In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states

ABSTRACT Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is

able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a

rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette

pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition

supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with

bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over

both pluripotency progression and morphogenesis pivots from WNT to MEK signals. Access through your institution Buy or subscribe This is a preview of subscription content, access via your

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subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS INDUCTION OF ROSETTE-TO-LUMEN STAGE EMBRYOIDS USING REPROGRAMMING PARADIGMS IN ESCS Article

Open access 16 December 2021 CELL FATE ROADMAP OF HUMAN PRIMED-TO-NAIVE TRANSITION REVEALS PREIMPLANTATION CELL LINEAGE SIGNATURES Article Open access 07 June 2022 FORMATIVE PLURIPOTENT STEM

CELLS SHOW FEATURES OF EPIBLAST CELLS POISED FOR GASTRULATION Article Open access 19 February 2021 DATA AVAILABILITY Figures 3 and 4 have associated RNA-seq and single-cell RNA-seq data

deposited in the GEO and are accessible through GEO series accession number GSE145727. Figure 6 has associated ChIP-seq data deposited in GEO with the accession number GSE112234 . Previously

published RNA-seq data that were re-analysed here are available from ArrayExpress under accession codes E-MTAB-2958 and E-MTAB-5147. Previously published ChIP-seq data that were re-analysed

here are available from the GEO under accession codes GSE23943 and GSE60204. Source data for Figs. 1, 2 and 4–8 and Extended Data Figs. 1, 3–6 and 8 are presented with the paper. All other

data supporting the findings of this study are available from the corresponding author upon reasonable request. CODE AVAILABILITY No custom computer code was used in this study. REFERENCES *

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  Google Scholar  Download references ACKNOWLEDGEMENTS We are grateful to the following colleagues for providing cell lines used in this study: B. Merrill (_Tcf7l1_–/–), A. Smith (RGd2), J.

Wysocka (_Tbx3_-RFP;_Oct6_-GFP), A. Simeone (_Otx2_KO and _Otx2_OE) and M. Kyba (A2lox.Cre). We thank M. Muraro from Single Cell Discoveries for help with single-cell sequencing and data

analysis, and R. van Albada and A. Korporaal for technical assistance. NWO ECHO.10.B1.064, TI Pharma D5-402, Marie Curie FP7-PEOPLE-2009-RG-256560, ZonMW 116006104 (all to D.t.B.), NWO-VIDI

864.12.007 (to H.M.) and FES NIRM (Dutch Innovation Award) supported this study. AUTHOR INFORMATION Author notes * These authors contributed equally: Alex Neagu, Emiel van Genderen, Irene

Escudero. AUTHORS AND AFFILIATIONS * Department of Cell Biology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands Alex Neagu, Emiel van Genderen, Irene Escudero, 

Lucas Verwegen, Dorota Kurek, Johannes Lehmann, Jente Stel, Alex Maas, Yang Ge & Derk ten Berge * Department of Molecular Biology, Faculty of Science, Radboud University, Radboud

Institute for Molecular Life Sciences (RIMLS), Nijmegen, The Netherlands René A. M. Dirks, Guido van Mierlo & Hendrik Marks * Division of Reproductive Medicine, Department of Obstetrics

and Gynaecology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands Cindy Eleveld & Esther B. Baart * Center for Biomics, Erasmus MC, University Medical Center

Rotterdam, Rotterdam, The Netherlands Alexander. T. den Dekker, Rutger W. W. Brouwer & Wilfred F. J. van IJcken * The Francis Crick Institute, London, UK Miha Modic * Department for

Neuromuscular Diseases, UCL Queen Square Institute of Neurology, London, UK Miha Modic * Institute of Stem Cell Research, Helmholtz Zentrum München, Neuherberg, Germany Micha Drukker *

Department of Laboratory Medicine, Laboratory of Hematology, Radboud University, Nijmegen Medical Centre and Radboud Institute for Molecular Life Sciences (RIMLS), Nijmegen, The Netherlands

Joop H. Jansen * Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna Biocenter (VBC), Vienna, Austria Nicolas C. Rivron * Department of Developmental Biology,

Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands Esther B. Baart Authors * Alex Neagu View author publications You can also search for this author inPubMed Google

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Scholar * Derk ten Berge View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS A.N., I.E., E.v.G., L.V., D.K., J.L., J.S., M.M. and D.t.B.

designed and performed experiments and analysed data. A.N., I.E., D.K., J.L., J.S., R.A.M.D., G.v.M., M.M., M.D., E.B.B., H.M. and D.t.B. prepared the manuscript. J.S., D.t.B., R.A.M.D.,

G.v.M. and H.M. designed, performed and analysed the ChIP-seq experiments. D.t.B., J.S., A.T.d.D., R.W.W.B. and W.F.J.v.I. performed and analysed the RNA-seq experiments. E.v.G., D.t.B. and

N.C.R. performed and analysed the single-cell sequencing experiments. A.N., C.E., E.v.G. and E.B.B. prepared and analysed the chromosome spreads. A.M. assisted with the mouse experiments and

Y.G. performed additional experiments. H.M. and J.H.J. designed, performed and analysed nucleoside mass spectrometry analyses. D.t.B. conceived the study and coordinated the work.

CORRESPONDING AUTHOR Correspondence to Derk ten Berge. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer

Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 NAIVE AND PRIMED MARKERS IN THE EMBRYONIC

ROSETTE. A,B, Counts of embryos of the indicated stages displaying a rosette or lumen and KLF4 (A) or OTX2 (B) expression status. A, and (B) show different litters. C, Individual colour

channels for Fig. 1h (representative of 5 experiments). D, Individual colour channels for Fig. 1i (representative of 8 experiments). Scale bars 20 µm. Source data EXTENDED DATA FIG. 2 WNT

AND MEK SIGNALS COUPLE SUCCESSIVE STEPS IN MORPHOGENESIS TO PLURIPOTENCY PROGRESSION. (A, B) Representative images of rosettes and aggregates generated by ESCs 48 hrs after seeding in BME in

the indicated conditions and stained for the indicated markers. A, KLF4 (green) and OCT6 (red). B, OTX2 (green) and KLF4 (red). 3 independent experiments with similar results. C, Rosettes

generated by ESCs 48 hrs after seeding in BME and stained for PODXL live or after fixation and permeabilization. Permeabilized samples showed PODXL staining, incorrectly suggesting presence

of a lumen, while live staining revealed that the PODXL was not exposed at the cell surface and no lumen was actually present (illustrative examples, repeated > 5 times). D, Examples to

illustrate criteria for calling ESC aggregates displaying a lumen, using live PODXL staining combined with phalloidin to reveal polarization (illustrative examples, repeated > 5 times).

E, Individual colour channels for Fig. 2n (representative of 3 experiments). F, Individual colour channels for Fig. 2o (representative of 4 experiments). Scale bars 20 µm (a–f). EXTENDED

DATA FIG. 3 WNT CONTROLS THE INITIAL PHASE OF THE NAIVE-PRIMED TRANSITION. (A, B) Immunostaining for the indicated markers of ESCs plated in EpiLC conditions (A) or N2B27 medium (B) for the

indicated number of days (single experiments). C, Dispersed colonies (dashed white circles) obtained after passaging of a blastocyst grown out in the presence of IWP2, MEK inhibitor and LIF

(LIM). 15 from 18 and 12 from 13 embryos yielded cell lines. D, Chimeras (brown), obtained after blastocyst injection of newly derived LIM cell lines (LIMA2, LIMB1 and LIMB3), with mate

(black) and pups (only for LIMA2). Brown pups indicate that germline transmission occurred. E, RT-PCR analysis of ESCs maintained for 6 passages in L2i or MEK/WNT-inhibited conditions and of

EpiSCs. n = 3 (LIF/2i and LIM) or n = 2 (EpiSC) biological replicates. Scale bars 20 µm (A,B), 100 µm (C). Source data EXTENDED DATA FIG. 4 MEK/WNT-INHIBITION MAINTAINS ROSETTE

CHARACTERISTICS. A, Immunostainings and western blot for the indicated markers of R1 ESCs maintained in L2i or LIM for 6 passages or as indicated (3 independent repeats with similar results;

western blot 1 experiment, full blot in Source Data). B, Plating efficiencies of LIM cells in the presence and absence of LIF (n = 3 biological replicates, R1, FN3 and IB10 cells). C,

Female SV8 L2i ESCs, LIM cells and EpiSCs stained for Xist by RNA-FISH. Puncta indicating Xist expression (arrowheads) are visible in both L2i and LIM cells, while only EpiSCs display Xist

clouds indicating X-chromosome inactivation (arrows) (two independent experiments). D, Doubling times of the indicated cell lines in the indicated conditions. p12 and p24 indicate passage

numbers of the cell lines. E, t-SNE plot of single cell RNA-Seq data, and log2 expression levels of the indicated genes plotted into the t-SNE plot. n = 159, 178, 102 (R1) and 184, 149 and

164 (IB10) cells for L2i, LIM and primed, respectively. F, RSCs were cultured for 10 days in the indicated conditions and immunostained for OTX2 (red). Representative images from 3

independent replicates. G, To determine apicobasal polarity, the Golgi apparatus was marked by Gm130 antibody staining, imaged by confocal microscopy and colorized according to z-stack

level. Basal side of the cells correspond to low z-levels (green), and apical side to high levels (red). The data show apical Golgi localization in LIM cells, indicating their apicobasal

polarity. Three experiments with similar results. H, Live tracking of individual cell locations during 24 hrs of L2i ESCs, LIM cells on serum-coating, and LIM cells on BME-coating. 80, 47

and 20 cells, respectively. Representative of 4 experiments. Scale bars 20 µm (a,g), 10 µm (c,f), 100 µm (h). Source data EXTENDED DATA FIG. 5 OTX2 ENABLES MEK-DRIVEN TRANSITION TO PRIMED

PLURIPOTENCY. A, RGd2 ESCs and RSCs were plated in primed conditions and analysed at several time points for GFP by flow cytometry. n = 3 independent replicates (3rd is shown in main Fig.

5a). B, ESCs and RSCs, carrying the Tbx3-RFP;Oct6-GFP reporters, were plated in primed conditions and analysed daily for expression of the reporters. Lines go through means, n = 3

independent experiments. C, Western blots showing OCT6 (only for IB10), ERK and phospho-ERK in IB10 and R1 ESCs and RSCs upon transfer to primed medium. Two (for OCT6) and three biological

replicates. Full blots in Source Data. D, Immunostaining for OTX2 in TRE-Otx2 ESCs following induction by doxycycline for the indicated duration (single experiment). Scale bar 40 µm. Source

data EXTENDED DATA FIG. 6 A PRIMED CHROMATIN LANDSCAPE IN ROSETTE-LIKE STEM CELLS. A, Immunofluorescence and western blot analysis for DNMT3L expression in the indicated cell types (3

independent repeats with similar results; western blot 1 experiment). B, Individual colour channels for Fig. 6b, right panel. Representative of 5 experiments. C, Representative input plot

for H3K27me3 ChIP-Seq for the bivalent cluster. (D,E) H3K4me3 (d) or H3K27me3 (E) ChIP-Seq correlogram of ESCs, RSCs and EpiLCs. Two biological repeats for each condition. (F, G) Average

intensity plots of H3K4me3 and H3K27me3 in the H3K4me3 (f) or H3K27me3 (g) enriched cluster. Two biological repeats for each condition. H, Screenshots from the UCSC genome browser showing

H3K4me3 (blue) and H3K27me3 (red) peaks on the _Pou3f1_ and _Fgf5_ genes. Scale bars 20 µm (a,b). Source data EXTENDED DATA FIG. 7 ROSETTE-LIKE STEM CELLS REMODEL PERICENTRIC

HETEROCHROMATIN. (A–D) Metaphase chromosome spreads of ESCs, RSCs and primed cells stained for the indicated markers. DAPI (blue), centromeres (red). E, Female RSCs (LIMA6) were cultured for

3 days in primed conditions and immunostained for the indicated markers. Arrowheads indicate H3K27me3-positive inactive X chromosomes demonstrating transition to primed pluripotency. F,

RSCs were cultured for 10 days in the indicated conditions and immunostained for the indicated markers. Five (a–d) or three (e, f) biological replicates. Scale bars 20 µm (a-d), 10 µm (e,

f). EXTENDED DATA FIG. 8 ROSETTE-SPECIFIC FORMATTING OF PERICENTRIC HETEROCHROMATIN. A, Counts of embryos of the indicated stages displaying pericentric H3K27me3 accumulation, and in which

the presence of a rosette, KLF4 or OTX2 expression is detected. Embryos are the same as those of Extended Data Fig. 1a, b. B, Confocal microscopy images of E5.1 embryo stained for H3K27me3

(green), centromeres (magenta), OTX2 (red), and DAPI (blue). Some H3K27me3-positive pHC foci are indicated by white arrows (light blue, overlap of H3K27me3 and DAPI). Representative example

from the embryos analysed in Extended Data Fig. 8a. Scale bars 20 µm. Source data SUPPLEMENTARY INFORMATION REPORTING SUMMARY SUPPLEMENTARY TABLES 1–6 Supplementary Table 1. Related to Fig.

3c,d. Excel file containing RNA-seq data of R1 ESC time course in LIM and primed conditions. Supplementary Table 2. Related to Fig. 3e–g. Excel file containing RNA-seq data of R1 and CGR8

ESCs in L2i, LIM, 35 h in primed, and 4 days in primed conditions. Differential gene expression fold change and significance were estimated from biological duplicates using DESeq2

Bioconductor package v.1.18.1. The DESeq2 package uses a Wald test to establish _P_ values, which undergo Benjamini and Hochberg adjustment for multiple testing. Supplementary Table 3.

Related to Fig. 4. Contribution to chimeras of male LIM-derived cell lines. Supplementary Table 4. Related to Fig. 4f. Excel file containing GO analysis of genes differentially expressed

between L2i ESCs and LIM cells (two biological replicates). The _P_ values were calculated from minimum hypergeometric scores from a ranked gene list using GOrilla. Supplementary Table 5.

Related to Fig. 6c–h. Excel file of genes with bivalent promoters in L2i ESCs, RSCs and Epiblast-like cells. Supplementary Table 6. Oligonucleotide primers, antibodies and cell lines used in

the study. SOURCE DATA SOURCE DATA FIG. 1 Statistical source data. SOURCE DATA FIG. 2 Statistical source data. SOURCE DATA FIG. 4 Statistical source data. SOURCE DATA FIG. 5 Statistical

source data. SOURCE DATA FIG. 5 Unprocessed western blots. SOURCE DATA FIG. 6 Statistical source data. SOURCE DATA FIG. 7 Statistical source data. SOURCE DATA FIG. 8 Statistical source data.

SOURCE DATA EXTENDED DATA FIG. 1 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 3 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 4 Statistical source data. SOURCE DATA

EXTENDED DATA FIG. 4 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 5 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 5 Unprocessed western blots. SOURCE DATA EXTENDED

DATA FIG. 6 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 8 Statistical source data. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Neagu,

A., van Genderen, E., Escudero, I. _et al._ In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states. _Nat Cell Biol_ 22, 534–545 (2020).

https://doi.org/10.1038/s41556-020-0508-x Download citation * Received: 25 October 2018 * Accepted: 20 March 2020 * Published: 04 May 2020 * Issue Date: May 2020 * DOI:

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