Isolation of human skin fibroblasts and generation of iPSCs
For control A, human dermal fibroblasts (HDFs) from the facial dermis of a 36-year-old Caucasian female (Cell Applications Inc.) were used to establish iPSCs (201B7; Passage 20–29, YA9; Passage 15–24). The 201B7 iPSCs were kindly provided by Dr. Yamanaka. A skin-punch biopsy from a healthy 16-year-old Japanese female obtained after written informed consent (Keio University School of Medicine) was used to generate the control B iPSCs (WD39; Passage 8–17). PA iPSCs (PA1, 9, and 22; Passage 10–19) and PB iPSCs (PB1, 2, 18, and 20; Passage 8–17) were generated from a 71-year-old Japanese female patient and a 50-year-old Japanese male patient, respectively, using the same methods used to generate control B iPSCs. The maintenance of HDFs, lentiviral production, retroviral production, infection, stem cell culture and characterization, and teratoma formation were performed as described previously[14, 15]. All of the experimental procedures for skin biopsy and iPS production were approved by the Keio University School of Medicine Ethics committee (Approval Number: 20-16-18) and Juntendo University School of Medicine Ethics committee (Approval Number: 2012068). hESCs (KhES-1; Passage 29–38 (kindly provided by Dr. Norio Nakatsuji) were cultured on feeder cells in iPS culture media.
In vitro differentiation of human iPSCs
Neural differentiation of iPSCs was performed as previously described with slight modifications (Okada et al., manuscript in preparation). Briefly, iPSC colonies were detached from feeder layers and cultured in suspension as EBs for about 30 days in bacteriological dishes. EBs were then enzymatically dissociated into single cells and the dissociated cells cultured in suspension in serum-free media (MHM) for 10 to 14 days to allow the formation of neurospheres. Neurospheres were passaged repeatedly by dissociation into single cells followed by culture in the same manner. Typically, neurospheres between passages 3 and 8 were used for analysis. For terminal differentiation, dissociated or undissociated neurospheres were allowed to adhere to poly-L-ornithine- and fibronectin-coated coverslips and cultured for 10 days.
Immunocytochemical analysis of iPSCs and neurons
For immunocytochemical analysis, cells were fixed with phosphate buffered saline (PBS) containing 4% paraformaldehyde (PFA) for 30 min at room temperature (RT). The cells were analyzed by immunofluorescence staining using antibodies to the following proteins: β-III-tubulin (1:1000, Sigma), NANOG (1:100, ReproCELL), OCT3/4 (1:200, Santa Cruz Biotechnology), SSEA-4 (1:200, Millipore), TRA-1-60 (1:200, Millipore), TH (1:100, Millipore), α-synuclein (1:500, Invitrogen), pα-synuclein (1:1000, Wako), cleaved-Caspase3 (1:500, Cell Signaling) and ComplexIII (C-III)-core I (1:200, Invitrogen). Cells were washed with PBS after incubation with the primary antibody, followed by incubation with an Alexa Fluor 488-, Alexa Fluor 555-, or Alexa Fluor 647-conjugated secondary antibody (1:500, Invitrogen). Images were obtained using Apotome (Zeiss) or LSM-710 confocal (Zeiss) microscopes.
PCR amplification of genomic DNA
Genomic DNA was purified from HDFs and iPSCs using a DNeasy kit (Qiagen). The PCR conditions used have been previously described[2, 42].
Reverse transcription (RT)-PCR
RNA isolation and reverse transcription (RT)-PCR were performed as previously described. The amount of cDNA was normalized to β-actin mRNA. Real-time RT-PCR was performed on a ABI PRISM Sequence detection System 7900HT (Applied BioSystems) using SYBR premix ExTaq (Takara). Primers for the detection of Oct4, the transgenes Oct4-tg, Sox2-tg, Klf4-tg and c-Myc-tg, and MAO-A, and -B have been previously described[10, 15].
To assess teratoma formation, iPSCs were injected into the testis of 8-week-old NOD/SCID mice (OYG International) as previously described. Eight weeks after transplantation, tumors were dissected and fixed with 4% PFA in PBS. Paraffin-embedded tissue was sectioned and stained with H&E. Images were obtained using a BZ-9000 (Keyence) microscope.
Genomic DNA was restricted, labeled, and purified using the Agilent Oligo CGH Microarray Kit (Agilent Technologies) according to the manufacturer’s protocol. Labeled genomic DNA was processed for hybridization on a 4x 180K microarray (Agilent Technologies). Processing was performed as instructed by the manufacturer. The genomic analysis was performed using Agilent Genomic Workbench ver. 6.0 software (Agilent Technologies).
Reduced GSH levels were measured according to the kit manufacturer’s protocol (GSH-Glo Glutathione Assay; Promega). Chymotrypsin-like proteasome activity was measured using a Cell-Based Proteasome-Glo Assay according to the manufacturer’s instructions (Promega). Briefly, neural cells (1.0 × 104) derived from neurospheres were seeded in triplicate into a white 96-well plate (Nunc). Prepared reagent (100 μl) was added to each well. After incubation for 10 min at RT, luminescence intensity was recorded. ROS levels were determined by measuring DCFH-DA fluorescence (Invitrogen). Briefly, neurons were incubated with 5 μM DCFH-DA and Hoechst (1:2000) for 30 min at 37°C, after which they were washed with PBS and then incubated in differentiation media. Fluorescence was measured by an In Cell Analyzer 2000 system (GE Healthcare Biosciences).
Differentiated neurons were harvested in MAPK lysis buffer containing proteinase inhibitor, and protein concentrations were measured by BCA assay (Thermo Scientific). Samples were diluted to yield equivalent protein concentrations and then 4 μg was denatured by the addition of 4X sample buffer (Invitrogen) supplemented with β-mercaptoethanol followed by boiling. Samples (7 μl/lane) were loaded onto a 4–20% SDS-polyacrylamide gradient gel. Membranes were incubated in blocking solution with the indicated primary antibodies at 4°C overnight. Immunoreactive proteins were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and then visualized by chemiluminescence (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Quantification of band intensities was performed using an RAS4000 system. The primary antibodies used were anti-NQO1 (1:1000, Abcam), anti-NRF2 (1:1000, Santa Cruz Biotechnology) and β-actin (1:5000, Cell Signaling).
CCCP and Baf A1 treatments
Neurons were cultured with 30 μM CCCP (Sigma-Aldrich) or DMSO, with or without 5 μM Baf A1 (Sigma-Aldrich), for 48 h. The cells were then fixed and stained for βIII-tubulin and C-III Core I, and counterstained with Hoechst. To quantify the IMM area of the neurons, the cytoplasmic area was extracted as shown in Figure 3C. The C-III Core I-positive signals within the extracted area were then converted to gray-scale and digitized. The IMM area was quantified from the digitized values using Image J software.
Tetramethylrhodamine ethyl ester (TMRE) staining
iPSC-derived neurons were incubated with 1nM TMRE (Invitrogen) for 15 min at 37°C and then observed under an Olympus IX81 microscope.
Cells were fixed with 2% glutaraldehyde/2% PFA in 0.1 M phosphate buffer (PB) (pH7.2), post-fixed with 1% OsO4 in 0.1 M PB (pH 7.2), blocked and stained with a 2% aqueous solution of uranyl acetate, dehydrated with a graded series of ethanol, and then embedded in Epon 812 (TAAB). Coverslips were detached and the embedded samples were placed under a stereomicroscope to identify the cells of interest. Ultrathin sections were cut with a Leica UC6 or UC7 ultramicrotome (Leica Microsystems) and then stained with uranyl acetate and lead citrate. Samples were observed with a Hitachi H7100 or HT7700 electron microscope.
Morphometric analysis was used to measure the volume density of mitochondria in the neuronal perikarya as previously described. Briefly, electron micrographs of neurons (n = 20, 23, 41, and 44 for control A (B7), control B (WD39), PA9 and PB2, respectively) were obtained at a magnification of ×7000. After enlarging to three times the original magnification, point-counting was carried out to determine the volume density using a double-lattice test system with 1.5 cm spacing. Mitochondria were classified as normal, abnormal, or undetermined. The abnormal mitochondria were defined as those with irregularly arranged cristae, or with a high electron-dense matrix. The volume density (Vv) of each type of mitochondrion was expressed as percent volume according to the following formula: Vv = (Pi/Pt) × 100 (%), where Pi is the number of points falling on each mitochondrial structure and Pt is the number of points falling on the neuronal perikarya.
Immunohistochemical analysis of autopsied brain tissue
The ethical committee of the Kitasato University School of Medicine and Juntendo University School of Medicine reviewed and approved the protocol for analysis of autopsied brain tissue. Patients and control subjects were informed of the study and gave written informed consent. Brain tissue from patient PA was obtained following her death at age 72; brain tissue from the father of patient PB was obtained when he died at age 70. Tissue was fixed with 10% formalin and then embedded in paraffin. Midbrain sections (6 μm thick) were cut, deparaffinized with xylene, and then rehydrated in ethanol. After being boiled and treated with H2O2, sections were subjected to immunofluorescence staining with antibodies to the following proteins: α-synuclein (1:500, Invitrogen), pα-synuclein (1:1000, Wako), and TH (1:1000, Calbiochem). After washing with PBS, sections were incubated with a biotinylated secondary antibody (1:500; Vector Laboratories Inc.) at RT for 1 hr followed by incubation with an avidin-biotin peroxidase complex (Vector Laboratories Inc.) for 1 hr. Immunoreactive proteins were visualized using 3,3-diaminobenzidine (DAB; Wako Pure Chemical Industries) and nuclear fast red staining. For immunofluorescence, FITC-conjugated and Cy3-conjugated secondary antibodies (1:500; Jackson Immunoresearch Laboratories) were used. Images were obtained using a BIOREVO (Keyence) and a confocal laser-scanning LSM710 (Zeiss) microscope.
Values represent the mean ± SEM. The Mann–Whitney U-test was used to evaluate differences between groups. A P value of < 0.05 was considered significant.