Disease-Causing Mitochondrial Heteroplasmy Segregated Within Induced Pluripotent Stem Cell Clones Derived from a Patient with MELAS

Abstract

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as MELAS demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multi-lineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. Upon comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared to isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases.

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

“…In fact, iPSC-derived NPCs retained the entire mtDNA profile of the initial fibroblasts, including even the hypervariable D-loop region. 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).…”

1α,25-Dihydroxyvitamin D3 Regulates Mitochondrial Oxygen Consumption and Dynamics in Human Skeletal Muscle Cells

“…OCR was measured following the sequential addition of oligomycin (0.5 g/ml), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (1 M), and rotenone (0.5 M)/antimycin A (1 M). Baseline respiration rate (basal OCR Ϫ rotenone/antimycin A OCR), coupled respiration rate (basal respiration rate Ϫ oligomycin OCR), maximal respiration rate (carbonyl cyanide p-trifluoromethoxyphenylhydrazone OCR Ϫ rotenone/antimycin A OCR), and oxidative reserve (carbonyl cyanide p-trifluoromethoxyphenylhydrazone OCR Ϫ basal respiration rate) were calculated (31). Oxygen uptake in isolated mitochondria was determined using an Oroboros Oxygraph (Innsbruck, Austria) as described previously (28).…”

Energy metabolism in neuronal/glial induction and in iPSC models of brain disorders

“…MELAS (Mitochondrial myopathy, Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes) [93] was recently investigated using iPSCs. This was accomplished by several groups [93][94][95][96][97][98]. A common feature identified by these studies was that the original heteroplasmy level present in the patient fibroblasts can undergo changes before or during reprogramming.…”