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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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DNA Methylation.A. X Chromosome DNA Methylation and XIST Expression. Methylation levels of genes in the X-chromosome (S6A Table) are shown on the heatmap. Hierarchical clustering was performed on the samples, as indicated by the dendrogram. The genes are ordered according to their location (from the beginning to the end of the chromosome). Samples that show loss of DNA methylation for the “Enz” cluster are highlighted in blue, those that show DNA methylation for the “Ecm” cluster are highlighted in pink, and for both clusters in mauve. Genes located in the regions of loss of DNA methylation are listed to the right of the heatmap. XIST expression is shown on the line graph, with the detection limit for the microarray indicated by the red line. B. DNA methylation at imprinted loci. Methylation levels for imprinted probes (S6B Table) are shown on the heatmap. Hierarchical clustering was performed on the samples, as indicated by the dendrogram. The genes are ordered according to chromosome location; genes are listed to the left. The inset at the right shows a detail of the NESP/GNAS complex locus, indicating the positions of the CpG sites that were hypermethylated (red triangle) vs. hypomethylated (green triangle) in the late passage samples relative to the NESP/GNAS and NESPAS exons. C, D, E. Heatmaps showing differential DNA methylation genes for early vs. late passage (C), mechanical vs. enzymatic passage (D), and Mef vs. Ecm substrate (E). In heatmap (C), the black boxes indicate genes for which the DNA methylation levels in the late passage MefMech (P103) samples was more similar to those in the early passage samples. Probes were selected by multivariate regression. Functional enrichments identified by GREAT analysis are shown to the right of the heatmaps, visualized using REVIGO [13]. Samples were arranged according to passage and culture method, and hierarchical clustering was performed on the genes only. In the functional enrichment results, the size of the node indicated the number of contributing GO terms, and color of the nodes indicates the FDR (darker color for lower FDR), and the edge length indicates the similarity between GO terms (shorter edge for more similar terms).
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pone.0118307.g008: DNA Methylation.A. X Chromosome DNA Methylation and XIST Expression. Methylation levels of genes in the X-chromosome (S6A Table) are shown on the heatmap. Hierarchical clustering was performed on the samples, as indicated by the dendrogram. The genes are ordered according to their location (from the beginning to the end of the chromosome). Samples that show loss of DNA methylation for the “Enz” cluster are highlighted in blue, those that show DNA methylation for the “Ecm” cluster are highlighted in pink, and for both clusters in mauve. Genes located in the regions of loss of DNA methylation are listed to the right of the heatmap. XIST expression is shown on the line graph, with the detection limit for the microarray indicated by the red line. B. DNA methylation at imprinted loci. Methylation levels for imprinted probes (S6B Table) are shown on the heatmap. Hierarchical clustering was performed on the samples, as indicated by the dendrogram. The genes are ordered according to chromosome location; genes are listed to the left. The inset at the right shows a detail of the NESP/GNAS complex locus, indicating the positions of the CpG sites that were hypermethylated (red triangle) vs. hypomethylated (green triangle) in the late passage samples relative to the NESP/GNAS and NESPAS exons. C, D, E. Heatmaps showing differential DNA methylation genes for early vs. late passage (C), mechanical vs. enzymatic passage (D), and Mef vs. Ecm substrate (E). In heatmap (C), the black boxes indicate genes for which the DNA methylation levels in the late passage MefMech (P103) samples was more similar to those in the early passage samples. Probes were selected by multivariate regression. Functional enrichments identified by GREAT analysis are shown to the right of the heatmaps, visualized using REVIGO [13]. Samples were arranged according to passage and culture method, and hierarchical clustering was performed on the genes only. In the functional enrichment results, the size of the node indicated the number of contributing GO terms, and color of the nodes indicates the FDR (darker color for lower FDR), and the edge length indicates the similarity between GO terms (shorter edge for more similar terms).

Mentions: In earlier studies, we observed that low XIST (X (inactive)-specific transcript) expression is associated with variable loss of DNA methylation on the X chromosome, and that approximately half of a large collection of female hPSC lines showed low XIST expression, including the WA09 hESC line [22]. In addition, we have observed progressive loss of DNA methylation on the X chromosome with time in culture in hiPSCs that have low XIST expression [22]. Consistent with these observations, in the present study there was low expression of XIST and focal absence of DNA methylation in several regions of the X chromosome in all conditions at all time-points (Fig. 8A; S6A Table). We also observed expansion of these regions at late passage in some, but not all, conditions. In fact, two regions on the X chromosome showed loss of DNA methylation associated with enzymatic passage (“Enz”) and with extracellular matrix substrate (“Ecm”); we note that the EcmEnz condition samples showed loss of DNA methylation in both of these regions. These results support the notion that the loss of XIST expression at early passage is followed by gradual and progressive loss of DNA methylation on the X chromosome, and that this can be influenced by culture conditions.


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)

DNA Methylation.A. X Chromosome DNA Methylation and XIST Expression. Methylation levels of genes in the X-chromosome (S6A Table) are shown on the heatmap. Hierarchical clustering was performed on the samples, as indicated by the dendrogram. The genes are ordered according to their location (from the beginning to the end of the chromosome). Samples that show loss of DNA methylation for the “Enz” cluster are highlighted in blue, those that show DNA methylation for the “Ecm” cluster are highlighted in pink, and for both clusters in mauve. Genes located in the regions of loss of DNA methylation are listed to the right of the heatmap. XIST expression is shown on the line graph, with the detection limit for the microarray indicated by the red line. B. DNA methylation at imprinted loci. Methylation levels for imprinted probes (S6B Table) are shown on the heatmap. Hierarchical clustering was performed on the samples, as indicated by the dendrogram. The genes are ordered according to chromosome location; genes are listed to the left. The inset at the right shows a detail of the NESP/GNAS complex locus, indicating the positions of the CpG sites that were hypermethylated (red triangle) vs. hypomethylated (green triangle) in the late passage samples relative to the NESP/GNAS and NESPAS exons. C, D, E. Heatmaps showing differential DNA methylation genes for early vs. late passage (C), mechanical vs. enzymatic passage (D), and Mef vs. Ecm substrate (E). In heatmap (C), the black boxes indicate genes for which the DNA methylation levels in the late passage MefMech (P103) samples was more similar to those in the early passage samples. Probes were selected by multivariate regression. Functional enrichments identified by GREAT analysis are shown to the right of the heatmaps, visualized using REVIGO [13]. Samples were arranged according to passage and culture method, and hierarchical clustering was performed on the genes only. In the functional enrichment results, the size of the node indicated the number of contributing GO terms, and color of the nodes indicates the FDR (darker color for lower FDR), and the edge length indicates the similarity between GO terms (shorter edge for more similar terms).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118307.g008: DNA Methylation.A. X Chromosome DNA Methylation and XIST Expression. Methylation levels of genes in the X-chromosome (S6A Table) are shown on the heatmap. Hierarchical clustering was performed on the samples, as indicated by the dendrogram. The genes are ordered according to their location (from the beginning to the end of the chromosome). Samples that show loss of DNA methylation for the “Enz” cluster are highlighted in blue, those that show DNA methylation for the “Ecm” cluster are highlighted in pink, and for both clusters in mauve. Genes located in the regions of loss of DNA methylation are listed to the right of the heatmap. XIST expression is shown on the line graph, with the detection limit for the microarray indicated by the red line. B. DNA methylation at imprinted loci. Methylation levels for imprinted probes (S6B Table) are shown on the heatmap. Hierarchical clustering was performed on the samples, as indicated by the dendrogram. The genes are ordered according to chromosome location; genes are listed to the left. The inset at the right shows a detail of the NESP/GNAS complex locus, indicating the positions of the CpG sites that were hypermethylated (red triangle) vs. hypomethylated (green triangle) in the late passage samples relative to the NESP/GNAS and NESPAS exons. C, D, E. Heatmaps showing differential DNA methylation genes for early vs. late passage (C), mechanical vs. enzymatic passage (D), and Mef vs. Ecm substrate (E). In heatmap (C), the black boxes indicate genes for which the DNA methylation levels in the late passage MefMech (P103) samples was more similar to those in the early passage samples. Probes were selected by multivariate regression. Functional enrichments identified by GREAT analysis are shown to the right of the heatmaps, visualized using REVIGO [13]. Samples were arranged according to passage and culture method, and hierarchical clustering was performed on the genes only. In the functional enrichment results, the size of the node indicated the number of contributing GO terms, and color of the nodes indicates the FDR (darker color for lower FDR), and the edge length indicates the similarity between GO terms (shorter edge for more similar terms).
Mentions: In earlier studies, we observed that low XIST (X (inactive)-specific transcript) expression is associated with variable loss of DNA methylation on the X chromosome, and that approximately half of a large collection of female hPSC lines showed low XIST expression, including the WA09 hESC line [22]. In addition, we have observed progressive loss of DNA methylation on the X chromosome with time in culture in hiPSCs that have low XIST expression [22]. Consistent with these observations, in the present study there was low expression of XIST and focal absence of DNA methylation in several regions of the X chromosome in all conditions at all time-points (Fig. 8A; S6A Table). We also observed expansion of these regions at late passage in some, but not all, conditions. In fact, two regions on the X chromosome showed loss of DNA methylation associated with enzymatic passage (“Enz”) and with extracellular matrix substrate (“Ecm”); we note that the EcmEnz condition samples showed loss of DNA methylation in both of these regions. These results support the notion that the loss of XIST expression at early passage is followed by gradual and progressive loss of DNA methylation on the X chromosome, and that this can be influenced by culture conditions.

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