Human-iPSC generation and culture. Human iPSCs were generated and cultured as reported22. This study was approved by the Ethics Committee of Osaka University Graduate School of Medicine (approval number 13123-823). Informed consent was obtained from the patients’ guardians in accordance with the Declaration of Helsinki. Briefly, iPSCs were induced from cord blood mononuclear cells of a male baby with DS using a Sendai virus (SeV) vector encoding OCT4, SOX2, KLF4, and c-MYC. iPSCs were maintained on mitomycin C (Merck)-inactivated mouse embryonic fibroblasts (MEFs) in human embryonic stem cell (hES) medium consisting of DMEM/F12 (Fujifilm Wako), KnockOut Serum Replacement (20%; Thermo Fisher Scientific), l-alanyl-l-glutamine (2 mM; Fujifilm Wako), MEM non-essential amino acid solution (1%; Fujifilm Wako), 2-mercaptoethanol (0.1 mM; Merck), and basic fibroblast growth factor (5 ng/mL; bFGF, Katayama Chemical), with or without 2 µg/mL Dox (Takara). To remove SeV, siRNA L527 (Gene Design) was mixed with Lipofectamine RNAiMAX (Thermo Fisher Scientific) and used to transfect iPSCs as described66, until complete removal of the SeV genome was confirmed by PCR and immunostaining with an anti-SeV-NP antibody. The iPSC cultures were passaged every 6 to 9 days.
The iPSCs used in this study were karyotyped by Chromocenter, Inc. via G-band analysis or Q-band analysis. Potency assays were performed via immunocytochemistry. Briefly, iPSCs fixed in phosphate-buffer solution (PBS) containing 4% paraformaldehyde (Fujifilm Wako) were immunostained using primary antibodies against OCT4 (1:200; Santa Cruz Biotechnology) and SSEA4 (1:200; Merck). Secondary Alexa Fluor 488- or Alexa Fluor 594-conjugated antibodies were used (1:500; Thermo Fisher Scientific).
Insertion of a Dox - inducible XIST transgene. Full-length human XIST cDNA was kindly provided by Dr. Chikashi Obuse (Osaka University, Osaka, Japan). Introduction of a Dox-inducible XIST transgene was performed as described previously, with modifications19. A zinc finger nuclease (ZFN) against the AAVS1 locus on chromosome 19 was designed to enable insertion of the 3G rtTA transgene, as described67. Insertion of a lox cassette into the DYRK1A locus was performed using the CRISPR–Cas9 system (Supplementary Table 4). Single-guide RNA (sgRNA) oligos for the CRISPR–Cas9 system were cloned into the BbsI sites of the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (Addgene, #42230). The pEF1α-3G rtTA-pA cassette was cloned from a pEF1α-Tet3G vector (Clontech, #631167). Full-length XIST cDNA with loxP and lox5171 sites was cloned into the pTRE3G vector (Clontech, #631168). On the day before transfection, iPSC colonies were dissociated into single cells using TrypLE Express (Thermo Fisher Scientific) in the presence of 10 µM ROCK inhibitor (Reagents Direct). Dissociated cells (1.0 × 106) were mixed with the donor vector (4 − 6 µg) and either a ZFN pair (left and right ZFNs, 0.5 µg each) or CRISPR–Cas9 (2 µg) and electroporated using the Neon Transfection System (setting: 1200 V, 20 ms, 2 pulses; Thermo Fisher Scientific). The electroporated cells were plated in 10-cm dishes with DR-4 IRR MEFs (Thermo Fisher Scientific). On day 4 post-electroporation, drug selection with G418 (150 µg/mL) or puromycin (0.5 µg/mL) was initiated according to the drug-resistance gene. The resulting colonies were selected on days 12–18. PCR-positive clones were further expanded. The lox cassette contained a positive/negative drug-selection marker (puroΔTK, encoding a fusion protein between the puromycin-resistance gene and a truncated version of herpes simplex virus type 1 thymidine kinase) between the loxP and lox5171 sites.
For Cre recombinase-mediated cassette exchange, 1.0 × 106 cells were electroporated with a Cre expression vector (4 µg) and a donor vector (8 µg), which contained XIST transgene with loxP and lox5171, as described above. The electroporated cells were plated, and on day 4 post-transfection, negative selection was initiated using 2 µM 1-[2-deoxy, 2-fluoro-8-d-arabinofuranosyl]-5-iodouracil. The resultant clones were analysed by PCR and positive clones were expanded.
Maintenance and differentiation of NPCs. NPC differentiation was performed according to a previously described protocol, with modifications14. Briefly, embryoid bodies (EBs) were cultured for 8 days in Costar six-well, ultra-low-attachment plates (Corning) in hES medium without bFGF, consisting of 2 µM dorsomorphin (Merck), 10 µM SB431542 (Tocris Bioscience), and 10 µM ROCK inhibitor. Next, the EBs were attached for 13 days to Matrigel (Corning)-coated dishes in neuronal medium (N2B27 medium) consisting of DMEM/F12, Neurobasal Medium (Thermo Fisher Scientific), N2 supplement (1⋅; Thermo Fisher Scientific), B27 without vitamin A (1⋅; Thermo Fisher Scientific), GlutaMAX (1⋅; Thermo Fisher Scientific), l-alanyl- l-glutamine (2 mM; Fujifilm Wako), MEM non-essential amino acids solution (1%; Fujifilm Wako), 2-mercaptoethanol (0.1 mM), ROCK inhibitor (10 µM), and bFGF (20 ng/mL). Neural rosettes appearing in the centres of the attached EB colonies were isolated with TrypLE Express and replated in Matrigel-coated dishes in N2B27 medium containing bFGF (20 ng/mL). The culture medium (with or without 2 µg/mL Dox) was changed every day and the cells were passaged every 3–5 days.
Transfection of the piggyBac vector into NPC. To enhance rtTA, DSCR3, or PIGP expression, NPCs were transfected with a piggyBac vector harbouring an additional gene (rtTA, DSCR3, or PIGP), which was generated from the PB-TA-ERN vector (Addgene, #80474). The resulting vector (2 µg) and a pCMV-hyPBase vector (2 µg; a kind gift from the Sanger Institute) encoding transposase we co-transfected into NPCs (4.0 × 106) using the Neon Transfection System (settings: 1200 V, 20 ms, 2 pulses). After clone selection with puromycin (0.5 µg/mL), NPCs with the additional gene were established.
Maintenance and differentiation of APCs. The protocol used for differentiating APCs from NPCs has been described in detail25. NPCs were dissociated with TrypLE Express, and 2 ⋅ 104 cells/well were plated on Matrigel-coated 24-well plates with Astrocyte Medium (ScienCell) supplemented with 10 µM ROCK inhibitor. This medium was changed every 2 days, and the cells were passaged every 4–6 days, with dissociation using TrypLE Express. In this study, APCs were passaged 7–8 times before performing the analysis. When necessary, NPCs were differentiated into APCs via Dox administration for five days. The Dox-treated cell lines were administered Dox for approximately 6 weeks, and the Dremov cell lines were administered Dox for approximately 3 weeks, followed by growth in culture for 3 weeks without Dox. Two XIST-Tri 21 iPSC lines were generated from a male baby with trisomy 21. Both iPSC lines were differentiated into NPC lines. The NPCs were independently transfected using the piggyBac vector encoding an rtTA to generate three lines. The NPC-derived APCs were subjected to qRT-PCR analysis, cell-proliferation assays, RNA-seq analysis, and ChIP-seq analysis. Three cDi21 lines, which were generated using a chromosome-elimination technique, were used as controls in this study.
Genome editing of the DYRK1A gene using the CRISPR–Cas9 system. DYRK1A targeting was performed using the CRISPR–Cas9 system. The sgRNA sequence was designed using CRISPR Direct (http://crispr.dbcls.jp/; Supplementary Table 4). The sgRNA oligos were cloned into the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector (Addgene, #42230). On the day before transfection, iPSC colonies were dissociated into single cells using TrypLE Express with 10 µM ROCK inhibitor. Cells were dissociated with TrypLE Express, after which 1.0 × 106 cells were mixed with CRISPR–Cas9 (2 µg) and the donor vector (6 µg), and electroporated using the Neon Transfection System (settings: 1200 V, 20 ms, 2 pulses). The electroporated cells were plated in 10-cm dishes with DR-4 IRR MEFs (Thermo Fisher Scientific). On day 4, drug selection with hygromycin (75 µg/mL) was initiated. The resulting colonies were selected on days 12–18. PCR-positive clones were further expanded. The sequences of the primers used for the genome-editing experiments are listed in Supplementary Table 5.
Transfecting siRNAs into APCs. Cultured APCs were transfected with siRNAs using Lipofectamine RNAiMAX for 1 day. The final lipofectamine RNAiMAX and siRNA concentrations were 3 µl/mL and 10 nM, respectively. siRNAs against DSCR3 (Thermo Fisher Scientific, #s20157) and PIGP (Thermo Fisher Scientific, #s27717) were used. Silencer Select Negative Control siRNA #1 (Thermo Fisher Scientific, #4390843) was used as a control siRNA.
Immunocytochemistry. Immunocytochemistry was performed as described previously, with some modifications68. Cells were fixed with PBS containing paraformaldehyde (4%) and permeabilised with PBS containing Triton X-100 (0.5%) for 15 min. After blocking with PBS containing foetal bovine serum (5%) for 30 min, the cells were incubated overnight at 4 °C with primary antibodies against H3K27me3 (1:200; Merck), PAX6 (1:100; Stemgent), SOX1 (1:100; R&D Systems), GFAP (1:1000; DakoCytomation), S100β (1:1000; Merck), CD44 (1:200; Merck), or vimentin (1:500; Merck). The cells were washed with PBS and incubated for 120 min with appropriate Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies (Thermo Fisher Scientific). The nuclei were counterstained with Hoechst dye (1:1000).
RNA fluorescence in situ hybridisation (FISH). RNA FISH was performed as previously described, with some modifications23. XIST-Tri 21 iPSC colonies were dissociated into single cells and cultured on coverslips for 2 days. The cells were fixed with PBS containing 4% paraformaldehyde and incubated with ice-cold CSK buffer (100 mM NaCl, 300 mM sucrose, and 10 mM PIPES, pH 6.8) containing Triton X-100 (0.5%). After rinsing, the cells were dehydrated in ice-cold ethanol (100%) and then air-dried. The XIST probe was labelled using a DIG-Nick translation Mix (Merck) according to the manufacturer’s protocol. Hybridisation reactions consisting of labelled products (0.1 µg), herring sperm DNA (5 µg; Fujifilm Wako), baker’s yeast transfer RNA (5 µg; Thermo Fisher Scientific), human Cot-1 DNA (3 µg; Thermo Fisher Scientific), and RNase Out (4 U/µL; Thermo Fisher Scientific) in hybridisation buffer (4 × saline-sodium citrate [SSC] buffer, dextran sulphate (20%, w/v), bovine serum albumin [BSA]; 4 mg/mL) were carried out overnight in humidified incubator at 37 °C. The cells were then subjected to stringent washes at 42 °C (three times in 2 × SSC/50% formamide and three times in 2 × SSC). The cells were blocked in 4 × SSC containing 4 mg/mL BSA and 0.1% Tween-20 at 37 °C prior to detection. Detection was performed in detection buffer (4 × SSC containing 5% BSA and 0.2% Tween-20) with a mouse anti-digoxigenin antibody (Thermo Fisher Scientific) for 50 min, followed by amplification with Cy3-conjugated antibody (Jackson ImmunoResearch Labs) for 50 min. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and the slides were mounted in VECTASHIELD Antifade Mounting Medium (Vector Laboratories). Images were captured using an LSM 710 confocal scanning laser microscope (Carl Zeiss) and processed using Volocity software (PerkinElmer).
qRT-PCR analysis. Total RNA was isolated from iPSCs, NPCs, and APCs with the NucleoSpin RNA II Kit (Macherey-Nagel). Reverse transcription was performed using ReverTra Ace qPCR RT Master Mix (Toyobo). qRT-PCR analysis was performed using Thunderbird SYBR qPCR Mix (Toyobo). Gene expression levels were normalised to the expression of β-actin (ACTB). The sequences of all primers used for qRT-PCR analysis are shown in Supplementary Table 6.
Single-cell cloning. XIST-Tri 21 iPSC colonies were dissociated into single cells using TrypLE Express, and then 1.0 × 103 cells were plated in a 10-cm dish with DR-4 IRR MEFs using iPSC culture medium containing 10 µM ROCK inhibitor, G418 (150 µg/ml) and Dox (2 µg/ml). On days 12–18, the resulting colonies were passaged to individual wells. These clones were expanded further, and on day 21 after Dox addition, they were fixed and assessed using an immunocytochemistry method.
Cell-proliferation assay. A Click-it EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher Scientific) was used to measure cell proliferation. At 1 or 2 days before adding the thymidine analogue EdU, APCs were dissociated with TrypLE Express, and 5 ⋅ 103 cells/well were plated into a Matrigel-coated 96-well plate (Greiner Bio-one). On the day of EdU treatment, the cells were cultured with EdU (10 µM) for 8 h. The cultured cells were fixed with PBS containing paraformaldehyde (4%) and then permeabilised with PBS containing TritonX-100 (0.5%). The cells were stained as instructed in the manufacturer’s protocols. Images were taken with an In Cell Analyzer 6000 (GE Healthcare), and EdU-positive cells were detected using the In Cell Developer Toolbox 1.9 (GE Healthcare). Cell-counting assays were performed according to a method similar to the protocol described above. At 1 or 2 days after plating the cells (5 ⋅ 103/well) in a Matrigel-coated 96-well plate, they were fixed. The cell nuclei were counterstained with Hoechst 33342 dye (1:1000). Images were taken with an IN Cell Analyzer 6000, and the stained nuclei were counted using the In Cell Developer Toolbox 1.9. To study the effect of DYRK1A inhibition on cell proliferation, cells (5 × 103) were seeded into culture plates and FINDY (Merck) was added. Two days after FINDY addition, proliferation assays were performed.
RNA-seq analysis. APCs were harvested at passage 8 for RNA-seq analysis, and total RNA was isolated from each sample using the NucleoSpin RNA II Kit. RNA-seq analysis was performed by DNA Chip Research, Inc. The integrity and quantity of the total RNA were measured with an Agilent 2100 Bioanalyzer RNA 6000 Nano Kit (Agilent Technologies). Total RNA obtained from each sample was subjected to sequencing library construction using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) with the NEBNext Poly(A) mRNA Magnetic Isolation Module, according to the manufacturer’s protocols. The quality of the libraries was assessed with the Agilent 2200 TapeStation High Sensitivity D1000 ScreenTape System (Agilent Technologies). The pooled library samples were sequenced using a NextSeq 500 instrument (Illumina), with 76-base-pair (bp) single-end reads. Sequencing adaptors, low-quality reads, and bases were trimmed with the Trimmomatic-0.32 tool. The sequence reads were aligned to the human reference genome (hg19) using TopHat 2.1.1 (bowtie2-3.2.0)69, which can adequately align reads (including splice sites) with the genome sequence. Files of the gene-model annotations and known transcripts were downloaded from the Illumina iGenomes website (http://support.illumina.com/sequencing/sequencing_software/igenome.html). These files were necessary for performing whole-transcriptome alignments with TopHat. The aligned reads were subjected to downstream analyses using StrandNGS 3.2 software (Agilent Technologies). The read counts for each gene and transcript (RefSeq Genes 2015.10.05) were quantified using the trimmed mean of M-value (TMM) method70. Plots were created using the ggplot2 (version 3.3.2) package in R 3.6 software.
ChIP-seq analysis. For ChIP-seq analysis, APCs were harvested at passage 8. Chromatin was prepared from the cells using the SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology) according to the manufacturer's protocols. Approximately 8 × 106 cells were used for each immunoprecipitation experiment. ChIP was performed using an antibody against H3K27me3 (Cell Signaling Technology). ChIP-seq analysis was performed by DNA Chip Research, Inc. The quality and quantity of the ChIP DNA and input DNA were measured with an Agilent 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies). For each sample, 10 ng of DNA was subjected to sequencing library construction using the NEBNext Ultra II DNA Library Prep Kit for Illumina, according to the manufacturer’s protocols. The quality of each library was assessed with an Agilent 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies). Pooled sample libraries were sequenced using NextSeq 500 in 76-bp single-end reads. Sequencing adaptors, low-quality reads, and bases were trimmed with the Trimmomatic 0.32 tool. The sequence reads were aligned to the human reference genome (hg19) using bowtie2-3.2 software. PCR duplicates were removed using Picard ver. 1.119 (http://picard.sourceforge.net). The aligned reads were subjected to downstream analysis using the MEDIPS (version 1.30) package in R 3.4 software. We calculated the short read coverage (extend value = 300) with genome-wide 100-bp bins using MEDIPS software. Differential coverage of the ChIP-seq data between groups of samples was detected based on the TMM method for genome-wide 100-bp sliding windows.
Short-tandem repeat (STR) analysis. To perform STR genotyping, DNA was extracted from iPSCs using a DNeasy Blood & Tissue Kit (Qiagen). To assess DYRK1A-targeted alleles, junctional PCR (homologous recombination+) and outside PCR (homologous recombination−) were performed using KOD FX Neo enzyme solution (Toyobo). PCR products or genomic DNA were subjected to PCR using PrimeSTAR MAX (Takara), a fluorescently labelled forward primer, and a reverse primer. The final PCR products were mixed with an internal lane standard 600 (Promega) and HiDi formamide (Thermo Fisher Scientific) and separated by capillary electrophoresis on an ABI 310 Genetic Analyzer (Thermo Fisher Scientific), per the manufacturer’s instructions. The primer sequences are shown in Supplementary Table 5.
Allele-specific SNP-silencing analysis. Total RNA was isolated from XIST-Tri 21 APCs (Dox-untreated cell lines and Dox-treated cell lines) using the NucleoSpin RNA II Kit. Reverse transcription was performed using ReverTra Ace qPCR RT Mix. With the resulting total cDNA, PCR was performed using primers that amplified a region containing an SNP (rs457705) on exon 8 of ETS2 on chromosome 21. The sequences of the primers used for ETS2 are provided in Supplementary Table 7.
Western blotting. Cells were lysed with RIPA Buffer (Fujifilm Wako) containing a mixture of protease inhibitors (Merck) and phosphatase inhibitors (Nacalai Tesque). Lysates containing equal amounts of protein were mixed with an appropriate amount of Laemmli sample buffer (2×; Bio-Rad Laboratories) and 2-mercaptoethanol and denatured at 95 °C for 5 min. The samples were separated by 7.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories). The membranes were washed with Tris-buffered saline containing Triton X-100 (0.05%) and incubated with Blocking One or Blocking One-P (Nacalai Tesque) buffer for 60 min. Mouse anti-STAT3 (1:1000; Cell Signaling Technology) and rabbit anti-Phospho-STAT3 (Ser727) (1:500; Cell Signaling Technology) were used as primary antibodies. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies (1:2500; Promega) were used as secondary antibodies. As a control, β-actin was detected with anti-β-actin pAb-HRP-DirecT (1:2000; MBL). Blots were visualised using Clarity Western ECL Substrate (Bio-Rad Laboratories). The stained membranes were scanned with ImageQuant LAS 4000 Biomolecular Fluorescence Image Analyzer (GE Healthcare). When necessary, the antibody was stripped with Restore Western Blot Stripping Buffer (Thermo Fisher Scientific). Quantification was performed using ImageJ software (http://imagej.nih.gov/ij/).
Statistical analysis. All statistical analyses were performed using the EZR software. Comparisons of two groups were made using Student’s t-test or Welch’s two-sample t-test. We evaluated multiple comparisons using one-way analysis of variance (ANOVA) or the Kruskal–Wallis test with Bonferroni’s correction. A P value of less than 0.05 was considered to reflect a statistically significant difference. The data presented are expressed as the mean ± standard error of the mean (SEM) or standard deviation (SD).
Data availability. The RNA-seq data and ChIP-seq data reported here are available in the DDBJ Sequenced Archive under accession numbers DRA010528 and DRA010529, respectively.