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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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Chromosome 12 detail in MefEnz.Detailed view of the duplications in chromosome 12 for MefEnz. A. The green areas indicate the regions of duplication that were identified by CNVPartition 3.2.0. The areas that were detected manually rather than by the software are indicated in a scale from grey to black. Estimates of the percentage of the population carrying the duplication were performed using the BAF distance for heterozygous SNPs as described [3]. B. LogR Ratio (LRR) and BAF plots of replicate A of MefEnz showed changes in genetic aberrations over time. C. The CNV data were further validated via TaqMan Copy Number Assays. Probe 1 corresponds to Hs03295596_cn, Probe 2 to Hs04404518_cn, Probe 3 to Hs03812366_cn, Probe 4 to Hs03841181_cn, Probe 5 to Hs06947059_cn, and Probe 6 to Hs06363301_cn. D. Karyotyping results confirmed the CNV data.
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pone.0118307.g004: Chromosome 12 detail in MefEnz.Detailed view of the duplications in chromosome 12 for MefEnz. A. The green areas indicate the regions of duplication that were identified by CNVPartition 3.2.0. The areas that were detected manually rather than by the software are indicated in a scale from grey to black. Estimates of the percentage of the population carrying the duplication were performed using the BAF distance for heterozygous SNPs as described [3]. B. LogR Ratio (LRR) and BAF plots of replicate A of MefEnz showed changes in genetic aberrations over time. C. The CNV data were further validated via TaqMan Copy Number Assays. Probe 1 corresponds to Hs03295596_cn, Probe 2 to Hs04404518_cn, Probe 3 to Hs03812366_cn, Probe 4 to Hs03841181_cn, Probe 5 to Hs06947059_cn, and Probe 6 to Hs06363301_cn. D. Karyotyping results confirmed the CNV data.

Mentions: An overview of the regions of CNV that arose in the WA09 hESC line when cultured in different conditions is shown in Fig. 3A (S2A-B Table). Since we acquired data at multiple time points over the course of the experiment, we were able to follow the accumulation of genetic aberrations. Most dramatically, we observed a duplication of a small segment of chromosome 20 in multiple cultures, which we have previously described as a region of recurrent duplication among a large collection of hPSCs [3]. This duplication was observed after passage 40 in at least one of the replicates in all of the conditions. Since the same duplication (with the same start and end locations) appeared in multiple replicates in more than one condition, it is likely that there was a small subpopulation of cells carrying this duplication within the original cell population that was undetectable by our analysis methods. We also observed duplications on chromosome 12 in the MefEnz condition (Fig. 3A; Fig. 4; S2A Table); recurrent duplications of all or part this chromosome in hPSCs have also been described in other studies [3,19].


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)

Chromosome 12 detail in MefEnz.Detailed view of the duplications in chromosome 12 for MefEnz. A. The green areas indicate the regions of duplication that were identified by CNVPartition 3.2.0. The areas that were detected manually rather than by the software are indicated in a scale from grey to black. Estimates of the percentage of the population carrying the duplication were performed using the BAF distance for heterozygous SNPs as described [3]. B. LogR Ratio (LRR) and BAF plots of replicate A of MefEnz showed changes in genetic aberrations over time. C. The CNV data were further validated via TaqMan Copy Number Assays. Probe 1 corresponds to Hs03295596_cn, Probe 2 to Hs04404518_cn, Probe 3 to Hs03812366_cn, Probe 4 to Hs03841181_cn, Probe 5 to Hs06947059_cn, and Probe 6 to Hs06363301_cn. D. Karyotyping results confirmed the CNV data.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4340884&req=5

pone.0118307.g004: Chromosome 12 detail in MefEnz.Detailed view of the duplications in chromosome 12 for MefEnz. A. The green areas indicate the regions of duplication that were identified by CNVPartition 3.2.0. The areas that were detected manually rather than by the software are indicated in a scale from grey to black. Estimates of the percentage of the population carrying the duplication were performed using the BAF distance for heterozygous SNPs as described [3]. B. LogR Ratio (LRR) and BAF plots of replicate A of MefEnz showed changes in genetic aberrations over time. C. The CNV data were further validated via TaqMan Copy Number Assays. Probe 1 corresponds to Hs03295596_cn, Probe 2 to Hs04404518_cn, Probe 3 to Hs03812366_cn, Probe 4 to Hs03841181_cn, Probe 5 to Hs06947059_cn, and Probe 6 to Hs06363301_cn. D. Karyotyping results confirmed the CNV data.
Mentions: An overview of the regions of CNV that arose in the WA09 hESC line when cultured in different conditions is shown in Fig. 3A (S2A-B Table). Since we acquired data at multiple time points over the course of the experiment, we were able to follow the accumulation of genetic aberrations. Most dramatically, we observed a duplication of a small segment of chromosome 20 in multiple cultures, which we have previously described as a region of recurrent duplication among a large collection of hPSCs [3]. This duplication was observed after passage 40 in at least one of the replicates in all of the conditions. Since the same duplication (with the same start and end locations) appeared in multiple replicates in more than one condition, it is likely that there was a small subpopulation of cells carrying this duplication within the original cell population that was undetectable by our analysis methods. We also observed duplications on chromosome 12 in the MefEnz condition (Fig. 3A; Fig. 4; S2A Table); recurrent duplications of all or part this chromosome in hPSCs have also been described in other studies [3,19].

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