hiPSC culture

hiPSCs were generated from an Asian female HOIL-1L-deficient patient and healthy controls and kindly donated by the Center for iPS Cell Research and Application (Kyoto University, Kyoto, Japan). Patient-specific (HOIL-1L_1, CiRA-j-0154B and HOIL-1L_2, CiRA-j-0154D) and healthy control hiPSCs (Control_1, CiRA-j-1616-A, Asian female volunteer) were established from the peripheral blood mononuclear cells (PBMCs) using episomal vectors containing reprograming factors30. Another control hiPSC line from Asian male (Control_2, 110F5) was established as described previously31. Each cell line stored in liquid nitrogen using STEM-CELLBANKER (Takara, Cat.# 11924) and once thawed in 37C water bath, it was maintained in mTeSR1 medium (Stem Technologies, Cat.# 85,850) as previously reported32. Each cell was stocked at less than 15 passages, and all experimentations were done between 20 and 45 passages. The sequence of RBCK1 gene was confirmed by Sanger sequencing at the beginning of key experiments. Pluripotency of CiRA-j-1616-A, CiRA-j-0154B and CiRA-j-0154D was evaluated by OCT3/4 and NANOG mRNA expression by TaqMan qPCR and pluripotency of control_2 was evaluated by quantitative PCR analysis of Oct 3/4, Sox2, Klf4, and c-Myc using SYBR green. Cells were passaged every 45days at 1:10 or 1:12 ratio using accutase (Nacalai Tesque, Cat.# 12679-54). Dissociated cells were seeded on Matrigel-coated 6-well plates. The medium was supplemented with 5M Y27632 (TOCRIS, Cat.# 1254), a Rho-associated kinase inhibitor, on the first day of each passage. All cell lines were authenticated by their name, checked their sterrility regularly, and monitored of mycoplasma contamination using by PCR kit (Minerva biolabs, Cat.# 11-9025).

CMs were differentiated from hiPSCs using a previously reported protocol33. Briefly, hiPSCs were seeded into a 12-well growth-factor-reduced (GFR) Matrigel-coated plate, grown for 4days at 37C in 5% CO2 and mTeSR1 medium, and allowed to reach 8090% confluency. On day 0 of differentiation, the medium was changed to differentiation media, which was RPMI containing 2% B27 minus insulin supplement (Gibco, Cat.# A18956-01) and 1012M CHIR99021 (Selleck, Cat.# S2924), a GSK3 inhibitor. After incubation for 24h, the medium was replaced with fresh differentiation medium. On day 3, the medium was replaced with differentiation medium containing 5M IWP-2 (TOCRIS, Cat.# 3533), a Wnt inhibitor. On day 5, the medium was replaced with fresh differentiation medium. On day 7, B27 minus insulin was replaced with a B27 supplement (Gibco, Cat.# 17504044). Differentiated hiPSC-CMs were purified in glucose-depleted lactate medium as described previously34.

C2C12 cells were kindly provided by Dr. Yuji Yamanashi (The Institute of Medical Science, The University of Tokyo)35. The growth medium was DMEM/F12 (Sigma-Aldrich, Cat.# D6421) containing 20% FBS, 2mM glutamine (Gibco, Cat.# 25030081), 100 units/mL penicillin, and 100g/mL streptomycin. Cells were incubated at 37C in a humidified incubator containing 5% CO2. Myoblasts were differentiated into myotubes in DMEM/F12 medium containing 2% horse serum (Gibco, Cat.# 16050122, Lot. 1968945)36,37.

Lenti-CRISPR v2 (Addgene, Cat. # 52961), which contains a puromycin resistance gene, carrying a guide RNA oligonucleotide (5-acctcacccttcagtcacgg-3 for Exon 5 of the Hoil-1l gene or 5-acgcagcaccacggcctcgc-3 for Exon 7 of the Hoil-1l gene) was constructed. HEK293T cells were transfected with the plasmids using Lipofectamine 2000 (Thermo Fisher, Cat.# 11668019). Viruses were harvested at 48h after transfection, and the media were filtered through a 0.45m PES filter. C2C12 cells were transduced with the viruses in medium containing 10g/mL polybrene. At 24h after transduction, puromycin selection was started. The selected cells were collected and KO of Hoil-1l was confirmed by Sanger DNA sequencing.

Myotubes differentiated from C2C12 cells on day 5 of differentiation were fixed in 4% paraformaldehyde (PFA) for 1h at 4C, permeabilized in 0.1% Triton X-100 for 10min at room temperature, and blocked in PBS containing 3% skim milk for 1h. Thereafter, myotubes were stained with an anti-MHC antibody (1:200, mouse monoclonal, R&D Systems, Cat.# MAB4470). The fusion index was calculated by dividing the number of nuclei in myotubes by the total number of nuclei in a field of view36. The MHC density was calculated by dividing the area occupied by MHC-positive myotubes by the total area of the field of view. The fusion index and MHC density were reported as averages of at least three fields of view (>500 total nuclei). Three independent experiments were performed for the calculation. For pluripotency marker analysis, undifferentiated hiPSC colonies were fixed in the same way, and fixed cells were stained with mouse anti-Oct3/4 (1:50, Santa Cruz Biotechnology, Cat.# sc5279) and anti-TRA1-81 (1:100, Millipore, Cat.# MAB4381) antibodies. Cells were then incubated with Alexa Fluor-conjugated secondary antibodies (1:1000) overnight at 4C.Nuclei were stained withHoechst 33342(1:1000, Invitrogen, Cat.#H3570). For immunofluorescence microscopy analysis of hiPSC-CMs, size and multinucleation were analyzed after around 5060days of differentiation and mitosis was analyzed after 20days of differentiation. hiPSC-CMs were replated onto GFR Matrigel-coated 24-well dishes, incubated at 37C in 5% CO2 for 72h, fixed in 4% PFA for 1h at 4C, permeabilized in 0.1% Triton X-100 for 10min at room temperature, and blocked in PBS containing 3% skim milk for 1h. Thereafter, hiPSC-CMs were stained with anti-cTnT (1:100, mouse monoclonal, Thermo Fisher, Cat.# MA5-12960) and anti-phospho-histone H3 (Ser10) (1:1000, rabbit monoclonal, Cell Signaling, Cat.# D7N8E) antibodies. After primary antibody treatment, cells were rinsed three times with PBS for 5min at room temperature and then incubated overnightat 4 with secondary antibodies diluted 1:1000 in PBS. Nuclei were stained with Hoechst 33342. For isotype controls, mouse IgG1 isotype (BD Biosciences, Cat.# 554121) and rabbit IgG isotype (BD Biosciences, Cat.# 550875) were used. All immunofluorescence analyses were performed using a BZ-710X microscope (Keyence). The TUNEL assay was performed using a Cell Death Detection Kit (Roche, Cat.# 11684795910) following the manufacturers protocol.

Myotubes at day 5 of differentiation were lysed in M-PER buffer (Thermo Scientific, Cat.# 78501) containing 1protease inhibitor and then incubated on ice. The samples were sonicated on ice for 30s. The lysates were incubated on ice for 10min and then centrifuged at 15,000rpm for 15min. Protein concentrations were determined using the Bradford assay. Thereafter, 30g of protein was loaded onto each lane of 10% SDS-PAGE gels. The membranes were probed with an anti-MHC antibody (1:100, R&D Systems, Cat.# MAB4470) in blocking buffer (5% BSA) at 4C overnight, washed, incubated in secondary antibodies for 1h at room temperature, developed using ECL western blotting substrate (Bio-Rad, Cat.# 1705060), and imaged using the ChemiDoc MP Imaging System (Bio-Rad). The blots were cut prior to hybridization with antibodies, and two replicates were done at the same time for Fig.1B and Supplementary Fig.2C as shown in supplementaryFig.5.

hiPSC-CMs were dissociated on the day of evaluation by incubating them in 0.25% trypsinEDTA for 1015min at 37C. They were fixed in Cytofix/Cytoperm solution (BD Biosciences, Cat.# 554714) for 20min at 4C, washed with BD Perm/Wash buffer (Cat.# 554723), stained with an anti-cTnT antibody (1:200, mouse monoclonal, Thermo Fisher, Cat.# MA5-12960) followed by Alexa Fluor-conjugated secondary antibodies, and analyzed using FACSverse (BD Biosciences). In cell cycle analysis, hiPSC-CMs after 20days of differentiation were gathered. After fixing and washing the hiPSC-CMs as described above, they were stained with an anti-cTnT antibody (1:200) and an anti-Ki67 antibody (1:400, rabbit monoclonal, Cell Signaling Technology, Cat. # 9129) followed by Alexa Fluor-conjugated secondary antibodies, and analyzed using FACSverse. Data were collected from at least 10,000 events. Data with>70% cTnT populations were used for all experimental analyses.

Total RNA was extracted from day 0 to day 7 of myotube differentiation using an RNeasy Mini Kit (Qiagen, Cat.# 74104) according to the manufacturers instructions. qPCR was performed using SYBR Green PCR Master Mix (Takara, Cat.# RR820) on a StepOnePlus system (Thermo Fisher Scientific) with the Ct method. GAPDH was used to standardize gene expression. Total RNA was extracted from hiPSC-CMs at 4560days after differentiation.

Hoil-1l-KO and control mouse embryos were generated as described previously14. Paraffin sections of E10.5 Hoil-1l null/+ and control littermate mouse embryos were deparaffinized and stained with H&E.

RNA was isolated from C2C12 cells using a RNeasy Mini Kit, according to the manufacturers instructions. RNA integrity was measured using an Agilent 2200 TapeStation and RNA Screen Tapes (Agilent Technologies). Sequencing libraries were prepared using a NEBNext Ultra II RNA Library Kit for Illumina (New England Biolabs) with the NEBNext Poly (A) mRNA Magnetic Isolation Module (New England Biolabs), according to the manufacturers protocol. Prepared libraries were run on an Illumina HiSeq X sequencing platform in 150bp paired-end mode. Sequencing reads were aligned to the GRCm38 mouse genome assembly using STAR (c.2.5.3). Mapped reads were counted for each gene using the GenomonExression pipeline (https://github.com/Genomon-Project/GenomonExpression). Normalization of the read counts of RNA seq data and differential expression analysis were performed using the Bioconductor package DESeq2 (version 1.26.0). Differentially expressed genes with a greater than twofold change and a false discovery rate less than 0.1 were filtered and evaluated. RNA seq data have been deposited with links to BioProject accession number PRJDB17426 in the DDBJ BioProject database.

GSEA was performed using software (version 4.0.3) from the Broad Institute. Normalized expression data obtained from RNA seq were assessed using GSEA software and the Molecular Signature Database (http://www.broad.mit.edu/gsea/). c5 ontology gene sets were used, and a false discovery rate less than 0.01 was considered to be statistically significant. Pathway enrichment analysis using g:Profiler and visualization of enrichment results in an enrichment map were performed using Cytoscape software (version 3.7.2) as described previously38.

Data are shown as meanSEMs, as indicated in the figure legends. All statistical analyses were performed using Welchs t-test with GraphPad Prism (version 9.00, GraphPad Software). P<0.05 was defined as significant.

Use of patient-derived iPSCs was approved by the Ethics Committee of Kyoto University (R0091 and G0687), and written informed consent obtained from the donor (or their guardians) in accordance with the Declaration of Helsinki.

View original post here:
HOIL-1L deficiency induces cell cycle alteration which causes immaturity of skeletal muscle and cardiomyocytes ... - Nature.com