1. Volume 18, Issue 5, Pages 625-636 (May 2016)
Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs 
Eunju Kang, Xinjian Wang, Rebecca Tippner-Hedges, Hong Ma, Clifford D.L. Folmes, Nuria Marti Gutierrez, Yeonmi Lee, Crystal Van Dyken, Riffat Ahmed, Ying Li, Amy Koski, Tomonari Hayama, Shiyu Luo, Cary O. Harding, Paula Amato, Jeffrey Jensen, David Battaglia, David Lee, Diana Wu, Andre Terzic, Don P. Wolf, Taosheng Huang, Shoukhrat Mitalipov 
Cell Stem Cell 
Volume 18, Issue 5, Pages (May 2016)
DOI: /j.stem
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2. Cell Stem Cell 2016 18, 625-636DOI: (10.1016/j.stem.2016.02.005)
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3. Figure 1
mtDNA Mutations in Skin Fibroblasts and FiPSC Lines of a 72-Year-Old A Subject
(A) Eleven point mutations were identified in PF, all at low-heteroplasmy levels. The average number of cells in PF was approximately 1 million.
(B) A total of 34 mutations were discovered in ten individual CF, with 6 at over 80% heteroplasmy.
(C) Distribution of low (<15%) and high (>15%) heteroplasmic mutations in individual CF.
(D) In a panel of ten individual fibroblast iPSC lines, 28 mutations were discovered, including seven variants at over 90% heteroplasmy. Gray rectangles indicate the four mtDNA mutation sites common for PF and FiPSC lines.
(E) Distribution of mutations in individual FiPSC lines.
(F) Significant increase in the number of mutations in per cell in fibroblast or iPSC clones, compared to the pooled population (p < 0.05, one-way ANOVA). The number of mutations in PF was calculated based on sampled 1 million cells, while CF and iPSC clones were each counted as a single cell. Error bars indicate mean ± SD.
(G) Venn diagram showing that only a small number of mtDNA mutations in FiPSCs or CF are shared with parental tissue, while the remaining variants represent novel mutations.
See also Figures S1 and S2; Table S1, Table S2, sheet 1, and Table S3, sheet 1.
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4. Figure 2
mtDNA Mutations in Skin Fibroblasts, Blood, and the iPSCs of a 72-Year-Old B Subject
(A) Sixteen mutations at low heteroplasmy levels were detected in the DNA of PF, while a panel of ten FiPSC lines carried nine mutations, including four that were homoplasmic. Gray rectangles define the mutations shared between PF and FiPSCs.
(B) Venn diagram showing only one mutation in FiPSCs shared with PF.
(C) All ten FiPSC lines carried between one and five high-heteroplasmy (>15%) mutations.
(D) Mutation distribution in whole blood and BiPSCs was similar to that in PF and FiPSCs. Six mutations at low-heteroplasmy levels were observed in blood, while BiPSC lines displayed 21 mutations, including four over the 80% heteroplasmy level.
(E) Venn diagram showing four mutations in BiPSCs shared with whole blood and the 17 novel variants.
(F) Distribution of mutations in individual BiPSC lines.
See also Figures S2 and S3; Table S1; Table S3, sheet 2; and Table S4, sheet 1.
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5. Figure 3 mtDNA Mutations in Young Subjects
(A) Two mtDNA mutations were discovered in PF and six were discovered in a panel of FiPSC lines of a 33-year-old subject. Both variants of PF were carried to FiPSCs.
(B) Three mutations were found in whole blood from this subject. Note, two the blood mutations, depicted by gray rectangles, were the same as in PF and FiPSCs.
(C) Mutation load in individual FiPSCs of a 33-year-old subject. Four lines carried high heteroplasmy (over 15%) mutations.
(D) Increase in the number of mtDNA mutations per cell and their heteroplasmy level in FiPSCs compared to PF. The results were averaged for young subjects (n = 5; ages, 24–50 years) (p < 0.05, Student’s t test). In heteroplasmy comparisons, N indicates number of shared PF and FiPSC mutations.
(E) Increase in number of mtDNA mutations per cell in PF and FiPSCs in elderly subjects (n = 5; ages, 60–72 years), compared to young participants (n = 5; ages, 24–50 years) (p < 0.05, Student’s t test).
Error bars indicate mean ± SD.
See also Figure S4 and Tables S1 and S3.
Cell Stem Cell  , DOI: ( /j.stem )
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6. Figure 4
Transmission and Distribution of Somatic mtDNA Mutations to iPSCs
(A) A total of 112 mtDNA mutations were discovered in parental cells (PF, CF, and blood) from 11 subjects. Of these, 39 variants (35%) were found in corresponding 130 iPSC lines. Among non-transmitted, transmitted, and novel mutations in iPSCs, comparable percentages of variants (68%, 69%, and 79%, respectively) were coding mutations in protein, rRNA, or tRNA genes. This suggests that most pathogenic mutations do not affect iPSC induction. However, certain coding mutations including in ND3, ND4L, and 14 tRNA genes were not detected in iPSCs, suggesting possible pathogenicity. n, the number of mtDNA mutations. Blue font genes were detected in parental cells.
(B–D) A total of 80 high heteroplasmic (>15%) variants were detected in the present study in 130 FiPSC or BiPSC lines from 11 subjects. (B) The majority of these variants (76%) were non-synonymous or frame-shift mutations in protein-coding genes or affected rRNA and tRNA genes. (C) More than half of the mutations (56%) were never reported in a database containing whole mtDNA sequences from 26,850 healthy subjects representing the general human population ( (D) Most mutations (90%) were never reported in a database containing sequences from healthy subjects with corresponding mtDNA haplotypes. freq., frequent.
See also Figure S5 and Tables S3 and S4.
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7. Figure 5 Functional Analysis of Somatic mtDNA Mutations in iPSCs
(A) Fibroblasts derived from mutant FiPSCs displayed reduced mitochondrial oxidative capacity compared to mutation-free controls. OCR were dependent on specific mtDNA mutations and heteroplasmy.
(B) OCR in fibroblasts differentiated from mutant iPSCs (iPSa-5 and iPSa-10), and parental skin fibroblasts were significantly low, compared to rescue NT-ESCs (NT2) from the 72-year-old (A) subject. Seahorse assay was performed under basal conditions and in response to 0.5 μg/ml oligomyocin, 1 μM fluorocarbonyl cyanide phenylhydrazone (FCCP), 0.5 μM rotenone, and 1 μM antimycin.
Error bars indicate mean ± SEM (n = 8–10). See also Figure S6 and Table S8.
Cell Stem Cell  , DOI: ( /j.stem )
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