Abstract
The blastocyst (the early mammalian embryo) forms all embryonic and extra-embryonic tissues, including the placenta. It consists of a spherical thin-walled layer, known as the trophectoderm, that surrounds a fluid-filled cavity sheltering the embryonic cells1. From mouse blastocysts, it is possible to derive both trophoblast2 and embryonic stem-cell lines3, which are in vitro analogues of the trophectoderm and embryonic compartments, respectively. Here we report that trophoblast and embryonic stem cells cooperate in vitro to form structures that morphologically and transcriptionally resemble embryonic day 3.5 blastocysts, termed blastoids. Like blastocysts, blastoids form from inductive signals that originate from the inner embryonic cells and drive the development of the outer trophectoderm. The nature and function of these signals have been largely unexplored. Genetically and physically uncoupling the embryonic and trophectoderm compartments, along with single-cell transcriptomics, reveals the extensive inventory of embryonic inductions. We specifically show that the embryonic cells maintain trophoblast proliferation and self-renewal, while fine-tuning trophoblast epithelial morphogenesis in part via a BMP4/Nodal–KLF6 axis. Although blastoids do not support the development of bona fide embryos, we demonstrate that embryonic inductions are crucial to form a trophectoderm state that robustly implants and triggers decidualization in utero. Thus, at this stage, the nascent embryo fuels trophectoderm development and implantation.
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Acknowledgements
We would like to thank J. Deschamps for providing the Cdx2-eGFP mice; M. Creyghton for providing V6.5 mouse ES cells; V. Prideaux, J. Garner and J. Rossant for providing the F4 mouse TS cell lines; A.-K. Hadjantonakis for providing the PDGFRα-H2B-GFP mouse ES cells; N. Schelling for helping with logistics; S. van der Elst for helping with FACS assays; A. de Graaf for helping with microscopes; L. Yvernogeau for helping with the imaging of embryos; B. El Haddouti for helping with uterus injections; H. Begthel for helping with histology; W. de Lau for helping with the TCF luciferase assay; C. Rabouille and H. Clevers for commenting on the manuscript. N.C.R. is grateful for support from The Netherlands Organisation for Scientific Research (NWO-ZonMw) Translational Adult Stem Cell grant (116005008). We apologize that all relevant publications could not be cited.
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N.C.R. conceived and directed the project, conducted the experiments and wrote the manuscript. J.F.-A. performed the single-molecule FISH assay, the qRT–PCR assay, contributed to generating the CRISPR knockout lines and to the RNA-sequencing assays. E.J.V. performed the high-content imaging assays. J.K. completed the blastocyst complementation assays and the in uteri transfers. J.V. helped prepare the libraries of single cells for RNA sequencing. J.-C.B. contributed to the RNA-sequencing assays. R.K.T. contributed to the design of the microwell array. A.v.O. contributed to the RNA-sequencing assays. C.A.v.B and N.G. helped to direct the project and contributed equally to the study. All the authors discussed and corrected the manuscript.
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N.C.R., E.J.V., C.A.v.B. and N.G. are inventors on the patent US14/784,659 and PCT/NL2014/050239 (April 2014).
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Extended data figures and tables
Extended Data Fig. 1 In vitro formation of blastoids.
a, Non-adherent hydrogel microwell arrays were formed by replica molding using polydimethylsiloxane (PDMS) stamps as previously described7,8,40. The array used for 12-well plates contains 1,000 cylindrical structures of 200 µm diameter and height. Upon cell seeding, owing to the non-adherent properties of the hydrogel, all cells slide into the microwells. Upon settling, the number of ES cells per microwell follows a distribution. Mean number per microwell: 5.2; half of the microwells contained between 4 and 6 cells per microwell (right, top). Upon aggregation of ES cells (24–36 h depending on the cell line), TS cells were seeded. Mean number per microwell: 12; half of the microwells contained between 10 and 14 cells (right, bottom; time point 0 h). b, After culture in blank medium (see Methods), TS and ES cells mostly arranged into non-organized structures forming trophoblasts cysts. TS cells cultured without ES cells (65 h) formed fewer cystic structures than a co-culture. Rarely, TS cells enclosed ES cells and formed regular cystic structures morphologically similar to the blastocyst (white arrow). n = 4 independent microwell arrays. Structures shown in b were taken out of the microwell array at 65 h. Scale bars, 100 µm. c, d, Optimization of the engulfment of ES cells by TS cells. c, Dosing the number of TS cells. ES cells were seeded at t = −24/36 h. At t = 0 h, different numbers of TS cells were seeded on top of the ES cell aggregates. After 24 h, the engulfment efficiency was defined by measuring the percentage of coverage of TS cells around ES cells. The most efficient yields were observed when more than 12 TS cells were added to ES cell cultures. n = 250 independent microwells. The centre value is the mean, error bars are s.d. d, Left, optimization of the concentration of Y27632. At t = 0 h, different concentrations of Y27632 were added. At t = 24 h, the engulfment efficiency was measured as the percentage of coverage of TS cells around ES cells. The most efficient yields were obtained with 20 µM Y27632. n = 250 independent microwells. The centre value is the mean. Right, optimized engulfment. Images of ES cells (red) engulfed by TS cells (green) using the optimized conditions (mean of 12 TS cells per microwell and 20 µM Y27632, 80% engulfment efficiency). Images are representative of three independent experiments. Scale bar, 100 µm. Error bars are s.d. e, Blastocoel area of blastocysts formed from E2.5 CBA × C57BL/6 morula, selected for initiation of compaction and cultured in M16 medium for 24 h along with antagonists of WNT (XAV939, 15 µM), PKA (H89, 10 µM) or DMSO (1:1,000, control). n = 10 independent blastocysts. P = 0.015 and P = 0.002, two-sided Student’s t-test. The centre values are medians. Errors bars are s.d. f, RNA-sequencing data for Wnt6 and Wnt7b, in E3.25 and E3.5 blastocysts, TS cells (TSC) and ES cells (ESC). TS cells were cultured in serum-free (TX) or serum-rich (TS) medium (see Methods). Differentiation was induced by the removal of growth factors. g, TCF luciferase assay for WNT activity in TS cells (see Methods). WNT secretion was blocked using IWP2 (2.5 µM). n = 4 independent biological samples of TS cell culture. *P = 0.045, **P = 0.0057, two-sided Student’s t-test. The centre values are medians. Errors bars are s.d. h, Induction of cavitation. Blastoids were defined as described in the Methods. ES and TS cells were seeded in serum-free TX medium including Y27632 (20 µM) and a WNT modulator. WNT3A-conditioned medium (50% of the total volume), CHIR99021 (3 µM), or the combination of CHIR99021 (3 µM) and XAV939 (15 µM) was added at the time of TS cells seeding (t = 0 h). n = 3 independent microwell arrays. *P = 0.017, two-sided Student’s t-test. The centre values are medians. Errors bars are s.d. i, Yield of blastoids depending on the initial ratio of TS to ES cells, at t = 0 h, within individual microwells (left). Yield of blastoids formed using three lines of TS cells representative of the scope of efficiency observed (right). Different lines were isolated upon CBA × C57BL/6 matings (F1-1, F1-2, derived by N.R.) and ICR × ICR matings (F4, provided by J. Rossant). n = 3 independent microwell arrays. The centre values are the mean. Error bars represent s.d. The red line represents the median of the three cell lines (5% of the total number of microwell per array). j, Bright-field images of a representative E3.5 blastocyst (top) and a blastoid (bottom). Scale bar, 50 µm.
Extended Data Fig. 2 Regulation of trophoblast cells.
a, The number of Cdx2 transcript reads per million mapped reads (r.p.m.) as measured in bulk samples of: ES cultured in 2i medium and TS cells cultured in TX medium, TS cells cultured in blastoid medium, and blastoids and blastocysts. Note that the blastoids and blastocysts comprise both trophectoderm and ICM cells, the latter expressing only very low levels of Cdx2. n = 2 independent biological samples (see Methods). b, Measurement of CDX2 fluorescent intensity in blastoids and blastocysts. Blastoids were fixed and stained with an anti-CDX2 antibody (see Methods). E3.5 blastocysts were used as a positive control. n = 15 independent blastoids or blastocysts. Error bars are s.d. c, The number of Stat3 transcripts per million reads as measured in bulk samples of ES and TS cells and blastocysts. d, Immunostaining for phosphorylated STAT3 in a representative blastocyst colony of ES and TS cells (see Methods). Scale bar, 50 µm. e, TS cells cultured in the presence of different concentrations of the STAT/GP130 pathway inhibitor SC144. Both growth and viability were affected by concentrations of at least 1 µM. f, The number of transcripts per million reads for the STAT pathway ligands Il11, Lif and Il6, along with their receptors, as measured in bulk samples of ES cells, TS cells and blastocysts. g, Whole-mount single-molecule FISH for Il11, in an E3.25 blastocyst. Scale bar, 50 µm. See Methods for further details.
Extended Data Fig. 3 Screen for regulators of CDX2 expression in TS cells.
a, Calibration of the cell lines. The assay is performed using CDX2–eGFP+ TS cells and as described in the schematic and the Methods. For the initial calibration of the assay, gating was set so that wild-type TS cells do not appear in the gate (non-specific fluorescence, left FACS plot). In that condition, after 48 h, the following appeared in the gate: 50% of the CDX2–eGFP+ cells cultured in blank TX medium; 88% of the CDX2–eGFP+ TS cells cultured in TX medium including FGF4 (25 ng ml−1) and TGFβ1 (2 ng ml−1); 10% of the CDX2–eGFP+ TS cells cultured in TX medium including FGF4 (25 ng ml−1), TGFβ1 (2 ng ml−1) and PD0325901. Biological triplicates show very minor variability (n = 3 independent biological samples, right). Error bars are s.d. Scale bar, 100 µm. b, Calibration of the assay and primary screen. For the primary screen of proteins and small molecules, the condition with TX medium including FGF4 and TGFβ1 was used as a positive control. The gating was set up such that 50% of these cells appear in the gate. The condition with TX medium without FGF4 and TGFβ1 was used as a negative control, and 20% of these cells appear in the gate. 8Br-cAMP (0.04 to 5 mM), IL-11 (4 to 500 ng ml−1), LIF (3 to 375 ng ml−1), BMP4 (1 to 125 ng ml−1), and IGF2 (1 to 125 ng ml−1) were added to the medium. The value is the measurement of a single sample. The typical s.d. for this assay is shown in a. c, Secondary screen: combinations of hits. 8Br-cAMP (1 mM), IL-11 (30 ng ml−1), or 8Br-cAMP (1 mM) + IL-11 (30 ng ml−1) were added to TX medium including FGF4 and TGF. The full blastoid medium was also tested (see Methods). d, e, Markers of multipotency, differentiation and epithelization in stimulated TS cells. TS cells were grown for 48 h in blank medium (see Methods), TX medium (including FGF4 and TGFβ1) or TX medium supplemented with 8Br-cAMP and IL-11. Representative bright-field images of TS cells grown for 48 h in TX medium or in blastoid medium are shown (d), along with gene expression as characterized using bulk RNA sequencing (e, n = 3). Scale bars, 1,000 µm.
Extended Data Fig. 4 Evolution of the number of cells in the blastoid compartments.
a, E3.5 and E4.5 blastocysts (n = 6) were immunostained using Nanog and PDGFRα antibodies, and nuclei were counterstained with DAPI (see Methods). b, Percentage of blastoids including GATA6+ cells from a total number of 14 blastoids. c, Number of cells in each of the blastoid or blastocyst compartments, namely trophoblast, embryonic and primitive endoderm, were counted based on an immunostaining for Nanog, GATA6 and PDGFRα. The number of trophoblast was counted based on the expression of constitutive GFP. The quantification was done at 48, 72 and 86 h. Note that, for most experiments presented herein, blastoids are harvested at 65 h (see Methods). The horizontal bars represent the mean number of cells. The coefficient of variation (relative s.d.) is 19.4% for E3.5 blastocysts and 23.6% for blastoids at 48 h. n = 6 independent E3.5 blastocysts and E4.5 blastocysts.
Extended Data Fig. 5 De novo derivation and developmental potential of blastoid stem cells.
a, De novo TS and ES cell lines were derived from CDX2-high and CDX2-low blastoids, and blastocysts (see Methods). The derivation was considered successful if colonies appeared on passage 2 after blastocyst or blastoid plating. The derivation efficiency of TS cells from CDX2-high blastoids was significantly higher (****P = 0.0001, Student’s t-test) than CDX2-low blastoids and similar to blastocysts (left). n = 22 independent blastoids. Error bars are s.d. The potential of de novo-derived cell lines to chimaerize embryos was assessed by immunohistochemistry, using an anti-GFP antibody targeting GFP+ TS cells or an anti-RFP antibody targeting H2B-RFP+ ES cells (middle). Autofluorescence (denoted by an asterisk), probably due to the presence of blood, occurs within the ecto-placental cone of chimaeric embryos but is lower than the fluorescence recorded from chimaeric embryo after the injection of GFP+ TS cells (right). The top image shows a chimaeric embryo after the injection of GFP+ TS cells. The bottom image shows a chimaeric embryo after the injection of H2B-RFP+ ES cells. Both embryos are imaged in the GFP channel. Note the presence of a small patch of autofluorescence (denoted by an asterisk) in the bottom image. Microscope settings were adjusted to limit the acquisition of autofluorescence, and images taken upon excitation with a 450–490 nm (green) laser. Scale bars, 100 µm. This experiment is a representation of three similar experiments. b, Immunohistochemistry using antibodies against CDX2, ELF5, TEAD4, ASCL2, HAND1 and proliferin, within tissue sections of a representative deciduae including a blastoid grown in utero. Scale bar, 100 µm. This experiment is a representation of three similar experiments. c, Percentage of implantation sites in uterus transferred with blastoids and vehicle medium at E3.3–E3.5, and explanted at E7.5. n = 5 mice. The value is the mean. Error bars denote the s.d. **P = 0.01, Student’s t-test. d, Uterus explanted at E7.5 after a physiological development (blastocyst-induced), the transfer of blastoids only in the left horn (blastoid-induced, also see the same uterus in Fig. 2h), and the instillation of oil (oil-induced). e, Histological sections of deciduae produced by blastocysts, blastoids and oil instillation, and stained using an anti-ALDH3A1 antibody. Scale bar, 100 µm. This experiment is a representation of three similar experiments.
Extended Data Fig. 6 Bulk and single-cell RNA sequencing of blastoids and blastocysts.
a, b, Bulk RNA sequencing. a, Transcription factor expression distance map. Differentially expressed transcription factors between the ES cells, TS cells, blastoids, virtual blastoids (see Methods), E3.25 and E3.5 blastocysts (P < 0.05, DESeq negative binomial distribution) were used to generate a non-supervised distance map of transcription factors selected according to the Riken transcription factor database. The scale is log2. b, Gene ontology (GO) and KEGG pathways. Genes differentially regulated (P < 0.05, DESeq negative binomial distribution) were analysed using DAVID38 and corresponding GO and KEGG pathways are presented. The full lists of genes related to the depicted GO terms are in Supplementary Table 1 sheet 1. c–e, Single-cell sequencing. c, Schematic depicting the origin of the single cells. t-SNE maps of single cells from ES cells, TS cells, blastoids, trophospheres and embryoid bodies. The colours represent the origin of cells assessed by FACS sorting indexes (Supplementary Table 1 sheet 8). d, Clusters of similar cells, as generated by the RACE-ID protocol36. The heat map on the right is the distance map of single cells. The clusters from the t-SNE map and from the heat map are identified using the same colour code. Cells were processed as described in the Methods. e, The Krt18 and Oct4 genes are markers of the blastocyst trophectoderm and ICM compartments, respectively41, which we confirmed are also valid to mark the compartments of blastoids using immunohistochemistry (KRT8/18 and OCT4) and H2B-RFP+ ES cells. This image is representative of three independent blastoid experiments. f, t-SNE map representation of key genes for the trophectoderm, trophoblast differentiation and placental cell types. g, Heat map showing expression of genes of interest: markers of the trophectoderm and ICM compartments by Krt18 and Oct4, as identified in e; markers of pluripotency and differentiation, previously identified in ES cells and blastocyst cells via single-cell sequencing42; genes previously identified as markers of WNT/β-catenin targets regulating a naive state of pluripotency43; transcription factors upregulated in totipotent cells44; genes previously identified as differentiation markers in ES cells via single-cell sequencing42; genes previously identified as markers differentiating ES cells from epiblast stem cells30; markers of trophoblast differentiation; and markers of trophectoderm morphogenesis. The scale is log10.
Extended Data Fig. 7 Immunohistochemistry of epithelial markers within blastoids.
a, Cross-section of a blastoid stained with an antibody against E-cadherin. Maximum projection of 5 images taken with a 1-µm step. b, Phalloidin staining of the cytoskeletal molecule F-actin and Hoechst staining of DNA show the localization of actin at cell–cell junctions. Maximum projection of 40 images taken with a 1-µm step. c, Antibody against the tight junction molecule ZO-1. Section of 0.5 µm (top). Maximum projection of 4 images taken with a 0.5-µm step (bottom). d, Antibody against the apical molecule PKCz and Hoechst staining of DNA. Section of 1 µm (top); close up (bottom). All pictures were taken with a PerkinElmer Ultraview VoX spinning disk microscope combined with a Leica SP8. Images are representative of three experiments.
Extended Data Fig. 8 Assays of embryonic and trophectoderm compartments interactions.
a, Specificity of the ES cell inductions. Yield of formation and representative images of arrays of blastoids resulting from the association of TS cells with ES cells, EpiSCs, human ES cells, COS7 or C2C12 cells. n = 3 independent microwell arrays. Centre depicts median values. Error bar denote s.d. b, Number of Fgf4 mRNA transcripts measured by RNA sequencing in TS cells, ES cells, EpiSCs and XEN cells (extra-embryonic endoderm cell lines). c, Inventory of signalling pathways induced in trophoblasts by the embryonic cells: KEGG pathways differentiating the trophoblasts from blastoids and trophospheres. The list is exhaustive and generated using the list of statistically differentiated genes (P < 0.05, DESeq negative binomial distribution) with the highest fold changes (the 1,500 most highly upregulated and most highly downregulated genes). See the SORT-seq method described previously33. d, Selected KEGG pathways and genes related to the MAPK signalling pathway, TGFβ signalling pathway, cell cycle, focal adhesion and hippo pathway. e, Trophectoderm morphogenesis. Blastoids were assessed based on our definition of cavitated trophoblast structures comprising ES cells, with a circularity greater than 0.9 (circularity = 4π(area/perimeter2)), and a diameter between 70 and 110 µm (see Methods). Yield of blastoids (percentage per microwell array) as a function of the initial mean number of ES cells per microwell array. Horizontal bars denote mean yield. Error bars indicate s.d. **P = 0.01, ***P = 0.001, one-way analysis of variance (one-way ANOVA) and Tukey’s test. n = 8 independent microwell arrays. f, The diameter of blastoids and trophospheres measured at 65 h. n = 50. ****P = 0.0001, two-sided Student’s t-test. Centre values depict median. Error bar denote s.d. Representative images are shown in Fig. 4a.
Extended Data Fig. 9 Markers, transcriptional and morphogenetic functions of TGFβ signalling.
a, Single-molecule FISH for Id2, Bmp4 and Nodal genes of the TGFβ pathway, in an E3.5 blastocyst counterstained with Hoechst. Images are representative of five independent blastocysts. Scales bars are 30 µm for Id2 and 50 µm for Nodal and Bmp4 b, CRISPR strategy for the generation of Nodal deletion in ES cells. c, Venn diagram of all genes regulated after exposure to BMP4, Nodal or both (synergy). RNA sequencing showed that BMP4 and Nodal regulated a similar number of genes (BMP4: 904 genes, Nodal: 926 genes, genes with P < 0.05 (DESeq negative binomial distribution), of which 30% overlapped (413 genes). The GO analysis is shown for each group. See also Supplementary Table 2 sheets 1–4. d, RNA-sequencing analysis of the WNT-related genes in trophospheres stimulated with activators of the TGFβ signalling pathway. All genes are significantly regulated (P = 0.03, DESeq negative binomial distribution). This included the ligand Wnt6 (1.6-fold, P = 0.006, DESeq negative binomial distribution), which is expressed primarily in the cells surrounding the blastocyst cavity10, the corresponding receptor Fzd7 (2.8-fold, P = 0.002, DESeq negative binomial distribution), the intracellular effector Tcf4 (also known as Tcf7l2; 1.7-fold, P = 0.001, DESeq negative binomial distribution), the negative-feedback regulator Axin1 (1.5-fold, P = 0.005, DESeq negative binomial distribution, top), and of the reported cooperative SMAD/β-catenin targets Msx2 (1.6-fold, P = 0.00003, DESeq negative binomial distribution)45 and Ctgf (3.6-fold, P = 0.003, DESeq negative binomial distribution)46 (bottom). See also Supplementary Table 2 sheets 1–4. e, RNA-sequencing analysis of the Tgfb-related gene Id2 in TS cells, ES cells, blastoids, trophospheres, blastocysts (left) and in trophospheres after stimulation with Nodal, BMP4 or both (see Methods) (right). n = 2 independent biological samples. The centre depicts the mean. Error bars denote the s.d. f, Number of transcripts measured by RNA-sequencing analysis for markers of epithelial development Cldn4, Krt8 and Krt18. n = 2 independent biological samples. The centre depicts the mean. Error bars denote the s.d. g, Yield of cystic structures for a combination of ES and TS cells as compared to TS cells alone (trophospheres) and trophospheres stimulated with BMP4 and Nodal. Horizontal bars denote the mean yield. Error bars are s.d. *P = 0.02, Student’s t-test. n = 3 independent microwell arrays (see Methods).
Extended Data Fig. 10 Generating and assaying blastoids with Klf6−/− TS cells.
a, Frequency distribution of the diameter of blastoids, trophospheres and trophospheres exposed to BMP4 and Nodal (µm). For all graphics, the red bar is the median as measured for blastoids. b, Number of trophoblasts within blastoids, trophospheres and trophospheres stimulated with activators of the TGFβ signalling pathway. n = 30 independent blastoids or trophospheres. P = 0.0001, one-way ANOVA. The same graph is shown in part in fig. 4b. c, Effect of small-molecule inhibitors on blastocyst cavitation. Morula were flushed from the oviduct of E2.5 CBA × C57BL/6 mice and cultured in M16 medium. After the initiation of morula compaction, inhibitors or DMSO (1:1,000, control) were added. LDN193189 (0.25 µM) was used to inhibit ALK2 and ALK3 (SMAD pathway). Blastocysts were imaged 24 h after exposure. n = 10 independent blastocysts. P = 0.07, two-sided Student’s t-test. Error bars denote s.d. d, Targeting strategy of KLF6 in TS cells and PCR gel of the obtained clones. e, Bright-field image of wild-type and Klf6−/− TS cells (top left). Immunostaining for CDX2 in Klf6−/− TS cells (top right). E-cadherin immunostaining (bottom left) and quantitative PCR with reverse transcription (qRT–qPCR) (bottom right) of wild-type and Klf6−/− TS cells. f, Representative pictures of blastoids and trophospheres, and trophospheres stimulated with 45 ng ml−1 BMP4 and 5 ng ml−1 Nodal. Red asterisks denote blastoids, which comply to our definition of cavitated trophoblast structures comprising ES cells, with a circularity greater than 0.9 (circularity = 4π(area/perimeter2)), and a diameter between 70 and 110 µm (see Methods). Comparable results were obtained in three repeated experiments. g, qRT–PCR for Krt8 in structures formed by combining ES cells and wild-type or Klf6−/− TS cells. All structures (blastoids and non-blastoids) were collected from the microwell arrays and tested. Horizontal bars indicate mean expression. Error bars denote s.d. n = 3 pools from independent microwell arrays. *P = 0.04, two-sided Student’s t-test.
Supplementary information
Supplementary Table 1
Supplementary Table 1 contains a summary sheet followed by 8 sheets with gene expression data and GO analysis related to Figure 3.
Supplementary Table 2
Supplementary Table 2 contains a summary sheet followed by 3 sheets with gene expression data and GO analysis related to Figure 4.
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Rivron, N.C., Frias-Aldeguer, J., Vrij, E.J. et al. Blastocyst-like structures generated solely from stem cells. Nature 557, 106–111 (2018). https://doi.org/10.1038/s41586-018-0051-0
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DOI: https://doi.org/10.1038/s41586-018-0051-0
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