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Genomic instability of human embryonic stem cells in mechanic and enzymatic passaging culture

ABSTRACT: hESC have morphologic, genetic and genomic alternatiions when cells cultured in different passaging condition. Here sub-karyotypic genome change in three different hESC lines were compared in two passaging methods.

ORGANISM(S): Homo sapiens

PROVIDER: GSE47914 | GEO | 2015/06/01

SECONDARY ACCESSION(S): PRJNA208331

REPOSITORIES: GEO

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Expression of four hESC lines in enzymatic and mechanic passaging methods

Project description:hESC have morphologic, genetic and genomic alternatiions when cells cultured in different passaging condition. Here transcriptome of four different hESC lines were compared in two passaging methods.

2015-06-01 | GSE47916 | GEO

Transcriptome of embryonic stem cells cultured in enzymatic and mechanic passaging methods [RNA-seq]

Project description:hESC have morphologic, genetic and genomic alternatiions when cells cultured in different passaging condition.

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Increased Risk of Genetic and Epigenetic Instability in Human Embryonic Stem Cells Associated with Specific Culture Conditions II

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

Increased Risk of Genetic and Epigenetic Instability in Human Embryonic Stem Cells Associated with Specific Culture Conditions II

2015-03-29 | GSE56834 | GEO

ESC (HES2, MEL1 or H9) grown in various conditions and FACs sorted for GCTM-2 and CD9

Project description:In the following experiment, three different hESC cell lines (HES2, MEL1 and H9) were grown in the presence of KOSR, KOSR or mTESR containing media respectively. KOSR (Knockout serum replacement medium) is a standard media allowing the growth of hESC without the need for manual passaging - Enzymatic passaging is used instread. mTESR (Ludwig et al., 2007) is a media allowing the growth of hESC on matrigel with enzymatic passaging. At day 7 after passaging, these cells were FACs sorted for the presence of GCTM-2 and CD9 into 4 distinct fractions (p4: GCTM-2-neg, CD9-neg; p5: GCTM-2-low, CD9-low; p6: GCTM-2-medium, CD9-medium and p7: GCTM-2-high, CD9-high). For each cell line-subfraction combination, RNA was harvested and subject to microarray. From each experiment (individual cell line), 4 samples were collected (p4, p5, p6 and p7) and these were subject to microarray. In each case the experiment was performed in triplicate

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ESC (HES2, MEL1 or H9) grown in various conditions and FACs sorted for GCTM-2 and CD9

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2015-03-10 | GSE66674 | GEO

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Project description:Human embryonic stem cells (hESC) show great promise for clinical and research applications, but genomic instability hampers the development of their full potential. Here we demonstrate that increased culture density causes medium acidification due to lactic acid accumulation. hESC are able to cope with this, but acquire increased DNA damage associated to DNA replication stress. Also, single-cell genomics analysis reveals a strong correlation of the occurrence of de novo CNVs and culture density after only five days of culture. However, by an improved control over the accumulation of metabolites through more frequent medium changes, we were able to counter the effect of culture density on DNA damage and CNVs. These data underline the importance of optimal culture conditions, even at short-term, in the light of bringing hESC to their full potential.

2016-02-02 | GSE58281 | GEO

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2015-03-10 | GSE66673 | GEO

Time course mRNA-seq of in vitro decidualization of the T-HESC model

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