Human induced pluripotent stem cells (hiPS cells) have incredible promise for therapeutic use. With genetically identical stem cell lines, it may become possible to replace cells or tissues that have been compromised by disease. Yet questions remain about the safety of hiPS cell lines. Does the induction of pluripotency alter the genome of the resulting cell? If so, what mutations could arise? A new study in Nature begins to shed light on these issues. Using whole-exome sequencing, Gore et al characterized somatic coding mutations in 22 hiPS cell lines that were reprogrammed using five different methods.
Patterns of Mutation
They identified and validated a total of 124 mutations, or roughly 5-6 per hiPS line. The majority of mutations (92/124, or 74%) were missense, nonsense, or splice-site variants predicted to alter protein sequences. Strikingly, some 50 of the affected genes were known to be mutated in human cancers, an enrichment that proved highly significant (p=0.0019). Among these were tumor-suppressor gene ATM, tyrosine kinase receptors NTRK1 and NTRK3, cell division proteins, and others. Furthermore, 14 of the 22 hiPS lines had acquired mutations in genes linked to Mendelian disorders.
Sources of Mutation
Where did all of these mutations come from? The authors considered two possible explanations:
- The mutations already existed in the fibroblast cells, having been acquired over the lifetime of the donors, or
- The mutations were newly acquired during or shortly after reprogramming, and became fixed hiPS cell populations.
The ages of the donors for this study ranged from 0 to 82 years, and did not correlate with mutational load. Further, the observed mutation rate was ten-fold higher than that of skin fibroblasts from the same patients grown in culture without reprogramming.
Uber-Deep Sequencing in Normal Fibroblasts
All of the somatic mutations in hiPS lines were heterozygous and fixed at roughly ~50%. Thus, the process for generating these mutations had already completed. It could be that some mutations were present at very low levels in the skin fibroblasts, and became fixed through clonal expansion. To investigate this possibility, the authors performed PCR and deep Illumina sequencing (10-million-x coverage) for 32 mutations in the original fibroblast DNA. They claim that for 17 of 32 mutations, they detected the variant allele at low frequency (0.003-10%) by comparing read counts between fibroblasts and negative controls to remove the noise. Personally, I’m leery of this result. We’re talking about detection of variants at levels far below the known error rate for Illumina sequencing (~0.1%), and that doesn’t even account for PCR. I know, I know, you can try to model the errors by looking at the negative controls, but I just don’t buy it.
Dead Ends: Reprogramming, Mutation, and Selection
Even if you believe the uber-deep read counts, roughly half of the mutations (15/32) are completely absent from fibroblasts. Also, different hiPS clones derived from the same line contained different sets of mutations. These observations suggest that a significant portion (at the very least) of somatic mutations occurred during reprogramming and subsequent culturing. This might have happened during a short window of elevated mutation rate, possibly due to transient repression of TP53, RB1, or other tumor suppressor genes. Selection, too, might have played a role by favoring mutations that facilitated induction or colony growth. Yet the colonies with mutations in tumor-suppressor genes had similar mutational loads to those without, and pathway analysis of the iPS-acquired mutations found no significant functional advantage.
We seem to leave this study with more questions than answers about mutations in hiPS cell lines. When do they occur, and by what mechanism? How do they consistently become fixed in the colony population? More studies are needed to shed light on these important issues.
Gore A, Li Z, Fung HL, Young JE, Agarwal S, Antosiewicz-Bourget J, Canto I, Giorgetti A, Israel MA, Kiskinis E, Lee JH, Loh YH, Manos PD, Montserrat N, Panopoulos AD, Ruiz S, Wilbert ML, Yu J, Kirkness EF, Izpisua Belmonte JC, Rossi DJ, Thomson JA, Eggan K, Daley GQ, Goldstein LS, & Zhang K (2011). Somatic coding mutations in human induced pluripotent stem cells. Nature, 471 (7336), 63-7 PMID: 21368825