TALEN-mediated shift of mitochondrial DNA heteroplasmy in MELAS-iPSCs with m.13513G>A mutation

Abstract

Induced pluripotent stem cells (iPSCs) are suitable for studying mitochondrial diseases caused by mitochondrial DNA (mtDNA) mutations. Here, we generated iPSCs from a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) with the m.13513G>A mutation. The patient's dermal fibroblasts were reprogrammed, and we established two iPSC clones with and without mutant mtDNA. Furthermore, we tried to decrease mutant mtDNA level in iPSCs using transcription activator-like effector nucleases (TALENs). We originally engineered platinum TALENs, which were transported into mitochondria, recognized the mtDNA sequence including the m.13513 position, and preferentially cleaved G13513A mutant mtDNA (G13513A-mpTALEN). The m.13513G>A heteroplasmy level in MELAS-iPSCs was decreased in the short term by transduction of G13513A-mpTALEN. Our data demonstrate that this mtDNA-targeted nuclease would be a powerful tool for changing the heteroplasmy level in heteroplasmic iPSCs, which could contribute to elucidation of the pathological mechanisms of mitochondrial diseases caused by mtDNA mutations.

Figure 1

Establishment of human iPSCs from MELAS patient with m.13513G>A mutation. (A) Morphological features and expression of human ESC markers. All A01 MELAS-iPSC clones are identical to human ESCs. All iPSC clones expressed the human ESC markers Oct3/4 (green), SSEA-4 (red) and Nanog (green) analyzed by immunocytochemical analysis. Phase, phase-contrast image. Scale bar, 200 μm. (B) RT-PCR analyses of pluripotency markers and reprogramming factors (Oct3/4, Nanog, Sox2, L-Myc, Lin28, Klf4 and c-Myc). β-actin serves as a loading control. (C) Karyotype analysis revealed that all hiPSCs had a preserved normal karyotype. (D) In vitro differentiation of MELAS-iPSCs into all three germ layers. Sox17 (endoderm, green), αSMA (mesoderm, red) and Tuj1 (ectoderm, green). Scale bar, 100 μm.

Figure 2

Characterization of mtDNA in A01 MELAS-iPSCs. (A) Genetic analysis of A01 fibroblasts and MELAS-iPSCs at passage 5 by Sanger sequencing. Arrows indicate m.13513 position. (B) RFLP analysis of A01 fibroblasts and MELAS-iPSCs. The 120 bp and 93 bp + 27 bp fragments indicate the presence of mutant and wild-type mtDNA, respectively. Heteroplasmy level of m.13513G>A mtDNA (C) analyzed by ARMS-qPCR and mtDNA copy number (D) in A01 fibroblasts and MELAS-iPSCs. MELAS-iPSCs were collected at the following passage numbers: #15 at p5 and p20; #58 at p5 and p20; #30 at p5, p12, p20, p31 and p43; #67 at p5, p12, p21, p33 and p45.

Figure 3

Generation of G13513A-mpTALEN. (A) Schematic illustration of target site of G13513A-pTALEN. Black and white boxes indicate RVDs of L-pTALEN and R-pTALEN, respectively. Letters beside box indicate TALE’s name. Black bar indicates spacer region of L-pTALEN(PKLB)/R-pTALEN(PKR) pair. (B) Scheme of SSA assay. The reporter plasmid encodes a part of the mtDNA sequence including m.13513G or m.13513A, named m.13513 G(WT)-2 or m.13513A(MUT)-2, which is sandwiched by two split inactive parts of the luciferase gene with overlapping repeated sequences. Following a double strand break caused by TALENs, a functional luciferase gene is generated by an SSA reaction. (C) Evaluation of SSA activity (Luc/RLuc) of pTALEN pairs. Blue and red bars reflect cleaving activity against m.13513 G(WT)-2 and m.13513A(MUT)-2, respectively. NC, negative control. Data are expressed as mean SEM (n = 3). (D) m.13513A(MUT) target specificity of each L-pTALEN/R-pTALEN pair. Data are expressed as mean SEM (n = 3). (E) Components of plasmids used to express L-mpTALEN(PKLB) and R-mpTALEN(PKR) monomers. (F) Western blotting of lysed HEK293T cells transfected with plasmid coding L-mpTALEN(PKLB) or R-mpTALEN(PKR) for 2 days. L-mpTALEN(PKLB) or R-mpTALEN(PKR) was detected using anti-Flag or anti-HA antibodies, respectively. GAPDH serves as a loading control. (G) Intracellular localization of mpTALENs analyzed by immunocytochemical analysis. L-mpTALEN(PKLB) or R-mpTALEN (PKR) was transiently expressed in HeLa cells. Two days after transfection, L-mpTALEN(PKLB) or R-mpTALEN(PKR) was stained using anti-Flag or anti-HA antibodies, respectively (red). Mitochondria were stained using anti-TOM20 antibodies (green). Nuclei were stained with DAPI (blue). Scale bar, 20 μm.

Figure 4

Decrease of m.13513G>A heteroplasmy level in MELAS-iPSCs by G13513A-mpTALEN. (A) Experimental scheme. MELAS-iPSCs (#67) subcultured at the same time were transfected with plasmids coding L-mpTALEN(PKLB) and R-mpTALEN(PKR) and EGFP (n = 3). EGFP-positive and live cells sorted at day 2 after transfection, named L-mpTALEN(PKLB)/R-mpTALEN(PKR), were compared with sorted cells transfected with twice the amount of plasmid coding L-mpTALEN(PKLB), named L-mpTALEN(PKLB)/L-mpTALEN(PKLB), and untreated cells. (B; left) m.13513G>A heteroplasmy level was analyzed by ARMS-qPCR. Data are expressed as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, Tukey’s test. (B; right) Sanger-sequence analysis. Arrow indicates m.13513 position. (C) MtDNA copy numbers of L-mpTALEN(PKLB)/R-mpTALEN(PKR) are represented relative to those of L-mpTALEN(PKLB)/L-mpTALEN(PKLB). Data are expressed as mean ± SEM (n = 3). ***p < 0.005, Student’s t-test.