Blastocyst-like cell aggregates and methods

Abstract

The present invention provides a method for generating blastoid cells or blastocyst-like structures by cell aggregation and culture, comprising culturing aggregates of human pluripotent stem cells (hPSCs) and trophoblast cells in 3D culture in a medium containing a HIPPO pathway inhibitor; blastoid cell and blastocyst-like cell aggregates obtainable by said method and uses thereof; similarly, a similar treatment of blastocysts for in vitro fertilization that promotes blastocyst development and implantation; similarly, a similar contraceptive treatment of human embryos that reduces blastocyst development and implantation.

Description

[Technical field]

[0001] The present invention relates to the generation of blastocyst-like structures by aggregation and culture of cells. [Background technology]

[0002] Due to the small number of human embryos studied and the difficulties of experimentally manipulating them physically and genetically, our understanding of early human embryonic development and implantation is very limited. To study these early developmental and implantation mechanisms, other experimental model organisms, usually mice, have been used. However, it is now clear that there are important differences between humans and other species in terms of morphology and the molecules involved, necessitating the study of human embryonic development.

[0003] European Patent Application No. EP2986711A1 relates to the generation of blast-like cells using at least one trophoblast cell and at least one pluripotent cell.

[0004] WO 2018 / 175691 A1 relates to the generation of totipotent cells.

[0005] WO 2020 / 262531 A1 describes the generation of primordial endoderm stem cells by culturing blastocysts.

[0006] RONGHUI Li et al., Cell, Elsevier, Vol.179(3),2019:687, describe the generation of blastocyst-like structures from a single stem cell type.

[0007] RIVRON Nicolas C et al., Nature, MacMillan Journ. Ltd., Vol.557(7703), 2018: 106-111, describe the generation of embryonic day 3.5 blastocysts from trophoblast cells and embryonic stem cells.

[0008] VRIJ Erik J. et al., bioRxiv, DOI:10.1101 / 510396, describes a combinatorial screen of proteins, GPCR ligands, and small molecules to rapidly induce embryoid bodies to form a three-dimensional primitive endoderm / epiblast-like niche.

[0009] KIME Cody et al., Stem Cell Reports, 13(3),2019:485-498, describe induced self-organizing 3D blastocyst-like cysts (iBLCs) generated from mouse pluripotent stem cell cultures.

[0010] In summary, there remains a compelling need for in vitro alternatives to the use of human embryos for research. Models that are widely available and amenable to simple genetic manipulation and high-throughput drug screening are required.

[0011] It is therefore an object of the present invention to provide such a model. Summary of the Invention

[0012] The present invention provides a method for generating blastoid or blastocyst-like cell aggregates, comprising culturing aggregates of human pluripotent stem cells (hPSCs) and trophoblast cells in 3D culture in a medium containing a HIPPO pathway inhibitor. These blastoid cells can be used for high-throughput genetic or pharmaceutical screening in drug development. The method can also be used to induce pregnancy. The components and molecules of the medium revealed by the use of blastoid cells can also be used to modulate blastocyst behavior, for example to improve blastocyst development and implantation during in vitro fertilization procedures.

[0013] The present invention further provides a kit suitable for culturing blastoid cells, comprising a HIPPO pathway inhibitor, a MEK inhibitor, and a TGF-β inhibitor, wherein one or more of these compounds can be combined in a medium for culturing human pluripotent stem cells (hPSCs).

[0014] The present invention further provides blast-like cell and blastocyst-like cell aggregates obtainable by the above method.

[0015] Also provided are blastoid and blastocyst-like cell aggregates comprising an outer epithelial monolayer of trophoblast-like cells (preferably characterized, e.g., by expression of GATA3 and CDX2) surrounding at least one fluid-filled cavity and at least one inner cell mass comprising epiblast-like cells (preferably characterized, e.g., by expression of Nanog and Oct4) and hypoblast-like cells (preferably characterized, e.g., by expression of GATA4), wherein the outer epithelial monolayer comprises polar-like trophoblasts expressing NR2F2.

[0016] The present invention further provides an in vitro method for increasing or testing the potential of blastoid cells or blastocysts to implant into a layer of endometrial cells, comprising stimulating the endometrium with a Wnt inhibitor, preferably XAV939 and / or LF3, and contacting blastoid cells or blastocysts with the layer of stimulated endometrial cells; the testing method further measures the level of attachment, invasion and differentiation into endometrial cells of trophoblasts, blastoid cells or blastocysts.

[0017] The present invention further provides a Wnt inhibitor for use in a method of increasing the probability of blastocyst implantation, e.g., during an in vitro fertilization procedure, comprising contacting a blastocyst with the endometrium in the presence of a Wnt inhibitor, or stimulating the endometrium with a Wnt inhibitor, prior to implantation of the blastocyst into the uterus or endometrium.

[0018] In this regard, the present invention provides a method for increasing the probability of blastocyst implantation, for example during an in vitro fertilization procedure, comprising contacting a blastocyst with the endometrium in the presence of a Wnt inhibitor or stimulating the endometrium with a Wnt inhibitor prior to implantation of the blastocyst into the uterus or endometrium.

[0019] Also provided is the use of a Wnt inhibitor to manufacture a pharmaceutical composition for mediating blastocyst implantation during an in vitro fertilization procedure, comprising, for example, contacting a blastocyst with the endometrium in the presence of a Wnt inhibitor, or stimulating the endometrium with a Wnt inhibitor, prior to implantation of the blastocyst into the uterus or endometrium.

[0020] Further provided is a HIPPO pathway inhibitor for use in a method for producing a blastocyst suitable for implantation, for example to improve the quality of blastocysts during in vitro fertilization, comprising treating an embryo at an early stage selected from the morula stage or the blastocyst stage with a HIPPO pathway inhibitor until the mature blastocyst stage, and growing the morula stage embryo to the blastocyst stage, or growing the blastocyst stage embryo to a more mature blastocyst stage.

[0021] In this regard, the present invention provides a method for producing a blastocyst, comprising treating an early stage embryo selected from 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage, or 16-cell stage, morula stage, or blastocyst stage with a HIPPO pathway inhibitor until mature blastocyst stage, and growing the morula stage embryo until blastocyst stage, or growing the blastocyst stage embryo until more mature blastocyst stage. The 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage, or 16-cell stage is also called cleavage stage. Also provided is the use of a HIPPO pathway inhibitor for the manufacture of a pharmaceutical composition for producing a blastocyst suitable for implantation, for example to improve the developmental potential of a blastocyst during an in vitro fertilization procedure, which includes treating an early stage embryo selected from the 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage, or 16-cell stage, morula stage, or blastocyst stage with a HIPPO pathway inhibitor until the mature blastocyst stage, and growing an embryo at the 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage, or 16-cell stage, or morula stage until the blastocyst stage, or growing an embryo at the blastocyst stage until the more mature blastocyst stage.

[0022] All aspects of the invention are relevant, and the disclosure of a particular embodiment of one aspect is relevant to the other aspects. For example, the disclosure of the treatment of blastoid cells or blastocyst-like cell aggregates in vitro can also be carried out for the treatment in vivo for the preparation of blastocysts for preparation in IVF treatment and the preceding treatment steps. Any of the compounds described with respect to the methods of the invention can be part of a kit. Kit components and kits can be used in the methods and treatments of the invention. [Brief description of the drawings]

[0023] [Figure 1]Formation of human blast-like cells. A. Human pluripotent stem cells (hPSCs) are dissociated into single cells and seeded into microwell arrays. Under certain conditions, hPSCs aggregate to form blast-like cells within 5 days. B. The percentage of microwells containing blast-like cells depends on the initial number of cells first seeded into the microwells. [Diagram 2] Modulation of the Hippo pathway using small molecule and genetic approaches controls the formation of human blast-like cells. The Hippo pathway can be inhibited using lysophosphatidic acid (LPA), NAEPA, and suppressing the YAP-TEAD complex using verteporfin, which respectively increase and decrease the formation of human blast-like cells. Inhibition of the Hippo pathway can be mimicked using genetic overexpression of YAP-WT, YAP-5SA (constitutively active), which increases the formation of human blast-like cells. Activation of the Hippo pathway can be mimicked by suppressing the formation of the YAP-TEAD complex using YAP-5SA+S94A (TEAD binding defective), which decreases the formation of human blast-like cells. Immunofluorescence staining of YAP protein showed nuclear localization only in trophectoderm-like cells, but not in epiblast-like cells (Nanog-positive cells) and hypoblast-like cells. [Figure 3-1]Changes in cell number and overall aggregate size during blast-like cell formation. A. When naive hPSCs are seeded into microwell arrays, each microwell contains an average of 45 cells. B. After 24 hours, cell aggregates contain an average of 45 cells, all of which express the epiblast transcription factor Oct4. At this time point, the cells do not express the trophoblast transcription factor GATA3. C. Between 24 and 84 hours, cell number increases from an average of 45 to an average of 80, and trophoblast cells expressing GATA3 appear. D. Blast-like cells are fully formed by 120 hours by generating analogs of three founding cell lineages: OCT4+ epiblast-like cells, GATA4+ hypoblast-like cells, and GATA3+ trophectoderm-like cells. Similar to human blastocysts, the average total number of cells is 120, with trophoblast cells being the most abundant lineage. E,F. Evolution of overall size of cell aggregates during blast-like cell formation. After 24 hours, the overall diameter of the cell aggregates is 65 micrometers. This average diameter gradually increases to 200 micrometers after 120 hours (E). Photographs of one representative aggregate stained with the nuclear dye Hoechst at 24, 84, and 120 hours (F). [Figure 3-2] Changes in cell number and overall aggregate size during blastoid cell formation (cont.). G. Once formed, human blastoid cells are composed of analogs of three founding lineages. Inner clusters of epiblast-like and hypoblast-like cells form, characterized by expression of Oct4 and Nanog, respectively, as well as Gata4. The outer layer of human blastoid cells is formed by a monolayer of trophectoderm-like cells, characterized by expression of Gata3 and Cdx2. H. After culturing human blastoid cells for 5 days, trophoblasts in contact with the inner clusters, called polar trophoblasts, begin to express NR2F2, whereas mural cells do not. Polar trophoblasts are known to mediate the initial attachment of human blastocysts to the uterus. Scale bar is 25 micrometers. [Figure 4]Evolution of the structure of cell aggregates during blastoid cell formation. A. After 24 h, the overall diameter of the cell aggregate is 65 micrometers, all containing cells expressing the transcription factor Oct4 (see also Figure 3B). Cells surrounding the aggregate express higher levels of the apical marker PKC. B. After 84 h, the outer cells have enhanced the formation of membrane domains expressing PKC, whereas the inner cells have not formed such domains. The top row shows a cross-section of the aggregate. The bottom row shows a complete 3D projection of the aggregate. C,D. The formation of membrane domains expressing PKC coincides with the appearance of cells expressing the trophoblast transcription factor Cdx2 and the formation of small fluid-filled cavities (C), which coalesce to finally form a unique cavity after 120 h (D). [Diagram 5] Trophosphere formation by preventing the formation of inner clusters. A. Inhibition of the STAT pathway using the small molecule SC144 or the Hippo pathway using the small molecule XMU-MP-1 results in the formation of blast-like cells with few or no inner cell clusters, but generates trophectoderm-like cells that form cysts with fluid-filled cavities. This indicates the importance of these pathways in balancing or maintaining the numbers of epiblast-like and hypoblast-like cells. B-D. The use of 3 μM of the STAT inhibitor SC144 for 4 days reduces blast-like cell formation and increases the formation of trophospheres with no or few epiblast-like and hypoblast-like cells. The use of 2 μM of the Hippo inhibitor XMU-MP-1 for 4 days results in the formation of trophospheres with few or no epiblast-like and hypoblast-like cells. [Figure 6-1]Formation of open-faced endometrial organoids and their stimulation to mimic the window of implantation. A. Open-faced endometrial organoids are formed by first expanding human endometrial organoids using 3D Matrigel culture as previously published. These human endometrial organoids are known to recapitulate molecular features of the window of implantation (WOI) when exposed to a combination of estrogen, progesterone, and cyclic adenosine monophosphate (cAMP). This combination is called EPC. In a second step, the organoids are dissociated and seeded in 2D to form an open-faced monolayer of endometrial cells. This open-faced monolayer facilitates the deposition of blastocyst- or blastoid-like cells, allowing the evaluation of their potential to recapitulate aspects of implantation in vitro. B. Wnt inhibition by either XAV939 (XAV) or (PKF118-310) PKF causes endometrial cells to increase expression of the gene PAEP, a known marker of WOI. C. Expression of additional markers of WOI, including LIF and SPP1, is also upregulated by Wnt inhibition and EPC stimulation compared to EPC stimulation alone. [Figure 6-2] Formation of open-face endometrial organoids and their stimulation to mimic the window of implantation (cont.). D. Immunofluorescence shows that open-face endometrial organoids contain subpopulations of (A) ciliated cells (acetylated α-tubulin positive cells), (E) glandular cells (FOXA2+ cells), and (F) proliferative cells (EdU incorporation positive cells). G. Endometrial cells stimulated with EPCs and Wnt inhibitors express higher levels of PAEP at the protein level compared to unstimulated organoids. [Figure 7-1]In vitro combination of human blastoid cells and open-faced endometrial organoids recapitulates features of blastocyst implantation into the uterus. A,B. Interaction between human blastoid cells and open-faced endometrial organoids requires stimulating endometrial cells. Human blastoid cells are unable to attach and invade unstimulated endometrial cells that do not mimic the window of implantation (WOI) (top), but are able to attach and invade stimulated endometrial cells (bottom). B. Combination of EPC stimulation and Wnt inhibition using inhibitors XAV939, IWP-2, PNU-74654, and LF3 increases the likelihood of endometrial cells interacting with human blastoid cells. C-E. Interaction between human blastoid cells and open-faced endometrial organoids requires a specific trophoblast state. Presence of the STAT inhibitor SC144 during human blastoid cell formation induces the formation of a trophosphere with limited or no epiblast- and hypoblast-like cells. The presence of Hippo inhibitor XMU-MP-1 during human blastoid cell formation induces the formation of trophospheres with limited or no epiblast-like and hypoblast-like cells. Human trophoblast stem cells (Okae et al, 2018, doi:10.1016 / j.stem.2017.11.004) can form cytotrophoblast aggregates that reflect the stage after implantation. SC144-trophosphere, XMU-MP-1-trophosphere, and cytotrophoblast aggregates are unable to attach and invade stimulated endometrial cells (C, D). Conversely, human blastoid cells are able to attach and invade stimulated endometrial cells, attaching via polar regions (D, E). F. Upon attachment and invasion, human chorionic gonadotropin (hCGB), a hormone used to assess clinical pregnancy, is detectable in the medium after 24 and 48 h (middle and right pregnancy test strips, respectively). Where human blastoid cells do not adhere to and invade unstimulated endometrial cells, levels are undetectable (left test strip). [Figure 7-2]In vitro combination of human blastoid cells and open-faced endometrial organoids recapitulates features of blastocyst implantation into the uterus (cont.). G. Immunostaining shows that the hormone hCGB is produced only by a portion of the attached blastoid cells. Immunostaining also shows that the attached blastoid cells form numerous cells positive for polar trophoblast and NR2F2, a marker for post-implantation trophoblast. These trophoblasts are distinct from the Oct4-positive cells characteristic of epiblast. The attached human blastoid cells contain trophoblast cells that express CK7, a marker for post-implantation trophoblast cells. H. Upon attachment and invasion of endometrial cells, the blastoid cells form cells positive for epiblast markers Oct4, Klf17, Nanog, and IFI16. [Figure 8]Triple-inhibited naive hPSCs efficiently form human blastocyst-like structures containing analogs of the three founder lineages. a. Schematic of the time frame of human peri-implantation development thus modeled. M / MC / B=Morula / Morula Compacted / Blastocyst. b. One-step protocol for human blastoid cell formation. N2B27: serum-free medium. PALLY: PD0325901, A83-01, hLIF, LPA, Y-27632. c. Phase contrast image of human blastocyst-like structures formed on non-adherent hydrogel microwell arrays after 96 hours. Each microwell is 200 μm in diameter. Scale bar: 400 μm. d. Phase contrast image of a representative human blastocyst-like structure harvested from a microwell. Scale bars: 200 μm (top) and 100 μm (bottom). e. Quantification of the percentage of microwells containing human blastocyst-like structures for different naive hPSC lines cultured in PALLY conditions with optimized LPA concentration (Yield (%) of blastocyst-like structures; see also morphometric definition of blastocyst-like structures in Methods; n = 3 microwell arrays; mean ± standard deviation. f, g. Immunofluorescence staining of epiblast (EPI) markers (yellow) NANOG (f) and OCT4 (g); TE markers (cyan) CDX2 (f) and GATA3 (g); and primitive endoderm markers (magenta) SOX17 (f) and GATA4 (g) in human blastocyst-like structures. Scale bar: 100 µm. h. Quantification of the absolute number of OCT4-, GATA3-, GATA4-positive cells based on immunofluorescence staining (left), and the proportion of cells belonging to individual lineages expressed as a percentage of the total cell number in blastocyst-like structures (96 h) (right). Representative immunofluorescence staining of the tight junction molecule ZO-1 (yellow), the adherens junction molecule CDH1 (magenta), and the apical molecule aPKC (cyan) in a representative human blastocyst-like structure. Scale bar: 50 μm. [Figure 9-1]Human blastocyst-like structures form analogs of the three preimplantation lineages. a,b. UMAP of the transcriptomes of single cells derived from blastocyst-like structures (24, 60, 96 h), naive hPSCs, primed hPSCs, and hTSCs (representing the post-implantation cytotrophoblast layer); individual cells are colored based on their origin (a) or unsupervised cluster affiliation (b). c. Expression levels of markers for each blastocyst lineage (trophectoderm (TE), epiblast (EPI), and primitive endoderm (PrE)). d. Unsupervised distance map generated using the top 30 genes enriched in clusters 0, 1, and 3 (as defined in UMAP (see b)). [Figure 9-2] Human blastocyst-like structures form analogues of the three preimplantation lineages (cont.). e,f. UMAPs of single-cell transcriptomes of blastocyst-like structures, naïve hPSCs, and primed hPSC-derived cells were integrated with published datasets of preimplantation, periimplantation (in vitro cultured blastocysts), and gastrulation stages (Carnegie stage 7, i.e. E16–19) human embryos. Individual cells are colored based on their origin in the human embryo (e), blastocyst-like structures, or stem cells (f). [Figure 10-1]Three lineages are formed according to the sequence and time of blastocyst development. a. Schematic showing the sequential lineage specification of human blastocysts. b. Immunofluorescence staining of YAP1 (yellow) and GATA2 (cyan) in aggregates of naive hPSCs cultured in PALLY medium for 60 h. Scale bar: 50 μm. c. Dose-dependent effect of LPA on the yield of blast-like cells. n=3 independent microwell arrays; mean ± standard deviation; one-way ANOVA and Dunnett's multiple comparison test, ** is P=0.0016; **** is P<0.0001. d. Measurement of the effect of overexpression of different variants of YAP1 on cavity formation events in early blast-like cells. n=3 experiments; mean ± standard deviation; one-way ANOVA and Dunnett's multiple comparison test, ns is not significant, *** is P=0.0004; **** is P=0.00004. [Figure 10-2] Three lineages are formed according to the order and time of blastocyst development. e. Quantification of total cell numbers per lineage in developing blastoid cells at three time points of development (24, 60, 96 h). Error bars: standard deviation, n: Epi: 11 blastoid cells at 24 h, 68 h, and 96 h; TE: 8, 14, and 15 blastoid cells at 24 h, 48 h, and 96 h, respectively; PrE: 9, 37, and 9 blastoid cells at 24 h, 48 h, and 96 h, respectively. f. Immunofluorescence staining of DX2 (cyan), NR2F2 (magenta), and NANOG (yellow) in a representative B4 stage human blastocyst (left) and blastoid cells (center). Quantification of the percentage of blastoid cells with a preferentially polar NR2F2 expression pattern (axis) compared to a preferentially mural NR2F2 expression pattern (inverted axis) (right). n = 4 independent experiments with 4–12 blastoid cells per experiment. Mean ± SD; One-way ANOVA and Tukey's multiple comparison test, *, P < 0.05; ***, P < 0.001. Scale bar: 50 μm. Error bar: standard deviation. [Figure 11-1]Human blast-like cells recapitulate aspects of implantation. a. Schematic of modeled implantation times (left). Open-faced endometrial layers (OFELs) primed with EPC / XAV939 for receptivity as an implantation assay (right). E2: beta-estradiol. EPC: E2, progesterone, cAMP. b. Representative phase-contrast images of blast-like cells (GFP+) 24 hours after deposition on unstimulated OFELs (top left) or stimulated OFELs (bottom left). Scale bar: 100 μm. Attachment efficiency of human blast-like cells (right). n=7 independent experiments from 3 different donors; mean ± standard deviation; unpaired two-tailed t-test, **** is P=4.5e-8. c. Representative image of recently attached human blast-like cells (12 ± 4 hours). Dotted lines outline the inner cluster of blast-like cells formed from GFP+ naive hPSCs (top). Scale bar: 100 μm. XZ plane (bottom) of immunofluorescence staining for NR2F2 (magenta) and OCT4 (yellow) in blastoid cells immediately after attachment. Scale bar: 5 μm. [Figure 11-2]Human blastoid cells recapitulate aspects of implantation (cont.). d. Intensity profile of immunofluorescence staining (NR2F2, OCT4) in blastoid cells immediately after attachment. n=10. e. Representative phase contrast images of trophospheres formed using 3 μM SC144 (top) or 2 μM XMU-MP-1 (middle) and aggregates of hTSCs deposited on stimulated OFELs (bottom). Scale bar: 100 μm. Attachment efficiency (right). n=3 independent experiments; mean ± SD; one-way ANOVA and Dunnett's multiple comparison test, **** is P<0.0001. f. Pregnancy test strips detecting human chorionic gonadotropin (CGβ) secretion into the medium of unstimulated OFELs with unattached blastoid cells and stimulated OFELs with attached blastoid cells (48 h on OFELs, see also ELISA assay in Fig. 21b). g. Immunofluorescence staining of OCT4 (yellow) and aPKC (grey) in human blastocysts (left) or blastoid cells (right) grown for 4 days in post-implantation culture conditions. Counterstained with phalloidin to label F-actin (cyan). Scale bar: 100 μm. h. Number of OCT4, GATA3, and GATA4 positive cells in blastoid cells grown for 6 days in post-implantation culture conditions (time equivalent = day 13). n=5. Mean ± standard deviation. [Figure 12-1] Naïve hPSCs form human blastocyst-like structures that contain analogues of the three founder lineages. a. Phase contrast images of naïve hPSCs cultured in PXGL medium and on a MEF feeder layer. Scale bar: 50 μm. b. Time-lapse phase contrast images of naïve hPSC aggregates cultured in microwell arrays either without LPA (PALY medium, top) or with 500 nM LPA (PALLY medium, bottom). Scale bar: 200 μm. [Figure 12-2]Naive hPSCs form human blastocyst-like structures containing analogues of the three founder lines (cont.). c. Quantification of the effect of initial cell number per microwell array on the yield of blastocyst-like structures. n=1 microwell array. d. Quantification of the effect of serial passaging of naive hPSCs on the yield of blastocyst-like structures. n=3 microwell arrays. Mean ± standard deviation. e. Quantification of the number of cells per microwell in blastocyst-like structures at seeding and after 96 h when cells were seeded at 3.0 × 104 cells per microwell array. n=190 microwells (seeded) and n=12 blastocyst-like structures (96 h). f. Fluorescent staining of DNA using Hoechst in representative naive hPSCs aggregated during the formation of blastocyst-like structures (96 h, left). Scale bar: 50 μm. Measurement of the distribution diameter of structures during the formation of blastocyst-like structures (right). At 0, 60, and 96 hours, n=15, 31, and 11, respectively. g. Pseudotime analysis of human preimplantation development showing expression of TE markers GATA2, GATA3, CDX2, and TACSTD2. Gene expression analysis was performed using a public data analysis tool (https: / / bird2cluster.univ-nantes.fr / demo / PseudoTimeUI / ). [Figure 12-3]Naïve hPSCs form human blastocyst-like structures containing analogues of the three founder lineages (cont.). h. Immunofluorescent staining for EPI marker NANOG (yellow), TE marker CDX2 (cyan), and primitive endoderm marker GATA4 (magenta) in a representative B4 stage human blastocyst in five representative blastocyst-like structures. Scale bar: 50 μm. i. Immunofluorescent staining for EPI markers (yellow) NANOG (top) and OCT4 (bottom); TE markers (cyan) CDX2 (top) and GATA3 (bottom); and primitive endoderm marker (magenta) GATA4. Counterstained with Hoechst (grey) to label DNA. Scale bar: 50 μm. j. Immunofluorescent staining for EPI marker OCT4 (yellow) and TE marker GATA2 (cyan) in blastocyst-like structures. Scale bar: 100 μm. k, Immunofluorescence staining of TE markers GATA3 (cyan) and TROP2 (magenta) in blastocyst-like structures. Scale bar: 100 μm. l, Immunofluorescence staining of TE markers GATA3 (cyan) and GATA4 (magenta) and PrE marker PDGFRa (yellow) in blastocyst-like structures. Scale bar: 100 μm. [Figure 12-4] Naive hPSCs form human blastocyst-like structures containing analogs of three founder lineages (cont.). m. Single optical section of immunofluorescent staining image of tight junction molecule ZO-1 (yellow), adherens junction molecule CDH1 (magenta), and apical molecule aPKC (cyan) in a representative human blastocyst-like structure. Scale bar: 50 μm. n. Representative time points of time-lapse images of naive cell aggregates cavitating into blastocyst-like structures showing cycles of cavity expansion and contraction (left) - quantification of blastocyst-like structures showing distinct expansion and contraction frequencies (right). n=1 microwell array. Scale bar: 100 μm. o. Phase contrast images of representative areas of microwell arrays showing blastocyst-like structures formed from naive hPSC and hiPSC lines. n>3. Scale bar: 100 μm. p. Quantification of the yield of blastocyst-like structures obtained from naive and primed H9 hPSCs, n=3 microwell arrays, mean ± SD. [Figure 13]Human blastocyst-like structures form analogs of preimplantation lineages. a. Flow cytometry analysis plots of cells isolated from blastocyst-like structures and stained for lineage-specific surface markers PDGFRa (PrE) and TROP2 (TE). Gates were used to sort analogs of EPI (double negative), TE (TROP2high), and PrE (PDGFRαhigh) prior to processing for single-cell RNA sequencing. Note that the gates did not exclude cells. This analysis was performed to correlate RNA measurements while ensuring representation of all cell types. b. UMAP of the transcriptome of a single cell isolated from a blastocyst-like structure showing expression levels of genes specific to each of the three blastocyst lineages (TE-trophectoderm, EPI-epiblast, and PrE-primitive endoderm). c-g. UMAP of single cells isolated from both blastocyst-like structures and embryos from E3 to E19. c. Color of cells derived from in vitro fertilized (IVF) embryos isolated from day 3 (E3) to day 7 (E7). This period includes only embryos at the preimplantation stage. d. Color of cells derived from IVF embryos isolated from day 6 (E6) to day 12 (E12). These blastocysts (E6) were cultured in vitro. Note that this annotation reflects days in culture and not developmental stage. e. Color of cells derived from gastrulation stage embryos isolated from day 17 (E17) to day 19 (E19). f. Expression levels of genes specific to each of the three blastocyst lineages (EPI, TE, and PrE). g. Coloring of cells showing unsupervised cluster affiliation. [Figure 14-1]Measurement of off-target cell generation in human blastocyst-like structures and naïve human pluripotent stem cells. a,b. UMAP of clusters formed from cells isolated from blastocyst-like structures (1, × 50 high-resolution clustering compared to Figure 2b) (a), showing expression levels of genes specific to the amniotic lineage (b). c. Origin of cells constituting cluster 11. d-h. UMAP of naïve hPSCs, primed hPSCs, cells isolated from blastocyst-like structures, and cells isolated from human embryos at stage CS7. d. Coloring of embryonic cells. e. Color coding based on stem cell origin. f. Display of expression levels of genes specific to each of the three blastocyst lineages (EPI-epiblast, TE-trophectoderm, and PrE-primitive endoderm). [Figure 14-2] Measuring off-target cell generation in human blastocyst-like structures and naïve human pluripotent stem cells (cont.). g. Coloring of individual cells based on unsupervised cluster affiliation. h. Coloring of cells previously identified as cluster 11 (see a, b). i. Quantification of the percentage of cells identified as abnormal based on their location within the UMAP (top) and cell annotation (bottom) in h for both naïve hPSCs (left) and cells isolated from blastocyst-like structures (right). Similar results were obtained based on their location within the UMAP (Figure 13c-e). j. Heatmap of markers of different lineages differentially expressed in cells of blastocyst-like structures and gastrulation stage embryos. [Figure 15]Cells within human blastocyst-like structures are transcriptionally similar to preimplantation lineages. a. Principal component analysis (PCA) plots of PC1 vs. PC2 (top) or PC1 vs. PC3 (bottom) calculated using the top 500 variable genes of the bulk transcriptomes of individual lineages of blastocyst-like structures (EPI, TE, and PrE); stem cell lines: naive hPSCs and primed hPSCs; hTSCs: blastocyst-derived hTSCs (bTS5), primed hPSCs and hTSCs (BAP and TM4 protocols); PrE-like stem cell lines (RACL or nEND cells); naive PSCs and TSCs re-derived from blastocyst-like structures (see extended methods). b. Heatmap of key blastocyst and postimplantation lineage markers differentially expressed between TE analogs (TROP2+) of blastocyst-like structures and hTSCs within the bulk transcriptome. c. Pseudo-time series analysis of human mature TE markers CGB5, CGB7, CGB8 and CGA. Gene expression analysis was performed using public data analysis tools (https: / / bird2cluster.univ-nantes.fr / demo / PseudoTimeUI / ). d. Heatmap of key pluripotency-related genes differentially expressed between EPI analogs (PDGFR- / TROP2-) in blastocyst-like structures and primed hPSCs. e. Heatmap of key pluripotency-related genes or PrE markers differentially expressed between PrE analogs (PDGFRα+) in blastocyst-like structures, naïve PSC-derived PrE-like cells and nEND cells. [Figure 16]Human blastocyst-like structures allow the derivation of stem cell lines. a. Immunofluorescence staining of pluripotency factors NANOG (yellow), OCT4 (magenta), SOX2 (cyan), and naive pluripotency factor KLF17 (yellow) in naive hPSC control (top) and naive hPSC derived blastocyst-like structures (bottom). Scale bar: 100 μm. b. Phase contrast images of blastocyst-like structures on microwell arrays formed from three rederived naive hPSC lines. Scale bar: 200 μm. c. Immunofluorescence staining of EPI marker (NANOG), TE marker (CDX2), and primitive endoderm marker (GATA4) in a representative second generation blastocyst-like structure. Scale bar: 100 μm. d. Immunofluorescence staining of GATA3 (cyan), post-implantation trophoblast marker CK7 (magenta) and CDX2 (yellow) in bTS5 hTSCs (top) and hTSCs derived from blastocyst-like structures (bottom). Scale bar: 100 μm. e. Phase contrast images of day 6 EVT differentiation from three hTSC lines derived from blastocyst-like structures. Scale bar: 150 μm. f. Immunofluorescence staining of trophoblast marker GATA3 (cyan) and EVT markers HLA-G (yellow) and CGβ (magenta) of day 6 EVT analogs derived from three hTSC lines derived from blastocyst-like structures. Scale bar: 100 μm. g. Phase contrast images of day 3 SCT analogs differentiated from three hTSC lines derived from blastocyst-like structures. Scale bar: 150 μm. h. Immunofluorescence staining of trophoblast marker GATA3 (cyan) and SCT markers SDC1 (yellow) and CGβ (magenta) of day 3 SCT analogs formed from hTSC line (clone 1) derived from blastocyst-like structures. Scale bar: 100 μm. i. Immunofluorescence staining of CGβ (magenta) counterstained with phalloidin (cyan) and Hoechst to mark actin and DNA, respectively (left), and immunofluorescence staining of SDC (yellow), CK7 (magenta), and CK7 (magenta) counterstained with Hoechst to mark DNA (right) of day 6 trophoblast organoids formed from hTSC line (clone 1) derived from blastocyst-like structures. Scale bar: 50 μm. j. Relative expression levels measured by RT-PCR of day 6 EVT (top) and day 3 SCT analogs (bottom) of the respective undifferentiated hTSC line derived from blastocyst-like structures.Expression levels were normalized to expression of GAPDH, n=1 biological replicate of 3 independent clones. [Figure 17-1] Development of the human trophectoderm analogue depends on aPKC and Hippo elements. a. Time-lapse microscopy frames of a B2 stage human blastocyst (left). Schematic showing differential Hippo activity in inner and outer cells of the developing blastocyst and molecular regulators of the Hippo signaling pathway (right). b. Phalloidin fluorescent (cyan) staining of F-actin in naive hPSC aggregates cultured in PALLY medium for 24 h (top) and 60 h (bottom). Counterstained with Hoechst to mark DNA. Scale bar: 50 μm. c. Immunofluorescent staining of aPKC (cyan) and YAP1 (yellow) in naive hPSC aggregates cultured in PALLY medium for 24 h (top) and 60 h (bottom). Counterstained with Hoechst to mark DNA. Scale bar: 50 μm. [Figure 17-2]Development of human trophectoderm analogues depends on aPKC and Hippo elements (cont.). d. Immunofluorescent staining of YAP1 (yellow) and GATA2 (cyan) in aggregates of naive hPSCs cultured in PALLY medium for 24 h. Scale bar: 50 μm. e. Immunofluorescent staining of YAP1 (yellow) and GATA3 (cyan) (top), and YAP1 (yellow) and NANOG (cyan) (bottom) in aggregates of naive hPSCs cultured in PALLY medium for 24 h (left) and 60 h (right). Counterstained with Hoechst to mark DNA. Scale bar: 50 μm. f. Immunofluorescent staining of YAP1 (yellow) and GATA3 (cyan) in blastoid cells cultured without (top) or with aPKC inhibitor (2 μM CRT0103390, bottom). Counterstained with Hoechst to mark DNA. Inset: Individual and merged channels of YAP1 and GATA3 in a single optical section, as well as maximum intensity projections of all optical sections. Scale bar: 50 μm. g. Quantification of blast-like cell yield in cultures in PALLY medium or PALLY medium supplemented with aPKC inhibitor (2 μM CRT0103390). n=3 independent microwell arrays. Mean ± SD; two-tailed unpaired t-test. *** is P=0.0002. h. Quantification of the percentage of GATA3+ cells in structures cultured in PALLY medium or PALLY medium supplemented with aPKC inhibitor (2 μM CRT0103390). n=7 blast-like cells in the group cultured in PALLY medium and n=12 aggregates in the group cultured in PALLY medium supplemented with CRT0103390. Representative results from three independent experiments. Mean ± SD; two-tailed unpaired t-test. ****P=1.79e-08. [Figure 17-3]Development of human trophectoderm analogues depends on aPKC and Hippo elements (cont.). i. Quantification of the dose-dependent effect of the LPA receptor agonist NAEPA on blast-like cell yield. PALY medium (hence LPA-free) was supplemented with NAEPA. n=3 independent microwell arrays. Mean ± SD; one-way ANOVA and Tukey's multiple comparison test. ****P<0.0001. j. Phase contrast images of representative naive hPSC aggregates cultured for 72 h in PALLY medium supplemented with doxycycline (100 ng / ml) and overexpressing different variants of YAP1. Naive hPSC aggregates were cultured in conditioned PALLY medium characterized by reduced LPA concentrations (5 nM). Scale bar: 100 μm. k. Measurement of the effect of verteporfin (an inhibitor of the YAP1-TEAD complex) on blast-like cell yield. n=3 independent microwell arrays. Mean ± SD; One-way ANOVA and Dunnett's multiple comparison test. **, p = 0.0010, ***, p = 0.00019, ****, P < 0.0001. l. Phalloidin fluorescent staining of F-actin (cyan) in naive hPSC aggregates cultured in PALLY medium for 60 h. Counterstained with Hoechst to mark DNA. Yellow arrows: formation of cavities. Scale bar: 50 μm. m. Immunofluorescent staining of aquaporin 3 (AQP3, cyan) and OCT4 (yellow) in naive hPSC aggregates cultured in PALLY medium for 36 h (left) or 96 h (right, blast-like cell stage). Scale bar: 50 μm. [Figure 18-1]Blastoid cells recapitulate the sequential specification of lineages that occurs during blastocyst development. a. Heatmap of mean count values ​​in the expression of TE genes upon formation of blastoid cell TE analogs. b-d. Immunofluorescence staining of GATA3 (cyan) and OCT4 (yellow) (b), or CDX2 (cyan) and NANOG (yellow) (c), or CDX2 (cyan) and KLF17 (yellow) (d) in naive hPSC aggregates cultured in PALLY medium for 24 h (top) or 60 h (bottom). Scale bar: 50 μm. e. Gene ontology terms associated with the genes differentially regulated in the late TE analogs of blastoid cells (cluster 10) compared to early TEs (cluster 2). [Figure 18-2] Blastoid cells recapitulate the sequential specification of lineages that occurs during blastocyst development (cont.). f. Heatmap of mean count values ​​of Wnt, TGF-β, and Notch signaling-related genes in cells from clusters 4 (naive hPSCs), 10, 2, and 5 (TE analogs), and 7 (TSCs). g. UMAP of single cells isolated from blastoid cells showing expression levels of polar trophectoderm-specific gene NR2F2. h. Immunofluorescence staining of CDX2 (cyan), NR2F2 (magenta), and NANOG (yellow) in blastoid cells. Scale bar: 100 μm. i. UMAP of single cells isolated from blastoid cells showing expression levels of polar trophectoderm-specific gene CCR7. j. Immunofluorescence staining of CCR7 (cyan) in blastoid cells. Counterstained with Hoechst to mark DNA. Scale bar: 50 μm. k. Heatmap of the average count values ​​of the top differentially regulated genes in cells from clusters 4 (naive hPSCs), 0 (EPI analog), and 9 (primed hPSCs). [Figure 18-3]Blast-like cells recapitulate the sequential specification of lineages that occurs during blastocyst development (cont.). l. Immunofluorescence staining of KLF17 (cyan) and OCT4 (yellow), or KLF4 (cyan) and OCT4 (yellow) (top), and SUSD2 (cyan) and NANOG (yellow), or IFI16 (cyan) and KLF17 (yellow) (bottom) in blast-like cells. Counterstained with Hoechst to mark DNA. Scale bar: 100 μm. m. UMAP of single cells isolated from blast-like cells showing expression levels of the X-chromosome activation-associated gene XACT. n. Flow cytometry analysis plot of cells isolated from blastocyst-like structures cultured in PALLY medium for 60 h and stained with lineage-specific surface markers PDGFRa (PrE) and TROP2 (TE). op, Immunofluorescence staining of OTX2 (cyan), GATA4 (magenta), and OCT4 (yellow) (o), and SOX17 (cyan), and GATA4 (magenta) (p) in naive hPSC aggregates cultured in PALLY medium for 60 h. Counterstained with Hoechst to mark DNA. Scale bar: 50 μm. q. Heatmap of mean count values ​​in PrE gene expression upon formation of blastoid cells PrE analogs. r. Heatmap of mean count values ​​of SMAD, MAPK, and Wnt signaling-related genes in cells of clusters 1, 6 (EPI analogs) and 8 (PrE analogs). [Figure 19]Human blastoid cells recapitulate aspects of implantation. a. Immunofluorescence staining of CDH1 (magenta) and ciliated cell marker acetylated α-tubulin (yellow) in OFEL (left). YZ plane shows apical position of cilia (right). Scale bar: 50 μm. b. Immunofluorescence staining of FOXA2 (yellow) showing endometrial glandular cells in OFEL. Scale bar: 50 μm. c. Immunofluorescence staining of PAEP (yellow) in unstimulated OFEL (left) and stimulated OFEL (right). d. qRT-PCR measurement of expression levels of implantation window markers in OFEL cultured in different media. Ctrl: control medium, E: estradiol, P: progesterone, C: cAMP, X: XAV-939. Expression levels were normalized to the housekeeping gene GAPDH and control conditions. n=2 independent experiments. Colors indicate data from three different donors. e. Heatmap of key cell cycle and secretory epithelial genes differentially expressed between stimulated and unstimulated OFELs in the bulk transcriptome. f. Staining for incorporated EdU (red) reflecting cell proliferation in stimulated OFELs (left). Scale bar: 50 μm. Quantification of the number of EdU+ cells in unstimulated and stimulated OFELs (right). Counterstained with Hoechst to mark DNA. n=4 independent experiments. Mean ± SD; unpaired two-tailed t-test, *** is P=0.0009. g. Quantification of blastoid cell attachment to OFELs prepared using endometrial organoids from three different donors. n=3 independent experiments for donor 1, n=2 independent experiments for donors 2 and 3. Mean ± SD; unpaired two-tailed t-test, ** is P=0.0011. h. Immunofluorescence staining of MUC1 (magenta), a glycoprotein highly expressed at the luminal epithelial surface of receptive endometrium with attached GFP+ blast-like cells (48 h after deposition on OFEL). Dashed lines indicate areas where trophoblast cells repelled endometrial cells. Scale bar: 200 μm. i. Quantification of blast-like cell attachment to unstimulated OFEL, stimulated OFEL, and OFEL additionally exposed to the contraceptive drug levonorgestrel (LNG, 10 μM). n=3 independent experiments.Mean ± standard deviation; one-way analysis of variance and Tukey's multiple comparison test, *, P = 0.0211, ***, P = 0.0006. [Figure 20-1] The status of the trophectoderm is important for the interaction with the endometrium during implantation. a. Representative image of human blast-like cells immediately after attachment to OFEL. Dotted lines outline the inner cluster of blast-like cells formed using GFP+ naive hPSCs (top). Immunofluorescent staining of NR2F2 (magenta) and OCT4 (yellow) in blast-like cells immediately after attachment to OFEL (bottom). b. Immunofluorescent staining of NR2F2 (magenta) and OCT4 (yellow) and respective fluorescence intensity profiles of a representative blast-like cell immediately after attachment to OFEL. Profiles were measured perpendicular to the attachment surface (right). Line width, 10 μm. Y-axis indicates normalized intensity. c. Quantification of the distance between the first peaks of the fluorescence intensity profiles of NR2F2 and OCT4. n=10 attached blast-like cells. Mean ± SD. d. Pseudo-sequence analysis of human preimplantation development showing expression of IL6, IL6R, GP130, and STAT3. Gene expression analysis is performed using public data analysis tools (https: / / bird2cluster.univ-nantes.fr / demo / PseudoTimeUI / ). e. Quantification of the dose-dependent effect of LIF on the yield of blast-like cells. n=2 (without Lif) and n=3 (all other conditions) independent experiments. Mean ± standard deviation. f. Immunofluorescence staining of NANOG (yellow) and CDX2 (cyan) (left), OCT4 (yellow) and GATA3 (cyan) (center), and CDX2 (cyan) and NR2F2 (magenta) (right) in representative trophospheres formed from blast-like cells exposed to SC144. Scale bar: 50 μm. g. Immunofluorescence staining of NANOG (yellow) and CDX2 (cyan) (left), OCT4 (yellow) and GATA3 (cyan) (right) in representative trophospheres formed from blast-like cells exposed to XMU-MP-1. Scale bar: 50 μm. [Figure 20-2]Trophectoderm status is important for interaction with the endometrium during implantation (cont.). h. Heatmap of key lineage-specific genes differentially expressed in the bulk transcriptomes of blastoid cells (TROP2 positive cells), trophosphere (SC144 or XMU) and TSC (2D or 3D) trophectoderm compared to naïve hPSCs. [Figure 20-3] The status of the trophectoderm is important for its interaction with the endometrium during implantation (cont.). i. PCA plots calculated using bulk transcriptomes of blastoid cells, hPSCs (naive, primed, or blastoid re-derived naive cell lines), TSCs (bTS5, blastocyst re-derived lines or human stem cell-derived TSC-like cells), and pluripotent stem cell-derived primitive endoderm-like cells (RACL or NACL cells). j. Immunofluorescence staining of CDX2 (cyan) (left), CK7 (magenta) and GATA3 (cyan) (right) in aggregates formed from bTS5 hTSCs. Counterstained with Hoechst marking DNA. Scale bar: 50 μm. k. Representative phase contrast image of aggregates of naive hPSCs deposited on stimulated OFEL. Scale bar: 100 μm. l. List of selected putative ligand-receptor pairs involved in crosstalk between polarized trophectoderm and endometrial epithelial cells. This list was generated by in silico ligand-receptor analysis of genes enriched in polarized trophectoderm and stimulated OFEL using Cellinker. [Figure 21]Human blast-like cells recapitulate aspects of the pre- and post-implantation progression up to day 13. a. Brightfield images of human blast-like cells (96 h) cultured for an additional 4 days on low-attachment plates in post-implantation culture conditions (left). Each row shows the time series of an individual blast-like cell over 4 days. Note that when transferred to IVC medium with different osmolarity compared to N2B27 medium with PALLY, the blast-like cells stably retain the cavity for at least 2 days (see Extended Methods for composition of post-implantation medium). Scale bar: 200 μm. Quantification of the percentage of blast-like cells retaining the cavity at each day of culture during the post-implantation stage (right). n=2 independent experiments. b. Immunofluorescence staining of the syncytiotrophoblast-associated marker CGβ (magenta) in GFP+ blast-like cells attached on stimulated OFEL (48 h after deposition) (left). Counterstaining with Hoechst marking DNA. Scale bar: 50 μm. ELISA measurement of the concentration of secreted protein CGβ in the medium of unstimulated OFEL with unattached blast-like cells and stimulated OFEL with attached blast-like cells at 24 and 48 h (right). n=3 independent experiments. Mean ± SD; one-way ANOVA and Tukey's multiple comparison test, **** is P=0.00006. c. Immunofluorescence staining of CDX2 (cyan), NR2F2 (magenta), and SOX2 (yellow) in blast-like cells grown for 4 days in post-implantation culture conditions. Scale bar: 100 μm. d. Immunofluorescence staining of OCT4 (yellow), CK7 (cyan), and GATA4 (magenta) in blast-like cells grown for 4 days in post-implantation culture conditions. Scale bar: 100 μm. e,f. Immunofluorescence staining of CGβ (magenta) and NR2F2 (cyan) (e) or HLA-G (magenta) and GATA3 (cyan) (f) in blast-like cells grown in post-transplant culture conditions for 4 days (e) or 6 days (f). Counterstained with Hoechst to mark DNA. Arrows point to HLA-G positive EVT-like cells. Scale bar: 100 μm. g. Immunofluorescence staining of CD24 (magenta) and SOX2 (yellow) in blast-like cells grown in post-transplant culture conditions for 6 days. Counterstained with Hoechst to mark DNA. Scale bar: 100 μm.h. Immunofluorescence staining of PODXL (magenta) and SOX2 (yellow) in blast-like cells grown in post-transplant culture conditions for 4 days. Counterstained with phalloidin to mark F-actin (cyan). Arrowheads point to the pro-amniotic-like cavity. Scale bar: 100 μm. i–k. Immunofluorescence staining of SOX2 (yellow), GATA3 (cyan), and CDX2 (magenta) (i), SOX2 (yellow), CDX2 (magenta), and TFAP2C (cyan) (j), OCT4 (yellow), GATA4 (magenta), and OTX2 (cyan) (k) in blast-like cells grown in post-transplant culture conditions for 4 days. Counterstained with Hoechst to mark DNA. Scale bar: 100 μm. l. Quantification of the number of cells belonging to the EPI, TE, or PrE lineages in blastoid cells grown for 4 days in post-implantation culture conditions on glass or OFEL. n=7 biological replicates. Mean ± SD. m. Immunofluorescence staining for OCT4 (yellow), GATA3 (cyan), and GATA4 (magenta) in blastoid cells grown for 6 days in post-implantation culture conditions, a time equivalent to day 13 for cultured human blastocysts (left). Scale bar: 100 μm. [Figure 22] Quantification of the percentage of microwells containing human blastocyst-like structures formed from aggregated naive hPSCs stimulated or not with LPA, and PD0325901, and A83-01 (triple inhibition). [Diagram 23] Phase contrast images of representative areas of microwell arrays showing blast-like cells formed by treatment of PLLY with two different TGFb signaling inhibitors, 1 μM A83-01 (top) or 1 μM SB431542 (bottom, abbreviated as "SB43") for 4 days. Quantification of the yield of blast-like cell structures. Dotted lines represent the yield of blast-like cells with the standard protocol (PALLY for 2 days, LY for 2 days). n=3 microwell arrays. Error bars: standard deviation. PLLY: 1 μM PD0325901, 500 nM LPA, 10 ng / ml hLIF, 10 μM Y-27632. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention includes a method for forming blastocyst-like cell aggregates, commonly referred to as blastoid cells, from human pluripotent stem cells (hPSCs). In contrast to in vivo blastocysts, blastoid cells can be produced in large quantities and are suitable for genetic and drug screening, but given that artificial blastocyst-like cell aggregates and blastoid cells cannot form or develop into human embryos, some of the ethical concerns associated with the manipulation of human embryos are alleviated.

[0025] Blastocyst-like cell aggregates and blastoid cells are potentially important human embryo models for biomedical insights such as drug safety / efficacy or early pregnancy treatments (e.g., improved IVF procedures and contraception).

[0026] The blastoid cells of the present invention are capable of forming a primary axis, whose epiblast induces the maturation of polarized trophectoderm, and thus acquire the potential to specifically adhere to hormonally stimulated endometrial cells. Such human blastoid cells are an accurate, scalable, versatile and ethical model for investigating human transplantation and development.

[0027] As used herein, the terms blastocyst-like cell aggregates, blastocyst-like structures and blastoid cells are used interchangeably to reflect tissues obtainable by the methods of the invention that model blastocysts rather than true blastocysts, and the term blastocyst refers to such embryos.

[0028] The blastoid cells recapitulate the three-dimensional morphological and molecular features of the human blastocyst, including the concomitant specification and spatial organization of tissues that reflect the three fundamental lineages that form the entire organism: the trophectoderm, epiblast, and hypoblast.

[0029] A high-fidelity and high-efficiency model of the human blastocyst would support scientific and medical advances. However, its predictive power depends on the ability to accurately reproduce the cellular specification and sequence of morphogenesis of the blastocyst according to its natural developmental pace. Accurate modeling not only ensures the formation of cells that reflect only the blastocyst stage, but also allows aspects of implantation and pre-implantation development to be reproduced in vitro.

[0030] The present invention also describes the treatment of (human) blastocysts to prepare them to improve the probability of implantation, for example during in vitro fertilization (IVF) procedures or when treating patients after natural conception to improve the chances of conception. Such treatments may be medical or therapeutic in nature to treat the human fetus or the recipient mother. With respect to such methods, the present invention also relates to the manufacture of therapeutic compounds (e.g., HIPPO pathway inhibitors) or pharmaceutical compositions comprising said compounds for use in therapy. The human embryo itself or its use for industrial or commercial purposes may not be part of the present invention.

[0031] In a central aspect, the present invention provides a method for generating blastoid or blastocyst-like cell aggregates, comprising culturing human pluripotent stem cells (hPSCs) and trophoblast cell aggregates in 3D culture in a medium containing a HIPPO pathway inhibitor. Preferably, the human pluripotent stem cells (hPSCs) are surrounded by, or become surrounded by, trophoblast cells.

[0032] According to the present invention, the formation of blast-like cells according to the present invention is achieved by forming three-dimensional aggregates of hPSCs and regulating the activity of the HIPPO pathway, which leads to the concomitant specification and three-dimensional self-organization of epiblast, trophectoderm, and hypoblast-like cells along with the formation of embryonic-non-embryonic axis.Therefore, HIPPO pathway inhibitors are used as a central aspect of the present invention to generate blast-like cells.

[0033] The present invention provides for the first time the induction of the simultaneous formation of three cell types from hPSCs: (i) epiblast-like cells, (ii) trophectoderm-like cells, and (iii) hypoblast-like cells, and their self-organization into structures morphologically and molecularly similar to human blastocysts, thereby recapitulating the 3D morphological changes constrained by simultaneous cell lineage segregation, morphogenesis, and tissue maturation reflecting the trophectoderm, epiblast, and hypoblast.

[0034] The resulting blastoid cells, when rendered receptive by stimulation with, for example, estrogen, progesterone, cAMP, and the Wnt inhibitors XAV939 and / or LF3, can actively interact with layers of endometrial cells in vitro or with the endometrium, the lining of the uterus, in vivo. As with the blastocyst, attachment, invasion, and differentiation of the blastoid cells into endometrial cells occurs primarily via the polarized trophectoderm, or epiblast cells, and the adjacent trophoblast. Upon implantation, the polarized trophoblast of the blastoid cells proliferates, differentiates, and produces human chorionic gonadotropin, the hormone used to indicate clinical pregnancy.

[0035] Thus, the method of the present invention is useful for the following applications: 1. Genetic and drug screening for understanding and management of early pregnancy; 2. Development of contraceptives and fertility drugs; 3.Development of culture conditions for IVF; 4. In vitro toxicity / safety assays for drug development; 5. In vitro formation of specific cells, tissues, and organs for in vitro assays and in vivo transplantation.

[0036] The present invention comprises culturing aggregates of human pluripotent stem cells (hPSCs) and trophoblast cells in 3D culture in a medium containing a HIPPO pathway inhibitor.Preferably, the human pluripotent stem cells (hPSCs) are surrounded by trophoblast cells in this method step, or the human pluripotent stem cells (hPSCs) are surrounded by trophoblast cells, for example, through a previous step, for example, any step of culturing aggregated hPSCs in a medium containing a HIPPO pathway inhibitor, where trophoblast cells may form around the hPSCs.

[0037] The terms "HIPPO pathway inhibitor" and "HIPPO pathway antagonist" are used interchangeably herein. The terms refer to compounds that reduce the activity of the HIPPO pathway. The HIPPO pathway is reviewed in Gumbiner and Kim, Journal of Cell Science (2014) 127,709-717 (incorporated herein by reference). The HIPPO pathway exerts an inhibitory effect on the ability of Hippo-Yes associated protein (YAP) to translocate to the nucleus. One such inhibitory effect is through phosphorylation of YAP, inhibiting YAP from entering the nucleus. Thus, a compound that prevents or reduces YAP phosphorylation in cells is a suitable HIPPO pathway inhibitor. Inhibiting the HIPPO pathway removes or reduces the inhibition on YAP, increasing the activity of YAP in the nucleus, which usually results in cell proliferation (see, for example, Figure 4 of Gumbiner and Kim). Thus, the HIPPO pathway inhibitor of the present invention can also be a YAP activator, i.e., resulting in increased YAP activity in the nucleus. Thus, HIPPO pathway inhibition includes YAP activation, and HIPPO pathway inhibitors include YAP activators. An example of YAP activation is overexpression of YAP in cells, for example, by using a recombinant nucleic acid expressing YAP as a YAP activator. Such a nucleic acid can be administered to cells, for example, using a vector, for YAP activation. One Hippo pathway inhibitor is XMU-MP-1 (Triastuti et al., Br J Pharmacol. 2019;176:3956-3971), which is preferably used at low concentrations according to the method for preparing blastoid cells and the method for preparing blastoid cells or blastocysts for transplantation into the endometrium of the present invention. XMU-MP-1 is a very potent Hippo pathway inhibitor. Large amounts of XMU-MP-1, for example, greater than 1 μM or about 2 μM or more in the medium, can be used to induce the formation of trophectoderm-like cells and form blastoid cells with a limited number of inner cells, or blastoid cells without inner cells (so-called trophospheres). XMU-MP-1 is a potent inhibitor of the Hippo pathway with more potent and longer-lasting effects than LPA.As a result, the aggregates form mostly trophoblasts at the expense of epiblast and hypoblast cell formation. Blast-like cells with inner cells are formed even with lower amounts of XMU-MP-1 in the medium, for example at about 1 μM or less. The results can generally be controlled by optimizing the concentration of the Hippo pathway inhibitor, the length of exposure of the cells to the Hippo inhibitor, the combination of the Hippo inhibitor with additional molecules, and the initial cell number. XMU-MP-1 is an inhibitor of MST1 / 2. The present invention provides the use of XMU-MP-1 or MST1 / 2 inhibitors in a method of generating trophospheres (which may lack inner cells) that cannot attach to or invade endometrial cells - the method may be in vitro; and / or in a contraceptive method. To obtain trophospheres, a sufficient amount of XMU-MP-1 and the exposure time of the cells to XMU-MP-1 may be used, for example, exposure of 2 μM or more for 4 days or more. Also provided is an MST1 / 2 inhibitor, preferably XMU-MP-1, for use as a contraceptive.

[0038] A preferred HIPPO pathway inhibitor is a ligand of the lysophosphatidic acid receptor (LPAR), and particularly preferred is lysophosphatidic acid itself (LPA, for example 1-oleoyl lysophosphatidic acid). Further preferred HIPPO pathway inhibitors and ligands of the lysophosphatidic acid receptor are NAEPA or OEA-P (oleoylethanolamide phosphate), N-[2-(phosphonooxy)ethyl]-9Z-octadecenamide. These are lysophosphatidic acid (LPA) mimetics. The ligand of the LPAR may be an activator or agonist of the LPAR. As an additional or alternative LPA ligand, any derivative of LPA can be used. The derivative of LPA is preferably a compound of formula 1:

[0039] [ka]

[0040] In the formula, R is C8 to C 24 -Alkyl, C8-C 24 -Alkenyl, or C8-C 24 -alkynyl. Preferably, R is C-, C 10 -, C 11 -, C 12 -, C 13 -, C 14 -, C 15 -, C 16 -, C 17 -, C 18 -, C 19 -, C 20 -, C 21 -, C 22 -, C 23 -alkenyl, -alkyl, or -alkynyl.

[0041] A preferred compound is (2-hydroxy-3-phosphonooxypropyl)(Z)-octadec-9-enoate.

[0042] The LPAR is preferably LPAR1, LPAR2, LPAR3, LPAR4, LPAR5, or LPAR6. A particularly preferred LPAR is LPAR2.

[0043] Further LPAR ligands are GRI977143 and any derivatives thereof disclosed in WO 2014 / 036038A1, which is incorporated herein by reference. Such ligands include compounds of formula 2:

[0044] [ka]

[0045] In the formula, A is

[0046] [ka]

[0047] where R is H, or substituted or unsubstituted phenyl; R1, R2, R3, R4, R5, and R6 are independently H, NO2, Br, Cl, or OCH3; B is C2-C8-alkyl or -alkenyl; and C is

[0048] [ka]

[0049] and optionally substituted with F, Cl, Br, NO2, NH2, OCH3, CH3, CO2H, or phenyl. For example, the compound may be 2-((9-oxo-9H-fluoren-2-yl)carbamoyl)benzoic acid, 2-((3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid, 4,5-dichloro-2-((9-oxo-9H-fluoren-2-yl)carbamoyl)benzoic acid, or 2-((9,10-dioxo-9,10-dihydroanthracen-2-yl)carbamoyl)benzoic acid.

[0050] Alternative or combinable HIPPO pathway inhibitors may be Mst1 inhibitors, Mst2 inhibitors, or combined inhibitors of Mst1 and Mst2, such as XMU-MP-1 (Triastuti et al., supra), or Lats kinase inhibitors, such as TRULI (Kastan et al., bioRxiv, 2020, doi.org / 10.1101 / 2020.02.11.944157). Lats kinase inhibitors may be ATP-competitive inhibitors of Lats kinase. Lats kinase is involved in YAP phosphorylation (Gumbiner et al., supra), and thus inhibition of Lats activity reduces YAP inactivation by phosphorylation and increases YAP activity in the nucleus.

[0051] The HIPPO pathway inhibitor may be a YAP activator, in particular a YAP activator that reduces or prevents YAP phosphorylation and / or promotes YAP entry into the cell nucleus. A further preferred HIPPO pathway inhibitor for use according to the present invention is verteporfin.

[0052] 3D cultures are cultures that allow tissue development in all three dimensions. In contrast, in 2D cultures, cells are induced to attach and grow on a surface and prevented from growing away from said surface, but such growth is not necessarily eliminated. 2D cultures may induce cell layer formation, such as monolayer, bilayer or multilayer, and / or two-dimensional cell growth; 3D cultures usually allow even growth in all directions, and of course cell tissues can develop their own orientation or axis of organization. Layer formation in 2D cultures may be induced by gravity and / or adhesion between cells or to a surface. Conditions for 2D and 3D cultures may be influenced by the type of surface, an attached surface in the case of 2D cultures or a non-attached surface in the case of 3D cultures, the medium, the absence (2D) or presence (3D), or the 3D matrix, such as a scaffolding gel structure, e.g., a hydrogel. 2D cultures may contain feeder cells as an attachment surface.

[0053] The medium for growing cells and tissues allows uninhibited development up to blastoid cells. The medium may include nutrients such as one or more carbohydrates, amino acids and salts. An example of a medium is B27N2 medium (Suenwoldt et al., Front. Mol. Neurosci. 10,2017:305). The medium preferably includes insulin. Alternatively or in combination, the medium may include holotransferin, selenite, corticosterone or progesterone, retinol, or combinations thereof. Such a medium may also be provided with the kit of the invention.

[0054] The method of the present invention includes providing aggregates of human pluripotent stem cells (hPSCs) and trophoblast cells. hPSCs are pluripotent cells that can differentiate into trophectoderm-like, epiblast-like, and hypoblast-like tissues. The suffix "like" indicates that although the tissues resemble trophectoderm, epiblast, and hypoblast, respectively, these tissues do not normally develop in the same way as in vivo situations, since the blast-like cells of the present invention are still artificial constructs. However, the "like" tissues of the blast-like cells of the present invention usually express the same expression markers as their in vivo counterparts and can be similarly identified.

[0055] The aggregates of human pluripotent stem cells (hPSCs) and trophoblast cells can be provided by common techniques such as those disclosed in WO 2014 / 171824 A1 or Okae et al., Cell Stem Cell 22, 2018: 50-63 (both of which are incorporated herein by reference). In a preferred method according to the present invention, the aggregates of hPSCs and trophoblasts can be generated by culturing the aggregated hPSCs in a medium containing any one selected from a HIPPO pathway inhibitor, a MEK inhibitor, and a TGF-β inhibitor. Preferably, a MEK inhibitor and a TGF-β inhibitor are used. Preferably, a HIPPO pathway inhibitor is also used.

[0056] In a particularly preferred embodiment, the "triple inhibition" of HIPPO pathway, MEK / ERK and TGF-β (as described above) is used to generate the blastoid cells of the present invention. In particular, the method of the present invention comprises culturing human pluripotent stem cells (hPSCs) and trophoblast cell aggregates in 3D culture in a medium containing a HIPPO pathway inhibitor. In the triple inhibition, the hPSCs and trophoblast aggregates are preferably generated by culturing the aggregated hPSCs in a medium containing a MEK inhibitor and a TGF-β inhibitor, and it is particularly further preferred that the HIPPO pathway inhibitor is also included at this stage.

[0057] Naïve human pluripotent stem cells (hPSCs) in which the Hippo, TGF-β, and MEK / ERK pathways were inhibited efficiently (>70%) form blastoid cells with three founder lineages (>97% trophectoderm, epiblast, and primitive endoderm) following the sequence and pace of blastocyst development, as shown below.

[0058] The triple inhibition disrupts the symmetry of the aggregated cells, leading to the formation of a unique inner cluster of epiblast and primitive endoderm cells that remain attached to one side of the trophoblast cyst. As a direct consequence, the asymmetry created within the cyst by the presence of this unique inner cluster induces localized maturation of the polarized trophoblast and orients its attachment to the endometrial cells. Thus, the blastoid cells of the present invention may have one localized inner cluster of epiblast and primitive endoderm cells attached to one side of the trophoblast cyst.

[0059] As disclosed in Guo et al., bioRxiv 2020, doi.org / 10.1101 / 2020.02.04.933812, MEK (also called ERK) inhibition, for example by a MEK inhibitor, can be used to induce the formation of trophoblasts from hPSCs. MEK inhibition is not necessary if cell aggregates containing trophoblast cells are already provided. If no trophoblast cells are present, a MEK inhibitor can be used to form trophoblast cells. The MEK inhibitor may be a MAPK inhibitor, for example SB202190.

[0060] The MEK inhibitor is preferably PD0325901. PD0325901 may be a compound of formula 3:

[0061] [ka]

[0062] Further or alternative MEK inhibitors may be selected from selumetinib, mirdametinib, trametinib, U0126-EtOH, PD184352 (CI-1040), PD98059, pimasertib, TAK-733, AZD8330 (AR-RY704), binimetinib, PD318088, SL327, refametinib, GDC-0623 (G-868), cobimetinib.

[0063] The TGF-β inhibitor may be an inhibitor of TGF-β type I receptor. The TGF-β inhibitor is preferably A83-1 (A83-01, A-83-01). A83-1 is a compound of formula 4:

[0064] [ka]

[0065] Further or alternative TGF-β inhibitors may be selected from SD-208, GW788388, SRI-011381, TP0427736, RepSox (E-616452, SJN 2511), LY2109761, SB505124, BIBF-0775, LY 3200882, galunisertib (LY2157299), bactosertib (TEW-7197, EW-7197), LY364947 (HTS 466284), SB525334, ITD-1, or SB431542. SB431542 is particularly preferred.

[0066] Aggregated hPSCs (due to the above process of generating aggregates of hPSCs and trophoblast cells) can be formed by seeding hPSCs and aggregating the seeded hPSCs by culturing in growth medium. The seeded hPSCs are preferably dissociated hPSCs. The hPSCs are dissociated, for example, by trypsinization. Dissociated hPSCs do not aggregate into a single connective tissue. However, the method of the invention allows them to aggregate later during development.

[0067] These method steps for generating different stages and intermediates during blast-like cell formation of the present invention can be combined, for example when it is desired to generate aggregates of hPSCs and trophoblast cells in situ. Thus, blast-like cells are formed from hPSCs in one culture. The same basal medium (such as B27N2) can be used for growth and nutrition, but different additional compounds are used in different steps. The method of the present invention can include a combination of the following steps: (i) seeding hPSCs and aggregating the seeded hPSCs by culturing in a growth medium to form aggregated hPSCs; (ii) culturing the aggregated hPSCs in a medium containing a MEK inhibitor and a TGF-β inhibitor to generate hPSCs and trophoblast cells; optionally, a HIPPO pathway inhibitor is also used in step (ii); (iii) culturing the hPSC and trophoblast cell aggregates in 3D culture in medium containing a HIPPO pathway inhibitor to generate blast-like or blastocyst-like cell aggregates.

[0068] The present invention includes combinations of steps (i), (ii) and (iii), but also combinations of steps (ii) and (iii), the latter of which may provide, for example, aggregated hPSCs (without the surrounding trophoblast cells that are the starting point for step (iii)).

[0069] In step (iii), MEK inhibitor and / or TGF-β inhibitor may not be used. In step (i) or step (ii), HIPPO pathway inhibitor may not be used. Also, in step (i), alternatively or in combination, MEK inhibitor and / or TGF-β inhibitor may not be used. In step (ii), the use of TGF-β inhibitor and MEK / ERK inhibitor is sufficient to form blastoid cells in step (iii).

[0070] The aggregated hPSCs (a step of generating aggregates of hPSCs and trophoblast cells) are preferably formed by seeding hPSCs and aggregating the seeded hPSCs by culturing them in growth medium for 0-64 hours, or 0-12 hours, or, as a preferred embodiment of step (i), 12-64 hours. The aggregation of the seeded hPSCs is an optional step, as the method will function without this step. In a preferred embodiment of step (i), optionally or alternatively, the growth medium comprises a ROCK inhibitor. A preferred ROCK inhibitor is Y27632 (Y-27632). Further or alternatively, the ROCK inhibitor may be selected from ZINC00881524, Thiazovivin, Fasudil, (HA-1077), GSK429286A (RHO-15), RKI-1447, Azaindole 1 (TC-S 7001), GSK269962A HCl (GSK269962B, GSK269962), Hydroxyfasudil (HA-1100), Netarsudil (AR-13324), Ripasudil (K-115), Y-39983 (Y-33075), KD025 (SLx-2119). The ROCK inhibitor increases or improves aggregation of seeded hPSCs.

[0071] Preferably, culturing the seeded hPSCs in a proliferation medium (e.g., step (i) above) comprises the steps of seeding 1-200 hPSCs, preferably 20-150 hPSCs, particularly preferably 30-120 hPSCs, and even more preferably 30-60 hPSCs in a vessel, and expanding the seeded hPSCs in a proliferation medium. This cell number leads to optimal blast-like cell formation in a subsequent step.

[0072] In a preferred embodiment, the treatment and / or growth of the seeded hPSCs is or has been done in a 2D culture environment, which may result in two-dimensional cell growth as described above.

[0073] Preferably, hPSCs in 2D culture (before 3D culture) are treated with MEK inhibitors and / or PKC inhibitors, for example as described in Guo et al., Development 2017, doi: 10.1242 / dev.146811. Wnt inhibitors and STAT activators can also be used in combination or alternatively as described above.

[0074] In a preferred embodiment, the PKC inhibitor is selected from Goe6983 (GOE6983) and Ro-31-8425, or a combination thereof.Further or alternative PKC inhibitors may be selected from enzastaurin (LY317615), sotrastaurin (AEB071), mitoxantrone (NSC-301739), staurosporine (CGP 41251), bisindolylmaleimide I (GF109203X, GO 6850), bisindolylmaleimide IX (Ro 31-8220), LXS-196 (IDE-196), VTX-27, midostaurin (pkc412, CGP 41251), chelerythrine, Go6976 (PD406976), CRT0103390.

[0075] In a preferred embodiment, the 2D cultured hPSCs are or have been treated with Wnt inhibitors and / or STAT agonists. Such treatments generate more naive hPSCs or ground state hPSCs. Such naive or ground state hSPCs are preferably used as hPSCs in the method of the present invention in step (i). Such treatments for generating more naive or ground state hPSCs are disclosed, for example, in Takashima et al., Cell 158(6),2014:1254-1269 or WO 2016 / 027099 A2 (both of which are incorporated herein by reference). The STAT agonist is preferably a STAT3 agonist, for example, LIF.

[0076] An example of a Wnt inhibitor is XAV939. XAV939 may be a compound of formula (5):

[0077] [ka]

[0078] Further or alternative Wnt inhibitors may be selected from LF3, PKF118-310, Wnt3A, Adavivint (SM04690), CCT251545, PNU-75654, IWP-2, IWP-3, IWR-1-endo, iCRT3, WIKI4, ICG-001, XAV-939 (NVP-XAV939), LGK-974 (NVP-LGK974, WNT974), MSAB, KYA1797K, JW55, or combinations thereof. LF3 and / or XAV939 are particularly preferred. Particularly preferred Wnt inhibitors for all embodiments of the present invention are XAV939, IWP2, PNU74654, and LF3.

[0079] In a particularly preferred embodiment, hPSCs may be pretreated in a medium containing inhibitors of MEK, Wnt, PKC, and an inhibitor against or activator of STAT (e.g., LIF), as described, for example, in Guo et al., Development 2017, doi:10.1242 / dev.146811 and Takashima et al. (supra). A medium called PXGL maintains hPSCs in a more naive state. This more naive state improves blast-like cell formation. Many culture conditions for rendering hPSCs naive are known in the art and can be used according to the present invention, for example, as described in WO 2016 / 027099 A2.

[0080] In a preferred embodiment, the aggregated cells (eg, step (ii) above) are cultured for at least 1 day, preferably at least 2 days.

[0081] The above embodiments are preferably combined, for example in one example the culture starting from hPSCs comprises culturing the cells for at least 5 days: the first 0-24 hours or day is in medium free of small molecule inhibitors (free of MEK inhibitors (e.g. PD0325901), TGF-β inhibitors (e.g. A83-01), STAT activators (e.g. LIF), and HIPPO pathway inhibitors (e.g. LPA)) (except for ROCK inhibitors, e.g. Y27632, which promote cell aggregation). This is an example of step (i) and everything stated above for step (i) also applies here. Between 0-24 hours or on day 2, treatment with MEK inhibitors (e.g. PD0325901), TGF-β inhibitors (e.g. A83-01), STAT activators (e.g. LIF), ROCK pathway inhibitors, and HIPPO pathway inhibitors (e.g. LPA) is initiated. This is an example of step (ii), and everything stated above for step (ii) applies here too. The same medium is used on day 3 (step (ii)). On day 4, the cells / aggregates are cultured in medium containing only ROCK pathway inhibitors and HIPPO pathway inhibitors (e.g., LPA) among the small molecule inhibitors discussed. For example, MEK inhibitors (e.g., PD0325901), TGF-β inhibitors (e.g., A83-01), STAT activators (e.g., LIF) are no longer used. This is an example of step (iii), and everything stated above for step (iii) applies here too. Usually, blastoid cells are fully formed on day 5. This method can be used, for example, in vitro to promote the development of blastocysts resulting from in vitro fertilization, and / or in methods to enhance fertility during the first weeks of pregnancy. Hippo inhibitors, preferably LPA or NAEPA, can be used to enhance the development and potential of blastocysts. Also provided is a Hippo inhibitor, preferably LPA or NAEPA, for use as a fertility enhancer. The Hippo inhibitor, preferably LPA or NAEPA, can be administered to a patient 1 to 12 days after conception, preferably 2 to 9 days after conception, particularly preferably 3 to 7 days after conception.

[0082] By changing the growth conditions, the preferred time for each step may vary. In a preferred embodiment, step (i) is 18-48 hours; preferably, step (ii) is 36-92 hours; preferably, step (iii) is 18-48 hours. These times are also preferred embodiments when step (i) or steps (i) and (ii) are not performed, e.g., when aggregated hPSCs or aggregates of hPSCs and trophoblast cells are used as starting points.

[0083] The hPSCs (human pluripotent stem cells) are preferably not human totipotent cells, ie, not human embryos.

[0084] The hPSCs may be from any cell line. Preferably, the cell line is selected from hESC H9, Shef6, HNES1, hiPSC cR-NCRM2, and hiPSC niPSC16.2.b.

[0085] Preferably, the independently selected cells, aggregated hPSCs, or aggregates of hPSCs and trophoblast cells are seeded or placed in microwells. The microwells may be used to control cell number. The microwells may be in an array to allow multiple parallel blast-like cell formations. Preferably, at least 2, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more, e.g., 50 or more blast-like cells are generated in parallel by the methods of the invention.

[0086] Preferably, the 3D culture, e.g., the culture of aggregates of hPSCs and trophoblast cells, is carried out by culturing in a non-adhesive container, preferably by culturing in a microwell, particularly preferably by culturing in a microwell comprising a non-adhesive surface made of a hydrogel.

[0087] In a preferred embodiment, the medium for culturing in 3D culture, in particular the culture of aggregates of hPSCs and trophoblast cells (such as step (iii) above) and / or the medium for culturing aggregated hPSCs (such as step (ii) above), further comprises a STAT3 agonist. The STAT3 agonist is preferably leukemia inhibitory factor (LIF). The LIF is preferably human LIF.

[0088] In a preferred embodiment, the hPSCs and trophoblasts (eg step (iii) above) are cultured for at least 16 hours, preferably at least 20 hours, even more preferably for at least 1 day, and possibly for at least 2 days.

[0089] Preferably, the cells, particularly hPSCs and trophoblast cells, are cultured at least until trophectoderm-, epiblast-, and hypoblast-like tissues are formed from the hPSC and trophoblast aggregates. Alternatively, or in combination, the culture can be performed at least until the embryonic-non-embryonic axis is formed.

[0090] Alternatively, culture of cells, particularly hPSCs and trophoblast cells, in the presence of the MST1 / 2 inhibitor XMU-MP-1, which promotes trophoblast formation and prevents the formation or maintenance of epiblast- and hypoblast-like cells, is performed until trophoblast cysts are formed that contain smaller inner clusters of epiblast- and hypoblast-like cells or no inner clusters of epiblast- and hypoblast-like cells if sufficient concentrations of XMU-MP-1 are included. These trophoblast cysts are referred to as XMU-MP-1-Trophospheres.

[0091] The present invention provides the use of XMU-MP-1 or MST1 / 2 inhibitors in a method for generating trophospheres (which may be devoid of inner cells) - the method may be in vitro; and / or in a contraceptive method. MST1 / 2 inhibitors, preferably XMU-MP-1, can be used for contraception. Also provided is an MST1 / 2 inhibitor, preferably XMU-MP-1, for use as a contraceptive. MST1 / 2 inhibitors, preferably XMU-MP-1, can be administered to a patient 1-9 days after conception, preferably 2-7 days after conception, particularly preferably 3-6 days after conception.

[0092] Alternatively, the culture of cells, particularly hPSCs and trophoblast cells, is carried out in the presence of the STAT inhibitor SC144, which prevents the formation and maintenance of epiblast-like and hypoblast-like cells, until trophoblast cysts are formed that do not contain inner clusters of epiblast-like and hypoblast-like cells or contain a reduced number of epiblast-like and hypoblast-like cells. These trophoblast cysts are called SC144-Trophospheres. As mentioned above, STAT agonists can be used in certain steps of the method of the invention. To prevent or limit blast-like cell formation by inner cells, STAT inhibitors such as SC144 can be used. STAT inhibitors, preferably SC144, can be used for contraception. STAT inhibitors, preferably SC144, for use as contraceptives are also provided. STAT inhibitors, preferably SC144, can be administered to the patient 1-9 days after conception, preferably 2-7 days after conception, particularly preferably 3-6 days after conception.

[0093] Trophoblasts are the cells that form the outer layer of the blastocyst or blastocyst, which in vivo develop into most of the placenta. The term trophectoderm refers to the epithelial cystic tissue that forms the outer layer of the blastocyst. In blastocysts, the outer tissue resembles this trophectoderm and is called trophectoderm-like tissue.

[0094] The epiblast is one of two distinct layers that arise from the inner cell mass of the mammalian blastocyst and leads to the embryo itself through differentiation into the three main germ layers, ectoderm, mesoderm, and endoderm, during gastrulation. The epiblast-like cells develop an inner tissue that resembles the epiblast and is called epiblast-like tissue.

[0095] The hypoblast is one of two distinct layers that arise from the inner cell mass of the mammalian blastocyst. The hypoblast gives rise to the yolk sac, which in turn gives rise to the chorion. In the blastocyst, an inner tissue similar to the hypoblast has developed and is called hypoblast-like tissue.

[0096] The expression patterns of epiblast-like, trophoblast-like, and hypoblast-like cells are clustered as shown in FIG. 9d. Epiblast-like cell expression markers are, for example, TDGF1, GDF3, SUSD2, POU5F1, PRDM14, DPPA4, and / or DNMT3L. Trophoblast-like cell expression markers are, for example, KRT19, CLDN4, GATA2, KRT18, and / or HAND1. Hypoblast-like cell expression markers are, for example, PDGFRA, COL4A1, COL4A2, GATA6, and / or LAMA1. Gene names and gene symbols are those of the genes as defined by the HUGO Gene Nomenclature Committee (www.genenames.org). Expression patterns can be determined, for example, by measuring mRNA expression.

[0097] The formation of the embryonic-non-embryo axis is a preferred embodiment as blastocyst and blastoid cell transplantation via trophoblast apposing the epiblast / hypoblast inner aggregates. These so-called polar trophoblasts are characterized by the expression of NR2F2+ and / or CCR7+. Preferably, the blastoid cells of the present invention comprise polar trophoblasts expressing NR2F2+ and / or CCR7+.

[0098] Preferably, the cells, particularly hPSCs and trophoblast cells, are cultured until three-dimensional cell aggregates of at least 100 μm, preferably at least 140 μm, even more preferably 180 μm-220 μm in overall diameter are formed by an outer epithelial monolayer of trophoblast-like cells surrounding a fluid-filled cavity and at least one inner cell mass comprising epiblast-like cells and hypoblast-like cells. The blast-like cells of the present invention may comprise any or all of these characteristics.

[0099] In a further embodiment, the blastoid cells produced by the method of the present invention may be seeded on a layer of endometrial cells.The seeding is preferably performed in vitro.The blastoid cells can be implanted in or on a layer of endometrial cells.This implantation is a process that the blastoid cells can carry out by themselves, unless it is artificially inhibited.

[0100] The layer of endometrial cells may be a monolayer.

[0101] Preferably, the endometrial cells or endometrium in the IVF method are treated with a compound selected from estrogen, estrone, estriol, ethinylestradiol, 17α-ethinylestradiol, mestranol, progesterone, progestin, cAMP, and Wnt inhibitors (preferably XAV939, IWP2 (also called IWP-2), PNU-74654, and LF3). Such treatment improves the receptiveness of blastoid cells or blastocysts to implantation in or on endometrial cells. Particularly preferred is treatment with a Wnt inhibitor, preferably XAV939, or any of the above Wnt inhibitors. This treatment with a Wnt inhibitor can be combined with treatment with estrogen, estrone, estriol, ethinylestradiol, 17α-ethinylestradiol, mestranol, progesterone, progestin, and / or cAMP.

[0102] Particularly preferred is the inhibition of Wnt to prepare the endometrial cell layer or endometrium of the uterus during in vitro fertilization for the implantation of blastocysts or blastocysts, which increases the receptivity of the endometrial cells or endometrium to implantation.

[0103] Seeding of blastoid cells into a layer of endometrial cells can be used to study the effect on implantation quality, efficacy, and / or inhibition. In general, any step of the method of the invention can be used to study the development or performance or properties of blastoid cells as a model of blastocyst development, or as a model of blastocysts, or performance or properties.

[0104] The method of the invention can be used to test or screen for candidate compounds and / or candidate gene mutations and / or environmental influences such as temperature that affect blastoid cell formation and / or implantation of blastoid cells into endometrial cell layers. Such a method may include treating the aggregates with at least one candidate compound and / or providing the aggregates with at least one candidate gene modification and carrying out the method of the invention. The effect of the method can be compared to a method that does not include at least one candidate compound and / or at least one candidate gene mutation and / or altered environmental influence, respectively, as a control comparison. Otherwise, to evaluate the effect of only at least one candidate compound and / or at least one candidate gene mutation and / or environmental influence, the control comparison is carried out as would be the case for a control.

[0105] For successful implantation and subsequent development to occur, separate tissues (polar trophoblast and mural trophoblast cells) are formed on either side of the trophoblast cyst. These tissues are believed to play different roles (e.g., adhesion, induction of proliferation) during interaction with the endometrium and uterine tissue. Thus, human blastocysts implant via the polar tissue. The method of the present invention can be used to study the development of these different tissues and thus implantation. Candidate compounds and / or candidate gene mutations and / or environmental influences can be studied if they affect this tissue formation or its implantation.

[0106] For successful implantation and subsequent development to occur, the inner clusters of epiblast-like and hypoblast-like cells secrete molecules that induce the outer trophoblast. These molecular inducers are believed to play various roles (e.g., proliferation, differentiation, mechanical action) to endow the trophoblast with the ability to interact with the endometrium and uterine tissue. Thus, human blastocysts implant through the pole tissue. The method of the present invention can be used to study the role of molecular inducers in endowing the trophoblast with the endometrium and uterine tissue. Candidate molecular inducers and / or candidate gene mutations and / or environmental influences can be studied if they affect these molecular inducers and their effects on the trophoblast or its implantation.

[0107] The present invention also provides a blast-like cell obtainable by the method of the present invention. The present invention provides a blast-like cell comprising an outer epithelial monolayer of trophoblast-like cells surrounding at least one fluid-filled cavity and at least one inner cell mass comprising epiblast-like cells and hypoblast-like cells, wherein the outer epithelial monolayer comprises polar trophoblasts expressing NR2F2. Any of the above characteristics, cell types, tissue types may be part of the blast-like cell of the present invention, such as the fluid-filled cavity, and may not have immobilized cells. It may also not include seeded cells or may include seeded cells.

[0108] The present invention further provides a kit suitable for culturing blastoid cells. Any of the above-mentioned components or combinations thereof may be included in the kit. The kit may preferably include a HIPPO pathway inhibitor, a MEK inhibitor, and / or a TGF-β inhibitor. These compounds are preferably combined in a medium for human pluripotent stem cells (hPSCs). The compounds can be used in any of the above steps. The kit preferably further includes a Wnt inhibitor, such as XAV-939. Any of the above-mentioned inhibitors can be used, and the preferred inhibitors shown are also preferred for the kit of the present invention. A particularly preferred HIPPO pathway inhibitor is LPA.

[0109] The kit may include any compound selected from PD0325901 (MEK inhibitor), Go6983 (PKC inhibitor), XAV-939 (Wnt inhibitor), A83-01 (TGF-β inhibitor), or a combination thereof, as preferred examples of MEK inhibitors, PKC inhibitors, Wnt inhibitors, and TGF-β inhibitors, respectively.

[0110] The kit may also include a ROCK inhibitor.

[0111] The kit may also include any growth factor selected from LIF, IGF-1, IL-6, IL-11, FGF2, FGF4, or a combination thereof. LIF may be used as described above. IGF-1 and / or IL-6 and / or IL-11 and / or FGF2 and / or FGF4 may be used to improve the growth of aggregated cells in the medium of the present invention in any one of steps (i), (ii), (iii), or a combination thereof.

[0112] The kit may also include a culture medium as described above. Particularly preferably, the kit includes insulin.

[0113] As discussed above, the present invention has shown that the use of Wnt inhibitors improves the implantation of blastoid cells into a layer of endometrial cells.Therefore, the present invention provides an in vitro method for increasing the possibility of implanting blastoid cells or blastocysts into a layer of endometrial cells, which comprises treating said blastoid cells or blastocysts with a Wnt inhibitor, preferably XAV939, and contacting said blastoid cells or blastocysts with a layer of endometrial cells.This method can also be used to test at least one candidate compound and / or at least one candidate gene mutation and / or environmental influence as described above, and test their effects on the development of blastoid cells at implantation or after implantation, or on endometrial cells after implantation, for example, compared with a control that does not contain at least one candidate compound and / or at least one candidate gene mutation and / or environmental influence, etc.

[0114] The improvements afforded by the use of Wnt inhibitors may also be used in vivo and / or in vitro during in vitro fertilization (IVF) procedures.

[0115] Provided is a Wnt inhibitor for use in a method for increasing the probability of blastocyst implantation during in vitro fertilization, comprising contacting blastocyst with endometrium in the presence of a Wnt inhibitor, preferably XAV939; preferably, wherein endometrium is contacted with Wnt inhibitor locally, systemically, or together with blastocyst.In this regard, the present invention provides a method for increasing the probability of blastocyst implantation during in vitro fertilization, comprising contacting blastocyst with endometrium in the presence of a Wnt inhibitor.Also provided is the use of a Wnt inhibitor for manufacturing a pharmaceutical composition for mediating blastocyst implantation during in vitro fertilization, comprising contacting blastocyst with endometrium in the presence of a Wnt inhibitor.

[0116] In vitro fertilization involves contacting an embryo with the endometrium, and the embryo may be developed in vitro to the blastocyst stage. During in vitro development, or during or shortly after contacting the blastocyst with the endometrium, a Wnt inhibitor is administered to the blastocyst or endometrium to increase the probability of implantation.

[0117] The discussed HIPPO pathway inhibitors also improve the probability of IVF or pregnancy, as discussed above with respect to blastoid cells. In the case of IVF, the blastocyst or any of its preceding stages, such as 1-cell, 2-cell, 4-cell, 8-cell, or 16-cell, or morula stage embryos, can be treated with HIPPO pathway inhibitors to increase the probability of pregnancy, or generally for the preparation of blastocysts. The present invention provides a HIPPO pathway inhibitor for use in a method for producing a blastocyst suitable for in vitro fertilization or suitable for increasing the probability of pregnancy, comprising treating an early stage embryo selected from the 1-cell, 2-cell, 4-cell, 8-cell, or 16-cell stage, or morula stage, or blastocyst stage, with a HIPPO pathway inhibitor, particularly preferably with NAEPA or a ligand of the lysophosphatidic acid (LPA) receptor, even more preferably with LPA, to the mature blastocyst stage, and growing the 1-cell, 2-cell, 4-cell, 8-cell, or 16-cell, or morula stage embryo to the blastocyst stage, or growing the blastocyst stage embryo to a more potent or more mature blastocyst stage. In that regard, the present invention provides a method for producing a blastocyst suitable for in vitro fertilization or suitable for increasing the probability of pregnancy, comprising treating an embryo at an early stage selected from the 1-cell, 2-cell, 4-cell, 8-cell, or 16-cell, or morula, or blastocyst stage with a HIPPO pathway inhibitor until a potential or mature blastocyst stage, and growing an embryo at the 1-cell, 2-cell, 4-cell, 8-cell, or 16-cell, or morula stage until a blastocyst stage, or growing an embryo at the blastocyst stage until a more potential or mature blastocyst stage.Also provided is the use of a HIPPO pathway inhibitor for the manufacture of a pharmaceutical composition for generating a blastocyst suitable for in vitro fertilization or for increasing the probability of pregnancy, comprising treating an early stage embryo selected from 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage, or 16-cell stage, or morula stage, or blastocyst stage with a HIPPO pathway inhibitor to a potential or mature blastocyst stage, and developing an embryo at 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage, or 16-cell stage, or morula stage to a blastocyst stage, or developing an embryo at a blastocyst stage to a more potential or mature blastocyst stage. For example, a Hippo inhibitor may be used by a patient to improve the development of a blastocyst in the uterus, for example by taking a HIPPO pathway inhibitor several days after conception and before implantation (e.g., 0-12 days, or 1-12 days, preferably 6-9 days). As mentioned above, to increase the probability of pregnancy, a Hippo inhibitor, preferably LPA or NAEPA, can be administered to the patient on days 0-12 after conception, preferably days 2-9 after conception, particularly preferably days 3-7 after conception. The probability of pregnancy is increased by promoting blastocyst development according to the present invention, which may develop a higher implantation ability and thus lead to pregnancy. The Hippo inhibitor may be administered intrauterinely.

[0118] Alternatively or additionally to the preceding paragraph, the supernatant or culture of blastoid cells can be used in place of or in addition to a HIPPO pathway inhibitor in this method of producing blastocysts suitable for IVF treatment or during IVF treatment or to increase the probability of pregnancy. As shown in EP2471538A1, blastocyst culture supernatant promotes pregnancy in blastocyst transfer by producing LPA, and the same can be done with blastoid cell supernatant. Thus, the present invention also provides a culture supernatant of the blastoid cells of the present invention for use in a method for producing a blastocyst suitable for in vitro fertilization treatment or for increasing the probability of pregnancy, the method comprising treating an early stage embryo selected from the 1-cell, 2-cell, 4-cell, 8-cell, or 16-cell, or morula, or blastocyst stage with a HIPPO pathway inhibitor, particularly preferably with a ligand of the lysophosphatidic acid (LPA) receptor, even more preferably with LPA, to the mature blastocyst stage, and growing the 1-cell, 2-cell, 4-cell, 8-cell, or 16-cell, or morula stage embryo to the blastocyst stage, or growing the blastocyst stage embryo to the more mature blastocyst stage. Further provided is a method for increasing the probability of pregnancy, whether by in vitro fertilization using blastoid cell culture supernatant or by transferring blastoid cell culture supernatant into the uterine cavity for in vitro fertilization or to increase the probability of natural conception. A blastoid cell culture supernatant or a component thereof can be implanted into the uterus and contacted with the blastoid cell culture supernatant or a component thereof in the uterine cavity. The blastoid cell culture supernatant or a culture can be administered to the patient 1-12 days after conception, preferably 2-9 days after conception (fertilization of the egg cell), particularly preferably 3-7 days after conception, to increase the probability of pregnancy. The probability of pregnancy is increased by promoting the development of the blastoid cell according to the present invention, which may develop a higher implantation ability and thus lead to pregnancy. The blastoid cell culture supernatant or a culture can be administered to the uterus.

[0119] Also provided is a method for producing LPA, comprising culturing the blastoid cells of the present invention, and collecting said LPA from the culture, preferably from the supernatant of the culture.The blastoid cells of the present invention have the same characteristics as the LPA-producing blastocysts described in EP 2471538 A1, so that the blastoid cells of the present invention can be used in the same manner as described for blastocysts in EP 2471538 A1.

[0120] The LPA produced may be any of LPA-C16:0, LPA-C16:1, LPA-C18:0, LPA-C18:1, LPA-C18:2, or a combination thereof. Any of these LPAs may be used as HIPPO pathway inhibitors of the present invention.

[0121] Any active agent described herein, such as a Wnt inhibitor or a Hippo pathway inhibitor, can be administered, for example, (1) in vivo and systemically, or (2) in vitro by exposing the embryo to the active agent before transfer to the uterus, or (3) in utero by co-implanting the molecule with the embryo during uterine transfer. Systemic administration can be, for example, orally (e.g., as a pastille, tablet, troche, lozenge, pill, gum, powder, or drinking solution), parenterally (e.g., injection, e.g., intravenously, or as a transdermal patch). The supernatant or culture of blastoid cells is preferably administered (2) in vitro by exposing the embryo to the active agent before transfer to the uterus, or (3) in utero by co-implanting the molecule with the embryo during uterine transfer.

[0122] Administration can be in a formulation that includes any one of pharmaceutical carriers, excipients, vectors, additives, or combinations thereof. The term "carrier" refers to a diluent, such as water, saline, excipients, or vehicles with which the composition can be administered. In the case of a solid or liquid composition, the carrier or additive in the pharmaceutical composition may include a binder such as SiO2, TiO2, microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), tragacanth gum, gelatin, starch, lactose or lactose monohydrate, alginic acid, maize (corn) starch, etc.; a lubricant or surfactant such as magnesium stearate or sodium lauryl sulfate; a glidant such as colloidal silicon dioxide; and a sweetener such as sucrose and saccharin. Preferably, the formulation includes a buffer or pH adjuster, such as selected from citric acid, acetic acid, fumaric acid, hydrochloric acid, malic acid, nitric acid, phosphoric acid, propionic acid, sulfuric acid, tartaric acid, or a combination thereof.

[0123] The phrase "more potent or more mature blastocyst stage" refers to the improvement in development and maturation by the HIPPO pathway inhibitor, which improvement is relative to a control or comparative blastocyst maintained or grown under the same conditions, except for the absence of the HIPPO pathway inhibitor used in accordance with the present invention.

[0124] The following numbered embodiments are preferred according to the present invention: 1. A method for generating blastoid or blastocyst-like cell aggregates, comprising culturing aggregates of human pluripotent stem cells (hPSCs) and trophoblast cells in 3D culture in medium containing a HIPPO pathway inhibitor. 2. The method of 1, wherein the hPSC and trophoblast aggregates are generated by culturing the aggregated hPSCs in a medium that also contains a MEK inhibitor and a TGF-β inhibitor, preferably a HIPPO pathway inhibitor. 3. The method of 2, wherein the aggregated hPSCs are formed by seeding hPSCs, culturing in growth medium, preferably aggregating the seeded hPSCs by culturing in growth medium for 0-64 hours, or 12-64 hours, and / or preferably, wherein the growth medium comprises a ROCK inhibitor, particularly preferably the ROCK inhibitor is Y27632. 4. The method of 3, wherein the seeded hPSCs have been treated with a MEK inhibitor and / or a PKC inhibitor prior to 3D culture, preferably during 2D culture, preferably further comprising a Wnt inhibitor and / or a STAT agonist, preferably wherein the treatment is during 2D culture. 5. The method of claim 4, wherein the PKC inhibitor is selected from Goe6983 and Ro-31-8425. 6. The method according to any one of 1 to 5, wherein the HIPPO pathway inhibitor is a ligand of the lysophosphatidic acid (LPA) receptor, preferably LPA and / or NAEPA, or verteporfin; and / or the MEK inhibitor is PD0325901; and / or the TGF-β inhibitor is A83-1 or SB431542. 7. The method according to any one of 1 to 6, wherein the medium for culturing in the 3D culture according to embodiment 2 and / or the medium for culturing the aggregated hPSCs further comprises a STAT3 agonist, preferably leukemia inhibitory factor (LIF). 8. The method according to any one of 1 to 7, wherein the 3D culture is by culturing in a non-adherent container, preferably by culturing in a microwell, particularly preferably by culturing in a microwell comprising a non-adherent surface made of a hydrogel. 9. The method according to any one of 3 to 8, wherein culturing hPSCs in the proliferation medium of embodiment 3 comprises placing 1 to 200 hPSCs, preferably 20 to 150 hPSCs, particularly preferably 30 to 120 hPSCs, and even more preferably 30 to 60 hPSCs in a container and proliferating the seeded hPSCs in the proliferation medium. 10. The method according to any one of 1 to 9, wherein the hPSCs and trophoblasts are cultured for at least 1 day, preferably at least 2 days. 11. A method according to any one of 2 to 10, wherein the aggregated cells according to embodiment 2 are cultured for at least 1 day, preferably at least 2 days. 12. The method of any one of 1 to 11, comprising culturing the cells until at least trophectoderm-like tissue, epiblast-like tissue, and hypoblast-like tissue are formed from the hPSC and trophoblast aggregates, preferably further until at least an embryonic-non-embryonic axis is formed. 13. The method according to any one of 1 to 12, comprising culturing the cells until formation of three-dimensional cell aggregates having an overall diameter of at least 100 μm, preferably at least 140 μm, even more preferably between 180 μm and 220 μm, formed by an outer epithelial monolayer of trophoblast-like cells surrounding a fluid-filled cavity and at least one inner cell mass comprising epiblast-like cells and hypoblast-like cells. 14. The method according to any one of 1 to 13, further comprising the steps of seeding the blast-like cells onto endometrial cells, and implanting the blast-like cells into or onto endometrial cells. 15. The method of claim 14, wherein the endometrial cells are treated with a compound selected from estrogen, estrone, estriol, ethinyl estradiol, 17α-ethinyl estradiol, mestranol, progesterone, progestin, cAMP, and a Wnt inhibitor (preferably XAV939, IWP-2, PNU-74654, and / or LF3). 16. A method according to any one of 1 to 15 for testing or screening for candidate compounds and / or candidate gene mutations influencing blastoid cell formation and / or engraftment of blastoid cells into a layer of endometrial cells, comprising treating with at least one candidate compound and / or providing an aggregate having at least one candidate gene modification, and carrying out a method according to any one of embodiments 1 to 15. 17. A kit suitable for culturing blastoid cells, comprising a HIPPO pathway inhibitor, a MEK inhibitor, and a TGF-β inhibitor; preferably incorporated into medium for human pluripotent stem cells (hPSCs). 18. A blast-like cell obtained by the method according to any one of 1 to 16. 19. An outer epithelial monolayer of trophoblast-like cells surrounding at least one fluid-filled cavity and at least one inner cell mass comprising epiblast-like cells and hypoblast-like cells, wherein the outer epithelial monolayer comprises polar trophoblasts that express NR2F2, and blast-like cells. 20. An in vitro method for increasing the potential of a blastoid cell or blastocyst to implant into a layer of endometrial cells, comprising treating the blastoid cell or blastocyst with a Wnt inhibitor, preferably XAV939, IWP-2, PNU-74654, and / or LF3, and contacting the blastoid cell or blastocyst with a layer of endometrial cells. 21. A Wnt inhibitor for use in a method of increasing the probability of blastocyst implantation, for example during in vitro fertilization or natural pregnancy, comprising contacting a blastocyst with the endometrium in the presence of the Wnt inhibitor, or stimulating endometrial cells with a Wnt inhibitor, preferably XAV939, IWP-2, PNU-74654, and / or LF3, in the absence of a blastocyst; wherein preferably the endometrium is contacted with the Wnt inhibitor locally, systemically, or together with the blastocyst, the Wnt inhibitor. 22. A HIPPO pathway inhibitor for use in a method for producing a blastocyst suitable for in vitro fertilization or for increasing the probability of pregnancy, comprising treating an early stage embryo selected from the 1-cell, 2-cell, 4-cell, 8-cell, or 16-cell, or morula, or blastocyst stage with a HIPPO pathway inhibitor, particularly preferably with NAEPA or a ligand of the lysophosphatidic acid (LPA) receptor, even more preferably with LPA, to the mature blastocyst stage, and growing the 1-cell, 2-cell, 4-cell, 8-cell, or 16-cell, or morula stage embryo to the blastocyst stage, or growing the blastocyst stage embryo to a more mature blastocyst stage. 23. The culture supernatant of blastoid cells according to embodiment 18 or 19 for use in a method for producing a blastocyst suitable for in vitro fertilization or for increasing the probability of pregnancy, comprising treating an early stage embryo selected from the 1-cell, 2-cell, 4-cell, 8-cell, or 16-cell, or morula, or blastocyst stage with supernatant from a culture of blastoid cells to the mature blastocyst stage, and growing an embryo at the 1-cell, 2-cell, 4-cell, 8-cell, or 16-cell, or morula stage to the blastocyst stage, or growing an embryo at the blastocyst stage to the more mature blastocyst stage. 24. A method for producing LPA, comprising culturing the blastoid cells according to embodiment 18 or 19, and collecting said LPA from the culture, preferably from the culture supernatant. 25. A method for forming trophospheres, comprising culturing hPSCs in the presence of an MST1 / 2 inhibitor, preferably XMU-MP-1, and / or a STAT inhibitor, preferably SC144. 26. The method according to claim 25, further performed as described in any one of embodiments 1 to 16. 27. A method of contraception comprising administering to a patient an MST1 / 2 inhibitor, preferably XMU-MP-1, and / or a STAT inhibitor, preferably SC144, and / or contacting an embryo in vivo with an MST1 / 2 inhibitor, preferably XMU-MP-1, and / or a STAT inhibitor, preferably SC144. 28. The method according to claim 27, wherein an MST1 / 2 inhibitor, preferably XMU-MP-1, and / or a STAT inhibitor, preferably SC144, is administered to the patient 1 to 9 days after conception, preferably 2 to 7 days after conception, particularly preferably 3 to 6 days after conception. 29. An MST1 / 2 inhibitor, preferably XMU-MP-1, and / or a STAT inhibitor, preferably SC144, for use in a method of contraception, preferably according to embodiment 27 or 28. 30. An MST1 / 2 inhibitor, preferably XMU-MP-1, and / or a STAT inhibitor, preferably SC144, for use in the manufacture of a contraceptive.

[0125] Throughout this disclosure, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0126] As used herein, approximation words such as, but not limited to, "about", "substantial", or "substantially" refer to a state that, when modified in such a way, is not necessarily absolute or complete, but is considered close enough to those skilled in the art to call the state as it exists. The extent to which the description is changed depends on how large a change can be introduced to allow those skilled in the art to recognize that the modified feature still has the necessary properties and capabilities of the unmodified feature. In general, subject to the above discussion, numerical values ​​in this specification modified by synonyms such as "about" may vary from the stated value, for example, by ±10%.

[0127] As used herein, the words "comprising" (and any form of including, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), or "including" (and any form of including and including and including and including and including and including and including and including and including and including and including and including and including and including and including and including and containing) are inclusive or open-ended and do not exclude additional, unmentioned elements or method steps. When the term "comprising" is used with respect to an element in combination with a numerical range of specific values ​​for that element, it means that the element is limited to that range, but "comprising" still relates to the optional presence of the other element. For example, an element with a range may have an implied condition that excludes the presence of the element in an amount outside that range. As used herein, the phrase "consisting essentially of" requires the specified integers or steps, as well as those that do not materially affect the nature or function of the claimed invention. As used herein, the limited term "consisting of" is used to indicate that only the recited elements are present.

[0128] The present invention will be further illustrated by the following examples, without however being limited to these embodiments of the invention.

[0129] Working Example Example 1: Formation of hPSC aggregates. The formation of blast-like cells depends on the aggregation of an optimal number of hPSCs. This can be achieved by seeding a specific number of hPSCs on a microwell array made of a non-adhesive hydrogel and containing a large number of microwells, for example, 200 micrometers (Figure 1A). We observed that human blast-like cells preferentially form from aggregates of 20–100 cells, which favors the formation of aggregates capable of forming cavities (Figure 1B). We quantify this phenomenon by measuring the number of structures that form cavities, and the number of blast-like cells, defined as three-dimensional structures with an overall diameter of 180–220 micrometers, formed by a unique fluid-filled cavity and an outer epithelial monolayer of trophoblast-like cells surrounding an inner cell mass composed of epiblast-like and hypoblast-like cells. The timing of molecular stimulation of the aggregates also determines the level of specification and self-organization of cells within this aggregate. We observed that by initially allowing the cells to aggregate in B27N2 medium and then switching the medium to B27N2 medium containing A83-1, PD0325901, and LIF after 24–48 h, the aggregates were sufficient to form outer trophoblast cysts containing the three cell types, as well as single inner cell clusters of epiblast and hypoblast cells. Under these conditions, human blastoid cells were formed within 5 days (Figure 1B).

[0130] Example 2: Inhibition of the HIPPO pathway by using either small molecules or genetic approaches. The activity of the HIPPO pathway is essential for blast-like cell formation. Stimulation of hPSC aggregates with the small molecules LPA or NAEPA, inhibitors of HIPPO pathway activity, triggers the transition from solid cell aggregates to cyst structures containing a single liquid-filled cavity. This liquid-filled cavity is lined with cells expressing trophectoderm markers, including CDX2 and GATA3, and is composed of a single cluster of cells expressing markers of the epiblast, including NANOG and OCT4, and markers of the hypoblast, including GATA4. The necessity of modulating the activity of the HIPPO pathway to form blast-like cells is also assessed by the effect observed upon treatment of hPSC aggregates with verteporfin, a YAP-specific inhibitor. This small molecule completely blocks cavity formation (1 μM) (Figure 2C). In accordance with the role of the HIPPO pathway in human blastoid cell formation, the HIPPO pathway effector YAP translocates to the nuclei of the outer trophectoderm-like cells, but not to the nuclei of the inner cell cluster (Figure 2E). Finally, overexpression of wild-type and constitutively active YAP (YAP 5SA) promotes the formation of cavities at the early stage (day 2) of blastoid cell formation (Figure 2D). Conversely, overexpression of YAP with a mutation in the TEAD binding site (YAP S94A), known to inhibit the activation of HIPPO target genes, does not show a positive effect on cavity formation. Thus, consistent with data generated using small molecules, the genetic activation of the trophectoderm program and the formation of the blastocoel-like cavity require regulation of the HIPPO pathway.

[0131] Example 3: Simultaneous specification and morphogenesis of trophectoderm-like, epiblast-like, and hypoblast-like tissues. Inhibition of the LIF-STAT pathway by treatment with the chemical inhibitor SC-144 induced the formation of monolayered, cavity-forming aggregates that did not form inner clusters of epiblast-like / hypoblast-like cells. Lineage specification maturation of lateral trophectoderm cells was also impaired by SC-144 treatment, indicating a critical role for cell-cell communication between the three lineages in blastoid cell formation.

[0132] Example 4: Spontaneous formation of embryo-non-embryonic axes. In human late blastocysts, the polar trophectoderm adjacent to the epiblast begins to express the transcription factor NR2F2, marking a region that mediates implantation into the uterus. Similarly, when cultured in vitro, the trophectoderm-like tissue of the blastoid cells spontaneously forms a distinct region marked by expression of NR2F2. Conversely, the mural trophectoderm on the opposite side of the fluid-filled cavity does not express NR2F2 (Figure 3H). Thus, similar to the blastocyst, the blastoid cells can spontaneously form a region that mediates implantation and form an axis.

[0133] Example 5: Differentiation of endometrial cells into recipient cells mimicking the window of implantation. To model blastocyst implantation in vitro, we defined conditions (the so-called window of implantation, WOI) under which organoid-derived endometrial cells could be deposited in 2D culture plates and stimulated with molecules that would differentiate into cells that line the uterus upon implantation. We found that inhibition of the Wnt signaling pathway using multiple small molecules, as well as hormones (estradiol, progesterone), induces an upregulation of expression of genes that are highly expressed during WOI (Figure 6). As seen using RTqPCR in endometrial cells cultured with XAV939, Wnt inhibition increases the expression of PAEP, SPP1, LIF, and DPP4. As shown using immunofluorescence, stimulated endometrial cells also express PAEP at the protein level, and the cells are proliferating, as seen by EdU incorporation. Endometrial cells also contain a subpopulation of ciliated cells that specifically express acetylated alpha-tubulin, as seen using immunofluorescence, and glandular cells that specifically express FOXA2.

[0134] Example 6: Transplantation of human blastoid cells into receptive endometrial organoids. Deposition of human blast-like cells on EPC / XAV939-treated endometrial cells results in the attachment of blast-like cells and their invasion into the endometrial layer (Figure 7). Upon attachment and invasion, blast-like cells maintain expression of the pluripotency transcription factor Oct4. If endometrial cells are not stimulated with EPC / XAV939, blast-like cells are barely able to attach and invade the endometrial layer. If blast-like cells are barely able to form inner clusters of epiblast-like and hypoblast-like cells (SC144-Trophospheres, XMU-MP-1-Trophospheres), they are barely able to attach and invade the endometrial layer. Aggregates formed from human trophoblast stem cells, which reflect the postimplantation cytotrophoblast layer (Okae et al. 2018), are unable to attach and invade the endometrial layer.

[0135] Example 7: Expansion method. Culture of human naïve pluripotent stem cells Experiments were performed using the following hPSC lines; hESC lines: H9, Shef6 and HNES1. hiPSC lines: cR-NCRM2 and niPSC 16.2.b. Naive H9 and H9-GFP lines were provided by Yasuhiro Takashima's laboratory. Other naive hESCs and hiPSCs were provided by Austin Smith's laboratory. Naive hPSCs were cultured on gelatin-coated plates containing a feeder layer of gamma-irradiated mouse embryonic fibroblasts (MEFs) in PXGL medium. PXGL medium is prepared using N2B27 basal medium supplemented with PD0325901 (1 μM, MedChemExpress, HY-10254), XAV-939 (1 μM, MedChemExpress, HY-15147), Goe 6983 (2 μM, MedChemExpress, HY-13689), and human leukemia inhibitory factor (hLIF, 10 ng / ml, in-house). N2B27 basal medium contained DMEM / F12 (50%, in-house), Neurabasal™ medium (50%, in-house), N2 supplement (Thermo Fisher Science, 17502048), B-27 supplement (Thermo Fisher Science, 17504044), GultaMAX™ supplement (Thermo Fisher Science, 35050-038), non-essential amino acids, 2-mercaptoethanol (100 μM, Thermo Fisher Science, 31350010), and bovine serum albumin solution (0.45%, Sigma-Aldrich, A7979-50ML). Cells were routinely passaged as single cells every 3–4 days.

[0136] Culturing primed pluripotent ESCs Primed H9 cells were plated on vitronectin XF (STEMCELL Technologies, 07180)-coated plates (1.0 μg / cm 2 ) using Essential 8 medium.

[0137] Microwell Array A microwell array consisting of 200 μm diameter microwells was imprinted in a 96-well plate.

[0138] Induction of blast-like cells and trophosphere Cultures of naïve or primed hPSCs were treated with Accutase™ (Biozym, B423201) for 5 min at 37°C, followed by gentle mechanical dissociation using a pipette. After centrifugation, the cell pellet was resuspended in PXGL medium supplemented with Y-27632 (10 μM, MedChemExpress, HY-10583). To eliminate MEFs, the cell suspension was transferred to gelatin-coated plates and incubated at 37°C for 70 min. After MEF exclusion, cell numbers were measured using a Countess™ automated cell counter (Thermo Fisher Scientific) and trypan blue staining to assess cell viability. Cells were then resuspended in N2B27 medium containing 10 μM Y-27632 (aggregation medium) and eluted at 3.0 × 10 4Cells were seeded onto microwell arrays contained in wells of a 96-well plate. Depending on the cell line and based on its aggregation tendency, cells were allowed to form aggregates within the microwells for a period ranging from 0 to 24 h. The aggregation medium was subsequently replaced with PALLY medium-N2B27 supplemented with PD0325901 (1 μM), A 83-01 (1 μM, Med-ChemExpress, HY-10432), hLIF (10 ng / ml), 1-oleoyl lysophosphatidic acid sodium salt (LPA), (500 nM, Tocris, 3854) and Y-27632 (10 μM). PALLY medium was refreshed every 24 h. After 48 h, PALLY medium was replaced with N2B27 medium containing 500 nM LPA and 10 μM Y-27632. At 96 h, blastoid cells are defined based on their morphological similarity to B6 stage human blastocysts as structures consisting of single-layered cysts with an overall diameter of 150–250 μm containing one inner cell mass. We also verified that, beyond morphology, blastoid cells form analogs of the three blastocyst cell lineages in a sequential and timely manner during blastocyst development. Blastoid cells were formed reproducibly with high efficiency, and no differences were observed based on the number of passages after resetting in PXGL culture conditions. The effects of LPA, NAEPA (Sigma-Aldrich, N0912) and verteporfin (Selleck Chemicals Llc, S1786) on the yield of blastoid cell formation were assessed by culturing naïve hPSC aggregates in PALY medium supplemented with the molecules daily from 0 to 96 h post-culture. Verteporfin treatment was performed without exposure to light. The effect of the aPKC inhibitor CRT0103390 (a gift from Kathy Niakan's laboratory) was assessed by culturing naive hPSC aggregates in PALLY medium supplemented with 2 μM CRT0103390 daily for 0–96 h. Trophosphere formation was induced by culturing naive hPSC aggregates in PALLY medium supplemented with 2 μM XMU-MP-1 (Med Chem Express, HY-100526) or 3 μM SC-144 (Axon, 2324) daily for 0–96 h.BSA concentrations were titrated within the range of 0-0.3% for individual cell lines used for blastoid and trophosphere formation.

[0139] Derivation of cell lines from human blastoid cells Induction experiments were performed with blast-like cells cultured for 96 h as described in the previous section. Blast-like cells were individually transplanted onto gelatin-coated 96-well plates with a feeder layer of gamma-irradiated MEFs. Naïve hPSCs were induced in PXGL medium. hTSCs were induced in human trophoblast stem cell (hTSC) medium (Okae, H. et al. Cell Stem Cell 22, 50-63.e6 (2018)). After 24 h of culture on the feeder, the blast-like cells attached and formed colonies within 1 week. Induction was considered successful after 3 passages after blast-like cell transplantation. For immunofluorescence assays, naïve hPSCs were cultured in Geltrex® (0.5 μL / cm 2 )-coated coverslips, and hTSCs were transferred to fibronectin-coated coverslips (5 μg / ml, Sigma Aldrich, 08012).

[0140] Formation of trophoblast organoids Organoid formation was performed using a blastoid cell-derived hTSC line. Organoids were cultured as previously described (Turco, MY et al. Nature 564, 263-267 (2018)) with some modifications. Colonies of hTSC were dissociated into single cells using 1× trypsin for 5 min at 37°C. After centrifugation, 200,000 cells were resuspended in 150 μl of Matrigel (Corning, 356231). Drops of 20 μl per well were placed into pre-warmed 48-well cell culture plates and placed upside down in an incubator for 20 min. Organoids were cultured in 250 μl of TOM medium (Advanced DMEM-F12, N2 supplement, B27 supplement minus vitamin A, PenStrep, N-acetyl-L-cysteine ​​(1.25 mM), L-glutamine (2 mM), A83-01 (500 nM), CHIR99021 (1.5 uM), recombinant human EGF (50 ng / ml), 10% R-Spondin 1 conditioned medium, recombinant human FGF2 (100 ng / ml), recombinant human HGF (50 ng / ml), PGE2 (2.5 μM)). Medium was changed every other day. Organoids were maintained in TOM medium until day 7 for SCT formation.

[0141] 2D trophoblast differentiation Differentiation of blastoid cell-derived hTSCs was performed as previously described (Okae, H. et al. Cell Stem Cell 22, 50-63.e6 (2018)) with some modifications. Prior to the experiments, hTSC lines were adapted to fibronectin coating (5 μg / ml, Sigma Aldrich, 08012) for at least three passages. For differentiation of EVT and SCT, cells were dissociated with TrypLE for 5 min at 37 °C, and cells were seeded at a density of 55,000 cells / well on 12-well plates. For SCT differentiation, plates were precoated with 10 μg / ml fibronectin and cultured in SCT medium (DMEM / F12 supplemented with 0.1 mM 2-mercaptoethanol, 0.5% penicillin-streptomycin, 1% ITS-X supplement, 7.5 mM A83-01, 2.5 mM Y27632, 4% knockout serum replacement, and 2 mM forskolin) for 3 days. For EVT differentiation, plates were precoated with Matrigel and cells were cultured in EVT medium (DMEM / F12 supplemented with 0.1 mM 2-mercaptoethanol, 0.5% penicillin-streptomycin, 1% ITS-X supplement, 2% Matrigel, 7.5 mM A83-01, 2.5 mM Y27632, 4% knockout serum replacement, and 100 ng / ml NRG1). After 3 days, the medium was replaced with EVT medium containing 0.5% Matrigel and without NRG1. The cells were cultured until day 6.

[0142] Culture of human preimplantation embryos Human embryos were thawed according to the manufacturer's instructions (Cook Medical: Sydney IVF Thawing kit for slow cooling, Vitrolife: RapidWarmCleave or RapidWarmBlast for vitrification). Human embryos frozen at the 8-cell stage were loaded into 12-well dishes (Vitrolife: Embryoslide Ibidi) containing non-sequential medium (Vitrolife G2 plus) under mineral oil (Origio: liquid paraffin) at 37°C in 5% O2 / 6% CO2 for 5 min.

[0143] Plasmid construction The cDNA sequences of hYAP1, hYAP1 5SA, and hYAP1 5SA+S94A were amplified from pQCXIH-Myc-YAP, pQCXIH-Myc-YAP-5SA, and pQCXIH-Myc-YAP-S94A plasmids, respectively. These YAP plasmids were gifts from Kunliang Guan (Addgene plasmids #33091, #33093, and #33094) (Zhao, B. et al. Genes Dev.21,2747-2761(2007)). Individual cDNA sequences were cloned into pDONR211 and subsequently cloned into PB-TAC-ERP2 using the Gateway (invitrogen) cloning strategy. PB-TAC-ERP2 was a gift from Knut Woltjen (Addgene plasmid #80478) (Kim, S.-I. et al. Methods Mol. Biol. 1357,111-131(2016)).

[0144] Cell transfection in human naive PSCs pCAG-PBase (5 μg) and PB-TAC-YAP1-ERP (5 μg) were electroporated at 5 × 10 in single cell suspension by NEPA21 electroporation (Nepa Gene Co. Ltd). 4 Electroporated naive hPSCs were transfected into cells in PXGL medium containing Y-27632 (10 μM) in Geltrex (0.5 μL / cm 2 The transformed cells were plated on 6-well plates coated with 5% ribosomal RNA (Pribosomal RNA, Thermo Fisher Science, A1413302). Puromycin (0.5 μg / ml, Sigma-Aldrich, P7255) was added to the PXGL medium from day 1 to days 3–4 to select for transformed cells. pCAG-PBase was a gift from Knut Woltjen.

[0145] Overexpression of YAP in naive hPSC aggregates Naive hPSC aggregates were formed from a naive H9 cell line integrated with a doxycycline-inducible cassette as described in the section above. Aggregates were cultured for 0–48 h in PALLY medium containing reduced concentrations of LPA (5 nM) together with 100 ng / ml doxycycline. Higher LPA concentrations masked the effects of genetic overexpression of YAP1 variants. The number of aggregates in which cavity formation had occurred was counted after 72 h.

[0146] Single-cell RNA-seq library preparation and sequencing To avoid over-representation of TE cells, blast-like cells were collected, dissociated, and cell suspensions were stained using antibodies against TROP2 and PDGFRa, which mark trophoblast cells and primitive endoderm, respectively. At the 96-h time point, blast-like cells were selectively harvested from the microwell array before dissociation according to the morphological criteria described above. Cells were FACS sorted into 384-well plates containing lysis buffer for Smart-seq2 and immediately frozen. Antibody staining was utilized to collect a specific number of TROP2+, PDGFRa+, and double-negative cells. An abutted FACS gate (DiVa 9.0.1) covered the entire spectrum and blast-like cells were not excluded. H9 naïve cells cultured on MEFs were stained using an antibody against SUSD2 and sorted by FACS. Dead cells were excluded by DAPI staining. Smart-seq2 libraries were generated as previously described with minor optimizations (Picelli, S. et al. Nat. Protoc. 9, 171-181 (2014)). Maxima H Minus reverse transcriptase (3U / reaction, Thermo Fisher Science, EP0751) was used for cDNA synthesis. Prepared libraries were sequenced on an S1 or SP flow cell using an Illumina Novaseq instrument in 50 bp paired-end mode.

[0147] Single-cell RNA-seq data analysis Smart-Seq transcriptome sequencing experiments were analyzed using genome sequences and gene annotations from Ensembl GRCh38 release 103 as reference.

[0148] For gene expression quantification, RNA-seq reads were first trimmed using trim-galore v0.6.6 and then aligned to the human genome (Ensembl GRCh38 release 103) using hisat2 v2.2.1. Uniquely mapped reads within genes were quantified using htseq-count v0.13.5 with the parameter -s no. TPM estimates were obtained using RSEM v1.3.3 with the parameter -single-cell-prior.

[0149] Further analyses were performed in R v4.0.3 using Seurat v4.0.1. Based on an initial assessment of quality control metrics per cell and identification of outliers using the median absolute deviation algorithm, we excluded cells with 2000 or fewer detected genes or a proportion of mitochondrial genes greater than 12.5%. Only genes detected in at least five cells were retained. Count data were log-normalized, the top 3000 highly variable were selected, and expression values ​​per gene across cells were standardized using Seurat's NormalizeData, FindVariableFeature, and ScaleData data functions. Principal component analysis (PCA) based on the standardized highly variable features was used for linear dimensionality reduction, shared nearest neighbor (SNN) graphs were constructed based on the dimensionality-reduced data, and graphs were partitioned using the SNN module optimization-based clustering algorithm at various resolutions using Seurat's RunPCA, FindNeighbors, and FindClusters with default settings. Cluster marker genes were identified with Wilcox likelihood ratio tests using the FindAllMarkers function. Uniform Manifold Approximation and Projection (UMAP) was used for visualization.

[0150] To integrate Smart-Seq experiments from multiple sources, we followed a procedure previously described (Zhao, C. et al. doi:10.1101 / 2021.05.07.442980). Published data from E-MTAB-3929 (human preimplantation embryos from embryonic day 3 to 7) Petropoulos, S. et al. Cell vol. 167 285 (2016)), GSE109555 (in vitro cultured blastocysts) were downloaded, and data from Carnegie stage 7 embryos were kindly provided by the authors (Tyser, RCV et al. bioRxiv (2020) doi:10.1101 / 2020.07.21.213512). For GSE109555, all data were preprocessed to obtain read counts per gene using the same protocol described for blastoid cells, including adaptations to accommodate UMI and CB information according to the authors' instructions (https: / / github.com / WRui / Post_Implantation). For GSE109555, 1000 cells randomly subsampled from the 3184 high-quality single cells described in the original publication were used. Cells belonging to hemogenic endothelial progenitors and erythroblasts were excluded. After evaluating quality control metrics per cell, cells with more than 2000 detected genes and a proportion of mitochondrial genes below 12.5% ​​were retained. Genes detected in at least five cells in any dataset were retained. Log-normalization was performed using computeSumFactors in the scran package v1.18.7, and batch-wise scaling normalization was performed using multi-BatchNorm in batchelor v1.6.3. The dataset was aligned using the fastMNN approach via SeuratWrappers v0.3.0 using log-normalized batch-adjusted expression values. The MNN low-dimensional coordinates were then used for clustering and visualization with Uniform Manifold Approximation and Projection (UMAP).

[0151] Culture and aggregate formation of human trophoblast stem cells Experiments were performed using the human blastocyst-derived hTSC line bTS5, provided by the Arima Takahiro laboratory. Cells were cultured in hTSC medium on plates coated with laminin 511 (5 μg / ml, BioLamina, LN511) as previously described. hTSC aggregates were formed as follows: colonies were dissociated into single cells using Accutase™ at 37°C for 5 min. Cells were resuspended in hTSC medium containing 10 μM Y-27632 and diluted at 3.0 × 10 4 Cells were seeded into the microwell arrays imprinted in the wells of a 96-well plate. The same medium (Okae, H. et al. Cell Stem Cell 22, 50-63.e6 (2018)) was refreshed daily. After 72 h, aggregates were used for both characterization and transplantation experiments.

[0152] Endometrial organoid culture Cryopreserved human endometrial organoids were provided by the Hossein Bahavand Laboratory (Royan Institute) within the framework of a collaboration agreement. Human endometrial organoids were established from healthy human donors following a previously described protocol (Boretto, M. et al. Development 144, 1775-1786 (2017)) with some modifications. Briefly, organoids were cultured in medium supplemented with 1x N2 supplement, 1x B27 supplement, 1x insulin-transferrin-selenium (in-house), Glutamax (1 μM), N-acetylcysteine ​​(1.25 mM, Sigma-Aldrich, A7250), nicotinamide (2.5 mM, Sigma-Aldrich, 72340), EGF (50 ng / ml, Peprotech, 100-47), bFGF (2 ng / ml, Peprotech, 100-18B), HGF (10 ng / ml, Peprotech, 315-23), FGF10 (10 ng / ml, Peprotech, 100-26), A83-01 (500 nM), and SB202190 (10 μM, Tocris, 1264) at 10%. The organoids were cultured in human endometrial expansion medium consisting of Rspo1-conditioned medium (made in-house) and 10% Noggin-Fc-conditioned medium (Heijmans, J. et al. Cell Rep. 3, 1128-1139 (2013)). Y-27632 (10 μM) was used for the first 2 days after passaging to prevent apoptosis. The medium was changed every 2 days and the organoids were passaged in TrypLE followed by mechanical dissociation every 7–9 days.

[0153] Hormonal stimulation of endometrial organoid and OFEL cultures Endometrial organoids were passaged as described in the previous section. Dissociated cells were resuspended in Matrigel supplemented with Y-27632 (10 μM), and the cell suspension was deposited in 48-well plates and cultured in Endometrial Expansion Medium for 2 days. Organoids were first stimulated with E2 (10 nM, Sigma-Aldrich, E2758) for 2 days, followed by treatment with E2 (10 nM), P4 (1 μM, Sigma-Aldrich, P8783), and cAMP (250 μM, Biolog, B 007) with or without XAV939 (10 μM) (EPC or EPCX, respectively) for 4 days. For OFEL culture, organoids were harvested from the Matrigel droplets using ice-cold DMEM / F12 and mechanical pipetting. Dissociate the organoids using TrypLE and mechanically triturate to generate single cells, and plate 3–4.5 × 10 cells per well in a 96-well glass-bottom plate (Cellvis, P96-1.5HN). 4 Cells were seeded at a density of 1000 x 1000 and cultured for 2–3 days with stimulation. For contraceptive treatment, levonorgestrel (LNG) (10 μM, Sigma-Aldrich, PHR1850) was added daily to the medium 2 days after hormonal stimulation and continued until the end of the experiment.

[0154] In vitro transplantation assay Confluent OFELs were prepared for transplantation assays at least 2 h prior to deposition of blastoid cells, trophospheres, naïve hPSCs, or hTSC aggregates by washing OFELs twice with DMEM / F12 and adding IVC medium (Xiang, L. et al. Nature 577, 537-542 (2020)). The structures were then transferred onto the OFELs using a mouth pipette under an inverted microscope. After 24–48 h, the medium was removed and the wells were washed with PBS and fixed using 4% formaldehyde for 30 min at room temperature, followed by processing for immunofluorescence staining. The percentage of attached structures was reported as the percentage of the total transferred structures.

[0155] In vitro culture of human blastoid cells in post-transplant conditions Human blastoid cells were selected using a mouth pipette, washed with CMRL1066 medium, and transferred to Matrigel-coated suspension culture plates or 96-well plates containing pre-equilibrated medium adapted from monkey blastocyst cultures (Ma, H. et al. Science 366, (2019).) with minor modifications as follows. On the first day, the culture medium was CMRL1066 supplemented with 10% (v / v) FBS, 1 mM l-glutamine (Gibco), 1x N2 supplement, 1x B27 supplement, 1 mM sodium pyruvate (Sigma), and 10 μM Y27632. After 24 h, half of the medium was replaced with new medium containing 5% Matrigel. After 48 h, 50% of the medium was replaced with new medium supplemented with 20% (v / v) FBS and 5% Matrigel. After 72 hours, half of the medium was replaced with fresh medium supplemented with 30% (v / v) KSR and 5% Matrigel. Thereafter, half of the medium was replaced daily and blastoid cells were cultured for up to 6 days. After 4 and 6 days of in vitro culture with 4% PFA, cultures were fixed for staining as described above.

[0156] ratio Comparative example of human preimplantation embryos The use of human embryos donated for research as surplus from IVF treatments was permitted by the French embryo research monitoring committee: Agence de la Biomédecine, under approval numbers RE13-010 and RE18-010. All human preimplantation embryos used in this study were obtained from and cultured at the Assisted Reproduction Unit of the Nantes University Hospital, France, which is authorized to collect embryos for research under Agence de la Biomédecine approval number AG110126AMP. The embryos used were initially created in the context of assisted reproduction cycles with a clear reproductive purpose and were subsequently donated for research voluntarily by patients after they had met their reproductive needs or tested positive for the presence of a monogenic disorder.

[0157] RNA extraction, cDNA synthesis, and qRT-PCR RNA was extracted using RNeasy mini kit (Qiagen, 74106) and cDNA synthesis was performed using Superscript III (Invitrogen, 18080093) enzyme. qPCR reactions were performed using GoTaq® qPCR Master Mix (Promega, A6001) on a CFX384 Touch Real-Time PCR Detection System (Bio-rad). Quantification was performed using Microsoft Office Excel by applying the comparative cycle threshold (Ct) method. Relative expression levels were normalized to GAPDH.

[0158] ELISA assay for CGβ detection Media from wells containing non-adherent or adherent blastoid cells were collected, centrifuged to remove debris, and stored at -80°C until use. Supernatants were subjected to CGβ ELISA (Abcam, ab178633) according to the manufacturer's instructions, in parallel with CGβ standards.

[0159] Ligand-receptor analysis To predict putative receptor-ligand interactions between polarized TEs and endometrial epithelial cells, we used the Cellinker web platform. Gene modules of endometrial epithelial cells that mark the transition to phase 4 of the menstrual cycle, together with genes in the NR2F2 module of late TEs, enriched genes of polarized TEs, and genes upregulated in stimulated OFELs, were used as queries to search for ligands and receptors in the database.

[0160] immunohistochemistry Samples were fixed with 4% formaldehyde for 30 min at room temperature. After fixation, the formaldehyde solution was removed and samples were washed at least three times with PBS. Samples were then permeabilized and blocked for at least 60 min using 0.3% Triton-X100 and 10% normal donkey serum in PBS. Samples were then incubated overnight at 4°C with primary antibodies diluted in fresh blocking / permeabilization solution. Samples were washed at least three times for 10 min each with PBS containing 0.1% Triton-X100 (PBST). Next, the wash buffer was replaced with Alexafluor-tagged secondary antibodies (Abcam or Thermofisher Scientific) along with nuclear dye Hoechst-33342 (1:500 or 1:300 for 2D or 3D samples, respectively, Life Technologies, H3570) diluted in PBST for 30 min at room temperature in the dark. Samples were then washed three times with PBST for 10 min each. For human blastocysts, samples were fixed at B4 or B6 stage according to the grading system proposed by Gardner and Schoolcraft, or at B3 or B4 + 72 hours of in vitro culture. Embryos were fixed with 4% paraformaldehyde for 10 minutes at room temperature and washed with PBS / BSA. Embryos were permeabilized and blocked in PBS containing 0.2% Triton-x100 and 10% FBS for 60 minutes at room temperature. Samples were incubated with primary antibodies overnight at 4°C. Incubation with secondary antibodies was performed for 2 hours at room temperature with Hoechst counterstaining. Samples were mounted in PBS in wells of glass-bottom microslides (Ibidi, 81507) for imaging. EdU staining was performed using the Click-iT EdU Alexa Fluor 64 Imaging Kit (Thermo Scientific, C10640) according to the manufacturer's instructions.

[0161] Microscopy and image analysis Phase contrast images were acquired using a Thermo Fisher Scientific EVOS cell imaging system and an inverted widefield microscope Axio VertA1. The number of blast-like cells or hollowed out structures was counted manually for each well. After 96 hours, blast-like cells were defined based on morphological parameters as described in the previous section. Fluorescent and time-lapse images were acquired on an Olympus IX83 microscope with a Yokogawa W1 spinning disk (software: CellSense2.3, camera: Hamamatsu Orca Flash® 4.0) or a Nikon Eclipse Ti E inverted microscope with a Yokogawa W1 spinning disk (software: Visiview 4.5.0.7, camera: Andor Ixon Ultra 888 EMCCD). Confocal images were analyzed and displayed images were exported using FIJI 1.53k or Bitplane Imaris 9.7.0 software. Bitplane Imaris software was used for cell counting. Cell count parameters were set to match the voxel size and fluorescence intensity, and the spot function in Imaris was used to obtain overall cell count data for each image. Note that large cavities within blastoid cells increase the depth of the imaging field, resulting in reduced signal from deeply located cells. Therefore, the counting data in Fig. 8H may be an underestimate, especially for trophectoderm cells. Quantification of the percentage of blastoid cells forming NR2F2 axes was performed manually. To do so, blastoid cells stained to detect NR2F2 expression were imaged using a confocal spinning disk microscope. Images were projected using the 3D project function in FIJI. A blastoid cell was classified as having an axis if NR2F2 expression was restricted to its polar half and there was no or low expression in the parietal half. An inverted pattern of NR2F2 expression was classified as an inverted axis. Blastoid cells with NR2F2 expression in both the polar and parietal halves were classified as not having an axis. Confocal immunofluorescence images of human blastocysts were obtained using a Nikon confocal microscope and a 20×Mim or 25×Silicon objective. Optical sections 1 μm thick were taken.Images were processed using Fiji (http: / / fiji.sc) and Volocity 6.3 visualization software. Nuclei were detected and counted using Volocity software.

[0162] Statistics and reproducibility All experiments were performed in at least three biological replicates unless otherwise stated in the methods and figure legends. Statistical analyses were performed using Graphpad prism 8.1.1 (330).

[0163] Example 8: Triple inhibition (Hippo / ERK / TGF). The blastocyst is formed within 3–4 days by generating the conceptus 3 founding lineages, namely epiblast (EPI, embryonic), trophectoderm (TE, extraembryonic), and primitive endoderm (PrE, extraembryonic) (Figure 8a). Peripheral cells become TE by inhibiting the Hippo pathway. Also, naive hPSCs (cultured in PXGL) efficiently form TE analogs by inhibition of the TGF-β and ERK pathways. Therefore, we aggregated naive hPSCs in non-adhesive hydrogel microwells and inhibited these three pathways (Figure 8b and Figure 12a–c). When exposed to lysophosphatidic acid (LPA, Hippo pathway inhibitor), A83-01 (TGF-β family receptor inhibitor) and PD0325901 (ERK inhibitor) in chemically defined medium containing LIF (STAT activator) and Y-27632 (ROCK inhibitor), blastocyst-like structures were efficiently formed (Fig. 8c-e, >70%, 150<φ<250μm, see full morphometric criteria in expansion method) and consistently formed (Fig. 12d, >20 passages). LPA significantly improved efficiency (Fig. 12b-d). Within 5 days, cell number (from 47+ / -9 to 129+ / -27) and overall size (from 65μm to 200μm) increased similarly to the range of day 5-7 blastocysts (stages B3-6) (Fig. 12e, f). TE cell analog (GATA2 + / GATA3 + / CDX2 + / TROP2+ ) undergo cycles of expansion and contraction (Fig. S12n), form and proliferate (Fig. S8f-h and Fig. S12g-l), establish adherens junctions (epithelial cadherin (CDH1)), and form apical-basal polarity (aPKC localization) and tight junctions (ZO-1 + , Fig. 8i and Fig. 12m). Strikingly, all blastocyst-like structures expressed EPI (OCT4+; mean = 27 + / - 13 cells; 26% of all cells) and PrE (GATA4 + / SOX17 + / PDGFRa + We isolated unique inner cell masses reflecting EPI (average = 7 + / - 5 cells; 7% of total cells) (Figure 8f-h and Figure 12i,j,l), indicating a disruption of symmetry after cell aggregate formation. Naïve hESCs (Shef6, H9, HNES1) and hiPSCs (niPSC 16.2.b, cR-NCRM2) of multiple lineages formed such structures with comparable high efficiency (Figure 8e and Figure 12o), whereas primed hPSCs reflecting EPI after transplantation did not (Figure 12p). Triple inhibition of the Hippo pathway (e.g., by LPA), MEK / ERK (e.g., by PD0325901), and TGF-b (e.g., by A83-01) was required for the generation of blastoid cells with cavities based on human cells (Figure 22). Figure 23 shows that an alternative TGF-b inhibitor (SB431542) also works.

[0164] Example 9: Formation of blastocyst analogs only. Single-cell transcriptome analysis showed that the blastocyst-like structures formed three major transcriptome states (Figures 9a, b and 13a) characterized by three founder lineage-specific genes, including GATA2 / GATA3 (TE), POU5F1 / KLF17 (EPI), and GATA4 / SOX17 (PrE) (Figures 9c, d and 13b). Comparison with cells from blastocysts, in vitro-cultured blastocysts, and gastrulation-stage embryos revealed that the cells were transcriptionally similar to the blastocyst state and distinct from the postimplantation state (Figures 9e, f and 13c-g). High-resolution clustering analysis (×50) isolated one cluster of non-blastocyst-like cells with a gene expression pattern reminiscent of post-implantation tissues (GABRP, ISL1, APLNR, CRABP2) (Fig. 14a-c), which appeared transcriptionally similar to amnion (annotated as non-neural ectoderm) and mesoderm (Fig. 14d-j). ​​This subpopulation constituted less than 3% of the cells (Fig. 14i). Of note, naïve hPSC cultures contained 5.6% of similarly differentiated cells (Fig. 14i). Bulk RNA sequencing analysis revealed no significant differences in the expression of isolated trophoblast analogs (TROP2 by flow cytometry) and that were similar to the amniotic blastocyst-like cells (Fig. 14c). + ) had an intermediate transcriptome between naive hPSCs and postimplantation-like trophoblast cells (hTSCs) (Fig. S15a). Furthermore, trophoblasts were enriched in blastocyst stage TE transcripts (ESRRB, GRHL1, OVOL1, GATA2, GATA3, TBX3, KRT19, CGA, CGB5, CGB7) but not in some postimplantation markers (SIGLEC6, DPP4) (Fig. S15b, c). The isolated EPI analog (TROP2 - / PDGFRa - The transcriptome of the isolated PrE analog (PDGFRa) resembled that of naive hPSCs (Fig. S15a), was enriched for blastocyst-stage EPI-specific markers (KLF17, ATG2A, SUSD2, TFCP2L1, ZFP57, DPPA2, UTF1, PRDM14), and was distinct from that of primed hPSCs (Fig. S15a, d). Finally, the isolated PrE analog (PDGFRa+ ) had an intermediate transcriptome between naive hPSCs and extraembryonic endoderm cell lines (nEND cells) (Fig. 15a) and were enriched in blastocyst stage PrE markers (early blastocyst: GATA6, MSX2, HNF4A. Late blastocyst: PDGFRA, GATA4, SOX17, HNF1B, FOXA2) and EPI genes (ARGFX, PRDM14, SOX2, NANOG, DPPA2, POU5F1) that were downregulated similarly to blastocysts (Fig. 15e). Blastocysts have the ability to establish stem cell lines. Similarly, naive hPSCs (NANOG) that can form second generation blastocyst-like structures (Fig. 16b, c) by blastocyst-like structures were enriched in PrE markers at the blastocyst stage (early blastocyst: GATA6, MSX2, HNF4A. Late blastocyst: PDGFRA, GATA4, SOX17, HNF1B, FOXA2) and EPI genes that were downregulated similarly to blastocysts (ARGFX, PRDM14, SOX2, NANOG, DPPA2, POU5F1) (Fig. 15e). Blastocysts have the ability to establish stem cell lines. Similarly, blastocyst-like structures were enriched in naive hPSCs (NANOG) that can form second generation blastocyst-like structures (Fig. 16b, c). + / SOX2 + / OCT4 + / KLF17 + ) (Fig. 16a), and hTSCs (CDX2 - / GATA3 + / CK7 + ) (Fig. 16d). Of note, derivation of PrE cell lines from human blastocysts has not been reported before. Overall, this model morphologically resembles a blastocyst (see criteria in the extension method), transcriptionally mirrors the blastocyst stage, and generated three lineages analogous to those that develop in a sequential and timely manner in the blastocyst, which we termed blastoid cells.

[0165] Example 10: Hippo inhibition. Knowledge of human blastocyst lineage separation is limited (Fig. 10a). However, inhibition of the Hippo pathway occurs in peripheral cells upon acquisition of the apical domain and is required for trophoblast specification (Fig. 17a). We tested whether blastoid cells employ this mechanism. Strikingly, atypical protein kinase C (aPKC) and F-actin expression domains appeared to be co-aligned in outer cells that also accumulated the Hippo downstream effector YAP1 in the nucleus (Fig. 17b, c). The nuclear location of YAP1 correlated with GATA2 / 3 expression and contrasted with NANOG expression, which became restricted to TE analogs (Fig. 10b and Fig. 17d, e). An aPKC inhibitor (CRT0103390) significantly prevented YAP1 nuclear accumulation and downregulated GATA3 + It reduced cell number and prevented blast-like cell formation (Figure 17f-h). Conversely, ligands of the LPA receptor (LPA and NAEPA), which inhibit the Hippo pathway, enhanced blast-like cell formation (Figure 10c and Figure 17i). Because inhibition of the Hippo pathway allows YAP1 to enter the nucleus, we tested whether engineered YAP1 levels and function would affect morphogenesis. Overexpression of wild-type or constitutively active YAP1(5SA) promoted cavity formation (Figure 10d). Interaction between YAP1 and TEAD transcription factors is required for downstream gene regulation. Accordingly, overexpression of YAP1 containing a mutation in the TEAD binding site (S94A) did not affect cavity formation (Figure 10d and Figure 17j), and verteporfin, a drug that disrupts the YAP1-TEAD interaction, prevented blast-like cell formation (Figure 17k). Cavity morphogenesis occurred through the apparent fusion of multiple fluid-filled cavities (Figure 17l). Aquaporin 3 (AQP3), the most highly expressed water transporter in human blastocysts, was initially observed in all cells (36 h) and then restricted to the TE analogs (96 h) (Figure 17m). Thus, similar to human blastocysts, trophoblast specification and morphogenesis of blastoid cells depends on aPKC, inhibition of the Hippo pathway, nuclear translocation of YAP1, and its ability to bind TEAD transcription factors.

[0166] Example 11: Proper Sequence of Events In the blastocyst, the trophoblast appears first (day 5-6, GATA2 + / DAB2 + ), PrE cells appear last (days 6-7, GATA6 + / ADM + This sequence is recapitulated in the blastoid cells that initially form trophoblasts (<24 h, DAB2) with altered levels of transcripts related to PKC and Hippo signaling (AKAP12, CAPZB, ULK4, MOB1a, AMOT, AMOTL2, LATS2, TEAD1). + , CDX2 + , and GATA2 + / 3 + , Fig. 10e and Fig. 18a). At the protein level, early TE-like cells initially express YAP1 核 / GATA2 + (24 hours), then CDX2 + / GATA3 +appeared while maintaining KLF17 / OCT4 but not NANOG expression (60 h) (Fig. 18b-d). Thereafter, OCT4 was undetectable (Fig. 8g and Fig. 12i). Genes related to SMAD, ERK, Notch, and Wnt signaling pathways were modulated during this process (Fig. 18e, f). Finally, polar trophoblast analogs matured as characterized by expression of OVOL1, GREM2, CCR7, SP6, and NR2F2 (Fig. 10f and Fig. 18g-j), upregulation of NR2F2 and CCR7, and downregulation of CDX2 (10f and Fig. 18h, j). The transcriptome of EPI analogues underwent an overtime progression characterized by the regulation of Nodal (NODAL, LEFTY1 / 2), and mTOR signaling-related genes (LAMTOR1 / 4 / 5, XBP1, SEC13, MLST8) as well as the X-chromosome activation-related gene XACT (Fig. 18k-m), while maintaining core blastocyst markers (POU5F1, NANOG, KLF17, SUSD2, KLF4, ARGFX, GDF3) (Fig. 10e and Fig. 18k,l). Subsequently, PrE analogues appeared (<60 hours), and GATA4, OTX2, and SOX17 were detected (72 hours) (Fig. 10e and Fig. 18n-p). While early PrE marker genes (GATA6, LBH, ADM, and LAMA1) were uniformly expressed among PrE analogs, some late PrE marker genes (CTSE, APOA1, PITX2, and SLCO2A1) were expressed only in a subpopulation, suggesting progression to a late blastocyst state (Fig. 18q). By 96 h, mature PrE analogs regulated SMADs (NODAL, BMP2 / 6, GDF3, ID1 / 2) and Wnt signaling-related transcripts (WNT3, RSPO3, LBH) and were enriched in transcripts regulating extracellular matrix organization, endothelial, and epithelial differentiation (LAMA1, LAMB1, LAMC1, COL4A1 / 2) (Fig. 18q,r). Overall, the blastoid cell lineages specified and progressed according to the sequence of blastocyst development.

[0167] Example 12: Distinct interactions with endometrial cells Human blastocysts initiate implantation in the uterus (days 7-9) via apposition and attachment of polarized TEs to the receptive endometrium (Figure 11a, left). We wondered whether blastoid cells could model this interaction. We seeded endometrial organoids (Boretto, M. et al. Development 144,1775-1786(2017)) in 2D to form an open-faced endometrial layer (OFEL) that promotes the deposition of blastoid cells (Figure 11a, right). Subpopulations were positive for acetylated α-tubulin, which marks ciliated epithelial cells (Figure 19a), and FOXA2, which marks glandular epithelial cells (Figure 19b). Exposure to estrogen (E2) and progesterone (P4) and Wnt inhibition opens the window for implantation. Thus, OFEL responded to E2, P4, cAMP, and XAV939 by upregulating the expression of genes that characterize mid-secretory endometrium (Fig. 19c-e) and decreasing proliferation (Fig. 19e, f). Notably, blast-like cells deposited on unstimulated OFEL did not adhere, whereas blast-like cells interacted with stimulated OFEL by adhering to and then repulsing endometrial cells, as occurs in the uterus (Fig. 11b and Fig. 19g, h). The contraceptive drug levonorgestrel impaired blast-like cell adhesion (Fig. 19i). We conclude that human blast-like cells can interact with hormonally receptive endometrium.

[0168] Example 13: Epiblasts signal gatekeeping interactions. Human blastocysts attach to the endometrium via the polar trophectoderm defined by contact with EPI. Similarly, blastoid cells initiated attachment through this region (Fig. 11c, d and Fig. 20a-c). We next tested the importance of the polar / EPI interface by forming trophospheres (without EPI). IL6 is highly expressed in polar TE, and transcripts of its receptor (IL6R, GP130) and effector (STAT3) are abundant in EPI (Fig. 20d). Consistent with a role for STAT signaling in EPI, blastoid cell formation efficiency increased with LIF concentration (Fig. 9e), while addition of a GP130 inhibitor (SC144) yielded trophospheres (Fig. 11e and Fig. 20f). Trophospheres were also obtained in the presence of a potent inhibitor of Hippo kinase MST1 / 2 (XMU-MP-1) (Fig. 11e and Fig. 20g). The transcriptomes of these trophospheres reflected early and late blastocyst trophoblasts, respectively (Fig. S20h, i). Both trophosphere types were unable to attach to the OFEL (Fig. S11e), and the post-implantation cytotrophoblast (CDX2 - / CK7 + ) or naïve hPSC aggregates (Fig. 11e and Fig. 20j, k). We conclude that signals from EPI ensure the maturation of polarized trophoblasts, which allows their interaction with endometrial cells. Based on transcriptome analysis and in utero data, we propose several pairs of molecules whose transcripts become more abundant upon stimulation of endometrial cells and maturation of polarized trophoblasts (Fig. 20l). These may mediate the first touch between the blastocyst and the uterus. Overall, we conclude that a preimplantation polarity-like state, whose maturation depends on EPI induction, gate-keeps the interaction of the blastocyst with the endometrium.

[0169] Example 14: Post-stage modeling (day 13). The morphology of the blastoid cells was stable for 2 days in peri-implantation culture conditions (Figure 21a). Clinical pregnancy is characterized by the detection of chorionic gonadotropin beta (CGβ) hormone. Upon attachment, the blastoid cells formed trophoblasts that expressed CGβ at levels detectable using standard pregnancy tests and ELISA (Figures 11f and 21b). NR2F2 + Polar trophoblast analogs proliferated while upregulating the preimplantation gene cytokeratin 7 (CK7) and reduced CDX2 expression (Fig. 21c, d). Some trophoblasts further differentiated into SCT and EVT expressing CGβ and HLA-G, respectively (Fig. 21e, f). EPI analogs maintained OCT4, SOX2, upregulated primed pluripotency marker CD24 (Fig. 11g and Fig. 21g), patterned cortical F-actin as during EPI epithelialization, and some blastoid cells expressed F-actin / PODXL. + / aPKC + The PrE analogs formed a pro-amniotic-like cavity rich in SOX2 and TFAP2C (Fig. 11g and Fig. 21h). A peripheral subpopulation of the EPI analogs also expressed CDX2 together with SOX2 or TFAP2C, suggesting an early amniotic analog (Fig. 21i,j). The PrE analogs were characterized by restricted expression of OTX2 (Fig. 18o,21k). In long-term culture (up to 6 days), the three lineages consistently proliferated up to a time equivalent to day 13 (Fig. 11h and Fig. 21l,m), but similar to the blastocysts, their organization did not reflect their developmental stage.

[0170] Consideration. The fidelity, efficiency, scalability, and versatility of this model make it important to study human blastocyst development and implantation. The blastoid cells of the present invention aid in the identification of therapeutic targets and contribute to preclinical modeling (e.g., IVF media complements, such as candidate LPA / NAEPA, or contraceptives, such as candidate SC144). Given the balance (balance of benefits and harms) and complementarity (pursuing goals using the least morally questionable means) of human embryology, blastoid cells provide an ethical opportunity to complement research using embryos.

[0171] Strikingly, stimulation of hPSC aggregates with the three inhibitors not only triggers the formation of trophoblast cells, but also the simultaneous formation of spatially organized trophoblast, epiblast, and primitive endoderm cells. The spontaneous organization of these three cell types is characterized by the formation of a trophoblast cyst that forms an outer layer and a fluid-filled lumen, as well as the formation of a unique inner cluster of epiblast and primitive endoderm cells that remains attached to one side of the trophoblast cyst. As a direct consequence, the asymmetry created within the cyst by the presence of this unique inner cluster induces the local maturation of polarized trophoblasts and orients their attachment to endometrial cells.

Claims

1. An in vitro method for generating blastoid or blastocyst-like cell aggregates, comprising culturing aggregates of human pluripotent stem cells (hPSCs) and trophoblast cells in a 3D culture medium containing a HIPPO pathway inhibitor, wherein the aggregates of hPSCs and trophoblasts are produced by culturing aggregated hPSCs in a medium containing a MEK inhibitor and a TGF-β inhibitor.

2. The method according to claim 1, wherein the aggregates of hPSCs and trophoblasts are produced by culturing the aggregated hPSCs in a culture medium further containing a HIPPO pathway inhibitor.

3. The method according to claim 1 or claim 2, wherein the aggregated hPSCs are formed by seeding hPSCs and culturing the seeded hPSCs in a growth medium.

4. The method according to claim 3, wherein the seeded hPSCs are treated with a MEK inhibitor and / or a PKC inhibitor.

5. The method according to claim 4, wherein the PKC inhibitor is selected from Goe6983 and Ro-31-8425.

6. The method according to any one of claims 1 to 5, wherein the HIPPO pathway inhibitor is a ligand for the lysophosphatidic acid (LPA) receptor; and / or the MEK inhibitor is PD0325901; and / or the TGF-β inhibitor is A83-1 or SB431542.

7. The method according to any one of claims 1 to 6, comprising the steps of: culturing cells until at least trophectoderm-like tissue, epiblast-like tissue, and hypoblast-like tissue are formed from the hPSC and trophoblast aggregates; and / or culturing cells until at least a three-dimensional cell aggregate having a total diameter of 100 μm is formed, which is formed by a liquid-filled cavity and an outer epithelial monolayer of trophoblast-like cells surrounding at least one inner cell mass containing epiblast-like cells and hypoblast-like cells.

8. The method according to any one of claims 1 to 7, further comprising the steps of stimulating endometrial cells with a compound selected from estrogen, estrone, estriol, ethinylestradiol, 17α-ethinylestradiol, mestranol, progesterone, progestin, cAMP, and a Wnt-inhibitor, and seeding blastoids onto a layer of stimulated endometrial cells to enable the blastoids to adhere to and penetrate the layer of endometrial cells.

9. The method according to any one of claims 1 to 8, comprising treating the aggregates with at least one candidate compound and / or giving the aggregates at least one candidate gene modification, and / or performing the method according to any one of claims 1 to 8, for testing or screening for candidate compounds and / or candidate gene mutations that affect blastoid formation and / or blastoid implantation into a layer of endometrial cells.

10. A kit suitable for culturing blastoids, containing a HIPPO pathway inhibitor, a MEK inhibitor, and a TGF-β inhibitor.

11. A blastoid obtained by the method according to any one of claims 1 to 9.

12. The blastoid according to claim 11, comprising at least one fluid-filled cavity and an outer epithelial monolayer of trophoblast-like cells surrounding at least one inner cell mass containing epiblast-like cells and hypoblast-like cells, wherein the outer epithelial monolayer comprises a polar trophoblast expressing NR2F2.

13. A blastoid comprising at least one fluid-filled cavity and an outer epithelial monolayer of trophoblast-like cells surrounding at least one inner cell mass containing epiblast-like cells and hypoblast-like cells, wherein the outer epithelial monolayer comprises a polar trophoblast expressing NR2F2.

14. A method for producing a blastocyst, comprising the steps of: treating an early stage embryo, selected from the 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage, or 16-cell stage, or the morula stage, or the blastocyst stage, with a HIPPO pathway inhibitor, particularly preferably with NAEPA or a ligand for the lysophosphatidic acid (LPA) receptor, and more preferably with LPA, until it reaches the mature blastocyst stage; and growing the embryo from the 1-cell stage, 2-cell stage, 4-cell stage, 8-cell stage, or 16-cell stage, or the morula stage, to the blastocyst stage, or growing the blastocyst stage embryo to a more mature blastocyst stage.

15. The method according to claim 3, wherein culturing in the growth medium includes culturing in the growth medium for 0 to 64 hours.

16. The method according to claim 3, wherein culturing in the growth medium includes culturing in the growth medium for 12 to 64 hours.

17. The method according to claim 3, wherein the growth medium comprises a ROCK inhibitor.

18. The method according to claim 17, wherein the ROCK inhibitor is Y27632.

19. The method according to claim 4, wherein the seeded hPSCs are further treated with a Wnt inhibitor and / or a STAT agonist.

20. The method according to claim 4, wherein the treatment is performed during 2D culture.

21. The method according to claim 6, wherein the HIPPO pathway inhibitor is LPA, NAEPA, or verteporfin.

22. The method according to claim 7, comprising the step of culturing cells until an embryo-non-hypocotyl is formed.

23. The method according to claim 7, wherein the total diameter is at least 140 μm.

24. The method according to claim 7, wherein the total diameter is 180 μm to 220 μm.

25. The method according to claim 8, wherein the Wnt inhibitor is XAV939, IWP-2, PNU-74654, or LF3.

26. The kit according to claim 10, wherein a HIPPO pathway inhibitor, a MEK inhibitor, and a TGF-β inhibitor are incorporated into a culture medium for human pluripotent stem cells (hPSCs).

Images