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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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Graphs illustrating the number (A) and total length (B) of genetic aberrations.Aberrations were identified in the samples using CNV analysis of high resolution SNP genotyping data from the WA09 hESC line. Duplications and deletions; regions of loss of homozygosity (LOH) were categorized as deletions and complex CNVs were categorized as duplications. The x-axis indicates the number of passages for the current study. All cultures started from a source culture that had been passaged for 37 passages. Genetic aberrations present in this source culture were not counted, and therefore at the start of the study, the number of duplications and deletions were set at 0. Of note, each culture condition was assigned a unique color that is consistent throughout the manuscript.
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pone.0118307.g002: Graphs illustrating the number (A) and total length (B) of genetic aberrations.Aberrations were identified in the samples using CNV analysis of high resolution SNP genotyping data from the WA09 hESC line. Duplications and deletions; regions of loss of homozygosity (LOH) were categorized as deletions and complex CNVs were categorized as duplications. The x-axis indicates the number of passages for the current study. All cultures started from a source culture that had been passaged for 37 passages. Genetic aberrations present in this source culture were not counted, and therefore at the start of the study, the number of duplications and deletions were set at 0. Of note, each culture condition was assigned a unique color that is consistent throughout the manuscript.

Mentions: In the hESC cultures, we compared the total number (Fig. 2A) and length (Fig. 2B) of duplications and deletions in each of the four culture conditions over time (see also Fig. 3A), as determined by SNP genotyping. Larger numbers of duplications were seen in the enzymatic passage conditions (MefEnz and EcmEnz), suggesting that dissociation to single cells during passaging might favor the accumulation of duplications. Between these two conditions, the MefEnz appeared to be associated with larger duplications (Fig. 2B). In contrast, the mechanical passaging conditions, MefMech and EcmMech, showed low numbers of duplications, most of which arose at very late passage. The same trends held for deletions, with the MefEnz and EcmEnz conditions showing higher frequencies of deletions, and the MefEnz condition showing the largest number of deletions at higher passages. These data indicate that Accutase-based enzymatic passaging is associated with higher rates of genetic instability.


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)

Graphs illustrating the number (A) and total length (B) of genetic aberrations.Aberrations were identified in the samples using CNV analysis of high resolution SNP genotyping data from the WA09 hESC line. Duplications and deletions; regions of loss of homozygosity (LOH) were categorized as deletions and complex CNVs were categorized as duplications. The x-axis indicates the number of passages for the current study. All cultures started from a source culture that had been passaged for 37 passages. Genetic aberrations present in this source culture were not counted, and therefore at the start of the study, the number of duplications and deletions were set at 0. Of note, each culture condition was assigned a unique color that is consistent throughout the manuscript.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118307.g002: Graphs illustrating the number (A) and total length (B) of genetic aberrations.Aberrations were identified in the samples using CNV analysis of high resolution SNP genotyping data from the WA09 hESC line. Duplications and deletions; regions of loss of homozygosity (LOH) were categorized as deletions and complex CNVs were categorized as duplications. The x-axis indicates the number of passages for the current study. All cultures started from a source culture that had been passaged for 37 passages. Genetic aberrations present in this source culture were not counted, and therefore at the start of the study, the number of duplications and deletions were set at 0. Of note, each culture condition was assigned a unique color that is consistent throughout the manuscript.
Mentions: In the hESC cultures, we compared the total number (Fig. 2A) and length (Fig. 2B) of duplications and deletions in each of the four culture conditions over time (see also Fig. 3A), as determined by SNP genotyping. Larger numbers of duplications were seen in the enzymatic passage conditions (MefEnz and EcmEnz), suggesting that dissociation to single cells during passaging might favor the accumulation of duplications. Between these two conditions, the MefEnz appeared to be associated with larger duplications (Fig. 2B). In contrast, the mechanical passaging conditions, MefMech and EcmMech, showed low numbers of duplications, most of which arose at very late passage. The same trends held for deletions, with the MefEnz and EcmEnz conditions showing higher frequencies of deletions, and the MefEnz condition showing the largest number of deletions at higher passages. These data indicate that Accutase-based enzymatic passaging is associated with higher rates of genetic instability.

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