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Genomic alterations in cultured human embryonic stem cells

Abstract

Cultured human embryonic stem cell (hESC) lines are an invaluable resource because they provide a uniform and stable genetic system for functional analyses and therapeutic applications. Nevertheless, these dividing cells, like other cells, probably undergo spontaneous mutation at a rate of 10−9 per nucleotide. Because each mutant has only a few progeny, the overall biological properties of the cell culture are not altered unless a mutation provides a survival or growth advantage. Clonal evolution that leads to emergence of a dominant mutant genotype may potentially affect cellular phenotype as well. We assessed the genomic fidelity of paired early- and late-passage hESC lines in the course of tissue culture. Relative to early-passage lines, eight of nine late-passage hESC lines had one or more genomic alterations commonly observed in human cancers, including aberrations in copy number (45%), mitochondrial DNA sequence (22%) and gene promoter methylation (90%), although the latter was essentially restricted to 2 of 14 promoters examined. The observation that hESC lines maintained in vitro develop genetic and epigenetic alterations implies that periodic monitoring of these lines will be required before they are used in in vivo applications and that some late-passage hESC lines may be unusable for therapeutic purposes.

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Figure 1: Comparison of copy number status between early- and late-passage hESC lines.
Figure 2: Gene promoter methylation in cultured hESCs.

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Acknowledgements

We thank J. Gearhart and C. Dang for comments and suggestions and J. LaDuca for carrying out FISH analysis. A.C. is supported by the Henry J. Knott Professorship in Genetic Medicine. A.M. is supported by the Sol Goldman Pancreatic Cancer Research Center at Johns Hopkins and a grant from the Maryland Cigarette Restitution Fund. A.C. and D.E.A. are supported by the Donald W. Reynolds Foundation Clinical Cardiovascular Research Center grant to Johns Hopkins University. A.F.G. is supported by the National Cancer Institute Early Detection and Research Network. The work related to lines BG01, 02 and 03 was partially supported by a grant from the US National Institutes of Health to BresaGen. The work related to lines SA001 and SA002/2.5 was partially supported by a grant from the US National Institutes of Health to Cellartis AB. A.C. is a paid member of the Affymetrix Scientific Advisory Board. The terms of this arrangement are being managed by Johns Hopkins University in accordance with its conflict of interest policies.

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Correspondence to Mahendra Rao or Aravinda Chakravarti.

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Competing interests

T.C.S. and S.N.B. are employed by BresaGen; their research is partially funded by BresaGen; and they may own stock options in BresaGen. J.C., S.A. and A. Colman are employed by ES Cell International; their research is partially funded by ES Cell International; and they may own stock options in ES Cell International.

Supplementary information

Supplementary Fig. 1

Comparison of copy number status between earlier and later passage hESC lines. (PDF 292 kb)

Supplementary Fig. 2

Confirmation of array-based copy number alterations in hESC lines. (PDF 157 kb)

Supplementary Table 1

Summary of SNP changes during cell culture of hES cells. (PDF 8 kb)

Supplementary Table 2

H1 has both reduced heterozygous calls, and reduced overall call rates in the deleted region on chromosome 18. (PDF 11 kb)

Supplementary Table 3

Summary of Q-MSP data. (PDF 13 kb)

Supplementary Methods (PDF 9 kb)

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Maitra, A., Arking, D., Shivapurkar, N. et al. Genomic alterations in cultured human embryonic stem cells. Nat Genet 37, 1099–1103 (2005). https://doi.org/10.1038/ng1631

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