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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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE40631 | GEO | 2012/09/05

SECONDARY ACCESSION(S): PRJNA174430

REPOSITORIES: GEO

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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. 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

2012-09-05 | E-GEOD-40631 | biostudies-arrayexpress

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

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

2015-09-11 | GSE72867 | GEO

Methamphetamine and HIV-Tat Alter Murine Cardiac DNA Methylation and Gene Expression [Methylation]

Project description:This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10d, 3mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change>1.5, p<0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides for calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. This study addresses how HIV-1 and methamphetamine comorbidities affect cardiac (left ventricle) gene expression and epigenetic nuclear DNA methylation.

2016-05-23 | GSE64158 | GEO

Methamphetamine and HIV-Tat Alter Murine Cardiac DNA Methylation and Gene Expression [Gene Expression]

2016-05-23 | GSE64157 | GEO

Gene expression profiles of Histone deacetylase inhibitors treated cord blood hematopoietic stem cells.

Project description:Gene expression profiling of primary cord blood hematopoietic stem cell (day 0, CD34+ cells), enriched control (untreated), Scriptaid and Valproic acid expanded CD34+ cells after a week in culture Individal cord blood CD34+ cells were processed individually and equal number of reisolated CD34+ cells from 3-4 samples were pooled after expansion to avoid the sample variations. Gene expression profiles of primary human cord blood CD34+ cells (day0), primary cells (PC) expanded in the presence or absence of histone deacetylase inhibitors (HDACIs) in serum containing cultures supplemented with a combination of cytokines (SCF, FLT3, IL3 and TPO) for 7 days.

2014-07-29 | E-GEOD-59803 | biostudies-arrayexpress

NanoLC-MSMS TMT comparison of in vitro cultured/native reticulocytes and erythrocytes

Project description:Triplicate samples of donor-matched native reticulocytes (CD71+ cells), erythrocytes (CD71- cells) and in vitro culture-derived reticulocytes (derived from CD34+ cells) were analysed through TMT-based quantitative proteomics to examine possible protein abundance changes underlying reticulocyte maturation.

2018-08-03 | PXD009023 | Pride

Gene expression analysis of human fetal liver hematopoietic stem and progenitor cells (HSPC) in culture

Project description:One of the long-standing goals in the field has been to establish a culture system that would allow maintenance of HSC properties ex vivo. In the absence of such system, the ability to model human hematopoiesis in vitro has been limited, and there has been little progress in the expansion of human HSCs for clinical application. To that end, we defined a mesenchyml stem cell co-culture system for expansion of clonally multipotent human HSPCs that are protected from apoptosis and immediate differentiation, and retain the HSPC phenotype. By performing a genome-wide gene expression analysis of purified HSPCs isolated at different stages of co-culture, we asked at the molecular level, to what degree hematopetic stem cell properties can be preserved during culture. This temporal gene expression data from in vivo derived- and ex vivo expanded human HSPCs will serve as a resource to identify novel regulatory pathways that control HSC properties, and to develop clinically applicable protocols for HSC expansion. Human CD34+ fetal liver cells were co-cultured on a subclone of OP9 stomal cells (OP9M2 sublemented with supportive cytokines (see below)). To distinguish between molecular changes acquired over prolonged culture versus immediately after exposure to culture, gene expression in isolated CD45+CD34+CD38-CD90+ HSPCs was assessed after 12 hours, 2 weeks and 5 weeks in culture. Cultured CD45+CD34+CD38-CD90+HSPCs were compared to freshly isolated CD45+CD34+CD38-CD90+HSPCs and their more differentiated CD45+CD34+CD38+CD90- downstream progenitor cells.

2013-01-31 | E-GEOD-34972 | biostudies-arrayexpress

DNA methylation profiles of freshly isolated CD34+ cells and upon expansion on either tissue culture plastic (TCP) or mesenchymal stromal cells (MSCs)

Project description:Hematopoietic stem and progenitor cells (HPCs) can be maintained in vitro, but the vast majority of their progeny loses “stemness” during culture. In this study, we have analyzed DNA methylation (DNAm) profiles of freshly isolated CD34+ cells and upon expansion on either tissue culture plastic (TCP) or mesenchymal stromal cells (MSCs). DNAm profiles of expanded CD34+ versus CD34- subsets reflected hematopoietic differentiation, whereas culture on TCP or MSCs had little impact. Notably, all cultured HPCs - even those which remained CD34 positive - acquired significant DNA-hypermethylation, particularly in up-stream promoter regions, shore-regions of CpG islands, and binding sides for PU.1 and RUNX1. Our results point to a coordinated epigenetic process which needs to be controlled to enhance self-renewal of HPCs in vitro.

2013-12-03 | GSE40799 | GEO

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