Isolation of human skin fibroblasts and generation of iPSCs
Human dermal fibroblasts (HDFs) were obtained from 4 Japanese SBMA patients and 3 controls: a 36-year-old Japanese male from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (TIG114), a 24-year-old Japanese male (YF), and a 39-year-old Japanese male (KA). HDFs were cultured in DMEM (Sigma-Aldrich, USA), 10% fetal bovine serum (FBS; Sigma-Aldrich, USA), 2 mM L-glutamine, and 1% penicillin/streptomycin. Then, 6×105 HDFs were transfected with 1 μg of each of the following: pCXLE-hOCT3/4-shp53 (OCT4 and shTP53), pCXLE-hSK (SOX2 and KLF4), and pCXLE-hUL (L-MYC and LIN28; a gift from Dr. Yamanaka). Plasmid transfection was performed using the Neon transfection system (Thermo Fisher Scientific, USA). After 6 days, the cells were harvested and plated on mitomycin-C-treated SNL murine fibroblast feeder cells in 0.1% gelatin-coated tissue culture dishes in human fibroblast medium. On the next day, the medium was changed to standard human embryonic stem cell (hESC) medium containing DMEM/F-12 (Wako, Japan), 20% knockout serum replacement (KSR) (Thermo Fisher Scientific, USA), 2 mM L-glutamine, 1% non-essential amino acids (NEAA) (Sigma- Aldrich, USA), 0.1 mM 2-mercaptoethanol (2-ME) (Sigma- Aldrich, USA), 0.5% penicillin/streptomycin, and 4 ng/mL recombinant human fibroblast growth factor-2 (FGF-2) (Peprotech, USA). When the colonies had grown to a sufficiently large size, they were picked and expanded in the same way as hESCs and hiPSCs. The properties of the established iPSC clones were evaluated, as described previously (Fig. 1, S1 and S2). Control iPSC clones, which were TIGE-9, TIGE-22, YFE-16, YFE-19, and EKA3, and the SBMA iPSC clones, which were SBMA1E-12, SBMA1E-18, SBMA2E-16, SBMA2E-44, SBMA3E-10, SBMA3E-11, SBMA4E-5, and SBMA4E-21, were established. The control iPSC clones (TIGE-9, YE-16, and EKA3) were previously reported [20, 21]. For the RNA sequence analysis, control iPSC clones (EKA3, TIGE-9, YFE-16 and YFE-19) and SBMA iPSC clones (1E-12, 2E-16, 3E-10, and 4E-5) were used and the data are presented as the average of four clones for the controls and SBMA.
iPSC culture and differentiation
iPSCs were differentiated into spinal MNs, as previously described [20]. iPSCs were maintained on mitomycin-C-treated SNL murine fibroblast feeder cells in 0.1% gelatin-coated tissue culture dishes in hESC medium and were used for MN induction. For differentiation, hESC/iPSC colonies were detached using a dissociation solution (0.25% trypsin, 100 μg/ml collagenase IV (Gibco), 1 mM CaCl2, and 20% KSR) and cultured in suspension in bacteriological dishes in standard hESC medium, after the removal of SNL feeder cells, with incubation for 1–2 h in gelatin-coated dishes. On day 1, the medium was changed to human embryoid body (hEB) medium containing DMEM/F-12, 5% KSR, 2 mM L-glutamine, 1% NEAA, and 0.1 mM 2-ME with 300 nM LDN-193189 (Sigma-Aldrich, USA), 3 μM SB431542 (Tocris, UK), and 3 μM CHIR99021 (FCS, USA). On day 2, the medium was changed to fresh hEB medium containing 300 nM LDN-193189, 3 μM SB431542, and 3 μM CHIR99021, and 1 μM retinoic acid (RA) (Sigma-Aldrich, USA). From day 4 to day 14, hEBs were cultured in hEB medium containing 1 μM RA and 1 μM purmorphamine (Calbiochem, Germany), and the medium was changed every 2–3 days. On day 14, hEBs were enzymatically dissociated into single cells using TrypLE Select (Thermo Fisher Scientific, USA). The dissociated cells were plated on poly-l-ornithine (PO) and recombinant mouse Laminin (Thermo Fisher Scientific, USA), or growth-factor-reduced Matrigel (33×dilution, thin coated; Corning)-coated dishes at a density of 5×104–1×105 cells/cm2 and cultured in motor neuron medium (MNM) consisting of media hormone mix (MHM) or KBM Neural Stem Cell medium (Kohjin Bio, Japan) [58] supplemented with 2% B27 supplement (Thermo Fisher Scientific, USA), 1% NEAA, 50 nM RA, 500 nM purmorphamine, 10 μM cyclic AMP (cAMP) (Sigma-Aldrich, USA), 10 ng/mL recombinant BDNF (R&D systems, USA), 10 ng/mL recombinant GDNF (R&D systems, USA), 10 ng/mL recombinant human IGF-1 (R&D systems, USA), and 200 ng/mL L-ascorbic acid (Sigma-Aldrich, USA) for up to 4 weeks in 5% O2 atmosphere. Half of the medium was changed every 2–3 days.
Immunocytochemistry
Cells were fixed in 4% paraformaldehyde for 15–25 min at room temperature. After blocking in blocking buffer (PBS containing 10% FBS and 0.3% Triton X-100), the cells were incubated with primary antibodies overnight at 4°C. The cells were then rinsed with PBS three times and incubated with Alexa 488-, Alexa 555-, or Alexa 647- conjugated secondary antibodies (Thermo Fisher Scientific) for 1 hour at room temperature. Nuclei were stained with 10 μg/ml Hoechst 33258 (Sigma-Aldrich, USA). The cells were then rinsed with PBS three times, mounted on slides, and examined using IX83 (Olympus, Japan). The primary antibodies used in these analyses were listed in Table S9, Additional file 3.
Teratoma formation assay
Each iPSC clone was harvested in dissociation solution, collected into tubes, and centrifuged, and the resulting pellets were suspended in hESC medium with 10 μΜ Y-27632 (Wako, Japan), which is a Rho-associated coiled-coil forming kinase (ROCK) inhibitor. Then, 1×105–5×105 cells were injected into the testes of NOD/ SCID mice (Charles River, USA). At 8–10 weeks after injection, the tumors were dissected and fixed with PBS containing 4% PFA. Paraffin-embedded tissue was sliced and stained with hematoxylin and eosin. Images were obtained using a BZ-9000 microscope (Keyence, Japan).
CAG repeat sizing
DNA was extracted using DNeasy Blood & Tissue kits (Qiagen, Germany). PCR amplification of the CAG repeat in the AR gene was performed using a fluorescent-labeled forward primer (5’-TCCAGAATCTGTTCCAGAGCGTGC-3’) and an unlabeled reverse primer (5’-GCTGTGAAGGTTGCTGTTCCTCAT-3’). Detailed PCR conditions were described previously [58]. For determining the CAG repeat numbers of each PCR product, capillary electrophoresis and direct sequencing were performed using the 3730xl DNA Analyzer (Thermo Fisher Scientific, USA). Fragment analysis was performed using Peak ScannerTM Software v1.0. Sanger sequencing was performed from both 5’ and 3’ sides using a forward sequence primer (5’- TGCGCGAAGTGATCCAGAAC-3’) and a reverse sequence primer (5’- TTGGGGAGAACCATCCTCAC-3’).
RNA isolation and quantitative RT-PCR analysis
RNA was isolated using a RNeasy mini kit (Qiagen, Germany) and then converted into cDNA using SuperScript III reverse transcriptase (Thermo Fisher Scientific, USA) and Oligo dT primers as described previously [58, 59]. Real-time quantitative RT-PCR were performed as previously described using SYBR Premix ExTaq II and the StepOnePlus or the QuantStudio Real-Time PCR system. The amount of cDNA was normalized to that of human- specific β-ACTIN mRNA. The primer sequences and PCR cycling conditions are listed in Table S10, Additional file 3.
Generation of the HB9e438::Venus lentivirus
HEK293T cells cultured in the Freestyle 293 expression medium (Thermo Fisher Scientific, USA) in 150 mm dishes were transfected with 16 μg of pSIN2-HB9e438-βglo-Venus (a variant of yellow fluorescent protein (YFP) with fast and efficient maturation [60]) or pSIN2-βglo-Venus and 10 μg of each of two packaging vectors (pCMV-VSV-G and pCAG-HIV-gp, kindly provided by Dr. Hiroyuki Miyoshi) in 200 μL of polyethylenimine (Polysciences, Inc., USA), and the medium was changed the next day. Three days after the medium change, the culture supernatant was harvested and centrifuged at 25,000 rpm for 90 min at 4 °C in an Optima LE-80 K ultracentrifuge (Beckman Coulter, USA). After discarding the supernatant, 80 μL of PBS/150 mm dish was added to the pellet, which was resuspended by repeated pipetting to obtain the HB9e438::Venus reporter lentivirus. Lentiviral infection was performed on day 3 or 4 of monolayer motor neuron differentiation. For lentiviral infection, HB9e438::Venus in Opti-MEM (Thermo Fisher Scientific, USA) was added to a motor neuron culture, followed by incubation for 2 h, after which the total medium was changed to MNM.
Flow cytometry
For flow cytometric analysis, iPSC-derived MNs were dissociated 4 weeks after infection with the HB9e438::Venus lentivirus using Tissue Dissociation Kits (Miltenyi Biotec) according to the manufacturer’s instructions. The dissociated cells (5×104–1×105 cells) were suspended in 50-100 μl of Hanks’ balanced salt solution (HBSS) (Thermo Fisher Scientific, USA) containing 2 % fetal bovine serum, 10 mM HEPES, and 1 μg/ml propidium iodide. The cells were then analyzed and sorted based on the expression of the HB9e438::Venus reporter using a FACSAria III cell sorter (BD Biosciences, USA).
RNA sequencing and data analysis
The yield and quality control of total RNA were measured using Nano Drop 2000c (Thermo Fisher scientific, USA) and Agilent RNA6000 Nano Kit (Agilent technologies, USA), respectively. The 2100 Bioanalyzer system (Agilent technologies, USA) was used to qualify RNA integrity number (RIN). RNA samples with sufficient RIN values (more than 9.2) were subjected to the generation of mRNA libraries using Illumina TruSeq protocols for poly-A selection, fragmentation, and adaptor ligation, according to the manufacturer’s instructions (TruSeq RNA Sample Prep Kit v2). Quantification of libraries was performed using Qubit3.0 (Thermo Fisher), Agilent 2200 TapeStaition System (Agilent), and qPCR analysis by Kapa Library Quantification Kit (TakaRa). The multiplexed libraries were sequenced as 75 nt pair end runs on an Illumina NextSeq500 system (San Diego, CA). Sequence reads were mapped to the reference human genome (GRCh38/hg38) using STAR (2-pass mode, version2.7.1a). We then excluded reads mapped to rRNA and tRNA regions. Annotation of the rRNA and tRNA regions were obtained from the UCSC Table Browser. Read counts of transcripts (feature counts [61]) were calculated using FeatureCounts of the Rsubread package. Ensembl gene annotation (Homo_sapiens.GRCh38.96.chr.gtf) was used for the transcript counts. DEGs based on the Wald test were analyzed using DESeq2 [62]. The expression data were grouped using a hierarchical clustering algorithm in Cluster 3.0 software (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm) [63] by average linkage with the Euclidean distance, and visualized by Java TreeView software (http://jtreeview.sourceforge.net/) [64].
Gene set enrichment analysis (GSEA)
GSEA is a computational method that determines if a priori defined set of genes show statistically significant, concordant differences between two biological states [23], and is accessible at http://www.broadinstitute.org/gsea/index.jsp. The expression profile data were analyzed using GSEA 4.0.1. The gene sets were downloaded from the Molecular Signatures Database (MSigDB) and C2 (curated gene sets: chemical and genetic perturbation (CGP) and Canonical pathway (CP)) and C5 (Gene ontology gene sets) were used for the GSEA.
Statistical analysis
For the statistical analysis in the quantitative RT-PCR, either the Student’s t-test or the Welch’s t-test were used. For the differential analysis of mRNAs between control and SBMA MNs using DESeq2, adjusted p-value for a false discovery rate (FDR) correction was performed by the Benjamini–Hochberg (B-H) method. The gene set enrichment analysis was performed using the Fisher’s exact test and corrected with the B-H FDR.