Mitochondria have a major role in energy production via oxidative phosphorylation1, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options2. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell)3,4. Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS)5, and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.
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The authors acknowledge the OHSU Embryonic Stem Cell Research Oversight Committee and the Institutional Review Board for providing oversight and guidance. We thank skin and oocyte donors and the Women's Health Research Unit staff at the Center for Women's Health, University Fertility Consultants and the Reproductive Endocrinology & Infertility Division in the Department of Obstetrics & Gynecology of Oregon Health & Science University for their support and procurement of gametes. We are grateful to M. Tachibana and A. Polat for help with derivation of PSCs and to M. Sparman for technical support. We are indebted to S. Gokhale for teratoma analysis and M. C. T. Penedo for microsatellite genotyping. We thank the staff at the Institute for Genomic Medicine Genomics Facility at UCSD for sequencing the RNA-seq libraries. Studies were supported by the Leducq Foundation, Mayo Clinic Center for Regenerative Medicine and OHSU and UCSD institutional funds. Work in the laboratory of J.C.I.B. was supported by the G. Harold and Leila Y. Mathers Charitable Foundation and the Leona M. and Harry B. Helmsley Charitable Trust (2012-PG-MED002).
The authors declare no competing financial interests.
Extended data figures and tables
a, Chromatographs showing mtDNA genotyping at 3243 position (arrow) in representative MELAS iPS cells. b, Chromatographs showing mtDNA genotyping at 8993 position (arrow). c, mtDNA at 13513 position (arrow) in representative iPS cells derived from Leigh syndrome patients. d, Chromatographs showing either wild-type A or mutant G allele at position 3243 in representative MELAS fibroblast clones. e, mtDNA genotyping demonstrated that all Leigh-iPS cell lines and Leigh-fib contain a G mutation allele at mtDNA position 8993. f, mtDNA genotyping demonstrated that Leigh-fib and Leigh-iPS1 cell lines contained a C mutant allele at position 4216 and a G mutant allele at position 8993, while Leigh-NT1 line carried oocyte mtDNA with a wild-type T allele at both positions.
a, MELAS-iPS1 and MELAS-iPS2 expressing NANOG detected by immunocytochemistry. Scale bars, 200 μm. b, Histological analyses of teratoma tumours produced after injections of MELAS-iPS1 and MELAS-iPS2 cells into SCID mice. Scale bars, 200 μm. c, Cytogenetic G-banding analysis confirmed that Leigh-NT1 and Leigh-iPS1 exhibited normal 46XY karyotypes and Leigh-NT2 exhibited a XXXY tetraploid karyotype. d, Leigh-NT1 and Leigh-iPS1cells expressed OCT4 and NANOG. Scale bars, 200 μm. e, Histological analyses of teratoma tumours produced after injections of Leigh-NT1 and Leigh-iPS1 cells into SCID mice. Scale bars, 200 μm. Haematoxylin and eosin staining of teratoma sections identify derivatives of ectoderm, mesoderm and endoderm.
a, OCR/ECAR ratio in MELAS-iPS cells. Mutant MELAS-iPS1 and MELAS-iPS3 displayed significantly decreased OCR/ECAR ratios compared to wild-type MELAS-iPS2 (P < 0.05), indicating a greater reliance on glycolysis (n = 9 per cell line, biological replicates). b, OCR/ECAR ratio in MELAS-iPS cell derived fibroblasts (n = 10 per cell line, biological replicates). c, Immunofluorescence analysis for neural progenitor markers in MELAS-iPS derived NPCs. Scale bar, 100 μm. d, Quantitative analysis of PSC (OCT4 and NANOG) or NPC (SOX1, NESTIN and PAX6) marker expression in MELAS-iPS cells and NPCs (n = 3 per cell line, biological replicates). e, OCR of MELAS-iPS cell derived NPCs (n = 6 per cell line, biological replicates). Error bars are mean ± s.e.m. Significance established with one-way ANOVA with Tukey's multiple comparison test.
a, OCR/ECAR ratio in Leigh-iPS1, Leigh-iPS2 and Leigh-NT1 derived fibroblasts, parental and oocyte donor fibroblasts (n = 9, 8, 10, 9 and 8 per cell line, respectively, biological replicates). b, Immunofluorescence analysis of Leigh-iPS1- and Leigh-NT1-derived skeletal muscle cells labelled with MF20 and myogenin antibodies. Scale bar, 100 μm. c, Cardiomyocyte differentiation efficiency in Leigh-iPS1 and Leigh-NT1 evaluated by FACS for CTnT-Alexa 647 and GATA4-FITC antibodies (n = 3 per cell line, biological replicates). Error bars are mean ± s.e.m. Significance established with one-way ANOVA with Tukey's multiple comparison test.
a, Immunofluorescence analysis of hESO-NT1 and hESO-8 derived NPCs with nestin and PAX6 antibodies. Scale bar, 100 μm. b, Metabolic profiles of NPCs differentiated from hESO-NT1 and hESO-8 (n = 6 per cell line, biological replicates). c, Immunofluorescence analysis of hESO-NT1 and hESO-8 derived cardiomyocytes with troponin I and NKX2.5 antibodies. Scale bar, 100 μm. d, Efficiency of cardiomyocyte differentiation in hESO-NT1 and hESO-8 evaluated by FACS analysis for CTnT-Alexa 647 and GATA4-FITC antibodies (n = 3 per cell line, biological replicates). e, OCR of hESO-NT1 and hESO-8 derived cardiomyocytes (n = 6 per cell line, biological replicates). Error bars are mean ± s.e.m. Significance established with Student's t-test.
Extended Data Figure 6 RNA-seq analyses of fibroblasts differentiated from MELAS and Leigh syndrome PSCs carrying wild-type and mutant mtDNA.
a, Heat map showing all differentially expressed 1,118 genes (adjusted P value < 0.05) between fibroblasts differentiated from mutant MELAS iPS cells (n = 4 from biological duplicates of MELAS-iPS2 and MELAS-iPS4) and wild-type MELAS iPS cells (n = 4 from biological duplicates of MELAS-iPS1 and MELAS-iPS3). b, Heat map demonstrating differentially expressed 2,950 genes (adjusted P value < 0.05) between fibroblasts derived from wild-type Leigh-NT1 (biological duplicates) and mutant Leigh iPS cells (n = 6 from biological duplicates of Leigh-iPS1, Leigh-iPS2 and Leigh-iPS3). c, Hierarchical clustering using Euclidean distance and average linkage using pvclust, which employs a multiple bootstrap resampling algorithm to calculate the approximately unbiased (AU, red) and bootstrap probability (BU, green) values for cluster distinctions. Hierarchical clustering showed that the Leigh-NT1 fibroblasts were similar to hESO-NT1, hESO-NT2, hESO-7 and hESO-8 fibroblasts. d, Mean log2 normalized counts ± s.e.m. for genes previously reported to be differentially expressed in MELAS cytoplasmic hybrid clones and involved in metabolic and stress response, signalling pathways and epigenetic modifying processes (wild type fibroblast; n = 14 from biological duplicates of 7 independent cell lines; mutant fibroblast n = 10 from biological duplicates of 5 independent cell lines).
Circular heat map displaying average expression levels for all mitochondrial genes grouped by sample differentiation status and presence or absence of a mutation in the mitochondrial genome (Fib mutant (including primary fibroblasts and PSC derived fibroblasts) with mutant mtDNA n = 14, biological duplicates of 7 independent cell lines; Fib wild type (including primary fibroblasts and PSC derived fibroblasts) with wild‐type mtDNA n = 14, biological duplicates of 7 independent cell lines; PSC mutant (undifferentiated IVF‐ESC, NT‐ESC and iPS cells) with mutant mtDNA n = 3; PSC wild type (undifferentiated IVF‐ESC, NT‐ESC and iPS cells) with wild‐type mtDNA n = 12). The expression of mtDNA‐encoded genes was similar irrespective of 3243A>G or 8993T>G mutations (adjusted P value >0.05).
This file contains Supplementary Tables 1-3. Table 1 contains a summary of mtDNA variants (SNPs) for MELAS-iPS1, MELAS-iPS2, MELAS-iPS3 and MELAS-fib defined by MiSeq. Table 2 contains a summary of mtDNA variants (SNPs) for Leigh-NT1, Leigh-iPS1 and Leigh-fib defined by MiSeq. Table 3 contains a summary of mtDNA variants (SNPs) for hESO-NT1, hESO-8 and HDF defined by MiSeq (total 12 SNPs differ between hESO-8 and HDF as indicated by red font). (XLSX 25 kb)
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Ma, H., Folmes, C., Wu, J. et al. Metabolic rescue in pluripotent cells from patients with mtDNA disease. Nature 524, 234–238 (2015). https://doi.org/10.1038/nature14546