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1 Full Methods Derivation of resc cell lines Mouse embryos were isolated from E5.5 - E /sv or ROSA129/Sv females mated with Oct4-ΔPE-GFP transgenic males with a mixed background of MF1, 129/sv, and C57BL/6J strains. The GFP expression of the reporter is under the control of Oct4 promoter and distal enhancer but lacks the proximal enhancer. This GFP transgene shows expression in the ICM of blastocysts and PGC in vivo, and in ES cells. ROSA129 is a knock-in mouse line with a LacZ reporter in the ROSA locus, where LacZ is ubiquitously expressed. Standard ES cell medium consists of DMEM/F12 medium plus 20% Fetal Calf Serum [Gibco, Cat. No: ] and 1000 U/ml LIF. Epiblast from E5.5 - E7.5 postimplantation embryos were obtained from transgenic mice and dissected with glass needles to remove the proximal region and the visceral endoderm, treated with 0.038% EGTA (in 1X PBS) for 10 min, followed by trypsin (0.05% trypsin-0.02% EDTA) for 5 min at room temperature. Epiblast cells were then dissociated into single cells by gently pipetting with a hand-pulled glass capillary of a diameter a little smaller than the epiblast fragment. These resulting single cell suspension was cultured in standard ES cell medium on mitomycin C treated mouse embryonic fibroblast (MEFs) feeder cells, which were pre-cultured (after mitomycin C treatment) for seven days before use. The density of feeder is X 10 4 cells per cm 2. The epiblast cultures grew efficiently and formed flat epithelial-like colonies after 4-7 days culture. The resulting colonies were treated with 1 mg/ml 1

2 collagenase for 10 min at room temperature and isolated from surrounding feeder cells by glass needles, and then transferred to another dish with ES medium. These isolated colonies were further dissociated into smaller clumps by mechanically cutting them into smaller pieces using glass needles; the colonies were passaged regularly on feeder cells at every 3-6 days interval. These colonies, referred to as cepi, could be passaged for at least 20 passages in ES medium following treatment with collagenase and mechanical dissociation. The morphology of cepi colonies was overall similar to that of EpiSCs and not ES cells; they were flatter and bigger than ES cell colonies and positive for alkaline phosphatase, but were negative for Oct4-ΔPE-GFP expression. Following culture of cepi cells for days, around GFP-positive cells appeared in cepi colonies. The clones bearing GFP-positive cells were isolated using glass needles, and passaged following treatment with collagenase and dissociated mechanically. When these GFP-positive cell clusters were approximately 200 μm diameter, they were treated with trypsin, which on subsequent culture produced GFP-positive colonies. We refer to these cells as rescs. These rescs have so far been propagated for more than 40 passages (1:10 to 1:20 dilution/ passage). The number of cell lines we derived from postimplantation embryos is summarised (see Supplementary Table 1). Derivation of EpiSC cell lines E6.5 epiblasts were obtained from 129 female mice mated with either X-GFP transgenic male mice 31 (On a mixed background of 129 & ICR strains) or Oct4-ΔPE-GFP transgenic male mice (On a mixed background of MF1, 129/sv, and C57BL/6J strains). Epiblast cells with the Oct4-ΔPE-GFP reporter were used to generate single cell suspension and cultured in chemically defined medium (CDM) 2

3 (ref. 4), supplemented with Activin A (20 ng/ml, Peprotech, Cat. No ) and bfgf (12 ng/ml, Invitrogen, Cat. No ). EpiSC colonies were formed 3-5 days after culture. EpiSC were passaged every 2-3 days using collagenase. The EpiSC cells with the X-GFP transgenic embryos were prepared as describe previously 4. X-GFP EpiSCs were FACS-sorted to get a pure GFP negative population. The purity of the GFP-negative EpiSCs was confirmed by visual inspection under fluorescence microscope. Then the GFP negative EpiSCs were used for reprogramming to rescs. The EpiSCs (Oct4-ΔPE-GFP EpiSCs at Passage 20 and GFP-negative X-GFP EpiSCs at passage 23) were treated by collagenase for 8min at room temperature. Then the EpiSC colonies were removed from the feeder layer and cut into small pieces by glass needle. After that they were transferred into new dishes with feeders and standard ES cell medium. After 10 to 20 days, we detected GFP-positive cells. When the GFP-positive cell clusters reached 100 to 200 μm in diameter, we dissociated them by treatment with trypsin into single cell suspension and passaged into new dishes with feeders and standard ES medium. Microarray analysis For microarray analysis, 2 μg total RNAs were converted to cdnas by reverse transcription. Then, double-stranded cdnas were subjected to in vitro transcription (IVT) to synthesize Digoxigenin-labeled crna using the Applied Biosystems NanoAmp RT-IVT protocol. The quality and yields of crnas were determined by Agilent s 2100 Bioanalizer and nanodrop spectrophotometer. Then, 10 μg of crnas 3

4 was fragmented by crna fragmentation buffer at 60 o C for 30 min and subsequently prepared for hybridization to the Applied Biosystems V2.0 Mouse Genome Microarray 32 in accordance with the Applied Biosystems Chemiluminescence Detection Protocol. Following hybridization, arrays were stained, washed and finally scanned using the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer. The full set of cdna microarray data were deposited at Gene Expression Omnibus ( The GEO reference numbers are: GSM to GSM Single-cell cdnas Preparation of Single-cell cdnas was performed following previous detailed description 33. Briefly, a single cell was picked using a capillary and lysed directly. Then the mrnas in the lysate were reversed transcribed into cdnas by poly(t) primer with V1 anchor sequence. After this, the non-reactive primers were degraded by treatment with Exonuclease I. Then a poly(a) tail was added to these first-stand cdnas at 3 -end by using Terminal Deoxynucleotidyl Transferase. Next, the second-strand cdnas were synthesized by using poly(t) primer with V3 anchor sequence. Finally these cdnas were evenly amplified by 20 cycles of PCR. The sequences of all related primers are available in Ref. 33. Real-Time PCR The cdnas from bulk amount (2 μg) of total RNAs or from amplified single cells were diluted 10 to 40 fold. Then 2 μl of diluted cdnas was used for each 20 μl real-time PCR reaction (1X Syber Green PCR Master Mix, 0.2 μm of each primer). All reactions were duplicated. The PCR was carried out as follows: first, 95 o C for 10 4

5 min to activate the Taq polymerase, followed by 40 cycles of incubation at 95 o C for 15 sec (for denaturation), and at 60 o C for 1 min for annealing and extension. Finally a dissociation step was run to exclude the possibility of non-specific amplification. Immunostaining Cells were briefly washed with PBS and fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were permeabilized for 30 min with 1% BSA and 0.1% Triton X-100 in PBS. Antibody staining was carried out in the same buffer at 4 C overnight. The slides were subsequently washed three times in 1% BSA, 0.1% Triton X-100 in PBS (5 min each wash), were incubated with secondary antibody for 1h at room temperature in the dark, washed once for 5 min in 1% BSA, 0.1% Triton X-100 in PBS and twice for 5 min in PBS. The slides were then mounted in Vectashield with DAPI (Vector Laboratories) and imaged using a BioRad Radiance 2100 confocal microscope. Primary antibodies used were: mouse monoclonal Oct4 (BD Biosciences, 1:200), rabbit polyclonal Nanog (Abcam, 1:100), rabbit polyclonal Sox2 (Abcam, 1:100), rabbit polyclonal H3K27me3 (Upstate, 1:500), rat monoclonal E-cadherin (TaKaRa, 1:40), and rabbit polyclonal N-cadherin (Abcam, 1:300). All secondary antibodies used were Alexa Fluor highly crossed adsorbed (Molecular Probes). LacZ Staining The embryos were isolated and washed twice in PBS. Then they were transferred to a cold solution (2% Formaldehyde, 0.2% Glutaraldehyde, 0.02% NP-40, 1mM MgCl 2, 0.01% Sodium deoxycholate in PBS) and fixed for 2 hours at 4 o C on a rocking platform. The embryos were washed three times in PBS for 2 min each, and stained in lacz staining solution (0.4 mg/ml Xgal, 4 mm Potassium ferrocyanide, 4 mm 5

6 Potassium ferricyanide, 1 mm MgCl 2, 0.02% NP-40, in PBS) at 30 o C for hours in dark. Alkaline phosphatase (AP) staining AP staining was carried out using AP staining kit from Roche according to manufacture's instructions. Briefly, the cells were fixed by 4% paraformaldehyde for 10 min, then they were stained by AP staining solution overnight at room temperature. Western Blotting The cells were lysed in RIPA lysis buffer (50 mm Tris (ph8.0), 150 mm sodium chloride, 1% NP40, 0.25% Sodium deoxycholate, 0.1% SDS). Equivalent amounts of protein extract were separated by 10% SDS-PAGE and transferred to a PVDF membrane (Amersham GE). Membranes were blocked with 5% BSA in TBS and incubated with primary antibody overnight at 4 C. Membranes were rinsed and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature, followed by chemiluminescent detection using ECL Western blotting detection reagents (Amersham GE). The primary antibodies used were: rabbit polyclonal STAT3 p-y705 (9131; Cell Signalling, 1:1500), and mouse monoclonal anti-tubulin (clone B-5-1-2; Sigma, 1:4000). Blocking of LIF-STAT3 signalling cepi cells and rescs were treated by 0.6 μm JAK inhibitor I (Calbiochem, Cat No: ) or vehicle for three days; the culture medium was changed everyday. Then the cepi cells were treated with collagenase for 10 min at room temperature and the colonies were isolated by glass needle and collected by pipette tips. The rescs were 6

7 treated with trypsin at 37 o C for min and the rescs were re-plated to gelatin-coated dish and incubate for 30 min to remove feeder cells. Then the cepi cells and rescs were lysed and extract total RNA by RNeasy Mini Kit (Qiagen). Bisulphite Sequence Analysis Bisulphite sequence analysis of the genomic DNA isolated from Epiblast, EpiSCs, cepi cells, rescs was carried out by EpiTectBisulfite Kit (QIAGEN). The primer sequences and PCR conditions for amplification of Stella and Rex1 sequences were as described previously 20,34, and for Peg1, Peg3, and Snrpn sequences were followed by Lucifero 35. The PCR products were cloned using pgem-t Easy Vector System I (Promega) and sequenced by Cogenics. 7

8 Supplementary Figure 1. Gene expression profile (Ct values) of cepi cells and rescs. (a-d) Single cell cdna real-time PCR of a cepi colony with the emerging Oct4- PE-GFP-positive rescs compared with rescs at Passage 4. Note upregulation of pluripotency specific genes in nascent rescs compared to cepi cells. (e) A cepi colony with emerging rescs; A culture of resc colonies (Passage 4) is shown in the adjacent panel. Supplementary Figure 2. Gene expression in cepi cells and rescs. (a) A culture of rescs with stella-gfp reporter indicated by white arrowhead; Expression of the reporter is detected in 20-30% of the cells, which is also the case in ES cells 20. (b) Immunostaining for Oct4, Nanog and Sox2 in cepi cells and rescs. Scale bar: 100 μm. Supplementary Figure 3. Unsupervised heatmap of cdna microarray of cepi cells, rescs at early (P4) and late (P24) passages, and ES cells. All of the raw and normalized data have been deposited at NCBI, GEO: GSM to GSM Supplementary Figure 4. LIF-STAT3 signalling for cepi cells, and rescs. (a) Western blot showing phosphorylated STAT3 in cepi cells, rescs and control ES cells. α-tubulin is used as loading control. (b) Response of cepi cells and rescs to JAK inhibitor after three days was monitored by real-time PCR analysis of marker genes. 8

9 Supplementary Figure 5. Epigenetic characterization of rescs. (a) X-chromosome inactivation of E6.5 epiblast in vivo. The inactivated X-chromosome is stained by anti-h3k27me3 antibody. (b) X-chromosome inactivation in EpiSCs. The inactivated X-chromosome is stained by anti-h3k27me3 and Ezh2 antibodies. (c) Imprinting status of Peg1, Peg3, & Snrpn in three resc cell lines: E6.5 cepi-derived rescs, E7.5 cepi-derived rescs, EpiSC-derived rescs determined by bisulphite sequencing. Scale bar: 20 μm. Supplementary Figure 6. Reprogramming of E6.5 epiblast-derived EpiSCs to generate rescs. (a) Derivation of resc line from Oct4- PE-GFP EpiSCs. Note the appearance of clusters of Oct4-GFP-positive cells in EpiSC colonies indicated by black arrowheads, and corresponding white arrowheads. Also note that the resc colonies are uniformly AP positive. (b) Derivation of resc line from X-GFP EpiSCs. Note the appearance of clusters of X-GFP-positive cells in EpiSC colonies. Scale bar: 100 μm. Supplementary Figure 7. Contribution of rescs to chimeric mice and germline transmission. (a) Progeny derived from chimera with rescs showing germline transmission indicated by black arrow. (b) E6.5 chimera with ROSA-lacZ rescs; Note contribution of early passage rescs both to the epiblast and to the extraembryonic ectoderm (ExE), a derivative of trophectoderm cells. (c) E6.5 chimera derived with ROSA-lacZ ES cells; Note contribution predominantly to the epiblast. (d) Blastocysts from wildtype female mice mated with a chimeric male with rescs carrying the Oct4- PE-GFP reporter. (e, f) E13.5 gonads of F1 embryos from wildtype female mice mated with a chimeric male with rescs carrying 9

10 Oct4- PE-GFP reporter. (g) A gonad from an E13.5 chimeric male embryo obtained as described above. Note contribution to germ cells in the gonad. (h) Wholemount lacz staining of E15.5 chimera (Left) with rescs carrying ROSA-lacZ reporter. A wildtype E15.5 embryo (Right) was used as a control. (i) E13.5 gonad of a chimeric embryo with EpiSC-derived rescs carrying Oct4- PE-GFP reporter. Note contribution to germ cells in the gonad. Supplementary Table 1. Derivation of rescs from postimplantation embryos. The embryos were isolated from E5.5 - E /sv or ROSA129/Sv females mated with Oct4- PE-GFP transgenic males. The epiblast part was dissected and digested into single cell suspension. cepi colonies emerged after 4-7 days in culture, and rescs were observed after days. Supplementary Table 2. Summary of origin, culture conditions, epigenetic status, and functional properties of different pluripotent stem cell lines. Supplementary Table 3. JAK Inhibitor I effect on the derivation of cepi & resc cell lines from mouse E6.5 embryos. Supplementary Table 4. Summary of production of adult chimeras from rescs. About resc from SQ1.1, SQ1R.4, and R5N lines were injected into C57BL/6 blastocysts. Then the blastocysts were transferred into uteri of pseudo-pregnant mice and full-term pups were obtained. The adult chimeric mice were determined by coat color (Agouti coat color is from the donor rescs). The male chimeric mice were mated with C57BL/6 female mice and the agouti coat color offspring (F1) confirmed 10

11 germline transmission of all three resc cell lines tested. Supplementary Table 5. Summary of production of E13.5 chimeric embryos from rescs. About rescs were injected into C57BL/6 blastocysts. Then the blastocysts were transferred into uteri of pseudo-pregnant mice and E13.5 embryos were collected. The green florescence emitted from primordial germ cells within the gonads proved the germline transmission for both early and late passages of rescs (Supplementary Fig. 7). Supplementary Table 6. Summary of production of E6.5 chimeric embryos from rescs and ES cells. About Oct4- PE-GFP and ROSA-lacZ double reporter rescs of early (Passage 3) and late (Passage 22 and Passage 40) passages were injected into C57BL/6 blastocysts. A control ROSA-lacZ reporter ES cell line was used in parallel. Then the blastocysts were transferred into uteri of pseudo-pregnant mice and E6.5 embryos were collected. LacZ staining of these embryos showed the contribution of rescs to either Epiblast or Extraembryonic Ectoderm (Supplementary Fig. 7b, c). 11

12 References 31. Hadjantonakis, A.K., Gertsenstein, M., Ikawa, M., Okabe, M., & Nagy, A. Non-invasive sexing of preimplantation stage mammalian embryos. Nat. Genet. 19, (1998). 32. Kuo, W.P. et al. A sequence-oriented comparison of gene expression measurements across different hybridization-based technologies. Nat. Biotechnol. 24, (2006). 33. Kurimoto, K., Yabuta, Y., Ohinata, Y., & Saitou, M. Global single-cell cdna amplification to provide a template for representative high-density oligonucleotide microarray analysis. Nat. Protoc. 2, (2007). 34. Lane, N. et al. Resistance of IAPs to methylation reprogramming may provide a mechanism for epigenetic inheritance in the mouse. Genesis 35, (2003). 35. Lucifero, D. et al. Methylation Dynamics of Imprinted Genes in Mouse Germ Cells. Genomics 79, (2002) 12

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20 Supplementary Table 1 Derivation of resc lines from mouse postimplantation embryos Transgenic lines Stage of embryos No. of embryos No. of cepi lines No. of resc lines Oct4-ΔPE-GFP E /22 (64%) 6/22 (27%) Oct4-ΔPE-GFP E /17 (59%) 4/17 (24%) Rosa-LacZ/Oct4-ΔPE-GFP E /28 (79%) 10/28 (36%) Oct4-ΔPE-GFP E /23 (30%) 5/23 (22%) 20

21 Supplementary Table 2. Origin, culture conditions, epigenetic status, and functional properties of different pluripotent stem cell lines. Cell Line Origin Culture Condition Chimera Formation X-inactivation Methylation of References Stella & Rex1 ES Blastocyst LIF, FCS Somatic and germline X-activation Unmethylated Ref. 5, 11 contribution hes Blastocyst bfgf, Activin A ND X-inactivation ND Ref. 31 EPL Blastocyst Conditioned medium No ND ND Ref. 12,13 E5.5 Epiblast FAB-SC Blastocyst bfgf, Activin A, BIO No* ND ND Ref. 14 EpiSC Epiblast (E5.5 - E6.5) bfgf, Activin A No X-inactivation Methylated Ref. 3, 4; This study resc Epiblast (E5.5 - E7.5) EpiSC LIF, FCS Somatic and germline X-reactivation Demethylated This study contribution ips Somatic cells LIF, FCS Somatic and germline X-reactivation Demethylated Ref. 8 contribution ND: Not Determined *: When FAB-SC are cultured in LIF ES medium, they convert to ES-like morphology and acquired the ability of chimera formation and germline transmission. 21

22 Supplementary Table 3 JAK Inhibitor I effect on the derivation of cepi & resc lines from mouse E6.5 embryos Condition cepi resc Standard (LIF + FCS) 8/15 (53%) 4/15 (27%) JAK Inhibitor I (No LIF) 0/15 0/15 22

23 Supplementary Table 4 Production of Chimeras from different resc lines No. of No. of No. of No. of Cell line cell passage injected blastocysts full term pups chimeras Male (coat rate) Female (coat rate) SQ1.1 P /18 (78%) 3/14 (21%) 2 (70%, 80%) 1 (40%) SQ1.1 P /16 (19%) 2/3 (67%) 1 (70%) 1 (10%) SQ1R.4 P /20(40%) 8/8(100%) 6 (80-90%) 2 (80%, 90%) R5N P /20(20%) 4/4(100%) 4 (70-90%) 0 All three resc lines tested gave to germline transmission. 23

24 Supplementary Table 5 Contribution of resc in E13.5 chimeric embryos Stage of No. of No. of No. of No. with embryo Cell line passage injected blastocysts collected embryos germline contribution E13.5 SQ1R.4 a P3 10 7/10 (70%) 5/7 (71%) E13.5 SQ1R.4 P /30 (87%) 16/26 (62%) E13.5 SQFW5 b P5 10 6/10 (60%) 2/6 (33%) a: E6.5 cepi-derived resc line (Oct4- PE-GFP/ROSA-lacZ). b: E6.5 EpiSC-derived resc line (Oct4- PE-GFP) 24

25 Supplementary Table 6 Contribution of resc in E6.5 chimeric embryos Stage of No. of No. of No. of No. of No. of No. of embryo Cell line passage injected blastocysts collected embryos chimeras contributed EXE uncontributed ExE E6.5 SQ1R.4 P /30 (80%) 18/24 (75%) 9/18 (50%) 9/18 (50%) E6.5 SQ1R.4 P /30 (77%) 20/23 (87%) 2/20 (10%) 18/20 (90%) E6.5 SQ1R.4 P /15 (87%) 13/13 (100%) 1/13 (8%) 12/13 (92%) E6.5 ES (Control) P /30 (83%) 23/25 (92%) 4/23 (17%) 19/23 (83%) 25