Polycomb repressive complex 2 shields naïve human pluripotent cells from trophectoderm differentiation

The first lineage choice in human embryo development separates trophectoderm from the inner cell mass. Naïve human embryonic stem cells are derived from the inner cell mass and offer possibilities to explore how lineage integrity is maintained. Here, we discover that polycomb repressive complex 2 (PRC2) maintains naïve pluripotency and restricts differentiation to trophectoderm and mesoderm lineages. Through quantitative epigenome profiling, we found that a broad gain of histone H3 lysine 27 trimethylation (H3K27me3) is a distinct feature of naïve pluripotency. We define shared and naïve-specific bivalent promoters featuring PRC2-mediated H3K27me3 concomitant with H3K4me3. Naïve bivalency maintains key trophectoderm and mesoderm transcription factors in a transcriptionally poised state. Inhibition of PRC2 forces naïve human embryonic stem cells into an ‘activated’ state, characterized by co-expression of pluripotency and lineage-specific transcription factors, followed by differentiation into either trophectoderm or mesoderm lineages. In summary, PRC2-mediated repression provides a highly adaptive mechanism to restrict lineage potential during early human development.

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Jie Wang, PhD Senior Editor Nature Cell Biology Tel: +44 (0) 207 843 4924 email: jie.wang@nature.comReviewers' Comments: Reviewer #1: Remarks to the Author: Kumar et al. investigate the role of Polycomb repressive complex 2 (PRC2) in human naïve and primed pluripotent stem cells.By performing MINUTE-ChIP of three histone modifications, the authors compare the epigenetic status between naïve and primed female H9 hESCs, in untreated and EZH2 inhibitortreated conditions.They show that naïve hESCs have higher levels of H3K27me3 and H2Aub genomewide compared to primed hESCs, and the majority of the H2Aub deposition is independent of H3K27me3.Unlike primed hESCs, naïve hESCs have elevated H3K27me3 on the X chromosomes, but this does not contribute to dosage compensation.The authors identify bivalent promoters which are specific to the naïve or primed state or present in both.Depletion of H3K27me3 resulted in changes in gene expression for a certain subset of bivalent genes including GATA3.Based on the derepressed genes in the naïve cells upon EZH2 inhibition, the authors suggest similarity to a rare population normally present in the naïve state and that PRC2 plays a role in counteracting gene expression of extraembryonic lineage markers although only a small fraction activates those in the treated naïve cells.
Overall, the data appear convincing and the presentation is robust (see below for a few visual improvements).Interestingly, the results presented in the current manuscript appear to correct and refine previous findings.Others showed lower global levels of H3K27me3 in naïve compared to primed, while Kumar et al. uses quantitative MINUTE-ChIP and identifies ~3-fold higher level, in line with recent mass-spectrometry-based data.However, some select loci, e.g., Dusp6, shows the same trend both in the current manuscript and Ref. 17. Furthermore, Ref. 14 states that "H3K27me3 peaks undergo genomic redistribution and become preferentially depleted from promoters and gene-body regions, rather than from intergenic regions".Secondly, PRC2 had been shown dispensable in naïve cells, as pluripotency markers did not change.In line with that, Kumar et al. also find that the cells grow the same way in the PRC2 inhibited state, but a fraction of cells lose pluripotency, referred to it as a stochastic event, and a subset of bivalent genes are derepressed.In their bulk assay, they see a loss of pluripotency and gain of new markers such as GATA3, though, in the discussion section, they state the majority of cells maintain pluripotency and do not gain GATA3.The latter point is worth addressing in more detail and possibly adjusting the title.If only a fraction turns trophectoderm genes on and transitions that way, then the title and some claims seem a bit misleading (more below).

Major comments:
-The authors put a major focus on the trophectoderm differentiation potential (see, e.g., title, discussion, though Figure 1-4 cover other aspects); moreover, the data shows (Figure 5a) that markers of multiple extraembryonic lineages are upregulated upon EZH2i (pointed out in a paragraph heading by the authors).With the current data presented, it is not possible to tell if lineage restriction by PRC2 in the naïve state is most prominent for the trophectoderm lineage; thus, the title is not perfectly fitting.
-The authors state that the overall morphology of EZH2i naïve cells does not change, and only a fraction expresses GATA3.Quantification of that fraction would be important to assess the extent of trophectoderm potential (Figure 5c, d).
-Co-staining pluripotency markers and TE markers (Gata3, Cdx2) can show if it is mutually exclusive or not.
-Figure 5e shows that H2Aub is still present over some of the now expressed loci, which seems to be in line with the heterogeneity in response.This should be quantified better.
-Staining for other extraembryonic lineage markers (e.g., Sox17 and Gata4 for PrE) and quantifying the fraction of cells gaining differentiation potential is recommended to determine the gain of differentiation potential of another extraembryonic lineage.Similar suggestion for amnion and yolk sac mesoderm markers.
-The authors compare the EZH2i condition to the intermediate population in naïve cells identified by Messmer et al.There, in single cells, GATA3 and other markers (Figure 4d, e) are co-expressed.If the similarity of intermediate cells to EZH2i naïve cells is present, as suggested by the authors, the marker genes would be co-expressed in single cells.Staining of these markers (ideally different extraembryonic lineages) or single-cell analysis is recommended to address that.
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-The authors identify naïve-specific and primed-specific sets of bivalent promoters.When analyzing the corresponding gene expression changes (Figure 3c), they conclude that EZH2i treatment in naïve cells derepresses naïve-only genes but not primed-only genes.However, they conclude that the same level of change for primed-only genes in EZH2i-treated primed cells is derepression.This conclusion is not supported by the data.A violin plot and heatmap of RNAseq are suggested to complement Figure 2b.Presenting the fraction of genes changing expression upon EZH2i treatment for both the naïve-specific and the primed-specific sets is suggested.
-For TFAP2C, a naïve-specific gene, Kumar et al. show that it is bivalent in the primed state, and depleting H3K27me3 leads to basal activation therein.It would be useful to include this gene in Figure 3e, as the authors base that analysis on this finding.They start by "To generalize this observation," however, as the data does not support it would work like this in general for naïve-specific pluripotency genes, it is important to highlight that in the text and avoid generalization.
-XIST expression seems lower in Figure 2c, and the H3K4me3 level is also lower in EZH2 treated cells, however in Figure 2g, it is indicated with black color that XIST does not change.Clarification would be useful.
-The EZH2i is a key component of the manuscript and while some analysis is provided (reads and IF), it would be important to show a western blot time course for the treatment.H3K27me3 would be most relevant but other could also be included.
-In Figure 1c the effect on DNA methylation is briefly assessed.Overall the chromHMM shows limited differences per features as the authors note.Some features lose indeed H3K27 methylation and gain DNA methylation, but others seem to maintain or even gain (especially the bivalent promoters) H3K27 and still gain DNA methylation.This needs to be broken down in more detail.
-The ChIPs are quantitative and it may be good to discuss some of the changes in enrichment further in the discussion (which is currently more of a recap).How does one have to think about the few-fold changes (up and down) mechanistically…more/less modified histones per locus or more cells that have the enrichment since it's a bulk assessment.
Minor comments: -In the text, there are instances where it is not entirely clear which condition is described (naïve versus primed or untreated versus treated).
-The wording H3K27me3 hypermethylation is a bit confusing.The lysine can't be further methylated, so its of course more about the local enrichment.
-Figure 2b: scale missing -Figure 2c is very small and has many details, while Figure 2d is almost the same size.This can be balanced better between panels.
-Figure 2d: color scale missing Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
-Figure 3a: data visualization in the alluvial plot and the corresponding main text does not help the reader to easily understand the presented results.The three groups described in the text could be shown in a more effective way.Also the panel is huge given its limited information.
-Figure 3c: instead of a violin plot for all genes, a violin plot for the class-specific genes is suggested.
-Figure 4c: The labels on the Venn diagram are unclear, and most numbers described in the corresponding main text do not fit the numbers on the plot, thus making it difficult to follow.
-Figure 4e: color scale missing -Figure 5a: indicate Gata3 on the plot (importantly for TE) and provide clearer annotation of gene names to corresponding dots.
Reviewer #2: Remarks to the Author: In their manuscript, Kumar et al. report that H3K27me3 is hypermethylated in naïve human PSCs and also a new naïve-specific set of bivalent promoters.By inhibiting EZH1/2, the enzymatic subunits of PRC2, H3K27me3 was depleted without changing the H3k4me3 status.Especially, PRC2 inhibition depleted naïve PSC-specific H3K27me3.Interestingly, trophectoderm genes were upregulated after the depletion of H3K27me3 in naïve human PSCs but not in primed human PSCs.The findings provide interesting insight on why naïve human PSCs can differentiate into trophectoderm.However, I have several concerns before publication.
One issue in that the manuscript confuses naïve-derived trophectoderm-like cells and primed-derived BMP-induced cells.The authors often refer to previous reports regarding primed PSC-derived BMPinduced cells but then perform experiments using naïve PSCs(p16-17).Naïve and primed PSCs are different cell types.In addition, recent reports suggest that BMP-induced cells are a counterpart of the amnion-like state, while naïve PSC-derived cells are trophoblast lineage cells (Guo et al., Cell Stem Cell 2021;Io et al., Cell Stem Cell 2021;Zhou et  Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
-The authors observed that EZH2i treatment caused naïve PSCs to express trophectoderm marker genes, but in Fig. 5a, amnion marker genes also seem to be upregulated.Since both amnion and trophoblast express many of the same genes, distinguishing the two by gene sets is difficult.The authors should consider PCA or UMAP using naïve (EZH2i +/-) and primed (EZH2i+/-) cells and human embryo data including trophectoderm and amnion.
-Other ES/iPS cell lines should be used to confirm their observations.Minor points -KLF2 and SOX11 are not shown in Fig. 1e.-Fig.5a Please show each gene set and the expression level in a table.Color codes look the opposite of what is described in the legend.
-Extended Data Fig. 3 c, d, e, Fig. 4 a, c.What are the color codes?
Reviewer #3: Remarks to the Author: In this manuscript, Kumar and collaborators aimed to clarify the molecular mechanisms that limit the conversion of human embryonic stem cells (hESCs) into extra-embryonic cell lineages.For that, they performed a quantitative epigenome profiling (MINUTE-ChIP), on H9 female hESCs, for three specific histone posttranslational modifications (H3K4me3, H3K27me3, and H2AK119ub), together with transcriptome analysis, in four different experimental conditions (naïve/primed-state plus/minus EZH2 inhibitor; EPZ-6438).By performing complex computational analysis, the authors provide an exhaustive characterization of the distribution of these histone modifications on naïve and primed hESCs.They use this information to categorize different genomic loci, according to the abundance of each histone mark, with a potential impact hESCs lineage restriction.Upon EPZ-6438 treatment, the authors analysed (1) the expression profile of lineage-specific genes and, (2) performed immunofluorescent of pluripotency factors (e.g.NANOG, SOX2, and OCT3/4) and GATA3, the master TF for trophectoderm development.These analyses enable the authors to postulate a role of the Polycomb Repressive Complex 2 restricting the lineage potential of hESCs.How the cell types are established and mantained during early human development, while other alternative lineages are restricted, is an open and interesting question for both fundamental and biomedical research.In this sense, the characterization of the epigenome dynamics in hESCs at different stages could provide relevant insights into the molecular mechanism controlling cell type specification in vivo.For that, we consider that this is a very useful resource manuscript, with intensive computational analysis, and with some relevant functional information included.
However, (1) we encourage the authors to provide a revision of the narrative of the text, to increase and facilitate understanding; and, (2) authors must provide additional information to strengthen their conclusions regarding the functional role of PRC2 in lineage restriction in hESCs.

Main points:
(1) The amount of interesting epigenomic and transcriptomic data generating or this manuscurpt is clear.However, we are afraid that, despite being a topic very much related to the research area in lur lab, it has been extremely difficult to follow the narrative of the manuscript, which eed to be 'drammatically' rewritten!In addition, using quantitative epigenome profiling, the authors provide a comprehensive categorization of different genomic features.These categories are characterized by gain/loss of histone marks and/or gain/loss of expression in different experimental conditions.To increase the readability of this resource, we would suggest including an informative table with all genomic features categorized in this study (active promoters, bivalent promoters, bivalent promoters de novo, primed-only, naïve-only, common bivalent, H3K4me-only promoters, etc), thus including their abundance and the criteria used for their categorization.
(2) As acknowledged by the authors in their discussion, the suggested functionality of PRC2 on restricting the trophectodermal lineage in naïve hESCs is certainly surprising, as two previous studies indicate a dispensable function of PRC2 for the maintenance of the naïve pluripotent state in hESCs (PMID: 28864533; PMID: 28939884).The authors provided two potential explanations for this inconsistency: (1) the stochasticity differentiation towards trophectoderm of a fraction of hESCs, which might be overlooked in the previous studies; or, (2) a more pronounced phenotype observed in this study by using an EZH2/1 inhibitor, in contrast to a previous study (PMID: 28939884) in which EZH1 expression was reduced in an EZH2-KO background.However, in the first case, the potential stochasticity is not evaluated in the current study.And, in the second case, the current study associates the functional impact of EPZ-6438 treatment to the loss of H3K27me3, although non-specific or secondary effects (e.g. the observed global lost H3K4me3) are not experimentally ruled out.Thus, considering that the role of PRC2 in sustaining naïve hESCs identity is an important conclusion raised by the authors, it needs to be further supported at the experimental level.
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.a) by single-cell RNA sequencing (naïve hESCs +/-EZH2 inh.) to evaluate the proportion of hESCs in naïve state that spontaneously transient to trophectoderm upon EZH2 inhibitor.b) Evaluating the functional impact of full KO of PRC2 core components SUZ12 or EED in naïve hESCs (loss of pluripotency markers, gain of trophectoderm lineage).Check figure references: -Figure 5a, "up" and "down" in the legend are switched -Extended Data Figure 10a is not a barchart, as it says in the figure description, and it does not show what the manuscript refers to.-In the results paragraph: "whereas primed-only bivalent genes were predominantly higher expressed (..) in naive (Fig. 3d -> 3c…) -In the discussion paragraph: "on the other hand, we observed an overall reduction of core pluripotency marker expression on the population level (Extended Data Fig. 7d -> 8c) Extra comments on the comparison of the two co-submissions: At the functional level, although both studies point towards an implication of PRC2 in pluripotent-totrophoblasts transition, there are relevant discrepancies among both studies.Zijlmans et al. findings, well-supported at multiple experimental levels, indidate a function of PRC2 as a barrier during trophoblast induction.This is because, in the absence of instructive signals, PRC2 inhibition does not cause spontaneous differentiation or loss of pluripotency marks in naïve hPSCs.This dispensable role of PRC2 in naïve culture conditions is an observation that seems in line with previously published studies (PMID: 28864533; PMID: 28939884).Only when cultured under trophoblast differentiation media, hPSCs transient more efficiently towards trophoblast cells in the presence of the EZH2 inhibitor.In contrast, Kumar et al. study shows that the treatment with EPZ-6438 for 7 days results in the spontaneous differentiation of a fraction of naïve hESCs (yet to be quantified) towards trophoblast cells, in the absence of inductive signals.This would suggest that PRC2 functions as the active blocker of trophoblast differentiation.We agree that the discrepancies might result from technical differences between both studies.
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Author Rebuttal to Initial comments
We would like to thank all reviewers for their insightful comments and suggestions.As we detail below, in addition to various additional analyses, we have performed major experiments to address the two key concerns:  -6).We have substantially rewritten the main text and also removed a number of sections that became peripheral to the overall scope of the manuscript.Due to the substantial editing, we note that the track-change version of the revised manuscript became quite fragmented, and we apologize for the inconvenience.
As detailed in the point-by-point responses below, we have adjusted the title of our manuscript to reflect the additional insight gained from scRNA-seq (but fully consistent with our prior bulk data), that mesoderm lineage cells are generated in addition to trophectoderm.
Reviewers' Comments: Reviewer #1: Remarks to the Author: Kumar et al. investigate the role of Polycomb repressive complex 2 (PRC2) in human naïve and primed pluripotent stem cells.By performing MINUTE-ChIP of three histone modifications, the authors compare the epigenetic status between naïve and primed female H9 hESCs, in untreated and EZH2 inhibitor-treated conditions.They show that naïve hESCs have higher levels of H3K27me3 and H2Aub genome-wide compared to primed hESCs, and the majority of the H2Aub deposition is independent of H3K27me3.Unlike primed hESCs, naïve hESCs have elevated H3K27me3 on the X chromosomes, but this does not contribute to dosage compensation.The authors identify bivalent promoters which are specific to the naïve or primed state or present in both.Depletion of H3K27me3 resulted in changes in gene expression for a certain subset of bivalent genes including GATA3.Based on the derepressed genes in the naïve cells upon EZH2 inhibition, the authors suggest similarity to a rare population normally present in the naïve state and that PRC2 plays a role in counteracting gene Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.expression of extraembryonic lineage markers although only a small fraction activates those in the treated naïve cells.
Overall, the data appear convincing and the presentation is robust (see below for a few visual improvements).Interestingly, the results presented in the current manuscript appear to correct and refine previous findings.Others showed lower global levels of H3K27me3 in naïve compared to primed, while Kumar et al. uses quantitative MINUTE-ChIP and identifies ~3-fold higher level, in line with recent mass-spectrometry-based data.However, some select loci, e.g., Dusp6, shows the same trend both in the current manuscript and Ref. 17. Furthermore, Ref. 14 states that "H3K27me3 peaks undergo genomic redistribution and become preferentially depleted from promoters and gene-body regions, rather than from intergenic regions".
We thank the reviewer for this assessment and we agree that our study corrects and refines conclusions drawn by prior non-quantitative studies.
Secondly, PRC2 had been shown dispensable in naïve cells, as pluripotency markers did not change.In line with that, Kumar et al. also find that the cells grow the same way in the PRC2 inhibited state, but a fraction of cells lose pluripotency, referred to it as a stochastic event, and a subset of bivalent genes are derepressed.In their bulk assay, they see a loss of pluripotency and gain of new markers such as GATA3, though, in the discussion section, they state the majority of cells maintain pluripotency and do not gain GATA3.The latter point is worth addressing in more detail and possibly adjusting the title.If only a fraction turns trophectoderm genes on and transitions that way, then the title and some claims seem a bit misleading (more below).This is an important question which must arise when bulk measurements are acquired from a cell population that is heterogeneous.We were also worried about this fact and we have thus carried out a comprehensive single-cell RNA-seq study (new Figs. 5 and  6).By integrating the profiles of naïve cultures with extensive embryo references we detect a small fraction (1.8%) of trophectoderm-like cells (TLC) and 1.0% mesoderm-like cells (MeLC) confirming a low grade spontaneous differentiation also within naïve culture conditions (Fig. 5).With EZH2i inhibition these two populations increased to 9.1% and 11. 7% respectively after 7 days.This is in good agreement with our immunfluorescent stainings of GATA3, which yielded ~12% GATA+ cells after 7d EZH2i treatment.The transcriptional analysis also resolved a distinct trajectory where cells first exit the naive "goundstate" and become "activated" with increased Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.lineage markers and reduced expression of pluripotency markers such as NANOG and SOX2, but not POU5F1.Following this activation, the cell trajectory bifurcates in two paths towards TLC and MeLC with corresponding induction of lineage markers such as HAND1, CDX1, TBXT, GATA2&3.In the last step where the treated cells adopt TLC and MeLC states POU5F1 is finally reduced most probably resulting in irreversible lineage commitment.Within 7 days, the majority of the treated cells have transitioned from the ground state into or beyond the activated state, suggesting that this is not just a stochastic event in a few cells but a robust response following the removal of an epigenetic barrier.

Major comments:
-The authors put a major focus on the trophectoderm differentiation potential (see, e.g., title, discussion, though Figure 1-4 cover other aspects); moreover, the data shows (Figure 5a) that markers of multiple extraembryonic lineages are upregulated upon EZH2i (pointed out in a paragraph heading by the authors).With the current data presented, it is not possible to tell if lineage restriction by PRC2 in the naïve state is most prominent for the trophectoderm lineage; thus, the title is not perfectly fitting.
We agree that this was an important question that remained inconclusive from our bulk RNA-seq data.Indeed, we have also found an alternative differentiation trajectory towards mesoderm, consistent with the upregulation of mesoderm markers we also observed in our prior Figure 5a (now expanded with additional lineages as Figure 3d).We have therefore followed the reviewer's suggestion and adjusted the title accordingly.
-The authors state that the overall morphology of EZH2i naïve cells does not change, and only a fraction expresses GATA3.Quantification of that fraction would be important to assess the extent of trophectoderm potential (Figure 5c, d).
The added scRNA-seq experiments address this question: between 2 and 4 days EZH2i treatment, the epiblast-like naive hESCs (ELC) population is largely maintained, still accounting for ~95% of all cells at these time points (Fig 5b).At 7 days EZH2i, the fraction of ELC is reduced but still remains the largest population (77%).211 out of 1613 cells assume a TE-like transcriptome whereas 219 cells assume a Mesodermlike transcriptome.As described in resonance above, the scRNAseq analysis further Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.reveals that EZH2 inhibition changes the majority of the cells within the naive cultures as they initiate lineage differentiation towards trophectoderm and mesoderm-like states.However this may not immediately result in a distinctive growth phenotype.Together, this explains why the naïve colonies still retain a normal morphology at 7 days EZH2i.
-Figure 5e shows that H2Aub is still present over some of the now expressed loci, which seems to be in line with the heterogeneity in response.This should be quantified better.
Indeed, we have in Fig 2d (previously Fig 3d) assessed the loss of H2Aub across two sets of bivalent genes, those that gain expression upon depletion of H3K27me3 and those that remain off: Only on the derepressed promoters we observed a 15% of H2Aub.Hence our data suggests a weak anticorrelation of transcriptional activity and H2Aub at bivalent promoters but does not resolve if the modest reduction in H2Aub is prerequisite or a consequence of gene activation.We have now also included H2Aub heatmaps in Figs.2e, 4a, EFig 12a -Staining for other extraembryonic lineage markers (e.g., Sox17 and Gata4 for PrE) and quantifying the fraction of cells gaining differentiation potential is recommended to determine the gain of differentiation potential of another extraembryonic lineage.Similar suggestion for amnion and yolk sac mesoderm markers.This is addressed by the added time-course scRNA-seq experiment already discussed in detail above.Mapping against a comprehensive reference annotation, we identify mesoderm and trophectoderm as the dominating lineages arising from EZH2i inhibition, with a very small percentage of amnion (Fig 5b).
-The authors compare the EZH2i condition to the intermediate population in naïve cells identified by Messmer et al.There, in single cells, GATA3 and other markers (Figure 4d, e) are co-expressed.If the similarity of intermediate cells to EZH2i naïve cells is present, as suggested by the authors, the marker genes would be co-expressed in single cells.Staining of these markers (ideally different extraembryonic lineages) or single-cell analysis is recommended to address that.This is addressed by the time-course scRNA-seq experiment already discussed in detail above.Consequently, the analysis of Messmer at.presented in the original 4d,e Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
became redundant and we have removed it in the revised manuscript.However, we have validated that the 'intermediate population' described there expresses similar markers as our TLC and MeLC populations, hence adding further support that these lineages are generated from naïve hESC cultures spontaneously at a low percentage (Extended Data Fig. 11d).
-The authors identify naïve-specific and primed-specific sets of bivalent promoters.When analyzing the corresponding gene expression changes (Figure 3c), they conclude that EZH2i treatment in naïve cells derepresses naïve-only genes but not primed-only genes.However, they conclude that the same level of change for primed-only genes in EZH2i-treated primed cells is derepression.This conclusion is not supported by the data.A violin plot and heatmap of RNAseq are suggested to complement Figure 2b.
These conclusions are based on comparing the behavior of naïve-specific and primed-specific bivalent genes sets to all genes using statistical analysis and both adjusted p values from Wilcoxon test and Cohen's d effect sizes are given in the text.
We have now also included these values in the figure.The statistical test determines if the naive and primed-specific bivalent gene sets are up-or down-regulated significantly as a group, hence individual fold-changes.E.g. the primed-specific bivalent gene set is significantly upregulated as a group in EZH2i-treated primed cells, but the same group of genes shows an non-significant response in EZH2i-treated naive cells (some genes are derepressed but others are downregulated).We have clarified this in the result section to Presenting the fraction of genes changing expression upon EZH2i treatment for both the naïve-specific and the primed-specific sets is suggested.
We have now expanded on this in Fig 3b.
-For TFAP2C, a naïve-specific gene, Kumar et al. show that it is bivalent in the primed state, and depleting H3K27me3 leads to basal activation therein.It would be useful to include this gene in Figure 3e, as the authors base that analysis on this finding.
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We have now included TFAP2C in Fig 2e (former 3e), genome track is now EFig 7a
They start by "To generalize this observation," however, as the data does not support it would work like this in general for naïve-specific pluripotency genes, it is important to highlight that in the text and avoid generalization.
We have substantially rewritten and streamlined the results section to provide a clearer flow and have also rephrased this.
-XIST expression seems lower in Figure 2c, and the H3K4me3 level is also lower in EZH2 treated cells, however in Figure 2g, it is indicated with black color that XIST does not change.Clarification would be useful.Indeed XIST is not significantly up-or downregulated even though it appears so in the genome track.There are several reasons for this: First, per-track normalization as done for genome browser visualization is not a precise way of normalizing.DESeq2 provides a statistical framework for properly normalizing datasets before calculating fold-changes.As a result, genome track and DESeq2 output may show small systematic differences.Second, the RNA-seq track is generated from one out of three biological replicates (always the same one to be consistent).We note that we did not explain this sufficiently in the figure legends since the ChIP-seq tracks show the combined triplicates.We have now clarified in the figure legends if individual replicates or a combined/average track is shown.In this particular case the replicates are quite variable in XIST expression and the tracks shown are thus not necessarily representative of the triplicate comparison (also the reason why DESeq2 returns no significant change).
Since we have shortened the discussion of X chromosome, we have also removed panel 2c from the figures, XIST is now only shown in chromosome overview EFig 4c.
-The EZH2i is a key component of the manuscript and while some analysis is provided (reads and IF), it would be important to show a western blot time course for the treatment.H3K27me3 would be most relevant but others could also be included.
We have now included a time course (0, 2, 4, 7 days EZH2i) as EFig 2e,f.The immunofluorescent staining demonstrates that H3K27me3 is consistently lost in all Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.cells with similar kinetics.
-In Figure 1c the effect on DNA methylation is briefly assessed.Overall the chromHMM shows limited differences per features as the authors note.Some features lose indeed H3K27 methylation and gain DNA methylation, but others seem to maintain or even gain (especially the bivalent promoters) H3K27 and still gain DNA methylation.This needs to be broken down in more detail.
We agree that this was only briefly touched upon in the text and did not provide new insight necessary to understand the further course of the manuscript.We decided to remove the DNA methylation panel and associated discussion entirely in the revised manuscript to streamline the text.
-The ChIPs are quantitative and it may be good to discuss some of the changes in enrichment further in the discussion (which is currently more of a recap).How does one have to think about the few-fold changes (up and down) mechanistically…more/less modified histones per locus or more cells that have the enrichment since it's a bulk assessment.
The scRNA-seq data helps addressing this question.In untreated cells, more than 96% of cells are homogeneous naïve and primed ESC according to the UMAP clustering.Hence the bulk ChIP and RNA-seq data comparing naïve and primed states quantitatively is valid in that it represents the average of a homogeneous population.After 7 days EZH2i, only 77% of cells remain in the ELC cluster, ~10% are MeLC and TLC.Hence it is clear that the bulk epigenome and transcriptome profiles represent this mixed population.However scRNA-seq, bulk RNA-seq and epigenome data agree very well on key trends, e.g. the downregulation of core pluripotency factors in the ELC population is mirrored in a reduction in bulk RNAseq and a loss of H3K4me3 at the respective promoters (EFig 7b).

Minor comments:
-In the text, there are instances where it is not entirely clear which condition is described (naïve versus primed or untreated versus treated).
We have carefully checked that the comparisons are clearly mentioned -The wording H3K27me3 hypermethylation is a bit confusing.The lysine can't Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.be further methylated, so its of course more about the local enrichment.
The term is commonly used for DNA methylation levels, for which the same caveat exists (DNA hypermethylation does not imply more methyl on a C).Technically speaking, de Clerk et. al, Scientific Reports (2019) also demonstrate by mass spectrometry that the increase in H3K27me3 in naive cells comes with a decrease in me1, hence the term may be more appropriate to use for H3K27me3 then for DNA methylation.In any case we have removed the wording in many instances because it implies a higher-than-normal level but H3K27me3 levels in naïve cells are just normal for this cell type.We have used it in one instance to describe the higherthan-normal H3K27me3 accumulation on the X chromosomes.
-Figure 2b: scale missing fixed -Figure 2c is very small and has many details, while Figure 2d is almost the same size.This can be balanced better between panels.fixed -Figure 2d: color scale missing fixed -Figure 3a: data visualization in the alluvial plot and the corresponding main text does not help the reader to easily understand the presented results.The three groups described in the text could be shown in a more effective way.Also the panel is huge given its limited information.
We have condensed the panel -Figure 3c: instead of a violin plot for all genes, a violin plot for the classspecific genes is suggested.
We believe it is important to interpret the class-specific changes against the background trend and hence we use the violin plot for all genes.Statistics on this comparison are now also included in the figure.
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-Figure 4c: The labels on the Venn diagram are unclear, and most numbers described in the corresponding main text do not fit the numbers on the plot, thus making it difficult to follow.
We have corrected this, ensuring that the numbers in the venn diagram are correctly referred to in the text.Supplementary Table 2 also contains annotations to reproduce the numbers and associated gene sets.
-Figure 4e: color scale missing We have replaced this with our own scRNA-seq data in Figure 6 -Figure 5a: indicate Gata3 on the plot (importantly for TE) and provide clearer annotation of gene names to corresponding dots.fixed (now Figure 3d) Reviewer #2: Remarks to the Author: In their manuscript, Kumar et al. report that H3K27me3 is hypermethylated in naïve human PSCs and also a new naïve-specific set of bivalent promoters.By inhibiting EZH1/2, the enzymatic subunits of PRC2, H3K27me3 was depleted without changing the H3k4me3 status.Especially, PRC2 inhibition depleted naïve PSC-specific H3K27me3.Interestingly, trophectoderm genes were upregulated after the depletion of H3K27me3 in naïve human PSCs but not in primed human PSCs.The findings provide interesting insight on why naïve human PSCs can differentiate into trophectoderm.However, I have several concerns before publication.
One issue in that the manuscript confuses naïve-derived trophectoderm-like cells and primed-derived BMP-induced cells.The authors often refer to previous reports regarding primed PSC-derived BMP-induced cells but then perform experiments using naïve PSCs(p16-17).Naïve and primed PSCs are different cell types.In addition, recent reports suggest that BMP-induced cells are a counterpart of the amnion-like state, while naïve PSC-derived cells are trophoblast lineage cells (Guo et al., Cell Stem Cell 2021;Io et al., Cell Stem Cell 2021;Zhou et al Nature 219).
We fully agree that naïve and primed stem cells are distinct in their potential to make trophectoderm cells and did not want to suggest that they are comparable.
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Our single-cell data now clarifies that our naïve cell-derived GATA3+ cells cluster with true trophectoderm lineage (a very small fraction with amnion) and also clearly shows that naïve cells do not transit through a post-implantation-like primed state (only a very small number of naïve cells maps to the primed cluster after EZH2i treatment) to give rise to trophectoderm.
-The authors mentioned the similarity of H3K27me3 hypermethylation in naïve human and mouse PSCs.If true, can they provide some explanation for why mouse ES cells are difficult to differentiate into trophectoderm-like cell type without transgene-expression?If exposed to PRC2 inhibitor, do naïve mouse PSCs differentiate into trophectoderm?PRC2 has been studied quite well and it is a general agreement that mESC are strictly pluripotent, hence neither naïve nor primed mESC can form trophectoderm. E.g. a study on naïve (2i ground state) mouse ESC with EED KO assessed transcriptional changes in the absence of PRC2 (van Mierlo et. al. , 2019).The reported changes were relatively minor, in line with the fact that ground state and naive mouse ESC in general (either LIF+serum or LIF+2i) are viable and maintain pluripotency.We have reanalyzed the published transcriptome dataset for TE markers and did not find a significant upregulation of these (with the exception of some induction of Krt7, Igf2, Gata2).Together, these observations suggest that the naïve mouse ESC culture does not fully resemble human naive conditions and/or the mechanism of TE induction is divergent between species.However, this is a common unknown in the field and not the scope of our study.Hence we have not further pursued this question.
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Main points:
(1) The amount of interesting epigenomic and transcriptomic data generating or this manuscript is clear.However, we are afraid that, despite being a topic very much related to the research area in our lab, it has been extremely difficult to follow the narrative of the manuscript, which needs to be 'dramatically' rewritten!
We have rewritten large parts of the manuscript, in some cases shortening or removing discussions that became peripheral to the main narrative.We have condensed  and 6).We hope this will provide a much more readable manuscript.
In addition, using quantitative epigenome profiling, the authors provide a comprehensive categorization of different genomic features.These categories are characterized by gain/loss of histone marks and/or gain/loss of expression in different experimental conditions.To increase the readability of this resource, we would suggest including an informative table with all genomic features categorized in this study (active promoters, bivalent promoters, bivalent promoters de novo, primed-only, naïve-only, common bivalent, H3K4me-only promoters, etc), thus including their abundance and the criteria used for their categorization.This is a very useful suggestion, we have now included this as Supplementary Table 3.
(2) As acknowledged by the authors in their discussion, the suggested functionality of PRC2 on restricting the trophectodermal lineage in naïve hESCs is certainly surprising, as two previous studies indicate a dispensable function of PRC2 for the maintenance of the naïve pluripotent state in hESCs (PMID: 28864533; PMID: 28939884).The authors provided two potential explanations for this inconsistency: (1) the stochasticity differentiation towards trophectoderm of a fraction of hESCs, which might be overlooked in the previous studies; or, (2) a more pronounced phenotype observed in this study by using an EZH2/1 inhibitor, in contrast to a previous study (PMID: 28939884) in which EZH1 expression was reduced in an EZH2-KO background.However, in the first case, the potential stochasticity is not evaluated in the current study.And, in the second case, the current study associates the functional impact of EPZ-6438 treatment to the loss of H3K27me3, although non-specific or secondary effects (e.g. the observed global lost H3K4me3) are not experimentally ruled out.Thus, considering that the role of PRC2 in sustaining naïve hESCs identity is an Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.important conclusion raised by the authors, it needs to be further supported at the experimental level.a) by single-cell RNA sequencing (naïve hESCs +/-EZH2 inh.) to evaluate the proportion of hESCs in naïve state that spontaneously transient to trophectoderm upon EZH2 inhibitor.
We thank the reviewer for this suggestion and have now performed scRNA-seq over a 2, 4, 7 day time course EZH2i treatment, and also used this data to quantify the fraction of cells transitioning to TE and Mesoderm.See also comments to Reviewer 1. b) Evaluating the functional impact of full KO of PRC2 core components SUZ12 or EED in naïve hESCs (loss of pluripotency markers, gain of trophectoderm lineage).
We thank the reviewer for this suggestion and have addressed this using an acute CRISPR/Cas9 knockout strategy that efficiently depleted EED across clonal naïve hESC colonies.The results presented in Fig. 4e-g, EFig 10b demonstrate GATA3 induction in 16.7% of cells in EED-targeted cultures, compared to 1.4% GATA3+ in untargeted, wildtype cultures.Hence, genetic targeting mirrors the pharmacologic inhibition with EZH2i.

Check figure references:
-Figure 5a, "up" and "down" in the legend are At the functional level, although both studies point towards an implication of PRC2 in pluripotent-to-trophoblasts transition, there are relevant discrepancies among both studies.Zijlmans et al. findings, well-supported at multiple experimental levels, indidate a function of PRC2 as a barrier during trophoblast induction.This is because, in the absence of instructive signals, PRC2 inhibition does not cause spontaneous differentiation or loss of pluripotency marks in naïve hPSCs.This dispensable role of PRC2 in naïve culture conditions is an observation that seems in line with previously published studies (PMID: 28864533; PMID: 28939884).Only when cultured under trophoblast differentiation media, hPSCs transient more efficiently towards trophoblast cells in the presence of the EZH2 inhibitor.In contrast, Kumar et al. study shows that the treatment with EPZ-6438 for 7 days results in the spontaneous differentiation of a fraction of naïve hESCs (yet to be quantified) towards trophoblast cells, in the absence of inductive signals.This would suggest that PRC2 functions as the active blocker of trophoblast differentiation.We agree that the discrepancies might result from technical differences between both studies.Thanks for pointing this out.We have now addressed this point through a time-course experiment where we assay the emergence GATA3+ cells following EZH2 inhibition with IF and the transcriptional changes on a global level using scRNAseq.This analysis revealed that even in untreated naïve cultures there is a low level of trophectoderm differentiation.After 7d EZH2 inhibition we can detect a robust increase in the trophectoderm-like population after.Our analysis also revealed that we also have mesoderm-like cells in the unperturbed naïve state which increases to 11.7% with 7d EZH2 inhibition.These populations emerged through a shared trajectory where most cells in the inhibited naive cultures actually initiate progressive differentiation towards these two populations.Of note, we see early gene expression changes within the Epiblast-like population (ELC) of naïve hESC treated with EZH2i already after 2-4 days (Fig 5b,f), but less than 6% of cells have fully differentiated at this point (Fig 5b), hence most significant population changes happen between day 4 and day 7 in our EZH2i time course.
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To add complementary support, we have performed additional experiments using a second inhibitor of PRC2 (EED226 targeting the EED regulatory subunit), and we have performed acute knockout of EED using CRISPR/Cas9.In both cases, we have observed the appearance of GATA3+ colonies comparable to our initial observations with EZH2i.Together, our data clearly supports the functional importance of PRC2 as an epigenetic barrier in naive cells preventing trophectoderm and mesoderm differentiation.

Decision Letter, first revision:
Subject: Your manuscript, NCB-E46376A Message: Our ref: NCB-E46376A 11th March 2022 Dear Dr. Elsässer, Thank you for submitting your revised manuscript "Polycomb Repressive Complex 2 shields naïve human pluripotent cells from trophectoderm and mesoderm differentiation" (NCB-E46376A).It has now been seen by the original referees and their comments are below.The reviewers find that the paper has improved in revision, and therefore we'll be happy in principle to publish it in Nature Cell Biology, pending minor revisions to satisfy the referees' final requests and to comply with our editorial and formatting guidelines.
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al Nature 2019).-The authors mentioned the similarity of H3K27me3 hypermethylation in naïve human and mouse PSCs.If true, can they provide some explanation for why mouse ES cells are difficult to differentiate into trophectoderm-like cell type without transgene-expression?If exposed to PRC2 inhibitor, do naïve mouse PSCs differentiate into trophectoderm?-Fig.3: Text and Figs do not match.Please revise.
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1)
We have added additional functional experiments that strengthen the claim that PRC2 shields naïve cells from differentiation.We have repeated and expanded our GATA3 staining experiments to include a second, orthogonal inhibitor of PRC2 (EED226, Fig 4b), performed the same experiment in another naïve hESC line (Extended Data Fig 10a), and we have performed an Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.acute genetic targeting of EED using synthetic CRISPR/Cas9 complex (Fig 4e-g, Extended Data Fig 10b).All of these experiments agree on the spontaneous induction of GATA3+ cells within 7d of PRC2 inhibition/deletion. 2) We have performed single-cell RNA-sequencing of a time course of EZH2 inhibition in naïve hESC, including controls in primed hESC and detailed analysis (Figs 5-6), which provide substantial new insights in addition to validating all our previous conclusions.In an effort to streamline the manuscript, we have combined original Figs 1 and 2 into new Fig 1 to accommodate an in-depth analysis of the new scRNA-seq data (Figs. 5 Fig. 2c (previously Fig 3c).Furthermore, we have added differential expression volcano plots for the EZH2i treatments of naïve (Fig 3a) and primed (EFig 7c) and for clarity the same groups as in Fig 2c are shown in the same colors in these plots.
Fig 1 and 2 into one Figure on the background of the substantial new data added (new Fig 5 Figure 10a is not a barchart, as it says in the figure description, and it does not show what the manuscript refers to.fixed -In the results paragraph: "whereas primed-only bivalent genes were predominantly higher expressed (..) in naive (Fig.3d -> 3c…) fixed -In the discussion paragraph: "on the other hand, we observed an overall reduction Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. of core pluripotency marker expression on the population level (Extended Data Fig. 7d -> 8c) fixed Extra comments on the comparison of the two co-submissions: Table, Video, or Note, and numbered continuously (e.g.Supplementary Figure 1, Supplementary Figure 2, Supplementary Table 1, Supplementary Table 2 etc.).
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Decision Letter, final requests:
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