Abstract
Individual induced pluripotent stem cells (iPSCs) show considerable phenotypic heterogeneity, but the reasons for this are not fully understood. Comprehensively analysing the mitochondrial genome (mtDNA) in 146 iPSC and fibroblast lines from 151 donors, we show that most age-related fibroblast mtDNA mutations are lost during reprogramming. However, iPSC-specific mutations are seen in 76.6% (108/141) of iPSC lines at a mutation rate of 8.62 × 10−5/base pair. The mutations observed in iPSC lines affect a higher proportion of mtDNA molecules, favouring non-synonymous protein-coding and tRNA variants, including known disease-causing mutations. Analysing 11,538 single cells shows stable heteroplasmy in sub-clones derived from the original donor during differentiation, with mtDNA variants influencing the expression of key genes involved in mitochondrial metabolism and epidermal cell differentiation. Thus, the dynamic mtDNA landscape contributes to the heterogeneity of human iPSCs and should be considered when using reprogrammed cells experimentally or as a therapy.
Highlights
Individual induced pluripotent stem cells show considerable phenotypic heterogeneity, but the reasons for this are not fully understood
We analysed highdepth mtDNA sequences derived from whole-genome sequence (WGS) data on 146 induced pluripotent stem cells (iPSCs) and 151 fibroblast lines obtained from 151 different donors aged 27–77 years recalled through the NIHR BioResource as part of the Human Induced Pluripotent Stem Cells Initiative (HipSci, http://www.hipsci.org/)
Concerns about heterogeneity between iPSC lines derived from the same donor have raised concerns about scientific reproducibility, and how generalisable the results are from the analysis of an individual cell line
Summary
Individual induced pluripotent stem cells (iPSCs) show considerable phenotypic heterogeneity, but the reasons for this are not fully understood. Analysing 11,538 single cells shows stable heteroplasmy in subclones derived from the original donor during differentiation, with mtDNA variants influencing the expression of key genes involved in mitochondrial metabolism and epidermal cell differentiation. 1234567890():,; There is a growing interest in the use of induced pluripotent stem cells (iPSCs) to model human disease mechanisms and in cell-based therapies, but iPSCs derived from the same tissue show considerable phenotypic heterogeneity that can affect their capacity to differentiate into organ-specific lineages. MtDNA mutations accumulate in somatic tissues throughout life, raising the possibility that mtDNA variation contributes to the molecular heterogeneity of human iPSCs. In keeping with this, several small studies have described mtDNA variants present in iPSC lines that were not detected in parental fibroblast lines, with some having detrimental effects on metabolism, including mitochondrial respiration in derived cardiomyocytes. Singlecell analysis shows that mtDNA variants define sub-clones that modulate gene expression within iPSCs and subsequent differentiated cell lineages
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