Culture and myogenic differentiation of C2C12 cells
C2C12 cells were grown and maintained in Dulbecco’s modified Eagle complete medium (DMEM; WAKO, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, TX, USA) and antibiotic-antimycotic mixture containing 100 units/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B (Nacalai Tesque, Kyoto, Japan) at 37 °C in the presence of 5% CO2. The myogenic differentiation of C2C12 cells was performed as described previously20. A schematic description of the myogenic differentiation protocol with C2C12 cells is shown in Fig. 1a. C2C12 cells were seeded at a density of 1 × 105 cells/dish in 6 cm-dish with 4 mL of DMEM (WAKO) supplemented with 10% FBS (Thermo Fisher Scientific) and the antibiotic-antimycotic mixture at 37 °C in the presence of 5% CO2. The medium was replaced the next day with DMEM containing 2% horse serum (HS; Sigma-Aldrich, MO, USA) and the antibiotic-antimycotic mixture. After 5 days of incubation, the myogenic differentiation was evaluated by microscopic observation based on myotube formation.
Establishment of Zip13-knockdown (KD) C2C12 cells
Either mouse Zip13-shRNA or non-specific (Scramble) DNA (see Supplementary Table S1 online) were inserted into the pSUPER.retro.puro vector plasmid (OligoEngine, WA, USA) according to the manufacturer’s instructions to generate Zip13-shRNA or Scramble plasmids. C2C12 cells were transfected with either Zip13-shRNA or Scramble plasmids using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacturer’s instructions and cultured in a medium containing 5 µg/mL of puromycin for 48 h. Then, the cells were collected, and the KD efficiency of Zip13 mRNA was examined by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis. The remaining cells were seeded by limiting dilution to generate a monoclonal cell line for both Zip13-KD and Scramble C2C12 cells. We examined the Zip13 mRNA expression in the twelve monoclonal cell lines and selected two clones (clone 6 and 7) according to the KD efficiency of Zip13.
Ethical approval
All experimental procedures used in the study were approved by the Ethics Committee of Tokushima Bunri University (approval number R2-19 and R3-16) and performed in accordance with the relevant guidelines and regulations. Written informed consent was obtained from two patients with EDSSPD3 (one female and one male), harboring a homozygous point mutation (c.221G>A, p.G64D) in exon2 of SLC39A13/ZIP13 gene (ZIP13G64D)4, for the use of their human dermal fibroblasts (HDFs).
Culture of HDFs
Healthy female (Caucasian, 36 years old) or male (Caucasian, 49 years old) DFs were purchased from Cell Applications, Inc., CA, USA. DFs from female and male EDSSPD3 patients4,21 were obtained from two subjects (one female and one male). These DFs were grown and maintained in DMEM (WAKO) supplemented with 10% FBS (Hyclone Laboratories, IA, USA), 1 × GlutaMAX (Thermo Fisher Scientific), and 100 units/mL penicillin and 100 µg/mL streptomycin (P/S; Thermo Fisher Scientific) at 37 °C in the presence of 5% CO2.
Generation of hiPSCs using an episomal vector
The hiPSCs were generated in an integration-free manner with episomal plasmid vectors (http://www.cira.kyoto-u.ac.jp/e/research/protocol.html)22. Briefly, healthy-, EDSSPD3-Female-, and EDSSPD3-Male-DFs were cultured in DMEM supplemented with 10% FBS (Hyclone), 1 × GlutaMAX (Thermo Fisher Scientific), and P/S (Thermo Fisher Scientific). Episomal plasmid vectors pCXLE-hOCT3/4-shp53-F, pCXLE-hUL, and pCXLE-hSK, which express human OCT3/4 and shRNA against p53, human L-MYC and LIN28, and human SOX2 and KLF4, respectively, were purchased from Addgene Inc., MA, USA and used for generation of integration-free hiPSCs. Three micrograms of the episomal plasmid mixture (1 µg of each plasmid) were electroporated into 3 × 105 HDFs using Neon transfection system (MPK5000; Thermo Fisher Scientific). The electroporation conditions used were 1,650 V, 10 ms, and 3 time pulses. The cells were cultured for 7 days after transduction at 37 °C in the presence of 5% CO2, and 5 × 104 cells were replated onto mitomycin-C (Kyowa Hakko Kirin, Tokyo, Japan)-treated SNL76/7 (SNL) feeder cell (DS pharma Biomedical, Osaka, Japan) layer in 60-mm dishes. Next day, the culture medium was replaced with primate embryonic stem (ES) cell medium (ReproCELL, Kanagawa, Japan) supplemented with 4 ng/mL recombinant human basic fibroblast growth factor (Wako, Osaka, Japan) and P/S (Thermo Fisher Scientific). The colonies were generated 20–30 days after plating, and those colonies similar to hESCs were selected and grown on mitomycin-C-treated SNL feeder cell layer.
Feeder-free culture of hiPSCs
The selected iPSCs were maintained under feeder-free condition in an uncoated manner using laminin fragments as previously described23. Briefly, the iPSC colonies from healthy or EDSSPD3 patients DFs (female and male) were dissociated with a detachment solution comprising 0.5× TrypLE select (Thermo Fisher Scientific) and 0.75 mM EDTA in Dulbecco's phosphate-buffered saline (DPBS) (-) (Nacalai Tesque) for 10 min at 25 °C after the removal of SNL feeder cells using CTK solution [2.5% trypsin (Thermo Fisher Scientific), 1 mg/mL collagenase IV (Thermo Fisher Scientific), 0.1 M CaCl2, and 20% knockout serum replacement (KSR; Thermo Fisher Scientific) in H2O]. The colonies consisting of single cells were suspended in StemFit medium (AK02N; Ajinomoto, Tokyo, Japan) supplemented with 10 µM Y27632 (WAKO), Rho-associated, coiled-coil containing protein kinase inhibitor, and P/S (Thermo Fisher Scientific) by pipetting. The cells were then seeded at 2 × 104 cells/cm2 onto cell culture plates with StemFit medium supplemented with 0.25 µg/cm2 iMatrix-511 (Nippi, Tokyo, Japan), laminin-511 E8 fragment, 10 µM Y27632, and P/S (Thermo Fisher Scientific), and the culture medium was replaced the next day with StemFit medium supplemented with P/S (Thermo Fisher Scientific). After 4 days of incubation, the culture medium was changed daily. The hiPSCs at 95% confluency were passaged in an uncoated manner using iMatrix-511. Then, each hiPSC line was maintained as a feeder-free culture on iMatrix-511.
For sequencing analysis of ZIP13-exon2 mRNA of EDSSPD3-iPSCs, total RNA was extracted from lysates of healthy or EDSSPD3 DFs and iPSCs (Female and Male each), respectively, using an RNeasy Mini Kit (Qiagen, GmbH, Germany). Total RNA was used to synthesize cDNA using SuperScript IV (Thermo Fisher Scientific) according to the manufacturer’s instructions. The synthesized cDNA was used as a template for PCR using human ZIP13-exon2 mRNA primers (see Supplementary Table S1 online). Each PCR amplicon was analyzed by Sanger sequencing.
Establishment of hiPSC lines expressing tetracycline-inducible human MyoD (hMyoD)
The tetracycline-inducible hMyoD-expressing iPSC lines (iPSCsMYOD) were established by the transduction of its gene cassette-inserted piggyBac (PB)-based vector as previously described13,16,18. Briefly, the plasmids for tetracycline-inducible hMyoD and mCherry and the neomycin resistant gene were constitutively prepared in PB vector (PB-hMyoD) or EF1a promoter-driven PB transposase gene in pHL vector (pHL-EF1a-hcPBase)13,16,18. The feeder-free maintained hiPSCs were treated with 10 µM Y27632 at least 2 h prior to the transduction experiment. hiPSCs were dissociated into single cells using a detachment solution, and the cells were resuspended in resuspension buffer R (Thermo Fisher Scientific). Two micrograms of PB-hMyoD and pHL-EF1a-hcPBase mixtures (1 µg each) were electroporated into 5 × 105 cells using Neon transfection system (Thermo Fisher Scientific). Electroporation conditions were 1,200 V, 20 ms, and 2 time pulses. Transfected hiPSCs were incubated as a feeder-free culture on iMatrix-511. After 2 days, the culture medium was replaced with StemFit medium supplemented with 2–4 mg/mL neomycin (Nacalai Tesque), and the cells were subjected to antibiotic selection. Each neomycin-selected hiPSC was cloned by limiting dilution. The established hMyoD-expressing hiPSC lines, named iPSCsMYOD, was confirmed based on mCherry expression in the presence of 1 µg/mL doxycycline (DOX; LKT Laboratories, MN, USA).
Correction of ZIP13G64D in EDSSPD3-Female-iPSCsMYOD by Cas9/sgRNA and single-stranded oligodeoxynucleotide (ssODN) electroporation
ZIP13G64D in EDSSPD3-Female-iPSCsMYOD was genome edited using homology-directed repair (HDR) combined with CRISPR-Cas9 system and ssODNs24. We designed ssODN containing silent mutations at positions 65 (TCC<TCA, Ser) and 66 (CTC<CTA, Leu) of amino acids to prevent Cas9 from recutting the ZIP13-exon2 in the genome. GGG site showed the sequence of protospacer adjacent motif for target of Cas9 nuclease. The day before transduction, EDSSPD3-Female-iPSCsMYOD were treated with 10 µM Y27632. hiPSCsMYOD were dissociated into single cells using a detachment solution, and the cells were resuspended in resuspension buffer R. For ribonucleotide protein (RNP) electroporation, 2 µg TrueCut Cas9 protein v2 (Thermo Fisher Scientific) and 500 ng scaffold-modified ZIP13G64D_sgRNA (Thermo Fisher Scientific; see Supplementary Table S2 online) were incubated for 5 min at 25 °C, and then 8 × 104 cells in resuspension buffer R, 3 µg Alt-R HDR-modified ZIP13G64D-corrected_ssODN (IDT, NJ, USA; see Supplementary Table S2 online), and 3 µM electroporation enhancer (IDT) were added. Cas9/sgRNA complex and ssODN mixture was electroporated into iPSCsMYOD using Neon transfection system. Electroporation conditions were 1,050 V, 20 ms, and 2 time pulses. Transfected EDSSPD3-Female-iPSCsMYOD were incubated as a feeder-free culture on iMatrix-511 with StemFit medium supplemented with 30 µM Alt-R HDR enhancer (IDT). The culture medium was replaced daily with StemFit medium. After 3 days of incubation, iPSCsMYOD were cloned by limiting dilution. The obtained clones were maintained as a feeder-free culture on iMatrix-511.
For sequencing analysis of human ZIP13-exon2, genomic DNA was extracted from lysates of transfected EDSSPD3-Female-iPSCMYOD clones using Geno Plus Mini kit (VIOGENE, New Taipei, Taiwan). The genomic DNA was used as a template for genomic PCR using human ZIP13-exon2 genome primers (see Supplementary Table S1 online). Each PCR amplicon was analyzed by Sanger sequencing. For separate sequencing analysis of individual alleles in a genome, each genomic DNA of ZIP13G64D-corrected EDSSPD3-Female-iPSCMYOD clone (repaired clone; RC) 1–3 was used as a template for genomic PCR using human ZIP13-exon2 genome primers for In-Fusion_pGEM (see Supplementary Table S1 online). Their PCR amplicons were inserted in pGEM-T easy vector (Promega, WI, USA) using In-Fusion cloning kit (Takara Bio, Siga, Japan) according to the manufacturer’s instructions. The PCR amplicon-inserted plasmids were cloned and analyzed by Sanger sequencing.
Myogenic differentiation of iPSCsMYOD
Differentiation of EDSSPD3-iPSCsMYOD into myocytes was induced by forced hMyoD expression under the control of DOX as previously described17,25. A scheme for myogenic differentiation protocol of feeder-free iPSCsMYOD with DOX-inducible hMyoD expression is shown in Supplementary Fig. S2 online. Briefly, the 24-well cell culture plate was coated with Matrigel Growth Factor Reduced Basement Membrane Matrix (Corning, NY, USA) at least 2 h prior to the differentiation experiment. Feeder-free cultured iPSCsMYOD were dissociated into single cells using a detachment solution, and 0.6 × 105 cells/well were seeded on the Matrigel-coated 24-well plate with StemFit medium supplemented with 10 µM Y27632. The culture medium was replaced the next day with primate ES cell medium. On day 2, 1 µg/mL DOX was added to the same medium, while on day 3 the latter was replaced with minimum essential medium Eagle, alpha modification (Nacalai Tesque) supplemented with 5% KSR (Thermo Fisher Scientific), 200 µM 2-mercaptoethanol (Thermo Fisher Scientific), and 1 µg/mL DOX (LKT Laboratories) on day 3. The mCherry expression of differentiated cells on day 3 was observed using a fluorescence microscope (BIOREVO BZ-9000; Keyence, Osaka, Japan). The medium was changed on days 4, 5, and 7. This myogenic differentiation was performed at 37 °C in the presence of 5% CO2 until day 8.
RT-qPCR
Total RNA of myogenic differentiated C2C12 cells or iPSCsMYOD were extracted from cell lysates using a Sepazol (Nacalai Tesque) or RNeasy Mini kit (Qiagen) used to synthesize cDNA with the PrimeScript RT Reagent Kit (Takara Bio) or SuperScript VILO (Thermo Fisher Scientific) according to the manufacturer’s instructions. The synthesized cDNA was used as a template for qPCR, which was performed using the SYBR Green real-time PCR Master Mix (TOYOBO, Osaka, Japan). The gene-specific primers used for qPCR are provided in Supplementary Table S1 online. PCR and data analyses were performed using a QuantStudio3 Applied Biosystems instrument (Thermo Fisher Scientific) or an Applied Biosystems StepOnePlus real-time PCR system (Thermo Fisher Scientific). The expression level of each mRNA was normalized to that of mouse β-actin or human glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Western blot analysis
The cells were lysed with RIPA buffer (Nacalai Tesque) supplemented with 0.1% bromophenol blue and 10% b-mercaptoethanol and boiled for 5 min. Cell lysates were then loaded onto a 6–10% polyacrylamide gel and subjected to SDS-PAGE. The separated proteins were transferred to a polyvinylidene fluoride microporous membrane (Millipore, MA, USA), and the membrane was blocked with 5% skim milk in tris-buffered saline with Tween 20. Respective primary and secondary antibodies (see Supplementary Table S3 online) were used to detect specific proteins. The signals were detected using Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate (Millipore). Signal intensities were measured using ImageJ software, and each protein level was normalized to that of b-ACTIN. Relative protein levels were then calculated. Uncropped full-length images of western blotting membrane can be seen in the supplementary information (Supplementary Fig. S4b and S5–8).
Alkaline phosphatase (ALP) staining
The hiPSCs colonies were fixed using 4% paraformaldehyde in PBS for 10 min at 25 °C. After washing with water, the colonies were photographed under a phase contrast using a fluorescence microscope (BIOREVO BZ-9000; Keyence). Then, ALP staining was performed using a Leukocyte Alkaline Phosphatase Kit (Sigma-Aldrich) according to the manufacturer's instructions. The stained colonies were photographed using the fluorescence microscope.
Immunofluorescence (IF) staining
The cells were fixed using 4% paraformaldehyde for 30 min at 4 °C and subsequently permeabilized by the addition of 0.3% Triton X-100 for 20 min at 25 °C. The cells were then washed with DPBS(-) and blocked using a stain buffer with FBS (BD Biosciences, NJ, USA). Respective primary and secondary antibodies (see Supplementary Table S3 online) were used to detect specific proteins. Cell nuclei were then stained using diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific). Wells were photographed using a fluorescence microscope (BIOREVO BZ-9000; Keyence).
Flow cytometry (FC)
Feeder-free maintained hiPSCs were dissociated into single cells using a detachment solution for 10 min at 25 °C, and then suspended in a stain buffer with FBS (BD Biosciences). The cells were stained with primary antibodies for 2 h at 4 °C, followed by a secondary antibody for 1 h at 4 °C. The antibodies used for this experiment are provided in Supplementary Table S3 online. Dead cells were excluded based on 7-amino-actinomycin D (7-AAD; BioLegend, CA, USA) staining. Finally, the stained cells were analyzed using a FACS Melody cell sorter (BD Biosciences), and the data were analyzed using FlowJo software (Tree Star, OR, USA).
Statistical Analysis
All results are presented as mean ± standard error of the mean (SEM). The statistical significance of differences between two groups was analyzed by Student’s t-test, whereas that between more than two groups was analyzed by one-way analysis of variance (ANOVA). Results were considered statistically significant at P < 0.05.