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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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Detail of recurrent deletions in chromosome 17.A. The short arm of the chromosome is enlarged and the regions that showed deletions in the MefEnz, EcmMech, and EcmEnz conditions are indicated by red bars. Blue lines enclose the common area among all the conditions and the genes in the common area are indicated in the lower part of the figure. Genes for which the level of expression correlated with copy number are highlighted by red squares. B. Graphs representing the expression levels of TP53, SENP3, and SOX15, as measured by gene expression microarray. The ideogram of the chromosome was from the U.S. Department of Energy Genome Programs.
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pone.0118307.g005: Detail of recurrent deletions in chromosome 17.A. The short arm of the chromosome is enlarged and the regions that showed deletions in the MefEnz, EcmMech, and EcmEnz conditions are indicated by red bars. Blue lines enclose the common area among all the conditions and the genes in the common area are indicated in the lower part of the figure. Genes for which the level of expression correlated with copy number are highlighted by red squares. B. Graphs representing the expression levels of TP53, SENP3, and SOX15, as measured by gene expression microarray. The ideogram of the chromosome was from the U.S. Department of Energy Genome Programs.

Mentions: Most strikingly, there were recurrent deletions on the short arm of chromosome 17 in four replicates of MefEnz (at passage 41 for one replicate and at passage 104 for three replicates), four replicates of EcmMech (at passage 147), and all six replicates of EcmEnz (at passage 41). These deletions all overlapped within a 158 kb region (Fig. 5A), and were associated with decreased expression of three of the 17 genes located in this region, as measured using HumanHT-12 BeadChip microarrays (Illumina). These three genes were SENP3 (SUMO1/sentrin/SMT3 specific peptidase 3), SOX15 (SRY-BOX 15), and TP53 (P53), a well-known tumor suppressor gene (Fig. 5B) [20]. Gene expression data for all of the genes in the deleted region are shown in S1 Fig. (please note that some probes are located in regions where two or three genes overlap, and that there are two probes each for CD68 and MPDU1).


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)

Detail of recurrent deletions in chromosome 17.A. The short arm of the chromosome is enlarged and the regions that showed deletions in the MefEnz, EcmMech, and EcmEnz conditions are indicated by red bars. Blue lines enclose the common area among all the conditions and the genes in the common area are indicated in the lower part of the figure. Genes for which the level of expression correlated with copy number are highlighted by red squares. B. Graphs representing the expression levels of TP53, SENP3, and SOX15, as measured by gene expression microarray. The ideogram of the chromosome was from the U.S. Department of Energy Genome Programs.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118307.g005: Detail of recurrent deletions in chromosome 17.A. The short arm of the chromosome is enlarged and the regions that showed deletions in the MefEnz, EcmMech, and EcmEnz conditions are indicated by red bars. Blue lines enclose the common area among all the conditions and the genes in the common area are indicated in the lower part of the figure. Genes for which the level of expression correlated with copy number are highlighted by red squares. B. Graphs representing the expression levels of TP53, SENP3, and SOX15, as measured by gene expression microarray. The ideogram of the chromosome was from the U.S. Department of Energy Genome Programs.
Mentions: Most strikingly, there were recurrent deletions on the short arm of chromosome 17 in four replicates of MefEnz (at passage 41 for one replicate and at passage 104 for three replicates), four replicates of EcmMech (at passage 147), and all six replicates of EcmEnz (at passage 41). These deletions all overlapped within a 158 kb region (Fig. 5A), and were associated with decreased expression of three of the 17 genes located in this region, as measured using HumanHT-12 BeadChip microarrays (Illumina). These three genes were SENP3 (SUMO1/sentrin/SMT3 specific peptidase 3), SOX15 (SRY-BOX 15), and TP53 (P53), a well-known tumor suppressor gene (Fig. 5B) [20]. Gene expression data for all of the genes in the deleted region are shown in S1 Fig. (please note that some probes are located in regions where two or three genes overlap, and that there are two probes each for CD68 and MPDU1).

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