For JAK inhibitor assay, Fgf4-induced stem cells were cultured without feeders for 48 h in trophoblast stem-cell culture medium supplemented with 0.6 μM JAK inhibitor (CalBiochem, 420097). As a control, ES cells were also cultured for 48 h in ES medium supplemented with 0.6 μM JAK inhibitor. After the JAK inhibitor treatment, cells were collected and their gene expression was analysed by RT–PCR. For MEK inhibitor assay, dissociated Fgf4-induced stem cells were plated in either LIF containing ES medium supplemented with 1 μM MEK inhibitor (PD025901) or FGF4 containing trophoblast stem cell medium supplemented with 1 μM MEK inhibitor for 48 h. As controls, dissociated Fgf4-induced stem cells were co-plated with 5% or 50% of ES cells into the same culture conditions. After the MEK inhibitor treatment, colonies that formed in each culture condition were counted.
Fgf4-induced stem cells were dissociated into single cells and were suspended in 0.5% BSA PBS. Suspended cells were Fc-blocked by treatment with 1 μg of mouse IgG per 10(to yhe power of 5) cells for 15 min at room temperature. PE-conjugated integrin α7 antibody (R&D system, FAB3518P, dilution at 1:10) was added into cell suspension, and cells were incubated for 30 min on ice. Finally, cells were rinsed with PBS three times and propidium iodide was added for dead cell elimination. As a control, Fgf4-induced stem cells in a separate tube were treated with PE-labelled rat IgG2B antibody. Integrin α7-positive and -dim cells were sorted by FACS aria II (BD).
RNA sequencing and ChIP sequencing analyses
RNA-sequencing of cell lines was performed with biological duplicate samples. Total RNA was extracted from T cells by the RNasy mini kit (Qiagen). RNA-seq libraries were prepared from 1 μg total RNAs following the protocol of the TruSeq RNA Sample Prep kit (Illumina) and subjected to the deep sequencing analysis with Illumina Hi-Seq1000. A cluster tree diagram of various cell types was obtained from hierarchical clustering of global expression profiles (log2 FPKM of all transcripts; FPKM, fragments per kilobase of transcript per million mapped reads). Complete linkage method applied to 1 − r (r = Pearson’s correlation between profiles) was used for generating the tree and 1,000 cycles of bootstrap resampling were carried out to obtain statistical confidence score in % units (also called AU P values). For the analysis that included morula and blastocyst embryos (only small amounts of RNA can be obtained from them), we used pre-amplification with the SMARTer Ultra Low RNA kit for Illumina Sequencing (Clontech Laboratories). Differentially expressed genes were identified by the DESeq package23.
ChIP-seq libraries were prepared from 20 ng input DNAs, 1 ng H3K4me3 ChIP DNAs, or 5 ng H3K27me3 ChIP DNAs using the KAPA Library Preparation kit (KAPA Biosystems). TruSeq adaptors were prepared in-house by annealing a TruSeq universal oligonucleotide and each of index oligonucleotides (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′, and 5′-GATCGGAAGAGCACACGTCTGAACTCCAGTCACXXXXXXATCTCGTATGCCGTCTTCTGCTTG-3′; where X represents index sequences).
Chromatin immunoprecipitation was performed as follows. Cells were fixed in PBS(-) containing 1% formaldehyde for 10 min at room temperature. Glycine was added to a final concentration of 0.25 M to stop the fixation. After washing the cells twice in ice-cold PBS(-), cells were further washed in LB1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) and LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA). Cells were then re-suspended in lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS). Lysates were prepared by sonication using COVARIS S220 in a mini tube at duty cycle = 5%, PIP = 70, cycles per burst = 200, and the treatment time of 20 min. Lysates from 2 × 106 cells were diluted in ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS). ChIP was performed using sheep anti-mouse IgG beads (Invitrogen) or protein A beads (Invitrogen) coupled with anti-histone H3K4me3 antibody (Wako, catalogue no. 307-34813) or anti-histone H3K27me3 antibody (CST, catalogue no. 9733), respectively.
After 4–6 h of incubation in a rotator at 4 °C, beads were washed five times in low-salt wash buffer (20 mM Tris HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), and three times in high-salt wash buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS). Target chromatin was eluted off the beads in elution buffer (10 mM Tris-HCl pH 8.0, 300 mM NaCl, 5 mM EDTA, 1% SDS) at room temperature for 20 min. Crosslink was reversed at 65 °C, and then samples were treated with RNaseA and proteinase K. The prepared DNA samples were purified by phenol-chloroform extraction followed by ethanol precipitation and dissolved in TE buffer.
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We thank S. Nishikawa and N. Love for discussion and M. Ohgushi, S. Kuraku, M. Eiraku, S. Ohtsuka and K. Kakiguchi for help with experimental planning, material preparation and analyses. Financial support for this research was provided by Intramural RIKEN Research Budget (H.O., T.W. and Y.S.), a Scientific Research in Priority Areas (20062015) to T.W., the Network Project for Realization of Regenerative Medicine to Y.S., and Department of Anesthesiology, Perioperative and Pain Medicine at Brigham and Women’s Hospital to C.A.V.
H.O. and Y.S. wrote the manuscript. H.O., Y.S., M.K., M.A., N.T., S.Y. and T.W. performed experiments, and M.T. and Y.T. assisted with H.O.’s experiments. H.O., Y.S., H.N., C.A.V. and T.W. designed the project.
Competing financial interests
The authors declare no competing financial interests.
Haruko Obokata or
Teruhiko Wakayama or
RNA-seq and ChIP-seq files have been submitted to the NCBI BioSample databases under accessions SAMN02393426, SAMN02393427, SAMN02393428, SAMN02393429, SAMN02393430, SAMN02393431, SAMN02393432, SAMN02393433, SAMN02393434 and SAMN02393435.
1. Extended Data Figure 1: Placental contribution of STAP cells. (295 KB)
a, Chimaeric mouse with STAP cells derived from CD45+ cells of B6GFP × 129/Sv mice (B6GFP, C57BL/6 line with cag-gfp transgene). Arrows indicate a placenta and a yolk sac. b, Cross-sections of yolk sac (top) and placenta (bottom). GFP-positive cells (arrows) were seen only in yolk sac and placenta of the STAP cell chimaera. Scale bars, 50 μm. c, Co-immunostaining showed that these GFP-positive cells (right) were found in the extra-embryonic endoderm-derived epithelial cells (pan-cytokeratin+ and overlying laminin+ basement membrane; left) of the yolk sac. Scale bar, 10 μm.
2. Extended Data Figure 2: Trophoblast differentiation potential of Fgf4-induced stem cells. (749 KB)
a, b, Immunostaining (cross-section) of placentae obtained in the blastocyst injection assay with GFP (constitutive)-labelled ES cells (upper) or Fgf4-induced stem cells (bottom). Brown shows pan-cytokeratin and red shows GFP (ES cell or Fgf4-induced stem cell contribution). Regions indicated in a are shown in b. Fgf4-induced stem cells contributed to all layers of placentae, whereas no contribution was observed with ES cells. a, Scale bars, 5 mm. b, Scale bars, 50 μm. c, Pluripotent marker expression of Fgf4-induced stem cells. Scale bars, 50 μm.
d, e, Effects of Fgf4 withdrawal from Fgf4-induced stem cell culture. Unlike trophoblast stem cells (d, left), which generated multi-nucleated large cells (arrow) in the absence of Fgf4, Fgf4-induced stem cells (d, right) simply stopped proliferation and gradually died on Fgf4 withdrawal. Scale bars, 50 μm. This finding suggests that placental differentiation of Fgf4-induced stem cells in vivo may involve more than just Fgf4 signal suppression. e, The number of 4N and 8N cells increased within 6 days of Fgf4 withdrawal in trophoblast stem cells but not in Fgf4-induced stem cells.
3. Extended Data Figure 3: Transcriptome analyses of STAP cells shown by heat maps. (494 KB)
a, Heat maps of expression profiles of top-ranked up- and downregulated genes in STAP cells (Oct4-GFP+ clusters converted from CD45+ cells) compared to ES cells. Their respective expression levels in STAP stem cells, trophoblast stem cells and Fgf4-induced stem cells are shown. Absolute expression values are scaled by log2. The genes expressed differentially between ES cells and STAP cells tended to show more similar expression profiles to ES cells in STAP stem cells and Fgf4-induced stem cells than in trophoblast stem cells. Expression of some early endodermal lineage genes such as Gata4 and Sox17 was moderately elevated in STAP cells as compared to ES cells, whereas its biological significance remains elusive (these genes are shown to be strongly expressed in Oct4-GFP-dim cells1). b, Heat maps of expression profiles of top-ranked up- and downregulated genes in ES cells compared to CD45+ cells and their respective expression levels in STAP cells.
The genes expressed differentially between CD45+ and ES cells tended to show similar expression profiles in ES cells and STAP cells. c, Heat maps of expression profiles of representative genes implicated in haematopoietic lineage development in CD45+, ES and STAP cells. No strong correlation was seen between CD45+ cells and STAP cells in their expression profiles (a similar tendency of no correlation was seen for the data in b).
4. Extended Data Figure 4: Transcriptome analyses for genes implicated in cell-cycle control and induced pluripotent stem-cell conversion. (452 KB)
a, Comparison of expression values of genes involved in cell-cycle control in ES and STAP cells; the G to M cell cycle phases (upper), the cell cycle checkpoint and cell cycle arrest (middle), and the cell cycle regulation (bottom) are shown. Expression level was measured by log2 of mean normalized counts. b, Heat map for upregulated genes in cells undergoing reprogramming by ‘Yamanaka factors’. c, Heat maps for upregulated genes in pre-iPS cells (top) and in partially reprogrammed cells by Yamanaka factors (bottom). Expression level was measured by log2 of mean normalized counts. Differentially expressed genes were identified by the DESeq package and only genes with a false discovery rate of 1% were selected for comparison, unless mentioned otherwise.
5. Extended Data Figure 5: Responses of Fgf4-induced stem cells to signal modifications. (333 KB)
a–f, JAK inhibitor treatment assay for Fgf4-induced stem cells. Fgf4-induced stem cells were cultured under feeder-free conditions and treated with 0.6 μM JAK inhibitor for 48 h. JAK inhibitor treatment assay eliminated ES cells (Oct4-GFP+) from the culture (a, b). The level of Oct4-GFP expression in Fgf4-induced stem cells, which was moderate, was maintained even after JAK inhibitor treatment (c, d; three independent experiments). Scale bar, 100 μm. e, f, For an additional control, Fgf4-induced stem cells were plated in trophoblast stem-cell medium containing Fgf4 together with Oct4-GFP ES cells that constitutively expressed BFP (the number of plated cells was one-tenth of that of plated Fgf4-induced stem cells). Whereas BFP-expressing colonies (ES-cell-derived) still expressed Oct4-GFP in trophoblast stem-cell culture medium after 2 days (e), no Oct4-GFP+ colonies from BFP-expressing ES cells were observed in the JAK-inhibitor-treated culture (f).
g, FACS analysis of integrin α7 expression in Fgf4-induced stem cells. Over 40% of Fgf4-induced stem cells strongly expressed both the pluripotency marker Oct4-GFP and the trophoblast marker integrin α7. The bottom panel shows an isotype control for integrin α7 antibody. In ES cells, integrin-α7-expressing cells were less than 0.1% (data not shown; three independent ES cell lines were examined).
6. Extended Data Figure 6: Characterization of ES-like cells converted from Fgf4-induced stem cells and comparison of STAP cells with early embryos. (316 KB)
a, Immunohistochemistry of ES-like cells for trophoblast and pluripotency markers. ES-like cells converted from Fgf4-induced stem cells no longer expressed the trophoblast marker (integrin alpha 7), but they did express the pluripotency markers (Oct4, Nanog and SSEA-1). Scale bar, 100 μm. b, Pluripotency of ES-like cells converted from Fgf4-induced stem cells as shown by teratoma formation. Those cells successfully formed teratomas containing tissues from all three germ layers: neuroepithelium (left, arrow indicates), muscle tissue (middle, arrow indicates) and bronchial-like epithelium (right). Scale bar, 100 μm. c, MEK inhibitor treatment assay for Oct4-gfp Fgf4-induced stem cells in trophoblast stem-cell medium containing Fgf4. No substantial formation of Oct4-GFP+ colonies was observed from dissociated Fgf4-induced stem cells in MEK-inhibitor-containing medium. Scale bar, 100 μm.
d, Cluster tree diagram from hierarchical clustering of global expression profiles. Red, AU P values. As this analysis included morula and blastocyst embryos from which only small amounts of RNA could be obtained, we used pre-amplification with the SMARTer Ultra Low RNA kit for Illumina Sequencing (Clontech Laboratories). e, f, Volcano plot of the expression profile of STAP cells compared to the morula (e) and blastocyst (f). Genes showing greater than 10-fold change and P value 1.0 × 10(to the power of−6 )are highlighted in red and are considered up- (or down-) regulated in the STAP cells.