The relevance of mitochondrial DNA variants fluctuation during reprogramming and neuronal differentiation of human iPSCs

Abstract

Summary The generation of inducible pluripotent stem cells (iPSCs) is a revolutionary technique allowing production of pluripotent patient-specific cell lines used for disease modeling, drug screening, and cell therapy. Integrity of nuclear DNA (nDNA) is mandatory to allow iPSCs utilization, while quality control of mitochondrial DNA (mtDNA) is rarely included in the iPSCs validation process. In this study, we performed mtDNA deep sequencing during the transition from parental fibroblasts to reprogrammed iPSC and to differentiated neuronal precursor cells (NPCs) obtained from controls and patients affected by mitochondrial disorders. At each step, mtDNA variants, including those potentially pathogenic, fluctuate between emerging and disappearing, and some having functional implications. We strongly recommend including mtDNA analysis as an unavoidable assay to obtain fully certified usable iPSCs and NPCs.

A call for consensus guidelines on monitoring the integrity of nuclear and mitochondrial genomes in human pluripotent stem cells

“…A study in 146 hPSC lines derived from healthy individuals observed that 76.6% of the lines displayed altered mtDNA mutation levels when compared to their parental somatic cells, with a mutation rate of $8.62 3 10 À5 /bp (Wei et al, 2021). Similar changes have been observed in hPSCs obtained from patients carrying pathogenetic mtDNA mutations (Palombo et al, 2021). Monitoring mtDNA integrity should therefore be included in the characterization of newly generated hPSC lines (Table 1).…”

“…The quantification of mtDNA changes can be performed by PCR, followed by Sanger sequencing, next-generation sequencing (NGS), or Nanopore sequencing. Primers for amplifying mtDNA molecules into two segments need to be located outside the regions involved in the generation of breakpoints underlying mtDNA deletions (Palombo et al, 2021). Alternatively, high-depth mtDNA sequences can be extracted from WGS (Wei et al, 2021).…”

Interpretable deep generative models for genomics

“…There has also been recent work studying the impact of mitochondrial heteroplasmy on iPSC differentiation potential. Several studies now suggest mtDNA integrity as mandatory iPSC selection criteria [86][87][88] . Heteroplasmy of several mutations have been linked to iPSC's ability to differentiate [87][88][89][90] .…”

“…Several studies now suggest mtDNA integrity as mandatory iPSC selection criteria [86][87][88] . Heteroplasmy of several mutations have been linked to iPSC's ability to differentiate [87][88][89][90] . Manipulating MT heteroplasmy through insertion of wild type mtDNA has been shown to revert diseased iPSC state and improve pluripotency 91 .…”

Implications of mitochondrial DNA mutations in human induced pluripotent stem cells

“…Harnessing this technology is not without its challenges, one of which is to understand why different clonal lineages derived from a single donor behave differently in different contexts. Recent studies [1][2][3][4][5][6][7] have now shown that iPSCs derived from somatic cells contain mitochondrial single-nucleotide variants (mtSNVs) that affect only a proportion of the total mitochondrial DNA (mtDNA) content (that is, they are heteroplasmic). Interestingly, the proportion of mutant mtDNA (heteroplasmy level or fraction) can differ markedly in the derived iPSC cell lines.…”