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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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Phenotypic assays.Horizontal brackets indicate significant differences between the specified conditions, as calculated using ANOVA (p-value<0.05). A. The MTT assay measures cell proliferation, with absorbance being correlated with cell growth. B. Doubling time was calculated from cell counts taken over 3 days (average of 3 replicates per condition). C. The EdU incorporation was determined via FACS, and reveals the cell cycle distribution. The graph shows the percent of cells in S phase. D. Relative telomerase activity is shown. E. Average number of telomere repeat fragments (TRF, average of 3 replicates) for each condition. F. Percent of apoptotic cells, determined using TUNEL-FACS. G. Example of an OCT4-positive focus in a teratoma section. H. Bar graph showing, for each culture condition, the percent of teratoma sections containing at least one OCT4-positive focus, and the average number of foci per section. The error bar indicates the standard deviation of counts from 6 (EcmEnz), 7 (MefMech), or 8 (MefEnz and EcmMech) sections. Raw counts can be found in S4 Table).
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pone.0118307.g006: Phenotypic assays.Horizontal brackets indicate significant differences between the specified conditions, as calculated using ANOVA (p-value<0.05). A. The MTT assay measures cell proliferation, with absorbance being correlated with cell growth. B. Doubling time was calculated from cell counts taken over 3 days (average of 3 replicates per condition). C. The EdU incorporation was determined via FACS, and reveals the cell cycle distribution. The graph shows the percent of cells in S phase. D. Relative telomerase activity is shown. E. Average number of telomere repeat fragments (TRF, average of 3 replicates) for each condition. F. Percent of apoptotic cells, determined using TUNEL-FACS. G. Example of an OCT4-positive focus in a teratoma section. H. Bar graph showing, for each culture condition, the percent of teratoma sections containing at least one OCT4-positive focus, and the average number of foci per section. The error bar indicates the standard deviation of counts from 6 (EcmEnz), 7 (MefMech), or 8 (MefEnz and EcmMech) sections. Raw counts can be found in S4 Table).

Mentions: To assess cell proliferation, we used the MTT assay, in addition to calculating doubling times. Both assays indicated that enzymatic passaging was associated with higher proliferation rates (Fig. 6A-B; S3 Table). Cell cycle distribution was determined by EdU incorporation and quantitative DNA staining with Pacific Blue, followed by FACS analysis (Fig. 6C; S3 Table). hESCs grown in the feeder-free conditions showed a higher level of EdU incorporation and a larger proportion of cells in S phase (p<0.05).


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)

Phenotypic assays.Horizontal brackets indicate significant differences between the specified conditions, as calculated using ANOVA (p-value<0.05). A. The MTT assay measures cell proliferation, with absorbance being correlated with cell growth. B. Doubling time was calculated from cell counts taken over 3 days (average of 3 replicates per condition). C. The EdU incorporation was determined via FACS, and reveals the cell cycle distribution. The graph shows the percent of cells in S phase. D. Relative telomerase activity is shown. E. Average number of telomere repeat fragments (TRF, average of 3 replicates) for each condition. F. Percent of apoptotic cells, determined using TUNEL-FACS. G. Example of an OCT4-positive focus in a teratoma section. H. Bar graph showing, for each culture condition, the percent of teratoma sections containing at least one OCT4-positive focus, and the average number of foci per section. The error bar indicates the standard deviation of counts from 6 (EcmEnz), 7 (MefMech), or 8 (MefEnz and EcmMech) sections. Raw counts can be found in S4 Table).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118307.g006: Phenotypic assays.Horizontal brackets indicate significant differences between the specified conditions, as calculated using ANOVA (p-value<0.05). A. The MTT assay measures cell proliferation, with absorbance being correlated with cell growth. B. Doubling time was calculated from cell counts taken over 3 days (average of 3 replicates per condition). C. The EdU incorporation was determined via FACS, and reveals the cell cycle distribution. The graph shows the percent of cells in S phase. D. Relative telomerase activity is shown. E. Average number of telomere repeat fragments (TRF, average of 3 replicates) for each condition. F. Percent of apoptotic cells, determined using TUNEL-FACS. G. Example of an OCT4-positive focus in a teratoma section. H. Bar graph showing, for each culture condition, the percent of teratoma sections containing at least one OCT4-positive focus, and the average number of foci per section. The error bar indicates the standard deviation of counts from 6 (EcmEnz), 7 (MefMech), or 8 (MefEnz and EcmMech) sections. Raw counts can be found in S4 Table).
Mentions: To assess cell proliferation, we used the MTT assay, in addition to calculating doubling times. Both assays indicated that enzymatic passaging was associated with higher proliferation rates (Fig. 6A-B; S3 Table). Cell cycle distribution was determined by EdU incorporation and quantitative DNA staining with Pacific Blue, followed by FACS analysis (Fig. 6C; S3 Table). hESCs grown in the feeder-free conditions showed a higher level of EdU incorporation and a larger proportion of cells in S phase (p<0.05).

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