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Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions.

Garitaonandia I, Amir H, Boscolo FS, Wambua GK, Schultheisz HL, Sabatini K, Morey R, Waltz S, Wang YC, Tran H, Leonardo TR, Nazor K, Slavin I, Lynch C, Li Y, Coleman R, Gallego Romero I, Altun G, Reynolds D, Dalton S, Parast M, Loring JF, Laurent LC - PLoS ONE (2015)

Bottom Line: 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, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs.Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation.The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures.

View Article: PubMed Central - PubMed

Affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America.

ABSTRACT
The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material 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, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-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 observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies.

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Differences in gene expression with time in culture, passaging method, and media type.Heatmaps showing differentially expressed genes for early vs. late passage (A), mechanical vs. enzymatic passage (B), and Mef vs. Ecm substrate (C). In heatmap (A), the yellow boxes indicate genes for which the expression levels in the late passage MefMech (P103) samples was similar to those in the early passage samples. Probes were selected by multivariate regression. Enrichments in KEGG pathways identified by functional enrichment analysis are shown. Samples were arranged according to passage and culture method, and hierarchical clustering was performed on the genes only. D. Diagram of the TP53 signaling pathway, showing genes downregulated in the late vs. early passage samples, and the Ecm vs. Mef substrate comparisons.
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pone.0118307.g007: Differences in gene expression with time in culture, passaging method, and media type.Heatmaps showing differentially expressed genes for early vs. late passage (A), mechanical vs. enzymatic passage (B), and Mef vs. Ecm substrate (C). In heatmap (A), the yellow boxes indicate genes for which the expression levels in the late passage MefMech (P103) samples was similar to those in the early passage samples. Probes were selected by multivariate regression. Enrichments in KEGG pathways identified by functional enrichment analysis are shown. Samples were arranged according to passage and culture method, and hierarchical clustering was performed on the genes only. D. Diagram of the TP53 signaling pathway, showing genes downregulated in the late vs. early passage samples, and the Ecm vs. Mef substrate comparisons.

Mentions: We saw significant effects of time in culture on gene expression for all culture conditions (Fig. 7A; S5A Table), although there were three clusters of genes for which the MefMech late passage samples were more similar to the early passage samples (yellow boxes, Fig. 7A). We observed very few effects dependent on the passage method (Fig. 7B; S5C Table) or substrate (Fig. 7C; S5E Table). Pathway analysis, performed on genes differentially expressed over time in culture, using DAVID identified significant changes (FDR <20%) in pathways associated with TP53 signaling, apoptosis, and cancer (Fig. 7A; S5B Table). Mapping the expression levels of the identified genes in the TP53 signaling pathway to a diagrammatic representation of the KEGG pathway, we noted that the expression pattern was consistent with down-regulation in TP53 function over time in culture (Fig. 7D). Inspecting the gene expression levels for these genes at different passages in the four culture conditions, we observed that the differential expression between the early and late passages was most pronounced in conditions carrying a TP53 deletion of in the late passages (MefEnz, EcmMech, and EcmEnz) (S8 Fig.). However, expression changes in the TP53 pathways were also seen in the condition in which deletions in TP53 were not observed (in the MefMech condition), indicating that loss of function of TP53 may occur through epigenetic as well as genetic changes (S3 Fig.).


Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions.

Garitaonandia I, Amir H, Boscolo FS, Wambua GK, Schultheisz HL, Sabatini K, Morey R, Waltz S, Wang YC, Tran H, Leonardo TR, Nazor K, Slavin I, Lynch C, Li Y, Coleman R, Gallego Romero I, Altun G, Reynolds D, Dalton S, Parast M, Loring JF, Laurent LC - PLoS ONE (2015)

Differences in gene expression with time in culture, passaging method, and media type.Heatmaps showing differentially expressed genes for early vs. late passage (A), mechanical vs. enzymatic passage (B), and Mef vs. Ecm substrate (C). In heatmap (A), the yellow boxes indicate genes for which the expression levels in the late passage MefMech (P103) samples was similar to those in the early passage samples. Probes were selected by multivariate regression. Enrichments in KEGG pathways identified by functional enrichment analysis are shown. Samples were arranged according to passage and culture method, and hierarchical clustering was performed on the genes only. D. Diagram of the TP53 signaling pathway, showing genes downregulated in the late vs. early passage samples, and the Ecm vs. Mef substrate comparisons.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4340884&req=5

pone.0118307.g007: Differences in gene expression with time in culture, passaging method, and media type.Heatmaps showing differentially expressed genes for early vs. late passage (A), mechanical vs. enzymatic passage (B), and Mef vs. Ecm substrate (C). In heatmap (A), the yellow boxes indicate genes for which the expression levels in the late passage MefMech (P103) samples was similar to those in the early passage samples. Probes were selected by multivariate regression. Enrichments in KEGG pathways identified by functional enrichment analysis are shown. Samples were arranged according to passage and culture method, and hierarchical clustering was performed on the genes only. D. Diagram of the TP53 signaling pathway, showing genes downregulated in the late vs. early passage samples, and the Ecm vs. Mef substrate comparisons.
Mentions: We saw significant effects of time in culture on gene expression for all culture conditions (Fig. 7A; S5A Table), although there were three clusters of genes for which the MefMech late passage samples were more similar to the early passage samples (yellow boxes, Fig. 7A). We observed very few effects dependent on the passage method (Fig. 7B; S5C Table) or substrate (Fig. 7C; S5E Table). Pathway analysis, performed on genes differentially expressed over time in culture, using DAVID identified significant changes (FDR <20%) in pathways associated with TP53 signaling, apoptosis, and cancer (Fig. 7A; S5B Table). Mapping the expression levels of the identified genes in the TP53 signaling pathway to a diagrammatic representation of the KEGG pathway, we noted that the expression pattern was consistent with down-regulation in TP53 function over time in culture (Fig. 7D). Inspecting the gene expression levels for these genes at different passages in the four culture conditions, we observed that the differential expression between the early and late passages was most pronounced in conditions carrying a TP53 deletion of in the late passages (MefEnz, EcmMech, and EcmEnz) (S8 Fig.). However, expression changes in the TP53 pathways were also seen in the condition in which deletions in TP53 were not observed (in the MefMech condition), indicating that loss of function of TP53 may occur through epigenetic as well as genetic changes (S3 Fig.).

Bottom Line: 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, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs.Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation.The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures.

View Article: PubMed Central - PubMed

Affiliation: Center for Regenerative Medicine, Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, United States of America.

ABSTRACT
The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material 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, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-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 observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies.

Show MeSH
Related in: MedlinePlus