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Lentiviral transduction of CD34+ cells induces genome-wide epigenetic modifications

ABSTRACT: Human CD34+ cells were cultured shortly in the presence of cytokines then with a gene transfer lentiviral vector (LV) expected to transduce cells but to have otherwise limited biological effects on the cells. At the end of the culture, the population of cells remained largely similar at the phenotypic level but some epigenetic changes were evident. Exposure of CD34+ cells to cytokines caused genome-wide DNA methylation changes. Surprisingly, the LV caused additional and distinct effects. Large-scale genomic DNA methylation analysis showed that balanced methylation changes occurred in about 200 genes following culture of CD34+ cells in the presence of cytokines but 900 genes were modified following addition of the LV, predominantly increasing CpG methylation. Epigenetic effects resulting from ex vivo culture and from the use of LV may constitute previously unsuspected sources of biological effects in stem cells and may provide new biomarkers to rationally optimize gene and cell therapy protocols. 3 replicates of control unstimulated (Group 1), cytokine stimulated, cytokine strimulated and treated with polybren and cytokine stimulated and transduced with lentiviral vector coding for GFP in the presence of polybren were analysed

ORGANISM(S): Homo sapiens

SUBMITTER: NIZAR TOULEIMAT

PROVIDER: E-GEOD-40631 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Lentiviral transduction of CD34+ cells induces genome-wide epigenetic modifications

Project description:Human CD34+ cells were cultured shortly in the presence of cytokines then with a gene transfer lentiviral vector (LV) expected to transduce cells but to have otherwise limited biological effects on the cells. At the end of the culture, the population of cells remained largely similar at the phenotypic level but some epigenetic changes were evident. Exposure of CD34+ cells to cytokines caused genome-wide DNA methylation changes. Surprisingly, the LV caused additional and distinct effects. Large-scale genomic DNA methylation analysis showed that balanced methylation changes occurred in about 200 genes following culture of CD34+ cells in the presence of cytokines but 900 genes were modified following addition of the LV, predominantly increasing CpG methylation. Epigenetic effects resulting from ex vivo culture and from the use of LV may constitute previously unsuspected sources of biological effects in stem cells and may provide new biomarkers to rationally optimize gene and cell therapy protocols.

2012-09-05 | GSE40631 | GEO

Transcription profiling by array of human cord-blood-derived CD34+ cells activated with cytokines

Project description:To generate normal control microarrays, RNA was isolated from 3 samples of cord blood derived-CD34+ cells activated with cytokines. We used Affymetrix expression profiles of CD34+ cells isolated from 3 normal donors (for LV-GFP and random sites) and activated in culture in the same conditions used for lentiviral transduction as control.

2010-08-01 | E-MEXP-2758 | biostudies-arrayexpress

DNA methylation response of hematopoietic stem cells to recombinant lentiviral vector infection

Project description:Human cord blood CD34+ cells respond to the infection with LV by modifying the methylation profile of a large number of CpG sites in their genome. Different vector preparations vary in their capacity to trigger a response; some induce sporadic modifications only while others trigger a strong response that systematically includes de novo methylation of the same 4126 CpG-s corresponding to 1059 genes Illumina Infinium 450K arrays were performed according to the manufacturer's directions on bisulfite converted gDNA extracted from CD34+ cells from cord blood. CpG methylation profiling on Illumina Infinium 450K arrays was performed.

2015-09-11 | E-GEOD-72867 | biostudies-arrayexpress

In Vitro Transformation of Primary Human CD34+ Cells by AML Fusion Oncogenes: Early Gene Expression Profiling Reveals Possible Drug Target in AML

Project description:Different fusion oncogenes in acute myeloid leukemia (AML) have distinct clinical and laboratory features suggesting different modes of malignant transformation. Here we compare the in vitro effects of representatives of major groups of AML fusion oncogenes on primary human CD34+ cells. For the 3 d and 8 d time points, the oncogenes were retrovirally expressed in primary human CD34+ cells, with cells transduced with empty retroviral vector as controls. As retroviral transduction requires days, for the 6 h time point the oncogenes were expressed from a pTracer-CMV/Bsd plasmid using nucleofection. Cells nucleofected with empty pTracer-CMV/Bsd vector were used as a control.

2014-05-01 | E-GEOD-57194 | biostudies-arrayexpress

lncRAN and mRNA expression signatures in SKOV3 cells: control vs. β-HCG transfected

Project description:To further development of the effects of β-HCG in ovarian cancer SKOV3 cells，we have employed lncRNA and mRNA microarray as a discovery platform to identify lncRNA and mRNA expression in β-HCG overexpressing ovarian cancer cells. SKOV3 cells were transfected with lentiviral vector with eGFP, encoding β-HCG and negative control vector (LV- β-HCG and LV-CON,) by using polybrene. The dysregulation of β-HCG was confirmed by using RT-PCR. RNA was extracted and detected by a lncRNA and mRNA microarray in LV-β-HCG and LV-CON SKOV3 cells. The different expression of lncRNA and mRNA in LV-β-HCG and LV-CON SKOV3 cells was analyzed to explore the mechanism that β-HCG affect ovarain cancer cells.

2018-11-06 | GSE122147 | GEO

lncRNA and mRNA expression signatures in OVCAR3 cells: control vs. miR-200a transfected

Project description:To further development of the effects of miR-200a in ovarian cancer OVCAR3 cells，we have employed lncRNA and mRNA microarray as a discovery platform to identify lncRNA and mRNA expression in miR-200a overexpressing ovarian cancer cells. OVCAR3 cells were transfected with lentiviral vector with eGFP, encoding miR-200a and negative control vector (LV- miR-200a and LV-CON,) by using polybrene. The dysregulation of miR-200a was confirmed by using RT-PCR. RNA was extracted and detected by a lncRNA and mRNA microarray in LV-miR-200a and LV-CON OVCAR3 cells. The different expression of lncRNA and mRNA in LV-miR-200a and LV-CON OVCAR3 cells was analyzed to explore the mechanism that miR-200a affect ovarain cancer cells.

2018-11-04 | GSE122123 | GEO

Cytokine receptor modulation by interleukin-2 broadly regulates T helper cell lineage differentiation

Project description:T helper (Th) cells control host defense to pathogens. IL 12R expression is required for Th1, IL-4RM-NM-1 for Th2, and IL-6RM-NM-1/gp130 for Th17 differentiation to allow responsiveness to IL-12, IL-4, and IL-6, respectively. IL-2 via STAT5 controls Th2 differentiation by regulating the Th2 cytokine gene cluster and Il4ra expression. Here we show that IL-2 regulates Th1 differentiation, inducing STAT5-dependent IL-12RM-NM-22 and T-bet expression, with impaired human Th1 differentiation when IL-2 was blocked. Th1 differentiation was also impaired in mouse Il2-/- T cells but restored by IL-12RM-NM-22 expression. Consistent with IL-2M-bM-^@M-^Ys inhibition of Th17 differentiation, IL-2 decreased Il6ra and Il6st/gp130 expression, and Il6st augmented Th17 differentiation even when IL-2 was present. Thus, IL-2 influences T-cell differentiation by modulating cytokine receptor expression to help specify/maintain differentiated states. Genome-wide mapping of STAT1,STAT4,STAT5A,STAT5B binding to their target genes in Th1 or human CD4+ cells was conducted

2011-04-24 | E-GEOD-27158 | biostudies-arrayexpress

Critical Role of STAT5 Transcription Factor Tetramerization for Cytokine Responses and Normal Immune Function (ChIP-Seq and RNA-Seq)

Project description:Cytokine-activated STAT proteins dimerize and bind to high-affinity motifs, and N-terminal domain-mediated oligomerization of dimers allows tetramer formation and binding to low-affinity tandem motifs, but the functions of dimers versus tetramers are unknown. We generated Stat5a and Stat5b double knock-in (DKI) N-domain mutant mice that form dimers but not tetramers, identified cytokine-regulated genes whose expression required STAT5 tetramers, and defined consensus motifs for dimers versus tetramers. Whereas Stat5- deficient mice exhibited perinatal lethality, DKI mice were viable, indicating that STAT5 dimers were sufficient for survival. Nevertheless, STAT5 DKI mice had fewer CD4+CD25+ T cells, NK cells, and CD8+ T cells, with impaired cytokine-induced proliferation and homeostatic proliferation of CD8+ T cells. DKI CD8+ T cell proliferation following viral infection was diminished and DKI Treg cells did not efficiently control colitis. Thus, tetramerization of STAT5 is dispensable for survival but is critical for cytokine responses and normal immune function. Genome-wide mapping of STAT5A,STAT5B binding in mouse WT and DKI T cells (cultured with or without IL-2 for 1 hr) was conducted. RNA-Seq is conducted in mouse CD8+ T cells (WT and DKI, non-treated or treated with IL-2/IL-15 for 4 hr, 24 hr, 48 hr and 72 hr)

2012-04-25 | E-GEOD-36882 | biostudies-arrayexpress

Pbx1-d Induces T cell Activation and a Decreased Apoptosis in Response to Retinoic Acids (RA)

Project description:The hypothesis was tested that the Pbx1-d isoform was responsible for the Sle1a.1 phenotypes in CD4+ T cells. Jurkat T cells were transfected with a lentiviral construct expressing Pbx1-d-GFP or control RFP. Pbx1-d over-expression reduced the percentage of late apoptotic cells in response to anti-CD3 and anti-CD28 stimulation as compared with control-Lin28-transfected cells. Overall, these data demonstrate that over-expression of Pbx1-d results in an activated/inflammatory phenotype and in a defective response to RA in Jurkat T cells, strongly suggesting that the increased expression of Pbx1-d is responsible for the Sle1a.1 phenotypes. Jurkat T cells (5 x 105 cells/ml) were transfected with an lentiviral (LV) vector expressing either control (RFP or Lin28) or Pbx1-d-GFP. Total RNA was extracted from GFP+ or RFP+ FACS-sorted Jurkat T cells transfected LV-GFP-Pbx1-d or LV-RFP, as well as non-transfected cells in 4 independent transfections per group.

2010-07-07 | E-GEOD-22799 | biostudies-arrayexpress

Deciphering molecular mechanisms of MEIS1-mediated prostate tumor suppression through RNAseq and ChIPseq - ChIPseq data

Project description:In order to identify direct gene targets and pathways regulated by MEIS1 in prostate cells and identify mechanisms of observed tumor suppression with MEIS1 expression, we performed Chromatin Immunoprecipitation and sequencing (ChIP-seq) of MEIS1 in the CWR22Rv1-LV-MEIS1 line. Also, given the dependence of HOXB13, and to enable determination of HOXB13-dependent vs. HOXB13-independent MEIS1 DNA binding, we performed parallel MEIS1 ChIP-seq in the CWR22Rv1-HOXB13ko-LV-MEIS1 line. LV-MEIS1 denotes cells with exogenous lentiviral expression of MEIS1, and they still express endogenous HOXB13. Knockout of HOXB13 was achieved by CRISPR and validated with western blotting. HOXB13ko-LV-MEIS1 denotes cells where the same lentiviral expression of MEIS1 was infected into the HOXB13ko cells, so these cells are positive for MEIS1 expression and negative for HOXB13.

2020-07-15 | GSE132716 | GEO

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