Reprogramming enriches for somatic cell clones with small scale mutations in cancer-associated genes

Abstract

Recent studies demonstrated that the mutational load in human induced pluripotent stem cells (hiPSCs) is largely derived from their parental cells, but it is still unknown whether reprogramming may enrich for individual mutations. 30 hiPSC lines were analyzed by whole exome sequencing. High accuracy amplicon sequencing showed that all analyzed small scale variants pre-existed in their parental cells and that individual mutations present in small subpopulations of parental cells become enriched among hiPSC clones during reprogramming. Among those, putatively actionable driver mutations affect genes related to cell cycle control, cell death and pluripotency, and may confer a selective advantage during reprogramming. Finally, a shRNA-based experimental approach was applied to provide additional evidence for the individual impact of such genes on the reprogramming efficiency. In conclusion, we show that enriched mutations in curated onco- and tumor suppressor genes may account for an increased tumor risk and impact the clinical value of patient-derived hiPSCs.

iPSC culture expansion selects against putatively actionable mutations in the mitochondrial genome

“…Hence, it is not surprising that during reprogramming clonal iPSC lines derived from the same parental cell population were observed to harbor different variants and heteroplasmy levels [8][9][10][11][12][13][14] . This unequal segregation of heteroplasmies between cells during reprogramming might arise from three sources acting on an inter-cellular and intra-cellular level with different forces depending on the external conditions, namely i) de novo mutations 8,10,15 , ii) genetic mosaicism in parental cell population leading to genetically distinct iPSC clones, and iii) random allele drift during genetic bottleneck 13,14,16 . Several recent studies report nuclear reprogramming as cause of de novo mtDNA mutations in iPSCs 8,10,15 .…”

“…This unequal segregation of heteroplasmies between cells during reprogramming might arise from three sources acting on an inter-cellular and intra-cellular level with different forces depending on the external conditions, namely i) de novo mutations 8,10,15 , ii) genetic mosaicism in parental cell population leading to genetically distinct iPSC clones, and iii) random allele drift during genetic bottleneck 13,14,16 . Several recent studies report nuclear reprogramming as cause of de novo mtDNA mutations in iPSCs 8,10,15 . However, Payne et al 17 introduced the concept of 'Universal Heteroplasmy', demonstrating that mosaicism of heteroplasmic mtDNA variants in somatic cells, albeit at low levels (< 1%), appears to be a universal finding among healthy individuals 17 .…”

“…However, Payne et al 17 introduced the concept of 'Universal Heteroplasmy', demonstrating that mosaicism of heteroplasmic mtDNA variants in somatic cells, albeit at low levels (< 1%), appears to be a universal finding among healthy individuals 17 . Recently, Deuse et al could confirm via single-cell sequencing of somatic cells that the heteroplasmy levels of variants obtained in bulk sequencing masks a wide range of heteroplasmies in the individual cells 15 . Accordingly, many reports show evidence that the majority if not all mtDNA variants in iPSCs are pre-existing in individual somatic parental cells and arise from this mosaicism in the corresponding parental cell population 13,14 .…”

“…Accordingly, many reports show evidence that the majority if not all mtDNA variants in iPSCs are pre-existing in individual somatic parental cells and arise from this mosaicism in the corresponding parental cell population 13,14 . Some mtDNA variants were pre-existing apparently at a very low frequency within the batch of parental cells but up to 100-fold enriched in a clonal iPSC line during reprogramming 13,15 .…”

“…After reprogramming, genetic drift and selection continue to form the mutational landscape of the mitochondrial genome during prolonged culture of iPSCs [11][12][13][14][15]37 . Although most studies report that mtDNA variants did not experience any substantial change of heteroplasmy level during long-term culture, there is also evidence for both positive 14,15 and negative selection of mtDNA variants [11][12][13] , without clear comprehension of the causes or mechanisms. On one hand, mitochondrial heteroplasmic variants were observed to arise in or dominate human iPSC and ESC lines during prolonged culture 14,15,37 .…”