Metabolic rescue in pluripotent cells from patients with mtDNA disease

Abstract

Mitochondria have a major role in energy production via oxidative phosphorylation, 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 options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). 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), 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.

Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders

“…Studies have indicated that the induction to iPSCs may be associated with heteroplasmic mtDNA alterations (Folmes et al, 2013;H€ am€ al€ ainen et al, 2013;Perales-Clemente et al, 2016;Prigione et al, 2011b). These variations appear to derive from the clonal origin of the iPSCs, because they also occur in clonally expanded fibroblasts (Kang et al, 2016;Ma et al, 2015). This means that the mtDNA sequence profile of patient-derived iPSC lines must be thoroughly assessed to ensure that the cells are suitable for use in drug discovery or other types of treatments.…”

“…To make useful models of mitochondrial diseases from NI NPCs, we needed to exclude the possibility that mtDNA had undergone changes during stages of induction, as can happen in the generation of iPSCs (Ma et al, 2015;Prigione et al, 2011b). We performed Sanger sequencing of the entire mtDNA, using long-range PCR with nested primers to exclude nuclear mtDNA pseudogenes.…”

Functional Human Oocytes Generated by Transfer of Polar Body Genomes

“…Research gamete donors were financially compensated for their time, effort, discomfort, and inconvenience associated with the donation process at rates similar to donors whose gametes were used for reproductive purposes. This specific IRB protocol was first approved in 2011 and then reviewed annually with modifications allowing the development, evaluation and testing, in vitro, of novel mitochondrial replacement techniques (see Data S1) (Kang et al, 2016;Ma et al, 2015;Tachibana et al, 2013a;Tachibana et al, 2013b).…”

Assisted reproductive technologies to prevent human mitochondrial disease transmission

“…The prospect of something similar in humans would potentially address carryover by enabling the isolation of patient-derived iPS cell lines that contained no affected mitochondria, either by selection or manipulation, and the recapitulation of meiosis from these lines to yield oocytes. Indeed, iPS cell lines containing exclusively wild-type mtDNA have previously been derived through spontaneous segregation from patients with Leigh syndrome and MELAS 69 . However, extrapolating in vitro oogenesis protocols from mouse to human is unlikely to be trivial; in vitro maturation (IVM) of immature oocytes to produce fertilizable oocytes in the mouse 70 took over two decades to translate to IVM in humans 71 .…”