Random Mutagenesis, Clonal Events, and Embryonic or Somatic Origin Determine the mtDNA Variant Type and Load in Human Pluripotent Stem Cells

Abstract

SummaryIn this study, we deep-sequenced the mtDNA of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) and their source cells and found that the majority of variants pre-existed in the cells used to establish the lines. Early-passage hESCs carried few and low-load heteroplasmic variants, similar to those identified in oocytes and inner cell masses. The number and heteroplasmic loads of these variants increased with prolonged cell culture. The study of 120 individual cells of early- and late-passage hESCs revealed a significant diversity in mtDNA heteroplasmic variants at the single-cell level and that the variants that increase during time in culture are always passenger to the appearance of chromosomal abnormalities. We found that early-passage hiPSCs carry much higher loads of mtDNA variants than hESCs, which single-fibroblast sequencing proved pre-existed in the source cells. Finally, we show that these variants are stably transmitted during short-term differentiation.

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

“…After reprogramming, genetic drift and selection continue to form the mutational landscape of the mitochondrial genome during prolonged culture of iPSCs 11–15,37 . 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 11–13 , 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 .…”

“…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 11–13 , 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 . Notably, single cell analysis has revealed heterogeneous heteroplasmies among cell populations and there were no indication of selection against potentially pathogenic variants during culture 14 .…”

“…However, at the same time this bottleneck might expose mutations to selective forces, and subtle selective pressures can exert maximal impact 16,21,23,25 . As of yet, it is not understood to what extent the segregation is driven by random allele drift or selection 14 . In contrast to the nuclear genome, selection on mutated mtDNA molecules can act on an inter-cellular or intra-cellular level.…”

“…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 8–14 . 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 .…”

“…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 . 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 .…”