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Methods
Cell culture and treatments. BG01V cells are embryonic stem cells that are easier to culture than BG01 cells without inadvertently causing cell differentiation. They are a derivative of BG01 cells with karyotypic abnormalities (49, +12, +17 and XXY) which retain embryonic stem cell markers and characteristics, and the ability to differentiate down a neural lineage ADDIN EN.CITE Dhara2008105251052517Dhara, S. K.Hasneen, K.Machacek, D. W.Boyd, N. L.Rao, R. R.Stice, S. L.Regenerative Bioscience Center, University of Georgia, Athens, GA 30605, USA.Human neural progenitor cells derived from embryonic stem cells in feeder-free culturesDifferentiation454-647652008Jan 318177420http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18177420 [49]. Differentiation was performed according to published protocols to obtain populations of NPs and astrocytes ADDIN EN.CITE Wilczynska2009105641056417Wilczynska, K. M.Singh, S. K.Adams, B.Bryan, L.Rao, R. R.Valerie, K.Wright, S.Griswold-Prenner, I.Kordula, T.Department of Biochemistry and Molecular Biology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA.Nuclear factor I isoforms regulate gene expression during the differentiation of human neural progenitors to astrocytesStem Cells1173-81275AnimalsAstrocytes/*cytology/metabolismCalcium-Binding Proteins/metabolismCell Differentiation/*geneticsCell LineExtracellular Matrix Proteins/metabolism*Gene Expression RegulationGlial Fibrillary Acidic Protein/metabolismHumansMiceModels, BiologicalNFI Transcription Factors/genetics/*metabolismNeurons/*cytology/metabolismProtein Isoforms/genetics/metabolismStem Cells/*cytology/metabolism2009May19418463http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19418463 Lawrence2006105271052717Lawrence, D. M.Seth, P.Durham, L.Diaz, F.Boursiquot, R.Ransohoff, R. M.Major, E. O.Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurologic Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.Astrocyte differentiation selectively upregulates CCL2/monocyte chemoattractant protein-1 in cultured human brain-derived progenitor cellsGlia81-91531AIDS Dementia Complex/immunology/metabolism/physiopathologyAstrocytes/cytology/immunology/*metabolismBinding Sites/physiologyCell Differentiation/genetics/*immunologyCells, CulturedCentral Nervous System/cytology/*embryology/metabolismChemokine CCL2/genetics/*metabolismChemotaxis/genetics/immunologyHIV-1/immunology/metabolismHumansJC Virus/immunology/metabolismNF-kappa B/genetics/metabolismNFI Transcription Factors/genetics/metabolismPhenotypePolyomavirus Infections/immunology/metabolism/physiopathologyPromoter Regions (Genetics)/geneticsStem Cells/cytology/immunology/*metabolismTrans-Activation (Genetics)/physiologyTumor Virus Infections/immunology/metabolism/physiopathologyUp-Regulation/genetics/*immunology2006Jan 116206198http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16206198 Adams2010119181191817Adams, B. R.Golding, S. E.Rao, R. R.Valerie, K.Department of Radiation Oncology, Virginia Commonwealth University, Richmond, Virginia, USA.Dynamic dependence on ATR and ATM for double-strand break repair in human embryonic stem cells and neural descendantsPLoS Onee1000154201020368801http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=20368801 [5, 27, 50]. Briefly, BG01V cells were cultured in ES medium as described in ADDIN EN.CITE Shin2006105231052317Shin, S.Mitalipova, M.Noggle, S.Tibbitts, D.Venable, A.Rao, R.Stice, S. L.Regenerative Bioscience Center, University of Georgia, Athens, Georgia 30605, USA.Long-term proliferation of human embryonic stem cell-derived neuroepithelial cells using defined adherent culture conditionsStem Cells125-38241AnimalsCell AdhesionCell CountCell Differentiation/drug effectsCell LineCell Proliferation/*drug effectsCell Survival/drug effectsCulture Media, ConditionedGrowth Substances/pharmacologyHumansMiceNeuroepithelial Cells/*physiologyOxygen/pharmacologyRosette FormationStem Cells/*physiologyTissue Culture Techniques/*methods2006Jan16100006http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16100006 Wilczynska2009105641056417Wilczynska, K. M.Singh, S. K.Adams, B.Bryan, L.Rao, R. R.Valerie, K.Wright, S.Griswold-Prenner, I.Kordula, T.Department of Biochemistry and Molecular Biology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA.Nuclear factor I isoforms regulate gene expression during the differentiation of human neural progenitors to astrocytesStem Cells1173-81275AnimalsAstrocytes/*cytology/metabolismCalcium-Binding Proteins/metabolismCell Differentiation/*geneticsCell LineExtracellular Matrix Proteins/metabolism*Gene Expression RegulationGlial Fibrillary Acidic Protein/metabolismHumansMiceModels, BiologicalNFI Transcription Factors/genetics/*metabolismNeurons/*cytology/metabolismProtein Isoforms/genetics/metabolismStem Cells/*cytology/metabolism2009May19418463http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19418463 [27, 54] consisting of Dulbeccos modified Eagles medium (DMEM)/F12 medium (GIBCO, Grand Island, NY) supplemented with 20% knockout serum replacement (KSR) (GIBCO), 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 U/ml penicillin, 50 g/ml streptomycin, 0.1 mM (-mercapto-ethanol, and 5 ng/ml basic fibroblast growth factor (b-FGF) (R & D Systems, Minneapolis, MN). Cells were expanded on dishes of decellularized MEF feeders. Derivation of NPs was performed as described ADDIN EN.CITE Shin2006105231052317Shin, S.Mitalipova, M.Noggle, S.Tibbitts, D.Venable, A.Rao, R.Stice, S. L.Regenerative Bioscience Center, University of Georgia, Athens, Georgia 30605, USA.Long-term proliferation of human embryonic stem cell-derived neuroepithelial cells using defined adherent culture conditionsStem Cells125-38241AnimalsCell AdhesionCell CountCell Differentiation/drug effectsCell LineCell Proliferation/*drug effectsCell Survival/drug effectsCulture Media, ConditionedGrowth Substances/pharmacologyHumansMiceNeuroepithelial Cells/*physiologyOxygen/pharmacologyRosette FormationStem Cells/*physiologyTissue Culture Techniques/*methods2006Jan16100006http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16100006 [54]. Briefly, cells were grown on laminin coated dishes in DMEM/F12 medium containing 15% FBS and 5% KSR followed by an additional 7 days in media containing (DMEM/F12, N2 supplement (GIBCO), penicillin/streptomycin, L-glutamine, 5 ng/mL of b-FGF, and 10 ng/mL leukemia inhibitory factor (LIF). The cells were cultured in DN2 media for an additional 7 days to obtain the NP population. NPs were propagated on poly-ornithine- and laminin-coated plates in Neurobasal A Medium (GIBCO) supplemented with B-27 (GIBCO), L-glutamine, and penicillin/streptomycin, 20 ng/mL b-FGF, and 10 ng/mL of LIF. Astrocytes were obtained by exposure of NPs to DMEM and FBS for 25 days on laminin-coated plates as described ADDIN EN.CITE Wilczynska2009105641056417Wilczynska, K. M.Singh, S. K.Adams, B.Bryan, L.Rao, R. R.Valerie, K.Wright, S.Griswold-Prenner, I.Kordula, T.Department of Biochemistry and Molecular Biology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA.Nuclear factor I isoforms regulate gene expression during the differentiation of human neural progenitors to astrocytesStem Cells1173-81275AnimalsAstrocytes/*cytology/metabolismCalcium-Binding Proteins/metabolismCell Differentiation/*geneticsCell LineExtracellular Matrix Proteins/metabolism*Gene Expression RegulationGlial Fibrillary Acidic Protein/metabolismHumansMiceModels, BiologicalNFI Transcription Factors/genetics/*metabolismNeurons/*cytology/metabolismProtein Isoforms/genetics/metabolismStem Cells/*cytology/metabolism2009May19418463http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=19418463 Lawrence2006105271052717Lawrence, D. M.Seth, P.Durham, L.Diaz, F.Boursiquot, R.Ransohoff, R. M.Major, E. O.Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurologic Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.Astrocyte differentiation selectively upregulates CCL2/monocyte chemoattractant protein-1 in cultured human brain-derived progenitor cellsGlia81-91531AIDS Dementia Complex/immunology/metabolism/physiopathologyAstrocytes/cytology/immunology/*metabolismBinding Sites/physiologyCell Differentiation/genetics/*immunologyCells, CulturedCentral Nervous System/cytology/*embryology/metabolismChemokine CCL2/genetics/*metabolismChemotaxis/genetics/immunologyHIV-1/immunology/metabolismHumansJC Virus/immunology/metabolismNF-kappa B/genetics/metabolismNFI Transcription Factors/genetics/metabolismPhenotypePolyomavirus Infections/immunology/metabolism/physiopathologyPromoter Regions (Genetics)/geneticsStem Cells/cytology/immunology/*metabolismTrans-Activation (Genetics)/physiologyTumor Virus Infections/immunology/metabolism/physiopathologyUp-Regulation/genetics/*immunology2006Jan 116206198http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16206198 [27, 50].
Cells were grown on Lab-Tek (Naperville, IL) glass slides. After treatment, cells were fixed with 3% paraformaldehyde, permeabilized with 0.5% Triton-X-100 in phosphate-buffered saline (PBS) and blocked with 10% non-fat dry milk/0.5% goat serum/PBS prior to exposure to primary antibodies. Subsequently, cells were incubated with primary antibodies at dilutions of 1:500 or isotype control non-specific sera (Chemicon) overnight at 4C in the blocking solut i o n , f o l l o w e d b y s e c o n d a r y a n t i b o d i e s A l e x a 4 8 8 g o a t a n t i - r a b b i t o r g o a t a n t i - m o u s e 5 4 6 F a b f r a g m e n t ( I n v i t r o g e n ) a t 1 : 5 0 0 d i l u t i o n , a n d n u c l e i c o u n t e r - s t a i n e d w i t h D A P I a t 1 g / m L . C e l l s w e r e i m a g e d a n d a n a l y z e d u s i n g a Z e i s s L S M 5 1 0 M e t a i m a g i n g s y s t e m ! " @
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