Increased Risk of Genetic and Epigenetic Instability in Human Embryonic Stem Cells Associated with Specific Culture Conditions
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Francesca Boscolo
PROVIDER: E-GEOD-56851 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them good sources of cells for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a systematic study over more than 100continuous passages to identify characteristics of culture conditions (including passage method, substrate, and media type) that influence the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. The predominant effects we observed were increased genetic instability with enzymatic passage, higher cell proliferation with feeder-free substrate, and variations among cultures in global gene expression and DNA methylation with time in culture. We observed recurrent duplications in two genomic regions that have been noted in earlier studies to be hotspots for duplication in hPSCs, as well as a previously unreported recurrent deletion of the tumor suppressor gene TP53 in all but one of the long-term culture conditions; the exception was the condition using mechanical passaging on feeder layers. The deletion of TP53 is associated with decreased mRNA expression of TP53, as well as alterations in the expression of several other genes in the TP53 pathway, which taken together indicate a decrease in the function of the TP53 pathway. Our results highlight the need for careful assessment of effects of culture conditions on cells intended for clinical therapies. Total RNA extracted at different passages from Human Embryonic Stem Cells in different culture conditions. Total DNA extracted at different passages from Human Embryonic Stem Cells in different culture conditions.
2015-03-29 | E-GEOD-34982 | biostudies-arrayexpress
Project description:Recently genome-wide association studies have identified significant association between Alzheimer’s disease and variations in CLU, PICALM, BIN1, CR1, MS4A4/MS4A6E, CD2AP, CD33, EPHA1 and ABCA7. However, the pathogenic variants in these loci have not yet been found. We conducted a genome-wide scan for large copy number variations (CNVs) in a dataset of Caribbean Hispanic origin (554 controls and 559 cases with late-onset Alzheimer’s disease) that was previously investigated in a SNP-based genome-wide association study using Illumina HumanHap 650Y platform. We ran four CNV calling algorithms and analyzed rare large CNVs (>100 Kb) to obtain high-confidence calls that were detected by at least two algorithms. In total, 734 such CNVs were observed in our dataset. Global burden analyses did not reveal significant differences between cases and controls in CNV rate, distribution of deletions or duplications, total or average CNV size; and number of genes affected by CNVs. However, we observed a nominal association between Alzheimer’s disease and a ~470 Kb duplication on chromosome15q11.2 (P=0.037). This duplication, encompassing up to five genes (TUBGCP5, CYFIP1, NIPA2, NIPA1 and WHAMML1) was present in 10 cases (2.6%) and 3 controls (0.8%). The dosage increase of CYFIP1 and NIPA1 genes was further confirmed by quantitative PCR. The current study did not detect CNVs (including common CNVs) that affect novel Alzheimer’s disease loci reported by large genome-wide association studies. However, since the array technology used in our study has limitations in detecting small CNVs, future studies must carefully assess novel AD associated genes for the presence of disease related CNVs. Case-control analysis, screening of large copy number variation in 559 Alzheimer cases and 554 control subjects of Caribbean Hispanic ancestry
2012-04-30 | E-GEOD-33528 | biostudies-arrayexpress
Project description:Given the association between mutational load and cancer, the observation that genetic aberrations are frequently found in human pluripotent stem cells (hPSCs) is of concern. Prior studies in hiPSCs have shown that deletions and regions of loss-of-heterozygosity (LOH) tend to arise during reprogramming and early culture, while duplications more frequently occur during long-term culture. For the corresponding experiments in hESCs, we studied two sets of hESC lines: one including the corresponding parental DNA; and the other generated from single blastomeres from four sibling embryos. Here, we show for the first time that genetic aberrations observed in hESCs can originate during preimplantation embryo development and/or early derivation. These early aberrations are mainly deletions and LOH, while aberrations arising during long-term culture of hESCs are more frequently duplications. Our results highlight the importance of close monitoring of genomic integrity and the development of improved methods for derivation and culture of hPSCs. Total DNA extracted at from human embryonic stem cells originated independently
2014-04-21 | E-GEOD-49452 | biostudies-arrayexpress
Project description:Induced pluripotent stem cells (iPSCs) have become an essential tool for both modeling how causal genetic variants impact cellular function in disease, as well as being an emerging source of tissue for transplantation medicine. Unfortunately the preparation of somatic cells, their reprogramming and the subsequent verification of iPSC pluripotency are laborious, manual processes that limit the scale and level of reproducibility of this technology. Here we describe a modular, robotic platform for iPSC reprogramming that enables automated, high-throughput conversion of skin biopsies into iPSCs and differentiated cells with minimal manual intervention. Using this platform, we demonstrate that automated reprogramming and the pooled selection of pluripotent cells results in high quality, stable, iPSCs. These lines display less line-to-line variation than either manually produced lines or lines produced through automation followed by single colony-subcloning. The robotic platform we describe will enable the application of iPSCs to population-scale biomedical problems including the study of complex genetic diseases and the development of personalized medicines. Two independent human fibroblast lines were reprogrammed, using modified mRNA, into induced pluripotent stem cells (iPSCs). The genomic stability of several cell lines was evaluated using SNP arrays. Three iPSCs originating from one fibroblast line were tested at passages 8 and 20, with two of these derived as picked (clonal) lines and the third being a pooled population. Five iPSCs originating from a second fibroblast line were tested at passages 8 and 20, with three of these derives derived as picked (clonal) lines and two derived as pooled populations. The iPSCs were compared against the original parental fibroblasts.
2015-08-03 | E-GEOD-69868 | biostudies-arrayexpress
Project description:The ideal genome sequence for medical interpretation is complete and diploid, capturing the full spectrum of genetic variation. Toward this end, there has been progress in discovery of single nucleotide polymorphism (SNP) and small (<10bp) insertion/deletions (indels), but annotation of larger structural variation (SV) including copy number variation (CNV) has been less comprehensive, even with available diploid sequence assemblies. We applied a multi-step sequence and microarray-based analysis to identify numerous previously unknown SVs within the first genome sequence reported from an individual. An Illumina SNP array experiment was performed according to the manufacturer's directions on DNA extracted from a lymphoblastoid cell line (HuRef).
2013-02-12 | E-GEOD-20284 | biostudies-arrayexpress
Project description:The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them good sources of cells for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, to on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments in hEScs involving over 100 continuous passages, , we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher cell proliferation, and persistence of OCT4-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observedculture-dependent variations in global gene expression and DNA methylation. Verification of the negative effects of enzymatic passaging and feeder-free conditions was performed in hiPSC cultures. Our results highlight the need for careful assessment of effects of culture conditions on cells intended for clinical therapies. Three independent hiPSC clones reprogrammed from human fetal dermal fibroblasts: HDF51iPS1, HDF51iPS7, and HDF51iPS11.
2015-03-29 | E-GEOD-56834 | biostudies-arrayexpress
Project description:Whole-genome DNA methylation was performed using the Infinium HumanMethylation27 assay (Illumina). DNA methylation scores for each locus were extracted from the GenomeStudio software (Illumina), used to generate beta-values and M-values for each CpG. Unsupervised classification was performed with ConsensusClusterPlus on beta-values, using Partition around medoids method and euclidean distance, based on top 5000 more variant probes, excluding those located on sexual chromosomes, and restricted to CpG Island. Correlations between genes methylation and expression, and between methylation and 17-hydroxy-progesterone secretion were performed using the M-value and Pearson correlation, using R V3.0.2
2020-08-12 | E-MTAB-6554 | biostudies-arrayexpress
Project description:Systemic lupus erythematosus (SLE) is a chronic-relapsing autoimmune disease of incompletely understood etiology. Recent evidence strongly supports an epigenetic contribution to the pathogenesis of lupus. To understand the extent and nature of dysregulated DNA methylation in lupus T cells, we performed a genome-wide DNA methylation study in CD4+ T cells from 12 lupus patients and 12 normal healthy controls. Cytosine methylation was quantified in 27,578 CG pairs located within the promoter regions of 14,495 genes. We identified 236 hypomethylated and 105 hypermethylated CG sites in lupus CD4+ T cells compared to normal controls, consistent with a global hypomethylation in lupus T cells. Further analysis identified hypomethylation in genes involved in connective tissue development including CD9, MMP9, and PDGFRA. Hypermethylated genes highlight “response to nutrients” ontology such as folate biosynthesis, suggesting a link between environmental factor and lupus and emphasizing the role of folate in DNA methylation. In addition, the transcription factor RUNX3 was hypermethylated in lupus CD4+ T cells. Protein-protein interaction maps identified a transcription factor, HNF4a, as a regulatory hub affecting a number of differentially methylated genes. Functional annotations such as apoptosis is also overrepresented. Further, our data indicate that the methylation status of certain genes predicts disease activity in lupus patients. This work provides a foundation to begin identifying novel pathogenic pathways in lupus T cells and developing novel epigenetic biomarkers for disease activity in lupus. We employed microarray-based technologies to perform a genome-wide DNA methylation assay and quantify CD4+ T cell DNA methylation levels at 27,578 CG sites spanning 14,495 genes of 11 lupus patients and 12 healthy controls.
2011-06-10 | E-GEOD-27895 | biostudies-arrayexpress
Project description:Although much is known about the pluripotency self-renewal circuitry, the molecular events that lead embryonic stem cells (ESCs) exit from pluripotency and begin differentiation are largely unknown. We found that the zinc finger transcription factor Snai1, involved in gastrulation and epithelial- mesenchymal transition (EMT) is already expressed in the inner cell mass of the preimplantation blastocysts. In ESCs Snai1 does not respond to TGFα or BMP4 signalling but it is induced by retinoic acid (RA) treatment, which induces the binding, on the Snai1 promoter, of the retinoid receptors RARγ and RXRα the dissociation of the Polycomb repressor compex 2 (PRC2) which results in the decrease of H3K27me3 and the increase of histone H3K4me3. Snai1 mediates the repression of pluripotency genes by binding directly to the promoters of Nanog, Nr5a2, Tcl1, c-Kit, and Tcfcp2l1. The transient activation of Snai1 in embryoid bodies induces the expression of the markers of all three germ layers. These results suggest that Snai1 is a key factor that triggers ESCs exit from the pluripotency state and initiate their differentiation processes. microarray analysis of embryonic stem cells (ESC) expressing Snail-ER at various time points of induction with 4-OHT
2015-01-12 | E-GEOD-57854 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2013-02-12 | E-GEOD-20290 | biostudies-arrayexpress