transgenic macrophages also behaved similarly to that of nontransduced cells showing that they were also physiologically normal.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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The lack of vector toxicity on cellular maturation is encouraging for future work with transduced hES-CD34 cells to derive other important differentiated cells like T cells and dendritic cells relevant for HIV studies.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Although there are numerous studies on hES cell differentiation into many important end stage mature cells, systematic work on hES cell hematopoietic differentiation and thorough characterization of end stage mature cells that participate in critical immune responses has just begun [21,27-29].
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Our current results established that physiologically normal macrophages could be derived from hES cells and that these cells have the potential for use in cellular and gene therapies.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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To our knowledge this is the first demonstration that hES cell derivatives can be used for infectious disease research.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Due to the extensive ability for hES cells to self-renew, large numbers of differentiated cells can be derived so that infection studies and evaluation tests can be carried out in a more standardized way.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Our results showing that both normal and transgenic derivative macrophages support HIV-1 infection points out to their utility for testing anti-HIV constructs transduced into hES-CD34 cells and pave the way for their application in stem cell based HIV gene therapy.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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So far a number of studies including our own have tested many gene therapeutic constructs in CD34 cells from conventional sources.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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These constructs include anti-HIV ribozymes, RNA decoys, transdominant proteins, bacterial toxins, anti-sense nucleic acids, and most recently siRNAs [36-50].
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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In addition, a number of cellular molecules that aid in HIV-1 infection such as cellular receptors and coreceptors CD4, CCR5 and CXCR4 have also been successfully tested in CD34 cell derived macrophages and T cells .
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Some of these approaches have progressed into clinical evaluations as well .
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Based on our current results, many of these novel anti-HIV constructs can also be tested in hES-CD34 cells for their potential application.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Although there are advantages of using hES cell derived CD34 cells for potential cellular therapies, transplantation of these cells constitutes an allogenic source with immune rejection as a major issue.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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However, a recent study using human leukocyte reconstituted mice suggested that hESCs and their derivative cell types were less prone to invoking an allogeneic response .
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Other recent studies demonstrated successful engraftment of primary and secondary recipients with hES cell derived hematopoietic cells in both immunodeficient mice and in vivo fetal sheep models adding further support that any obstacles could be overcome .
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Moreover, multiple novel strategies to avoid immune-mediated rejection of hES cell-derived cells have been proposed .
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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It is not too far in the future that even autologous hES cells may be derived from specific individuals for deriving CD34 cells which can be used for cell replacement therapy.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Phenotypically normal and functionally competent macrophages could be derived from hES-CD34 cells.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Since these cells are susceptible to HIV-1 infection, they provide a uniform source of macrophages for viral infection studies.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Based on these results, it is also now feasible to transduce hES-CD34 cells with anti-HIV genes such as inhibitory siRNAs and test their antiviral efficacy in down stream differentiated cells such as macrophages which are among the primary cells that need to be protected against HIV-1 infection.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Thus, the potential utility of hES derived CD34 hematopoietic cells for HIV-1 gene therapy can be evaluated.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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The NIH approved human ES H1 cell line was obtained from WiCell (Madison, Wisconsin).
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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hES cell colonies were cultured on mouse embryonic fibroblasts (MEF) (Chemicon, Temecula, CA) in the presence of DMEM-F12 (Invitrogen, Carlsbad, CA) supplemented with 20% KNOCKOUT serum replacement with 1 mM L-glutamine, 1% Nonessential Amino Acids, 0.1 mM β-mercaptoethanol, 0.5% penicillin/streptomycin, and 4 ng/ml human basic fibroblast growth factor.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Culture medium was replaced daily with fresh complete DMEM-F12.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Mature colonies were subcultured weekly by digesting with collagenase IV as previously described .
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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A VSV-G pseudotyped lentiviral vector (SINF-EF1a-GFP) containing a GFP reporter gene (kindly supplied by R. Hawley, George Washington University) was used for hES cell transductions as previously described (30, 58).
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Generation of the pseudotyped vector in 293T cells and its concentration by ultracentrifugation were described previously .
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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For vector transduction, the undifferentiated hES cells were prepared into small clumps of 50–100 cells with enzyme digestion as done for routine passaging of cells.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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The cell clumps were incubated with the vector for 2 hrs in the presence of polybrene 6 ug/ml.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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A secondary cycle of transduction was done by adding fresh vector and incubating for another 2 hrs.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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The general vector titers were 1 × 10and the multiplicity of infection was 10.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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The transduction efficiency was about 50%.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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The transduced colonies were cultured on MEF like above.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Undifferentiated hES cells were cultured on S17 mouse bone marrow stromal cell monolayers to derive cystic bodies containing CD34+ hematopoietic progenitor stem cells.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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hES cell cultures were treated with collagenase IV(1 mg/ml) for 10 minutes at 37°C and subsequently detached from the plate by gentle scraping of the colonies.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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The hES cell clusters were then transferred to irradiated (35 Gy) S17 cell layers and cultured with RPMI differentiation medium containing 15% FBS (HyClone, Logan, UT), 2 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% MEM-nonessential amino acids, and 1% penicillin/streptomycin.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Media was changed every 2 to 3 days during 14–17 days of culture on S17 cells .
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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After allowing adequate time for differentiation, hES cystic bodies were harvested and processed into a single cell suspension by collagenase IV treatment followed by digestion with trypsin/EDTA supplemented with 2% chick serum (Invitrogen, Carlsbad, CA) for 20 minutes at 37°C.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Cells were washed twice with PBS and filtered through a 70 uM cell strainer to obtain a single cell suspension.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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To assess the levels of CD34 cells in the bulk cell suspension, cells were labeled with PE conjugated anti-CD34 antibody (BD Biosciences, San Jose, CA) and analyzed by FACS.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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To purify the CD34 cells, Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech, Auburn, CA) was used following the manufacturer's protocol.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Isolated CD34 hematopoietic progenitor stem cells were then analyzed by FACS as mentioned above to determine cell purity.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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For comparative experiments, human CD34 hematopoietic progenitor cells were also purified from fetal liver tissue as described above.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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CD34 cells were cultured initially in semisolid media to derive myelomonocytic colonies followed by liquid culture in cytokine supplemented media as described below.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Purified CD34+ progenitor cells (~2.5 × 10to 4.0 × 10) were placed directly into Methocult semisolid medium (Stem Cell Technologies, Vancouver, BC), mixed, and cultured in 35 mm plates.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Myeloid colonies were allowed to develop for 12–15 days.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Upon differentiation and proliferation, myelomonocytic colonies were harvested by the addition of 5 ml DMEM containing 10% FBS, 10 ng/ml each GM-CSF and M-CSF.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Cells (~10) were placed in a 35 mm well and allowed to adhere for 48 hours.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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At two and four days post-harvest, medium was replaced with fresh complete DMEM supplemented with 10 ng/ml GM-CSF and M-CSF.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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By 4–5 days, cells developed into mature macrophages which were used for subsequent phenotypic and functional characterization.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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To determine if nontransduced and lentiviral vector transduced hES cell derived macrophages display normal macrophage surface markers, FACS analysis was performed using respective fluorochrome conjugated antibodies.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Fetal liver derived CD34+ cells as well as nontransduced and transduced hES cell derived macrophages were evaluated in parallel.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Cells were scraped from their wells, washed two times with PBS, and stained with the following antibodies: PE-CD14, PE-HLA-DR, PECY5-CD4, PECY5-CCR5, PECY5-CXCR4 (BD Biosciences, San Jose, CA).
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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A blocking step was first performed by incubating the cells with the respective isotype control for 30 minutes at 4C before staining with the respective cell surface marker antibodies.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Isotype control staining was used to determine background levels.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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FACS analysis was performed on a Beckman-Coulter EPICS XL-MCL flow cytometer with data analysis using EXPO32 ADC software (Coulter Corporation, Miami, FL).
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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A minimum of 8,000 cells were analyzed in each FACS evaluation.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Physiological roles of macrophages include phagocytic and immune related functions.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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To determine if hES cell derived macrophages were functionally normal, a stimulation assay to determine upregulation of the costimulatory molecule B7.1 was performed.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Activated macrophages upregulate the expression of B7.1 upon activation with various stimuli.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Accordingly, fetal liver CD34, nontransduced hES, and GFP-alone transduced hES cell derived macrophages were stimulated by the addition of LPS (5 ug/ml) to the cell culture medium.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Twenty-four hours post-stimulation, cells were stained with an anti-B7.1 antibody labeled with PE-Cy5 (BD Biosciences, San Jose, CA) and analyzed by FACS.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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To assess the hES cell derived macrophages' phagocytic function, 5 ug/ml of fluorescently labeled E. coli Bioparticles(Invitrogen, Carlsbad, CA) were added directly to the cell culture medium.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Four hours later, macrophages were washed six times with PBS and fresh medium with 10 ng/ml GM-CSF and M-CSF was added.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Twenty-four hours later, cells were analyzed by FACS for the presence of ingested Bioparticleswhich can be detected in the PE (FL2) channel.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Lentiviral vector transduced Magi-CXCR4 cells, a HeLa cell derivative with no phagocytic capacity, were used as non-phagocytic cell controls and similarly exposed to E. coli Bioparticles Human ES cell derived macrophages were also analyzed for their ability to secrete two major cytokines, IL-1 and TNF-α, upon external stimulation.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Accordingly, macrophages were stimulated with 5 ug/ml of LPS during culture.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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On days 1, 2, and 3 post-stimulation, cell culture supernatant samples were collected and analyzed by a QuantikineELISA kit (R&D Systems, Minneapolis, MN).
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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Non-stimulated supernatants were also analyzed for basal levels of cytokine secretion.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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To determine if hES cell derived macrophages can be infected with HIV-1 and support viral replication, cells were challenged with a macrophage R5-tropic BaL-1 strain of HIV-1.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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An m.o.i.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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of 0.01 in the presence of 4 ug/ml polybrene was used.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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At different days post-infection, culture supernatants were collected and assayed for p24 antigen by ELISA.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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To quantify viral p24 levels, a Coulter-p24 kit (Beckman Coulter, Fullerton, CA) was used.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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The author(s) declare that they have no competing interests.
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PMC1462997
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Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy.
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JA and SB contributed equally to this work.
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