Cell culture of MERRF iPSCs

The 6 lines of human iPSCs used in this study were generated from the skin fibroblasts established from 3 patients harboring the MERRF syndrome-specific m.8344A > G mutation. Among them, two patients are the members of a Taiwanese MERRF family, a 15-year-old girl (M1-iPSCs) and her 13-year-old sister (M2-iPSCs), and the third patient is a 25-year-old female (M3-iPSCs) as described previously [12, 14, 16]. M1 patient had poor learning ability in childhood and developed myoclonic epilepsy at 12 years of age, and exhibited severe clinical symptoms, including unsteady gait with tremor, intermittent myoclonus and polyneuropathy [17]. Her early asymptomatic sister M2 had a later onset after tissue cells had been collected despite carrying the m.8344A > G mutation. M3 patient was diagnosed with progressive ataxia and myoclonic epilepsy at 13 years of age [18]. She had suffered from progressive weakness and atrophy of extremities for six to seven years until the tissue biopsies were obtained. The primary cultures of skin fibroblasts were established in our laboratory before 1995 from these patients who had been diagnosed to have MERRF syndrome by Dr. Yuh-Jyh Jong at the Department of Pediatrics, Kaohsiung Medical University Hospital, Kaohsiung and by Dr. Chin-Chang Huang at the Department of Neurology, Chang Gung Memorial Hospital, Lin-Kou, respectively [17, 18]. The clinical specimens of the patients were provided by the clinicians according to the guidelines set by each of the hospitals. Among all iPSCs lines, M1-iPSC sublines (M1Low and M1High iPSCs) and M2-iPSCs (M2High iPSCs) were generated at Taipei Veterans General Hospital and have been used for the studies reported previously [12]. M3-iPSCs sublines (M3Low and M3Med iPSCs) were generated from another MERRF patient in collaboration with Prof. Patrick C. H. Hsieh at the Institute of Biomedical Sciences, Academia Sinica supported by Taiwan Human Disease iPSC Service Consortium [14]. Prof. Y. C. Hsu at Mackay Medical College kindly provided us the normal iPSCs (IBMS-iPSC-02-07), which were generated from the peripheral blood of a healthy 22-year-old Asian male volunteer and are commercially available from the Food Industry Research and Development Institute, Hsinchu, Taiwan. The generation of iPSCs from skin fibroblasts of MERRF patients was approved by the Institutional Review Board (IRB) of Mackay Memorial Hospital, Mackay Medical College, and Taipei Veterans General Hospital, respectively. For this study, the IRB waived the informed consent requirement for the use of these iPSCs cell lines. The ability of the MERRF iPSCs to differentiate into three germ layers in vitro and form teratoma and three germ layers in vivo was confirmed during characterization of iPSCs [12, 14]. The iPSCs were expanded in Gibco™ StemFlex™ medium on Geltrex-coated plates at 37 °C in a humidified CO2 incubator. For the first 24 h after seeding, the growth medium was supplemented with 10 μM Rho-associated protein kinase (ROCK) inhibitor Y27632 (Adooq, Irvine, CA, USA).

Total DNA extraction

Total cellular DNA was isolated by phenol/chloroform extraction. The cell pellet was re-suspended in the TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.3) containing 1% SDS, 0.25 mg/ml RNase A and 0.4 mg/ml proteinase K and incubated at 56 °C overnight. After repeated extraction with phenol/chloroform, DNA was precipitated with ice-cold 75% ethanol, air-dried, and dissolved in distilled water for further analysis.

Quantitative analysis of the m.8344A > G mutation by PCR–RFLP

The proportion of mtDNA with the m.8344A > G mutation in cultured cells was determined by PCR–RFLP using a forward primer L8150 (8150–8169): 5′-CCGGGGGTATACTACGGTCA-3′ and a mismatched reverse primer MR28 (8372–8345): 5′-GGGGCATTTCACTGTAAAGAGGTGCCGG-3′ as previously described [17]. The primers were designed to create a Nae I restriction site after PCR amplification of the DNA encompassing the putative m.8344A > G mutation in the tissue or cultured cells from MERRF patients. The 223-bp PCR product could be cleaved by Nae I into 197-bp and 26-bp fragments.

Whole genome sequencing of mtDNA by NGS

Whole genome sequencing of mtDNA was performed by collaboration with The Union Clinical Laboratory (Taipei, Taiwan) using the MiSeq Next-Generation Sequencing Systems. Briefly, the full-length mtDNA was amplified by a long-range PCR method with minor modifications [19]. The PCR was performed using Platinum SuperFi II DNA polymerase (Invitrogen, Carlsbad, CA, USA) with 50 ng of genomic DNA as the template, and the thermal cycler conditions include 30 cycles of 95 °C for 20 s and 65 °C for 8 min. PCR products (16.6 kb fragments) were cleaned-up by using the Beckman Coulter™ Agencourt AMPure XP (Beckman, Brea, CA, USA). Approximately 100 ng of PCR products were sheared to approximately 120 base pairs using the M220 Focused-ultrasonicator, and library construction and sequencing were then carried out using the KAPA HyperPrep Kit and MiSeq Reagent Kit v2 (Agilent Technologies, Santa Clara, CA, USA). The mtDNA variant calling and annotation were finally performed by the mitochondrial genome mapping (rCRS, GenBank: J01415.2) [20] and databases annotation was carried out according to the manufacturer’s instructions.

Immunofluorescence staining

Cells were fixed with 4% PFA, permeabilized and blocked with 0.3% Triton X-100, 6% bovine serum in the TBS buffer. Samples were incubated overnight at 4ºC with the primary antibodies against SEEA4 (Thermo, MA1-021-D488, 1:100), SOX2 (Thermo, MA1-014-D488, 1:100); Nestin (BioLegend, 656812, 1:100); SOX1 (Abcam, ab8775, 1:200); MAP2 (Merck Millipore, AB5622, 1:200), and TUJ1 (Abcam, ab224978, 1:200), respectively. Alexa-Fluor-Dye secondary antibodies were applied (1:500) at room temperature for 1 h. After staining, cells were washed twice and dried. Stained cells were photographed and images were acquired under a microscope of Tissue FAXS i4 SCAN (Zeiss, Dublin, CA, USA).

Western blot analysis

The cells were lysed with lysis buffer B (50 mM HEPES, 4 mM EDTA, 2 mM EGTA, 1% Triton X-100) containing a protease inhibitor cocktail (Bioman, Taipei, Taiwan). An aliquot of 30 μg proteins were separated on 10% SDS-PAGE and blotted onto a piece of the PVDF membrane (Pall Corporation, Port Washington, NY, USA). After blocking with skim milk, the membrane was incubated with primary antibodies and then reacted with horseradish peroxidase conjugated with anti-rabbit IgG and anti-mouse IgG, respectively. The protein bands were visualized on an imaging system (Gel Doc XR+ system, Bio-Rad Laboratories, Inc., Hercules, CA, USA) by using an ECL chemiluminescence reagent according to the manufacturer’s instruction and each protein signal was quantified by an Image Lab software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Measurement of mitochondrial respiration

The Seahorse XFe24 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA) was used to measure the OCR of cells in real-time at 37 °C with sequential injection of 1 μM oligomycin A (OA), 2 μM FCCP, and 2.5 μM antimycin A plus 2 μM rotenone (AA/Rot), respectively, to examine the effect of these compounds on respiration. Oligomycin A was injected to assay the ATP-coupled mitochondrial respiration rate. Injection of FCCP provided maximal mitochondrial respiration. Finally, antimycin A and rotenone were injected to estimate non-mitochondrial respiration rate. The maximal respiratory rate was calculated as the difference between the OCR after FCCP injection and the lowest OCR was reached after AA/Rot addition. OA-sensitive (ATP-coupled) respiration rate was calculated as the difference between the basal OCR and the OCR after OA injection.

Determination of the intracellular ROS level

Intracellular H2O2 levels was measured by staining cells with 20 μM 2,7-dichlorofluorescin diacetate (DCFH-DA) (Thermo Fisher Scientific, Waltham, MA, USA), respectively, at 37°C for 20 min. Cells were resuspended in 50 mM HEPES buffer (pH 7.4) and subjected to analysis on a flow cytometer (FACSAria III, BD Biosciences, San Jose, CA, USA). The intensity of emitted fluorescence at 525 nm was recorded for DCF.

Cortical neuronal differentiation of iPSCs

Differentiation of iPSCs to mature cortical-like excitatory neurons was performed by the two-step neural differentiation method. Combination of the small-molecule inhibitors of SMAD and GSK-3β with commonly used neural supplements can drive rapid production of neural stem cells or progenitor cells (iNSCs/iNPCs) and terminal differentiation of neurons, respectively, from iPSCs. Direct induction of iPSCs to expandable iNSCs was achieved by using the neural induction medium (Merck Millipore, Burlington, MA, USA) according to the manufacture’s protocol with modifications. On day 10 of neural induction, iNSCs were harvested and expanded. Cells were reseeded in StemPro NSC serum-free expansion medium (Gibco, Carlsbad, CA, USA) consisting of neurobasal medium supplemented with B27 supplement of bFGF, and l-glutamine. Cells were either expanded for banking or used for neuron differentiation between 3 and 8 passages. For terminal neuron differentiation, iNSCs were plated on poly-l-ornithine (Sigma Aldrich Chemical Co., St. Louis, MO, USA) and laminin (Gibco, Carlsbad, CA, USA) at 1–2 × 104 cells/cm2 with the differentiation medium (Merck Millipore, Burlington, MA, USA) supplemented with 0.5 mM dibutyryl cAMP and 0.2 mM ascorbic acid phosphate. Differentiation medium was refreshed every 2 days for the duration of 7–70 days. Mature neurons were harvested for characterization of protein markers by immunofluorescence staining and Western blot analysis of the protein expression levels of neuron-specific genes.

Measurement of electrophysiological activity and electrical stimulation

Human iPSC-derived iNSCs were seeded at 6 × 104 cells in a 24-well microelectrode array (MEA) plate (Axion Biosystems, Atlanta, GA, USA) containing 16 electrodes for the assay of neuron differentiation and maturation. All procedures were conducted by following the user’s manual of Axion Biosystems. MEAs system can capture the extracellular field potentials of electrically active cells by recording their neuronal activity [21]. To examine the spontaneous electrical activity of differentiated neuron, we collected the activity data from neuronal networks for 8 min by MEA every two or three days starting from day 14 after neuronal differentiation (3–4 replicates per experiment). Spike and burst rates represented the overall activity of the neuronal network, with more spikes and bursts corresponding to a higher electric activity. Network events include synchronous network spikes and bursts in which more than 35% electrodes captured the activity simultaneously. For electrical stimulation, after differentiation for 8 weeks the neurons received current injection at 50 μA/1000 μs/1000 mV every 5 s for five consecutive stimulations.

Statistical analysis

Statistical analysis was performed by the Microsoft Excel 2013 statistical package and the data are presented as means ± SEM of the results obtained from 3 independent experiments. The significance level of the difference between control and the experimental groups was determined by Student's t test. A difference is considered significant when *p value < 0.05, **p value < 0.01, and ***p value < 0.001.

Linear regression analysis

Linear regression analysis was performed by the Microsoft Excel to estimate the correlation between the m.8344A > G mutation load and biological function data. Regression output values including the coefficients, R-squared values and p-values are summarized in the Additional file 1: Table S1.