Method Details

To establish dox-inducible quadruple fluorescent knock-in reporter system, the mouse ESC line KH2 containing the dox-inducible activator M2rtTA in the Rosa26 locus and a flip-in system in the collagen (Col1a1) locus (Beard et al., 2006) was Sequentially targeted with targeting constructs either containing RBGpA or Neomycin selection cassette. RBGpA was introduced into pNTKV-frt-loxP (Dawlaty and van Deursen, 2006) vector using HpaI and HindII restriction sites using specific primers (For Sequences see Methods S1). Four different fluorescent reporters (Nanog-2A-EGFP, Utf1-2A-tdTomato, Esrrb-2A-TagBFP and Elf5-2A-EYFP-NLS) were introduced into the 3′UTR of the Nanog, Utf1, Esrrb or Elf5 genes and targeted clones were isolated either by FACS or Neomycin selection. The targeting vectors were designed by cloning the 5′arms and 3′arms using HpaI and SacII or SmaI respectively. 2A-tdTomato for Utf1 and 2A-TagBFP for Esrrb were introduced into pNTKV-RBGpA using HpaI and HindII restriction sites. gRNA for all loci was synthesized and cloned into pX330 (addgene #42230) using BbsI sites. The targeting constructs together with corresponding gRNA were transfected into the targeting cells using TransIT-LT1 Transfection Reagent (Mirus Bio) according to manufacturer’s instructions. Correctly targeted clones for Utf1 and Esrrb were selected by FACS and verified by Southern blot and PCR, respectively. For Southern blot (Utf1 locus), DNA was digested with NheI and probed for the 3′ region and PCR for Esrrb was done using primer pair A (Figures S1F and S1G). LacIq gene together with constitutively active Ubiquitin promoter (Ubb) were amplified by PCR (for primer Sequence see Methods S1) and cloned into pBS31-RBGpA (Beard et al., 2006) plasmid, using EcoRI site. The resulting construct was co-transfected together with flippase containing plasmid into KH2-NgUrEb ESCs and LacIq expressing clones were isolated following Hygromycin selection as previously described (Beard et al., 2006). The targeting construct for Elf5 locus was designed by cloning the 5′arm and 3′arm with HpaI and SacII/SmaI respectively and 2A-EYFP-NLS with HpaI and BglII into pNTKV-frt-loxP (Dawlaty and van Deursen, 2006). The positive colonies for Elf5 correctly targeted locus were picked after 10-12 day of G418 selection at 500μg/ml (Millipore) and verified by primer pair B (Figures S1J–S1L). The correctly targeted clone was transfected with Cre-expressing vector (FUW-zeo-Cre) for 10-12 days of selection of Zeocin (Invivogen) at 500μg/ml and correctly targeted clones were verified by primer pair C (Figures S1J–S1L) and Sequenced.

All dox-inducible factors were generated by cloning the open reading frame of each factor, obtained by reverse transcription with specific primers (for primer Sequences see Methods S1), into the pMINI vector (NEB) and then restricted with EcoRI or MfeI and inserted into the FUW-TetO expression vector. The mouse Eomes and Esrrb-2A-EGFP or 2A-tdTomato constructs were generated by cloning the ORF of the genes (without stop codon) into FUW-TetO-2A-EGFP or FUW-TetO-2A-tdTomato expression vector with EcoRI site (for primer Sequences see Methods S1). For infection, replication-incompetent lentiviruses containing the various reprogramming factors and rations (GETM 3:3:3:1, GTMS: 3:3:1:3, GETMS 2:2.5:2:1:2.5, OSKM 3:3:3:1 or STEMCCA cassette) were packaged with a lentiviral packaging mix (7.5 μg psPAX2 and 2.5 μg pGDM.2) in 10cm plate of 293T cells and collected 48, 60, and 72 hr after transfection. The supernatants were filtered through a 0.45μm filter, supplemented with 8 μg/ml of polybrene (Sigma), and then used to infect MEFs. Six hours following the third infection, medium was replaced with fresh DMEM containing 10%FBS. Eighteen hours later, medium was replaced to either TSC reprogramming medium (RPMI supplemented with 20%FBS, 0.1mM β-mercaptoethanol, 2mM L-glutamine, in house mouse recombinant FGF4 (equivalent to 25ng/ml), 1 μg/ml heparin (Sigma-Aldrich), and 2 μg/ml doxycycline) or ESC reprogramming medium (DMEM supplemented with 10%FBS, 0.1mM β-mercaptoethanol, 2mM L-glutamine, 1%non-essential amino acids, in-house mouse Leukemia inhibitory factor (mLif), and 2 μg/ml doxycycline). TSC or ESC reprogramming medium was replaced every other day for 20 days, followed by 10 days in TSC culturing medium or 2i/L culturing medium-respectively. Ten days (or as indicated in the figure) after dox removal, plates were screened for primary iTSC or iPSC colonies. For OSKM reprogramming, excluding Figure 1 that shows reprogramming to iPSCs with separate OSKM plasmids, all OSKM reprogramming experiments were performed using STEMCCA cassette. Reprogramming efficiency was measured by FACS and colony number. For iPSC or iTSC clone isolation, a single iPSC/iTSC colony was trypsinized (0.25%), and plated in a separate well in a 6-well plate on feeder cells. Wells were followed and medium was replaced every other day for five to ten passages, until stable colonies developed.

Cells were washed twice with PBS and trypsinized (0.25%) and filtered through mesh paper. Flow cytometry analysis was performed on a Beckman Coulter and analyzed using Kaluza Software. All FACS experiments were repeated at least three times, and the bar graph results are presented as a mean ± standard deviation of two biological duplicate from a typical experiment. For cell cycle analysis, GETMS-iTSCs grown in differentiation media for the indicated time points were trypsinized and fixed with 95% ice-cold ethanol. Cells were stained with propidium iodide (PI) staining solution (50 μg/ml PI [BD]; 0.1% [v/v] Triton X-100 [Sigma-Aldrich]; 0.2mg/ml RNaseA [Sigma-Aldrich] in PBS) for 30 min at room temperature. Flow cytometry analysis was performed on a Beckman Coulter and analyzed using Kaluza Software.

Blastocyst injections were performed using CB6F1 host embryos. After priming with PMSG (M.I.P. Veterinary) and hCG (Merck) hormones and mating with CB6F1 males, embryos were obtained at 3.5dpc (blastocyst stage), and then injected with 10–20 ES/iPS/iTS cells with a flat tip microinjection pipette with an internal diameter of 16 mm (Origio) in a drop of FHM medium (Zenith Biotech, ZEHP-050) covered by mineral oil. Shortly after injection, blastocysts were transferred to 2.5dpc pseudopregnant CD1/ICR females (10-15 blastocysts per female). Chimeric embryos and placentas were isolated at E13.5 and observed by fluorescent microscope (Nikon Eclipse T!).

ZHBTc4 ESCs were kindly provided by Professor Austin Smith. Cells were infected with either Esrrb-2A-EGFP or Eomes-2A-EGFP and bright EGFP-positive clones were isolated and treated according to Niwa et al., (2000) to induce transdifferentiation. Briefly, cells were cultured on feeder cells in ESC medium until formation of colonies. Doxycycline (2 μg/ml) was added, and medium was changed to 70:30% MEF conditioned TSC media (MEF-CM) on day 4 after dox induction. Pellets were collected every 24 hours for 5 days, and mRNA was purified for qPCR analysis. To validate formation of stable TS-like or XEN-like colonies, the cells were passaged five times after the transdifferentiation experiment.

A total of 5x10⁶ GETMS-iTSCs were resuspended in 100 μL CM containing in house mouse recombinant Fgf4 (equivalent to 25ng/ml) and 1 μg/ml heparin (Sigma-Aldrich) and injected subcutaneously into male athymic nude mice. 6-7 days later, lesions were dissected, fixed in 4% paraformaldehyde overnight, embedded in paraffin, and sectioned (4mm). Sections were stained with H&E and analyzed by a certified pathologist.

ZHBTc4 ESCs were plated in ESC medium (without CHIR & PD). The next day, cells were transfected with 2.5μg PX330 vector containing Esrrb gRNA (for gRNA Sequences see Methods S1), and 0.5μg Puromycin resistance plasmid, using TransIT-LT1 transfection reagent (Mirus). 48 hours later, medium was changed to selective ESC medium with CHIR, PD and Puromycin (2μg/μl). Resistant colonies were picked, and Esrrb knockdown was validated by Sequencing, immunostaining, sm-FISH and Western Blot.

Total RNA was isolated using the Macherey-Nagel kit (Ornat). 500–2000 ng of total RNA was reverse transcribed using iScript cDNA Synthesis kit (Bio-Rad). Quantitative PCR analysis was performed in duplicates using 1/100 of the reverse transcription reaction in a StepOnePlus (Applied Biosystems) with SYBR green Fast qPCR Mix (Applied Biosystems). Specific primers flanking an intron were designed for the different genes (for primer Sequences see Methods S1). All quantitative real-time PCR experiments were repeated at least three times, and the results were normalized to the expression of GAPDH and presented as a mean ± standard deviation of two duplicate runs from a typical experiment.

Cells were fixed in 4% paraformaldehyde (in PBS) for 20 minutes. The cells were rinsed 3 times with PBS and blocked for 1hr with PBS containing 0.1% Triton X-100 and 5% FBS. The cells were incubated overnight with primary antibodies (1:200) in 4°C. The antibodies are: anti-Gata6 (Abcam, ab22600), anti-sox17 (Santa Cruz, sc-130295), anti-Esrrb (Perseus Proteomics, PP-H6705-00), anti-Eomes (Abcam, ab23345), anti-Cdx2 (Biogenex, CDX2-88), anti-Nanog (Bethyl, A300-379A) and anti-Elf5 (Santa Cruz, SC-9645) in PBS containing 0.1% Triton X-100 and 1%FBS (1:200 dilution). The next day, the cells were washed 3 times and incubated for 1hr with relevant (Alexa) secondary antibody in PBS containing 0.1% Triton X-100 and 1% FBS (1:500 dilution). DAPI (1:1000 dilution) was added 10 minutes before end of incubation. For western blot, cell pellets were lysed on ice in lysis buffer (20 mM Tris-HCl, pH 8, 1mM EDTA pH 8, 0.5% Nonidet P-40, 150mM NaCl, 10% glycerol, 1mM, protease inhibitors (Roche Diagnostics)) for 10 min, supernatant were collected and 40μg protein were suspended with sample buffer and boiled for or 5 min at 100C^o, and subjected to western blot analysis. Primary antibodies: anti-Gata3 (Abcam, ab106625), anti-Tfap2c (Abcam, ab110635,), anti-Esrrb (Perseus Proteomics, PP-H6705-00), anti-Myc (Abcam, ab32072), anti-Eomes (Abcam, ab3345), and anti-actin (Santa cruz, Sc-1616). Blots were probed with anti-mouse, anti-goat, or anti-rabbit IgG-HRP secondary antibody (1:10,000) and visualized using ECL detection kit.

Chromatin immunoprecipitation (ChIP) assay was performed as previously described (Mendenhall et al., 2013). Briefly, cells were fixed for 10min at RT with a final formaldehyde concentration of 0.8%. Formaldehyde was quenched with glycine at a final concentration of 125mM. The cells were then lysed with lysis buffer (100mM Tris-HCl, 300mM NaCl, 2% Triton® X-100, 0.2%v sodium deoxycholate and 10mM Cacl2) supplemented with EDTA-free protease inhibitor (Roche, 11873580001) for 20min in Ice. The chromatin was digested by adding MNase (Thermo Scientific, 88216) for 20min at 37°C and MNase was inactivated by adding 20mM EGTA. The fragmented chromatin was added to pre-bounded Dynabeads (A and G mix, Invitrogen, 10004D/ 10002D) using H3K27ac antibody (Abcam, ab4729), H3K4me2 antibody (Millipore, 07-030), Esrrb antibody (Perseus Proteomics, PP-H6705-00) or Igg (santa cruz biotechnology, sc-2025). Samples were then washed twice with RIPA buffer, twice with RIPA high salt buffer (NaCl 360mM), twice with LiCl wash buffer (10mM Tris-Hcl, 250mM LiCl, 0.5% DOC, 1mM EDTA, 0.5% IGEPAL) and twice with 10mM Tris-HCl pH = 8. DNA was purified by adding RNase A (Thermo Scientific, EN0531) and incubated for 30 min at 37°C and then with Proteinase K (Invitrogen, 25530049) for 2h. The DNA was eluted by adding 2X concentrated elution buffer (10mM Tris-HCl, 300mM NaCl, 1% SDS, 2mM EDTA) and reverse crosslinked overnight at 65°C. The DNA was then extracted using AMPure XP beads (Beckman Coulter Genomics, A63881). Chip sample libraries were prepared according to Illumina Genomic DNA protocol.

Total RNA was isolated using the QIAGEN RNeasy kit. mRNA libraries were prepared using the SENSE mRNA-Seq library prep kit V2 (Lexogen), and pooled libraries were Sequenced on an Illumina NextSeq 500 platform to generate 75-bp single-end reads.

ATAC-Seq library preparation was performed as previously described (Buenrostro et al., 2013). Briefly, 50,000 cells per replicate (two biological replicates per line) were incubated with 0.1% NP-40 to isolate nuclei. Nuclei were then transposed for 30 min at 37°C with adaptor-loaded Nextera Tn5 (Illumina, Fc-121-1030). Transposed fragments were directly PCR amplified and Sequenced on an Illumina NextSeq 500 platform to generate 2 × 36-bp paired-end reads.

Reads were mapped to the mouse genome (mm9) using TopHat (Trapnell et al., 2009) with default parameters. Expression levels were estimated in six conditions (MEFs, OSKM, GTMS, GETM, GETMS_ESM, and GETMS_TSM, in biological duplicates) using cufflinks (Trapnell et al., 2010) with default parameters. ESC and bdTSC RNA-Seq data were similarly reanalyzed from (Benchetrit et al., 2015) (GEO accession, database: {"type":"entrez-geo","attrs":{"text":"GSE64684","term_id":"64684"}}GSE64684).

Gene expression levels (FPKM) for 23,309 genes were analyzed to remove genes with very low (< 10) or very high (> 1000) variance, retaining 7,446 genes. These were then standard normalized and PCA was applied using MATLAB (R2015b, “pca” function). For a closer examination of the three-day reprogramming dynamics, PCA was rerun on the same genes, using six libraries (MEFs, OSKM, GTMS, GETM, and GETMS in ESC and TSC media, in duplicates).

For transcriptional analysis of the various iPSC/ESC clones, raw reads (fastq files) were quality-trimmed and adapters removed with cutadapt (version 1.12). The processed fastq files were mapped to the mouse transcriptome and genome using TopHat (v2.0.14). The genome version was GRCm38, with annotations from Ensembl release 84. Quantification was done using htSeq-count (version 0.6.0). Genes with a sum of counts less than 10 over all samples were filtered out, retaining 21568 genes. Normalization was done with the DESeq2 package (version 1.16.1). PCA was applied and visualized in R (version 3.4.1).

Cluster analysis. Gene expression levels (FPKM) for 23,309 genes in eight RNA-Seq libraries were analyzed. Genes with FPKM lower than 2 in all eight conditions were removed, remaining with 13,309 genes. These were log2 transformed, and then multiplicatively normalized to maximal expression of 1 per gene. Genes were then clustered using spectral clustering algorithm with K = 19 (Ng et al., 2001). Briefly, this clustering algorithm computes the distance between each pair of genes, and embeds the data in a graph, whose nodes corresponds to genes and edges correspond to the similarity (or adjacency) of their expression patterns. Then, K densely connected components ( = K clusters of similarly expressed genes) are identified (using K-means, with 100 random starting points). K was selected as an optimal tradeoff between generalization (fewer clusters) and specificity – as shown in Figure 4C each cluster offers a unique expression profile.

Paired-end reads were mapped using Bowtie (Langmead, 2010) to the mouse genome (mm9), using max insert size of 2000. Only unique hits with up to 3 mismatches were retained. Genome-wide paired-end read coverage was then calculated and normalized to a total primary data size of 150M bases (using UCSC’s bigWigInfo program). ATAC-Seq peaks were called using the MACS2 function bdgpeakcall (Zhang et al., 2008), with min-length and max-gap parameters set to 500 bp. The top 50K peaks were then selected for each experiment, and annotated using HOMER’s annotatePeaks.pl program (Heinz et al., 2010), using default parameters. Metagene plot was created from the ChIP-Seq bigwig files using deepTools plotProfile.

For each peak, we used UCSC’s bigWigAverageOverBed program with -minMax option to compute the maximal ATAC-Seq signal for each of the eight conditions. These peaks were then united and cluster analysis was performed as described above, with K = 18. Metagene and heatmap plots were created from the ATAC-Seq bigwig files using deepTools (Ramírez et al., 2016).

The clustered ATAC-Seq peaks (Intronic+distal peaks among top 50K ATAC-Seq peaks, Figure 4D) were systematically analyzed for enriched transcription factor binding sites using HOMER (Heinz et al., 2010). Specifically, we compared the genomics Sequences at GETMS accessible regions against accessible Sequences from the GETM ATAC-Seq peaks as background. This was done using HOMER’s findMotifsGenome.pl program. In addition, we compared the motif enrichment of each cluster versus the overall enrichment in all other classes of intronic and distal ATAC-Seq peaks. This analysis highlighted several motifs, associated with various transcription factors including Esrrb (1e-128) and others (Table S4). We then identified the ATAC-Seq peaks that contain the Esrrb motif (HOMER’s “findMotifsGenome.pl –find esrrb.motif” option), and used GREAT to analyze the specific annotations (GO and others) enriched with those genes.

Paired-end reads were mapped to the genome (mm9) using Bowtie (Langmead, 2010) using max insert size of 1000, mapping unique hits with up to 3 mismatches. Genome-wide paired-end read coverage was calculated and normalized to 20M reads. ChIP-Seq peaks were called using MACS (Zhang et al., 2008), using a p value threshold of 1e-3, shift size of 2Kb, short local window of 10Kb and long local window of 50Kb. Differential peaks were identified using DiffBind version 2.4.8 (Stark and Brown, 2011). Annotation was performed using ChIPseeker version 1.12.1 (Yu et al., 2015). Data for MEF control were taken from database: {"type":"entrez-geo","attrs":{"text":"GSE36292","term_id":"36292"}}GSE36292 (Chang et al., 2014).

Reads from our study and three studies (Li et al., 2017, Parenti et al., 2016, Zhao et al., 2015) describing a XEN-like signature (database: {"type":"entrez-geo","attrs":{"text":"GSE73631","term_id":"73631"}}GSE73631, {"type":"entrez-geo","attrs":{"text":"GSE77550","term_id":"77550"}}GSE77550, and {"type":"entrez-geo","attrs":{"text":"GSE97721","term_id":"97721"}}GSE97721) were aligned to the Ensembl mouse genome version mm9 (NCBI37) using STAR 2.5.2b (default parameters). Uniquely aligned reads in BAM format were annotated against the protein-coding mRNA regions using SeqMonk v 1.38.2 platform (Babraham Bioinformatics, Cambridge UK). Raw counts per mRNA, strand-specific, merging isoforms were quantified using SeqMonk. Differential expression analysis was using R (version 3.4.1) and packages DESeq2 and EdgeR. The Venn diagram and heatmap were generated by comparing day 3 RNA-Seq data of MEFs, GETMS, GETM and GTMS induced cells and aligning them with genes related to OSKM-induced XEN cells (Parenti et al., 2016) or chemically-induced XEN cells. Each study was processed independently to obtain upregulated differentially expressed genes of MEF and chemically-induced XEN cells or MEF and OSKM-induced XEN cells. In our study we tried to exclude all genes that were upregulated in GETM-induced cells. For each study, upregulated differentially expressed genes identified using DESeq2 and EdgeR packages were merged as one list. A Venn diagram was constructed from the four lists to explore the overlap between the four studies. We identified 96 genes that were upregulated in our study and shared at least once with another study. The significance of the overlaps between our study and the other three studies were explored by the hypergeometric test and showed p value < 2.834E-16, 2.874E-07, and 7.835E-11.