Unknown,Transcriptomics,Genomics,Proteomics

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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Francesca Boscolo

PROVIDER: E-GEOD-56834 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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

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