Unknown,Transcriptomics,Genomics,Proteomics

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Genomic analysis of hESC pedigrees enables identification of de novo genetic alterations and determination of the timing and origin of mutational events

ABSTRACT: 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

ORGANISM(S): Homo sapiens

SUBMITTER: Francesca Boscolo

PROVIDER: E-GEOD-49452 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Genomic analysis of hESC pedigrees identifies de novo mutations and enables determination of the timing and origin of mutational events.

Ben-Yosef Dalit D Boscolo Francesca S FS Amir Hadar H Malcov Mira M Amit Ami A Laurent Louise C LC

Cell reports 20130912 6

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 human induced pluripotent stem cells (hiPSCs) have shown that deletions and regions of loss of heterozygosity (LOH) tend to arise during reprogramming and early culture, whereas duplications more frequently occur during long-term culture. For the corresponding experiments in human embryonic stem cells (hESC ...[more]

PMID: 24035391

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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.

Dataset Information

Genomic analysis of hESC pedigrees enables identification of de novo genetic alterations and determination of the timing and origin of mutational events

Publications

Genomic analysis of hESC pedigrees identifies de novo mutations and enables determination of the timing and origin of mutational events.

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