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 mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (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 multilineage 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. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with 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.

Keywords: Induced pluripotent stem cells; MELAS syndrome; Mitochondria; Mitochondrial DNA; Regenerative medicine.

Figure 1. MELAS derived dermal fibroblasts contain a mutation in G13513A and demonstrate reduced oxygen reserve capacity

Mitochondrial morphology revealed by transmission electron micrographs demonstrated distinctive patterns (A). MELAS fibroblasts demonstrated smaller mitochondria compared to BJ control fibroblasts in both the major and minor axis (B). Mitochondrial function based on in vitro oxygen capacity in response to a – 0.5 µg/mL oligomyocin, b – 1 µM FCCP and c – 0.5 µM rotenone (C). Genotype of the MELAS fibroblasts was determined with restriction fragment length polymorphism (RFLP) analysis with a focus on the disease causing position (G13513A) demonstrated 100% wild-type (Wt) sequence in control fibroblasts and heteroplasmy of 53% Wt sequence (144 bp) and 47% G13513A sequence (170bp) in MELAS fibroblasts that was unchanged despite three weeks of culturing in either glucose (glu) or galactose (Gal) containing media. Clonal expansion of fibroblast (FB) subpopulations revealed that ~20% of expandable clones from the original patient-derived MELAS sample contained Wt sequences (red boxes) after 5–8 weeks of in vitro cell growth (D). Alternative sequence (orange color) reads at position G13513A was confirmed to be ~50% heteroplasmy with next-generation sequence analysis within the MELAS sample compared to 100% Wt sequence (blue color) of the control BJ fibroblasts (E). Values are mean ± SEM, * represents P < 0.05 versus BJ Wt.

Figure 2. Patient-specific iPS cells derived from primary dermal fibroblasts

Upon nuclear reprogramming, MELAS-derived fibroblasts acquired embryonic stem cell-like compact colonies within 4 weeks. Three MELAS iPS cell lines (M-iPS1-3) expressed cell surface antigens SSEA-3 and Tra-1–60, as well as the nuclear transcription factor Nanog (A). Patient-derived iPS cell lines (denoted in shades of blue bars) demonstrated upregulation of endogenous stemness genes according to quantitative RT-PCR compared to MELAS fibroblasts (denoted in red bars; B). Spontaneous in vitro differentiation within embryoid bodies converted M-iPS1-3 into differentiated tissue (M-Diff1-3; denoted in shades of orange) demonstrating a significant increase in the lineage specific markers for ectoderm, mesoderm, endoderm along with NKX2.5 gene expression for early cardiac tissue (C). Examination of differentiated progeny using electron microscopy highlighted mature mitochondria and the appearance of specialized subcellular structures such as cardiac sarcomeres (D). Genetic fingerprinting established the identity of each clone originating from the MELAS cell population in contrast to the genetically distinctive profile of control BJ cell lines according to twelve short tandem repeat (E). Values are mean ± SEM, * represents P < 0.05 versus MELAS FB, and & represents P < 0.05 versus M-iPS.

Figure 3. Mitochondrial transformation in iPS cells results in reduced oxygen dependence

Mitochondrial acquire spherical cristae poor structures in all three M-iPS cell lines (A, top) and elongated complex morphology in differentiated tissues (A, bottom). Mitochondrial structures were indistinguishable between iPSC clones 1–3 with MELAS-derived iPS cell lines having similar major and minor axis lengths compared to wild-type (WT) BJ iPS (denoted in black and white bars) and embryonic stem cells (denoted by 95% confidence intervals highlighted in red lines). Upon differentiation (M-Diff), two of the three progeny acquired longer major axes (B). Metabolic function according to in vitro cellular oxygen consumption for M-iPS cells profiles were similar to control BJ Wt iPS cells and hESC for all components of oxygen consumption (C). M-iPS cell lines (*) demonstrated significant reduction in mtDNA copy number according to PCR-based amplification and quantification as compared to parental MELAS fibroblasts (FB) denoted by 95% confidence interval highlighted by red line and demonstrated similar reduction compared to BJ-derived iPS cells. The mitochondrial copy number of all iPS cell lines was equivalent to the low levels of hESC (denoted by 95% confidence interval highlighted by blue line). Differentiation of iPS cells provoked a dramatic re-activation of mtDNA copy number in progeny from all three MELAS-derived iPS cell lines (&) and BJ-derived control iPS cells to levels above the original MELAS fibroblasts (#; D). RFLP analysis of the G13513A complex I mutation indicated that M-iPS1 and 2 have similar levels of heteroplasmy as the starting material, while the mutation could not be detected in M-iPS3, with consistent results obtained after in vitro differentiation (M-Diff; E). Values are mean ± SEM, * represents P < 0.05 versus MELAS FB, & represents P < 0.05 versus M-iPS, and # represents P < 0.05 versus BJ iPS.

Figure 4. Nuclear reprogramming yields disease-free, isogenic clones from MELAS patient

Utilizing next-generation sequencing of the whole mitochondrial genomes of MELAS fibroblasts, M-iPS cells (M-iPS 1,2,3), and differentiated progeny (M-Diff 1,2,3), sequence alignments highlights the unique differences in patient-derived samples compared to control fibroblasts with mitochondrial genome labeled on the perimeter of the circle plot and color coded to coding regions. The control fibroblasts (BJ Wt FB) demonstrated alternative reads (orange) in hypervariable regions but not in disease causing position of G13513A illustrated in the innermost ring of the circle plot. Sequence analysis confirmed the presence of Wt (reference sequence denoted in blue) and mutant sequence (alternative sequence denoted in orange) in patient-derived fibroblasts (MELAS FB) of nearly 50% at position 13513 along with a unique profile of hypervariable regions illustrated in the second smallest ring of the circle plot. Two iPS clones (M-iPS1, 56% Wt sequence; M-iPS2, 54% Wt sequences), along with their respective differentiated progeny (M-Diff1, 55% wt sequence; M-Diff2, 61% wt sequence) maintained the equivalent heteroplasmy of the parental fibroblasts. Sequence analysis confirmed the absence of G13513A mutation in M-iPS3 as well as differentiated progeny (M-Diff3) as noted in the all blue bar without orange bars at the disease-causing position without variation in the remaining hypervariable regions across the mitochondrial genome.

Figure 5. Reduced mtDNA heteroplasmy improves cardiogenesis in patient-specific iPS cells

Analysis of heteroplasmy after 1 year in culture of the M-iPS2 clone compared with MELAS fibroblasts until senescence (A). Heteroplasmy in the mtDNA remains unchanged following 25 days of in vitro differentiation compared to the heteroplasmy of iPS cells (B). Light microscopy of differentiated progeny highlighted increased 3-dimensional differentiated areas within M-Diff2 p44 compared to M-Diff2 p21 (C). Low and high passage patient-derived iPS cell line (denoted in shades of dark and light blue bars respectively) demonstrated downregulation of endogenous stemness genes according to quantitative RT-PCR in differentiated progeny compared to undifferentiated iPS cells (denoted in orange bars; D). RT-PCR demonstrates an increase in TBX5, TNNI3 and significant increase in MYH6 gene expression upon side-by-side differentiation analysis (E). Values are mean ± SEM, * represents P < 0.05 versus respective M-iPS2, and # represents P < 0.05 versus M-Diff2 p21.