Exome sequencing

Whole-exome sequencing (WES) was performed as previously reported30. In brief, after DNA extraction, exome capture was performed with the TruSeq Exome Capture Kit (Illumina), and sequencing for the three participants (patient and both parents) was conducted using the NextSeq500 mid output system (Illumina) with a 75-bp paired-end run. The sequences were aligned to the human reference genome (GRCh37), and variant calling was performed using the Genome Analysis Toolkit (GATK V3.5, Broad Institute)31. Variants were first annotated by Variant Studio (V3.0, Illumina) and wANNOVAR (http://wannovar.wglab.org/)32. Candidate variants were checked with ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/). The pathogenicity of variants was classified according to the ACMG guidelines33.

Karyotype analysis

Karyotype analysis of iPSCs was conducted as follows: iPSCs that reached eighty percent confluence in 35 mm dishes were harvested after adding 10 µL of colcemid for 15 hours. Then, 2 ml 0.28% KCl was added twice for 50 minutes. Then, 2 mL methanol/acetic acid (3/1) was added for fixation. G banding was stained using 0.25% trypsin followed by Wright’s solution (pH 7.0 Gurr buffer/Wright’s = 3/1).

iPSC derivation, culture, and drug treatment

PBMCs from whole blood were separated by Ficoll-Hypaque density gradients and stored in liquid nitrogen. iPSCs were derived from the PBMCs of the patient and the patient’s father. PBMCs were reprogrammed using Sendai virus according to the manufacturer’s instructions. For feeder-free culture, iPSCs were maintained in StemFlex completed medium containing StemFlexTM Basal Medium (Gibco, Cat. No. A33493-01) and StemFlexTM Supplement (10X) (Gibco, Cat. No. A33492-01) on Matrigel hESC-qualified Matrix (Corning, Cat. No. 354277) at 37 °C in a humidified atmosphere containing 5% CO2. To prepare Matrigel-coated dishes, Matrigel hESC-qualified matrix supplemented with 25 mL of DMEM/F-12, HEPES (Gibco, Cat. No. 11330032) was added to 6-well plates (1 mL/well) or 100 mm dishes (7 mL/dish) and incubated at 37 °C for 2 hours. iPSCs were subcultured when the cells were in log phase, and the cells were detached by using Accutase® (Innovative Cell Technologies, Inc., Cat. No. #AT-104) and incubated in an incubator at 37 °C and 5% CO2 for 5 min. The detached cells were centrifuged at 1000 rpm (Kubota 2010 tabletop centrifuge) for 5 min. Before seeding the cells, Matrigel matrix supplemented with DMEM/F-12 was aspirated from the culture dish. iPSCs were seeded on a Matrigel-coated dish containing StemFlex complete medium and 10 μM Y-27632 dihydrochloride (Rock inhibitor) (Sigma–Aldrich, Cat: Y0503-5MG) at 37 °C with 5% CO2, and then the medium was changed every day. For RG7834 and cordycepin treatment, iPSCs were maintained in StemFlex complete medium with 1 µM RG7834 (MedChemExpress (MEC), Cat. No.: HY-117650A), 1 µM Cordycepin (MedChemExpress (MEC), Cat. No.: HY-N0262) or dimethyl sulfoxide (Sigma–Aldrich, Cat: D4540-100ML) as a control on Matrigel hESC-qualified matrix. The cells were subcultured as mentioned before.

DNA sequencing and genetic analysis

DNA sequencing of gDNA from PBMCs and iPSCs from healthy donors and DC patients was performed by polymerase chain reaction (PCR) amplification of the specific region of exon 14 for DKC1. The primers used for PCR and Sanger sequencing are listed in Supplementary Table 2. Each PCR (100 µl) contained 100 ng of DNA, 1X KAPA HiFi Fidelity Buffer, 0.3 mM KAPA dNTP, 0.3 µM each primer, and 0.005 U/μl KAPA HiFi DNA Polymerase (Kapa Biosystems, Cat: KK2102). The reactions were carried out in a T100TM Thermal Cycler (Bio Rad) under the following conditions: one cycle at 95 °C for 3 min, followed by 30 cycles of 20 s at 98 °C, 15 s at 50 °C and 30 s at 72 °C, with a final cycle at 72 °C for 1 min. PCR products were purified using a PCR extraction kit (TOOLS, Cat: TT-B14-3) according to the manufacturer’s instructions.

Genomic DNA extraction and qPCR

Genomic DNA was prepared from iPSC pellets (5 × 106 cells) with GenEluteTM Mammalian Genomic DNA Miniprep Kits (Sigma–Aldrich, Cat. No: G1N350-1KT) according to the manufacturer’s instructions. To evaluate telomere lengths, polymerase chain reaction (PCR)-based telomere length analysis methods have been developed. In this study, telomere length was quantified by comparing the amount of the telomere amplification product (T) to that of a single-copy gene (S, 36B4). The T/S ratio was then calculated to yield a value that correlated with the average telomere length. Each reaction contained 5 ng/μl genomic DNA or standard templates mixed with iQ™ SYBR® Green Supermix (Bio–Rad, Cat. No. 1708882) and primers. qPCR was performed in a CFX96TM Real-Time System, C1000 TouchTM Thermal Cycler (Bio–Rad), with the following cycling conditions: initial denaturation at 95 °C for 10 min and 40 cycles at 95 °C for 15 sec and 60 °C for 1 min. Graphing and statistical analysis of qPCR results were performed using Prism 8 (GraphPad). Standard telomere oligonucleotides, standard single-copy gene (36B4) nucleotides, and primers used for qPCR are listed in Supplementary Table 3.

Terminal restriction fragment (TRF) analysis

Genomic DNA (2.2 µg) from iPSCs was digested with Hinf I (New England Biolabs, Cat. No: #R0155S) and Rsa I (New England Biolabs, Cat. No: #R0167L) restriction enzyme in 10X CutSmart® Buffer (NEB, Cat. No: #B7204S) at 37 °C overnight. The digested gDNA fragments were separated on a 1% SeaKem® LE agarose gel (Lonza, Cat. No: 50002) by electrophoresis at 120 V for 12 h, followed by capillary transfer to a HybondTM-N+ nylon transfer membrane (GE Healthcare, Cat. No: RPN303B) in 10X SSC for 14.5 h. DNA was subsequently crosslinked to the membrane twice at 120 mJ in a UV Stratalinker 1800 (Stratagene, 254 nm, 120 mJ). The blot was prehybridized in Church buffer at 65 °C for an hour and then hybridized with 32P-α-dCTP-labelled (TTAGGG)3 overnight. The blot was exposed to a phosphor image screen (Fujifilm) at room temperature overnight. Phosphor images were obtained with an Amersham Typhoon 5 scanner (Cytiva). The telomere length images were quantified and analysed by ImageQuantTL software (Cytiva). All blots derive from the same experiment and were processed in parallel.

Single-cell RNA sequence capture, library construction, and sequencing

Single-cell capture and downstream library construction were performed using Chromium Next GEM Single Cell 3′ Reagent Kits v 3.1 (10x Genomics; PN-1000121, PN-1000127, and PN-1000213) for PBMCs from the DC patient and age-matched controls, according to the manufacturer’s protocol. Briefly, a total of approximately 8600 single cells, 50 µl of barcoded gel beads and 45 µl partitioning oil were loaded into Chromium Next GEM Chip G to generate nanoliter-scale gel beads-in-emulsion (GEMs). Afterwards, the polyadenylated mRNAs were reverse transcribed inside each GEM, and the full-length cell-barcoded cDNA was amplified via PCR to generate sequencing libraries. Library quality was assessed by using the Qubit 4.0 Fluorometer (Thermo Scientific) and a Qsep100TM system (Bioptic, Taiwan) to determine the library concentration and library size, respectively. In general, fragments of approximately 450–500 bp in size were expected for single-cell 3ʹ gene expression libraries. The effective concentrations of the library were assessed by Q-PCR. The qualified libraries were pooled according to the effective concentration and expected data volume. The library was sequenced on Illumina NovaSeq 6000 sequencers according to read length: 28 bp Read 1 (16 bp single-cell barcode, 10x barcode; 12 bp unique molecular identifier, UMI), 91 bp Read 2 (transcript insert or feature barcode in the case of the cell hashing library), and 8 bp i7 Index (sample index). TruSeq Read 1 and TruSeq Read 2 are standard Illumina sequencing primer sites used in the paired-end sequencing of single-cell 3ʹ gene expression libraries.

Single-cell gene expression analysis

In the Chromium Single-Cell Software Suite, Cell Ranger (cellranger count) was used to perform sample demultiplexing and generate feature-barcode matrices. Sequences were mapped onto the human reference genome (GRCh38) provided by 10X Genomics. Multiple samples were aggregated by “cellranger aggr” without depth normalization. Unique molecular identifier (UMI) count matrices were imported into R (v3.6.0) and processed with the R package Seurat (v3.2.1)34,35. Log-normalized expression values were obtained by the “NormalizeData” function of the Seurat package. Specifically, “LogNormalize” was set by default in this function, and the gene counts for each cell were divided by the total counts and multiplied by the scale.factor (default = 10,000) and then natural-log transformed. Further analysis, including quality control, the identification of highly variable genes (HVGs), dimensionality reduction, and standard unsupervised clustering algorithms, was performed using the Seurat package. Cells were filtered out before downstream analysis if (1) the percentage of mitochondrial genes was >20% or (2) the number of genes was <200 or >Q3 + 1.5 IQR of the population. HVGs, which are often used to keep the most informative variations in the scRNA-seq data36,37, were set in the “FindVariableFeatures” function of the Seurat package; the number of HVGs was defined by the median gene number in the population, while “nfeatures = 2000” was defined if the median was below 2000. Visualizing a high-dimensional single-cell dataset is critical for interpretation of the results. Cell clustering was performed by using dimensional reduction techniques and t-distributed stochastic neighbour embedding (tSNE)38,39. Note that in Seurat, both tSNE and UMAP were performed after PCA (“RunPCA” function). The “RunTSNE” and “RunUMAP” functions in Seurat were set with “dims = 1:20”. To evaluate the batch effect, batch mixing was used to quantify the extent of intermingling of cells from different batches40,41. If necessary, the mutual nearest neighbour (MNN)40 method was used for batch effect correction with the “fastMNN” function of SeuratWrappers (v0.3.0). To identify the cell types captured by scRNA-seq in an unbiased fashion, an automatic annotation method, SingleR (v1.0.1), was performed7; this method correlates each cell with reference transcriptomic datasets independently. Differentially expressed gene (DEG) analysis of the two conditions was performed in Python using sSeq42 (total UMI count <900) and edgeR43,44 (total UMI count >900), which is based on negative binomial distribution and asymptotic beta testing, respectively42,43,44. We followed the method as stated in Cell Ranger and the Loupe Cell Browser, and the source code is available in the 10X Genomics GitHub repository (https://github.com/10XGenomics/cellranger).

RNA extraction and reverse transcription PCR

Total RNA was isolated from the iPSC pellets (1 × 107 cells) using Ambion TRIzol® Reagent (Life Technologies, Cat. No: 15596018) according to the manufacturer’s instructions, followed by DNase I (New England Biolabs, Cat: M0303 L) treatment at 37 °C for 60 min. For cDNA synthesis, the reverse transcription reaction was performed according to the manufacturer’s instructions of the SuperScriptTM IV First-Strand Synthesis System (Thermo Fisher Scientific, Cat. No. 18091050). In brief, 2 µg of total RNA was primed with 50 µM Oligo d(T)20 primer or 50 ng random hexamers in 10 mM dNTP mix and DEPC-treated water. PCRs were carried out in a T100TM Thermal Cycler (Bio-Rad) under the following conditions: one cycle at 65 °C for 5 min and 4 °C for 1 min. Then, 1 µl of SuperScriptTM IV RT was added to the reaction mixture containing ribonuclease inhibitor (TOOLS, Cat: TTG-RI01), 5 mM DTT, and 1X SSIV Buffer, followed by incubation at 50 °C for 1 h, 80 °C for 10 min, 37 °C for 1 min and 4 °C for 1 min. Finally, 1 µl of RNase H (2 U/µl) was added to the samples to remove RNA at 37 °C for 20 min, 65 °C for 10 min, and 4 °C for 10 min. RT–PCR was performed with primers targeting pluripotent stem cell markers (OCT4, SOX2, NANOG) using a standard Taq Reaction Buffer Pack (New England Biolabs, Cat. No. B9014S and M0273S) in a T100TM Thermal Cycler (Bio-Rad) under the following conditions: one cycle at 95 °C for 2.5 min, followed by 35 cycles of 30 s at 95 °C, 30 s at 55 °C and 1 min at 68 °C, with a final cycle at 68 °C for 5 min. The primers used for RT–PCR are listed in Supplementary Table 4.

qRT–PCR

Quantitative reverse transcription-polymerase chain reaction (qRT–PCR) was performed with the SYBR Green method. The 25-fold diluted oligo dT or random hexamer priming cDNA was amplified with the primers shown in Supplementary Table 5 and was performed with the CFX384TM Real-Time PCR System, in a C1000 TouchTM Thermal Cycler (Bio-Rad) using iQ™ SYBR® Green Supermix (Bio-Rad, Cat. No. 1708882). The results were normalized to the GAPDH, ATP5β, and HPRT reference genes and measured by CFX Maestro software (Bio-Rad). Graphing and statistical analysis of qRT–PCR results were performed using Prism 8 (GraphPad).

Northern blotting

Total RNA (10 µg) was separated on a 4% polyacrylamide (29:1) gel containing 8 M urea at 20 W for 1 h and then transferred to a Hybond-N+ nylon transfer membrane (GE Healthcare, Cat. No: RPN303B) at 400 mA for 1 h in 0.5X TBE buffer. RNA was cross-linked to the membrane in a Stratalinker (Stratagene, 120 mJ). The blot was prehybridized in Church buffer at 65 °C (for hTR) or at 42 °C (for oligonucleotide probe) for an hour. Hybridizations with radiolabelled probes were performed in Church buffer at 65 °C (for hTR, probes were generated by nick translation of a polymerase chain reaction (PCR) fragment with 32P-α-dCTP) and 42 °C (for oligonucleotide probe against 7SL, which was labelled with 32P-γ-ATP by T4 PNK kinase). The oligonucleotide sequences are listed in Supplementary Table 6. All blots derive from the same experiment and were processed in parallel.

Cell lysis and Western blotting

All iPSC pellets were lysed in lysis buffer containing 0.5% CHAPS, 50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10% glycerol, 5 mM DTT, and 1 mM PMSF. Total lysates were incubated at 4 °C for 1 h on a rotator, and insoluble material was removed by centrifugation at 21,130 × g at 4 °C for 10 min. Protein concentration was measured using the protein assay dye (Bio-Rad, Cat. No. 5000006). Twenty micrograms of protein were resolved on 4–20% Bis-Tris gradient gels (GenScript, Cat. No: M00657) at 180 V for 40 min and then transferred to polyvinylidene fluoride (PVDF) membranes (BIO-RAD Immun-Blot®, Cat. #1620177) at 100 V for 1 h. Five percent skim milk in washing buffer was used as a blocking reagent. The prestained protein ladder (Omics Bio, Cat: 02101-250) was used as a marker; ɑ-tubulin (1:5000, ABclonal, Cat. No: AC012) was used as a loading control. Cytiva software was used. The antibodies used in this study are listed in Supplementary Table 7. All blots derive from the same experiment and were processed in parallel.

Cytoplasmic/nuclear fractionation

iPSCs (2 × 106) were washed with DPBS (Biological Industries, Cat. No: 02-023-1A) and lysed in 50 µl of fresh buffer A (0.05% Triton X-100, 10 mM HEPES-KOH, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 10% glycerol, 0.34 M sucrose, 1 mM DTT, supplemented with protease inhibitor cocktail) at 4 °C for 10 min. The cytosolic fraction was collected and clarified at 500 × g at 4 °C for 5 min twice. The cell monolayer was then washed three times with DPBS and resuspended in a new buffer A. Both nuclear and cytoplasmic fractions were analysed by Western blotting as mentioned before. The antibodies used in this study are listed in Supplementary Table 7.

Immunofluorescence assay

The iPSCs were fixed with 4% paraformaldehyde at RT for 20 min, permeabilized with 0.1% Triton X-100 at RT for 5 min, and blocked with 1% BSA in DPBS at RT for an hour, followed by incubation with the primary antibody against dyskerin (H-3) (Santa Cruz Biotechnology, Cat. No. sc-373956, 1:500 dilution) at 4 °C overnight. The slide was washed three times with DPBS and then incubated with fluorescein (FITC)-conjugated AffiniPure goat anti-mouse IgG (H + L) (Jackson ImmunoResearch, Cat. No. 115-095-003, 1:100 dilution) secondary antibody at 37 °C for 1 h. Nuclei were stained with bisbenzimide H 33258 (Sigma–Aldrich, Cat. No. B2883-1 g, 1:1000 dilution) for 10 min at 4 °C after washing the cells with DPBS. After the indicated treatments, coverslips were mounted on glass slides with FluoromountTM Aqueous Mounting Medium (Sigma–Aldrich, Cat. No. F4680-25ML) and photographed under an Axio Imager 2 fluorescence microscope (ZEISS). Acquired images were quantified by using ImageJ/Fiji software. Graphing and statistical analysis of the immunofluorescence assay results were performed in Prism 8 (GraphPad). The antibodies used in this study are listed in Supplementary Table 8.

Telomerase activity assay

Telomerase activity reactions were performed as 10 µl reactions with: 50 mM Tris-HCl pH 8.0, 50 mM KCl, 1 mM MgCl2, 1 mM spermidine, 5 mM DTT, 1 mM dATP, 1 mM dTTP, 10 µM dGTP, and 0.75 µM 32P-dGTP (3000 Ci/mmol), 1 µM telomeric primer (TTAGGG)3, and 2 µg cell extract at 37 oC for 2 hours. Reactions were stopped with 10 µl of 1 mg/ml proteinase K. DNA was extracted with phenol/chloroform equilibrated with 50 mM NaOAc (pH 7.0) and ethanol precipitated with 2.5 M ammonium acetate and 10 µg of glycogen at −80 oC overnight. Reactions were then centrifuged for 20 min at 14,000 r.p.m., and the pellets were washed with 1 ml of 70% ethanol. The dried pellets were then resuspended in 5 µl of 80% formamide loading buffer. Reaction products were analysed on a 10% polyacrylamide (19:1) gel containing 8 M urea. All blots derive from the same experiment and were processed in parallel.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.