An in vitro method of producing a blastocyst-like 3D structure of cells forming a model of monochorionic twins

Abstract

The disclosure relates to the field of generating blastocyst-like 3D structure of cells or models. In particular the disclosure relates to an in vitro method of producing a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures. The disclosure also relates to the blastocyst-like 3D structure of cells so obtained and to several uses of the same, in particular as model of monozygotic and monochorionic twinning. Methods of screening of candidate agents that modulate the mammal embryonic development are also disclosed.

Description

[0001]P136664PC00 Title: An in vitro method of producing a blastocyst-like 3D structure of cells forming a model of monochorionic twins Field of the disclosure The disclosure relates to an in vitro method of producing a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures. The disclosure also relates to a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures obtained or obtainable by such a method. The disclosure moreover relates to a culture of differentiated 3D cell aggregate structures. Additionally, the disclosure relates to particular uses of such blastocyst-like 3D structure of cells or culture of differentiated 3D cell aggregate structures. The disclosure further relates to an in vitro screening method for identifying an agent or a physical and / or mechanical mean that modulates the mammal embryonic development. Background of the disclosure Twins are two offspring produced by the same pregnancy. They can be either dizygotic (fraternal), or monozygotic (identical). Dizygotic twins derive from two eggs that were independently fertilized by two different sperm cells. On the other hand, monozygotic twins result when a unique egg is fertilized to form a zygote and then this zygote divides to form two separate embryos. Most (around 75 %) of the monozygotic twins are monochorionic twins, which means that they share the placenta. There can be also monozygotic dichorionic (or polychorionic) twins (25 %) that have separated independent placentas. It is known that pregnancies of monozygotic (identical) twins are at increased risk of developing complications such as fetal and intrauterine growth restriction, fetal anemia, preterm labor (in up to 50% of cases) and congenital anomalies. Specifically, among monozygotic twins, the monochorionic twins (i.e. share the placenta) are at risk for circulation abnormalities like twin-to-twin transfusion syndrome (a disparity in blood flow between the twins), leading to health complications. Natural identical twin embryos are not available for research due to their low incidence rate (3-4 in every 1000 births). The alternative, human embryo cloning, is ethically charged and prohibited in many countries. Thus, there is currently no way to study how monochorionic twins are formed and start developing, since monochorionic twins can only be identified during the course of pregnancy. How such placental abnormalities come to be and whether this can be prevented is currently unknown, mainly because there are currently no cell models to study the monochorionic twins. Recently, the development of blastocyst-like 3D cell structures or aggregates, also called blastoids, has emerged as a powerful tool to elucidate the mechanisms behind the embryonic development of mammals. Generically, blastoids result from pluripotent stem cells cultured first in particular culture mediums that allow the cells to propagate and then to capacitate (i.e., to start differentiation towards the three founding lineages of the early embryonic development). Effectively, the blastoids generate the three founding lineages (trophectoderm, epiblast and primitive endoderm), resembling the blastocyst stage of the embryonic development. Blastoids have, thus, an outer-layer of cells called the trophoblast or trophectoderm, which will eventually form the fetal portion of the protective placenta, that surrounds a fluid-filled cavity known as blastocoel. In most cases blastoids have one group of cells called the inner cell mass (ICM) inside the blastocoel, the group of cells containing cells of embryonic lineage forming epiblast-like structures and cells of extraembryonic lineage forming hypoblast-like structures. However, in few cases blastoids have two or more distinguishable group of cells occurring in the blastocoel, resembling the development of monochorionic twins. This occurrence of two or more distinguishable group of cells in the blastocoel of a blastocyst-like 3D structure of cells does percentagewise not differ much from the percentages of naturally occurring identical twin embryos in human pregnancies. The ICM, that contains from 50 to 150 cells in the actual blastocyst as well as in the blastoids, will evolve to the epiblast and the hypoblast, which will give rise to the embryo proper and the yolk sac, respectively. However, it is widely accepted that blastoids are not able to form or develop into mammal (e.g. human) embryos, which alleviates any ethical concern associated with the human embryo manipulation. Kagawa et al. (2021) in Human blastoids model blastocyst development and implantation, Nature, Vol. 601, 600-605, disclose this potentiality of the blastoids in the study of embryo development. The authors formed a model of the human blastocyst that specifically generated and spatially patterned cellular analogues of the blastocyst stage, and which enabled the model to mimic aspects of implantation. To do this, naïve pluripotent stem cells (PSC) were grown in PXGL medium first and later in N2B27 medium containing 10 μM Y-27632 (aggregation medium). The cells were seeded onto a microwell array included into a well of a 96-well plate and placed in a hypoxic chamber (5% CO2, 5% O2) for the whole period of blastoid or trophosphere formation. The potential of human naive stem cells to generate blastoids that show high fidelity to the human embryo, and that fulfil key criteria for an experimental model system, was studied by Yanagida et al., in Naive stem cell blastocyst model captures human embryo lineage segregation, Cell Stem Cell.2021 Jun 3; 28(6): 1016–1022.e4. Transcriptome analyses confirmed segregation of trophectoderm, hypoblast, and epiblast from naïve pluripotent stem cells with high fidelity to the human embryo. The authors seeded propagated pluripotent stem cells at a ratio of 50-200 cells per well (diameter around 5 mm, to 6-7 mm) in an ultra-low attachment multiple- well plate (Corning Coster). Once the cells formed three-dimensional aggregates, they were manually transferred into a non-adherent, ‘U’- bottomed 96-well (Greiner) containing pre-warmed N2B27 supplemented with 0.5 μM A83-01. This way, single epiblast population of cells with naïve features were obtained. The European patent application with publication number EP2986711 (IMBA- Institut für Molekulare Biotechnologie GmbH) is also another example that discloses a method for the formation of blastoids. Mouse embryonic stem cells (7 cells per microwell) and trophoblasts cells (17 cells per microwell) are combined to form a cell aggregate and let it to evolve to a double-layered structure, in which the trophoblast cells form the outer layer that surrounds the inner layer of cells derived from the mouse embryonic stem cells. None of the previously commented documents refers to models of blastocyst that can be used for the study of monozygotic and monochorionic twins. In particular the occurrence of a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures is not reported. Thus, there is still a need of adequate in vitro tools to study this twin related phenomenon with the aim to elucidate the mechanisms underlying the complications associated with the same. Summary It is an object of the disclosure to provide a method of producing a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures. Additionally or alternatively, it is an object of the disclosure to provide a cell product, such as a culture of cells, containing a workable percentage of blastocyst-like 3D structures of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures. A workable percentage in this regard is considered an occurrence percentage that is at least cost effective. That is, given the average costs of the current available methods of obtaining blastocyst-like 3D structures of cells, the natural or uninfluenced occurrence of approximately 2-3 blastocyst-like 3D structures of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures in 1000 blastocyst-like 3D structures of cells formed is far from cost effective. Preferably, the workable percentage is at least 1% of the total number of produced blastocyst-like 3D structures of cells, as this at least 1% allows an average occurrence of at least one blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures in a method employing a wells plate or other type of cell culture container in which approximately 100 or more distinct cell cultures can be grown simultaneously. In other words, the occurrence percentage of 1% enables the use of rather conventional means and methods in producing blastocyst-like 3D structures of cells for obtaining a workable number of blastocyst-like 3D structures of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures. Specialized or more costly means are not necessary to this end. To meet one or more of the objects, the disclosure provides for an in vitro method of producing a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures, the method comprising: providing at least 150 of pluripotent stem cells (PSC’s) in a confined culture space in which the PSC’s are kept spatially constrained in a microvolume of culture medium at a cell density between 0.5x103cells to 2.0x104cells per mm3culture medium, allowing the PSC’s to aggregate into an aggregate structure comprising at least 150 cells, adding an inhibitor of the Hippo pathway in the culture medium containing the aggregate structure in a concentration of at least 1µM, and differentiating the PSC’s in the aggregate structure to form the blastocyst-like 3D structure of cells; and to optionally isolate the blastocyst-like 3D structure of cells. Three-dimensional cell aggregates of PSC are commonly formed, as the skilled person in the art will recognize, by seeding the indicated numbers of PSC in a confined culture space in which the PSC’s are kept spatially constrained in a microvolume of culture medium. For example, the cells may be seeded in a cell culture container of low dimensions such as a microwell, which impairs to the cells spatial constraints that make them aggregate to form an aggregate structure of the indicated number of cells per cell culture container with low dimensions. The aggregate structures will further proliferate in this container or another if the aggregate is transferred, and will differentiate to more complex three-dimensional structures depending on the composition of the differentiation medium. The cell culture container of low dimensions may be part of a larger container, for example a well of a 96 wells plate, wherein for example multiple cell culture containers of low dimensions are defined by the upper surface of the bottom wall of the well, for example the well having micro pockets in the bottom wall, each micro pocket defining a confined culture space in which the PSC are kept spatially separated from other micro pockets. The confined culture space contains a microvolume of cell culture medium, which may be fluidically communicating with a larger volume of cell culture medium, for example a cell culture medium provided in the well in an amount exceeding the volume of all confined culture spaces in that well. Although a low number of PSC cells, theoretically even a single PSC cell, can be seeded in the confined culture space for further growth and differentiation to obtain a blastocyst-like 3D structures of cells, it was found that the number of PSC cells provided in the confined culture space and allowed to aggregate is a relevant factor to increase the percentage of blastocyst-like 3D structures of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures occurring. A higher number of cells in the confined culture space stimulates aggregation due to the higher spatial constraints for each individual cell, and results in relatively larger average aggregate structures containing more cells. Particularly, at the indicated number of cells per confined culture space, the formation of one or more cell aggregates comprising at least 150 cells per aggregate structure is favored. These aggregate structures are more likely to proliferate and differentiate to the more complex blastocyst-like 3D structure of cells (cavitated). Additionally, a higher cell density in the culture medium in the confined culture space and higher number of cells in the aggregate structure, i.e. larger aggregate structure, may affect the effect of a compound provided in the culture medium on each cell or some cells. This applies particularly to cells positioned centrally within the larger aggregate structures, i.e. more distant from or shielded from direct contact with the culture medium. As will be shown by the examples herein, it was found that the addition of a Hippo pathway inhibitor in a particular concentration of at least 1µM to the formed aggregate structures as a result of the PSC cell seeding numbers and density in the culture medium is a direct factor to increase the occurrence of blastocyst-like 3D structures of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures. Optionally, the inhibitor of the Hippo pathway is lysophosphatidic acid (LPA). As to the number of PSC cells provided in the confined culture space, the number of 150 cells is found to be the minimum to obtain at least a workable percentage of blastocyst-like 3D structures of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures occurring. However, a number of 200 PSC cells provided in the confined culture space increases this percentage to about 6-10% of all aggregate structures formed after differentiation with 1µM LPA, and a number of 300 PSC cells provided in the confined culture space increases this percentage to about 9-12% of all aggregate structures formed after differentiation with 1µM LPA. As to the concentration of inhibitor of the Hippo pathway added to the cell culture medium containing the aggregate structures, the concentration of 1µM is found to be the minimum to obtain at least a workable percentage of blastocyst-like 3D structures of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures occurring. However, all concentrations between 1-10 µM of an inhibitor of the Hippo pathway result in at least a workable percentage of blastocyst-like 3D structures of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures occurring. Addition of a Hippo pathway inhibitor to the cell culture medium containing the aggregate structures in a concentration between 2 µM -5 µM gives good percentages of blastocyst-like 3D structures of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures occurring, particularly when added to aggregate structures formed after providing 200 respectively 300 SPC cells to the confined culture space. Optionally in the method at least 200 pluripotent stem cells (PSC’s) are provided in the confined culture space in which the PSC’s are kept spatially constrained in a microvolume of culture medium at a cell density between 0.5x103cells to 2.0x104cells per mm3culture medium, and the inhibitor of the Hippo pathway added to the culture medium containing the aggregate structure is added in a concentration of at least 2.5µM. The disclosure relates, thus, to a method to form blastocyst-like 3D structure of cells from mammalian pluripotent stem cells (such as human pluripotent stem cells-hPSCs). As indicated, blastocyst-like 3D structure of cells formed from PSCs are not able to form or develop into mammal (e.g. human) embryos, alleviating any ethical concern associated in the particular case of departing from human pluripotent stem cells. For this reason, all along this description the terms blastocyst-like 3D structure of cells (as synonymous of blastoid), blastocyst-like stage, or even embryonic- like stage or embryonic-like development stage are used, since the herewith disclosed structures are not actual blastocysts or embryos although resembling certain embryonic development stages. In addition, it is widely known that pluripotent stem cells (naive or induced) are nowadays available from methods that do not suppose or originally come from the destruction of human embryos. For example, human pluripotent stem cells, such as human embryonic stem cells, can be obtained from parthenogenetically activated oocytes. Or, more preferably, pluripotent stem cells are induced from other cell types. In another aspect the disclosure provides a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures obtained or obtainable by an in vitro method as disclosed herein. The in vitro method provided herein, in any of its alternative definitions, also allows to obtain other differentiated three-dimensional cell aggregate structures that can be of interest, such as blastocyst-like 3D structures of cells with a single ICM. Thus, by means of the method disclosed herein a cell culture can be obtained derived from cultured pluripotent stem cells that comprises several three-dimensional cell aggregate structures of interest. Thus, in a further aspect, the disclosure provides a culture of differentiated three-dimensional cell aggregate structures comprising: - from 2 % to 25 % of a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures; - from 5 % to 60 % of a blastocyst- like 3D structure of cells having cells of embryonic lineage forming one distinguishable epiblast-like structure and cells of extraembryonic lineage forming one distinguishable hypoblast-like structure; all the percentages in relation to the total number (100 %) of three-dimensional cell aggregates in the culture. This culture of differentiated three-dimensional cell aggregates is optionally provided in a scaffold or support or container with non-cell adherent properties. Therefore, in another aspect of the disclosure provided is a container made of a non-cell adherent material and comprising the culture of differentiated three-dimensional cell aggregates described herein. The present disclosure is based on the surprising finding that particular cell numbers when culturing pluripotent stem cells in a spatially confined culture space, i.e. a particular cell density, to form cell aggregates, or when departing from pluripotent stem cell aggregates with a particular number of cells, affect the occurrence percentage of blastoids having at least two distinct ICM when the cells are in a differentiation culture medium adequate for the formation of embryoids. The disclosure relates to the use of an in vitro method of culturing and differentiating of a pluripotent stem cell, in particular mammalian pluripotent stem cell, in which the pluripotent stem cell is cultured, in particular in suspension, in a differentiation culture medium comprising at least one inhibitor of the TGFβ pathway and at least one inhibitor of the ERK pathway, at particular seeded cell densities to allow the formation of three-dimensional cell aggregates, for the preparation and optional isolation of a blastoid comprising at least two ICM. Alternatively, the disclosure relates to the use of an in vitro method of culturing and differentiating of a pluripotent stem cell, such as a mammalian pluripotent stem cell, in which a pluripotent stem cell aggregate comprising a particular amount of cells per aggregate is cultured, particularly in suspension, in a differentiation culture medium comprising an inhibitor of the Hippo pathway in the culture medium containing the aggregate structure in a concentration of at least 1µM and at least one inhibitor of the TGFβ pathway and at least one inhibitor of the ERK pathway. It is another aspect of the disclosure to provide a particular use of the in vitro method as disclosed herein, for the preparation and isolation of blastoids comprising at least two ICM. This aspect can also be formulated as the use of an in vitro method of culturing and differentiating of a mammal pluripotent stem cell for the preparation and optional isolation of a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures, in which the used in vitro method comprises the step of providing PSC aggregates from 150 to 300 cells / aggregate in a container with a cell differentiation culture medium, which medium comprises an inhibitor of the Hippo pathway in the culture medium containing the aggregate structure in a concentration of at least 1µM and at least one inhibitor of the TGFβ pathway and at least one inhibitor of the ERK pathway, and to allow to differentiate into a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures. A further aspect of the disclosure is the use of an in vitro method of culturing and differentiating of a pluripotent stem cell, such as a mammalian pluripotent stem cell, for the preparation and optional isolation of a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures, in which the used in vitro method comprises the steps of: (a) providing a culture of pluripotent stem cells; (b) providing the pluripotent stem cells in a confined culture space in which the PSC’s are kept spatially constrained in a microvolume of culture medium at a cell density from 0.5x103cells / mm3 to 2.0x104cells / mm3 culture medium; (c) to allow the pluripotent stem cells to proliferate in a cell differentiation culture medium in the container, which differentiation culture medium comprises an inhibitor of the Hippo pathway in the culture medium containing the aggregate structure in a concentration of at least 1µM and at least one inhibitor of the TGFβ pathway and at least one inhibitor of the ERK pathway, to obtain a culture of three-dimensional cell aggregates that comprise a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures; and (d) to optionally isolate the a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures. In other words, in step (c) the pluripotent stem cells are allowed to proliferate and differentiate, in particular from 3 to 5 days, to a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures. Yet another aspect of the disclosure is the use of the blastocyst- like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures, or the culture of three-dimensional cell aggregates, all as herein defined, for the screening of a candidate agent, or alternatively a physical and / or mechanical mean, with the capacity to modulate (i.e., inhibit or to promote / enhance) the mammalian embryonic development. Also another aspect of the disclosure is the use of the a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures, or the culture of three- dimensional cell aggregates, all as defined herein, as an in vitro model of the mammalian embryonic development of a blastocyst stage comprising at least two ICM. Finally, another aspect of the disclosure is an in vitro screening method for identifying an agent or a physical and / or mechanical means that modulates the mammalian embryonic development, the method comprising: (a) providing a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures, or a culture of differentiated three-dimensional cell aggregates, both as defined herein; (b) to contact the a blastocyst-like 3D structure of cells or culture of the previous step with a candidate agent, or alternatively, to subject the a blastocyst-like 3D structure of cells or culture to a physical and / or mechanical means; (c) detecting a change in mammalian embryonic development compared to a control to determine the candidate agent’s modulatory activity, or the modulatory activity of the physical and / or mechanical mean. Brief Description of the Drawing The disclosure will further be elucidated on the basis of exemplary embodiments which are represented in a drawing. The exemplary embodiments are given by way of non-limitative illustration. It is noted that the figures are only schematic representations of embodiments of the disclosure that are given by way of non-limiting example. In the drawing: Figure 1 shows twin blastoid formation, wherein: Figure 1A is a Brightfield image of a twin blastoid generated in microwells from naïve ESC (HNES1) and naive iPSC (HDF75 iPSC)s; Figure 1B is a schematic showing the twin formation process; Figure 1C is a Brightfield image of a representative set of twin blastoids; Figure 1D is a bar diagram showing yield of blastoids; Figure 1E is a bar diagram showing twin blastoids measured as the percentage of blastoids of all aggregates formed at various concentrations of lysophosphatidic acid ([LPA]) and with different starting cell densities (100 cells / microwell in blue, 200 cells / microwell in red and 300 cells / microwell in green (n=3),with the error bars representing the standard deviation (SD); Figure 1F is a diagram with projected area of singleton and twin blastoids generated from 200 cells and 300 cells, each supplemented with 2.5 µM LPA (200c and 300 respectively, pooled from 3 independent experiments); Figure 1G is an image showing maximum projection of a twin blastoid with immunofluorescent staining for KLF17 (red) and OCT4 (green); Figure 1H is an image showing maximum intensity projection of twin blastoid for OCT4 (green) and SOX17 (red); Figure 1I is a bar diagram with quantification of the number of twin blastoids with SOX17+ cells in either both, one of two ICM (1 ICM) or none of the ICMs; Figure 1J is an image showing maximum intensity projection of a twin blastoid with immunofluorescent staining for GATA3 (red) and OCT4 (green); Figure 1K is an image showing maximum intensity projection of a twin blastoid with immunofluorescent staining for NR2F2 (red) and OCT4 (green); Figure 1L is a bar diagram with quantification of the number of twin blastoids with NR2F2+ cells in either both, one of two ICM (1ICM) or none of the ICMs. All scale bars in the images depict100µm. Figure 2 shows development and morphology of twin blastoids, wherein: Figure 2A shows immunofluorescent images of ICM splitting stages, scale bar 100µm; Figure 2B is a heatmap depicting cyst expansion of 20 twin blastoids over time. The time point of ICM splitting is indicated with a bold box; Figure 2C is a boxplot showing spread of time between cavitation and ICM splitting; Figure 2D is a diagram with angular distance (α) between the distinct ICMs within a single cyst in twin blastoids generated from 200 and 300 cells; Figure 2E is a diagram with the ratio of total ICM area / cyst area per structure in singleton and twin blastoids generated from 200 and 300 cells per microwell, supplemented with 2.5 µM LPA (200c and 300c, respectively; Kruskal-Wallis test, P<0.0001, twin-twin comparison P<0.0001, n≥20); Figure 2F is a diagram with cell counts of 200c blastoids. Cell counts were performed with immunofluorescent imaging after TrypLE dissociation. Total number of cells in per individual singleton and twin blastoid (Welch's t-test, P=0.0017, n=20); Figure 2G is a diagram with Cell counts of 200c blastoids. Cell counts were performed with immunofluorescent imaging after TrypLE dissociation. Percentage of OCT4+ and SOX17+, indicative of the epiblast and hypoblast lineages respectively, over the total number of cells per individual singleton or twin blastoid (Welch ANOVA, OCT4+-OCT4+ comparison P=0.80, SOX17- SOX17 comparison P=0.21, n=20); Figure 2H is a diagram with cell counts based on high resolution confocal images of intact 200c blastoids. Frequency distribution of the number of OCT4+ cells per ICM between two ICMs within the same twin blastoids (bin size 0.1, n=50); Figure 2I is a diagram with cell counts based on high resolution confocal images of intact 200c blastoids. Total number of OCT4+ cells per blastoid, counted based on high resolution images (Mann-Whitney test, P=0.013, 20 singletons, 27 twin blastoids); Figure 2J is a diagram with cell counts based on high resolution confocal images of intact 200c blastoids. Ratio of SOX17+ / OCT4+ cells in singleton blastoids, total numbers per twin blastoid (ICM1+ICM2) and per individual ICM in twin blastoids (ICM1 and ICM2; Kruskal-Wallis test, P=0.064, n=14); Figure 3 is a bar diagram that illustrates the type and percentages of three-dimensional cell aggregates obtained from the culturing of pluripotent stem cells at different cell ratio per confined culture space (microwells). The different represented cell aggregates are called multiple cysts, trophospheres, embryoid bodies (EB), blastoids with oversized ICM (oversized ICM: ICM with more than 150 cells), blastoids with multiple ICM (multiple ICM; i.e., at least two ICM), and blastoids with a single ICM (blastoid), wherein: Figure 3A shows the results from the seeding of human embryonic stem cells (hESC); Figure 3B shows the results from the seeding of induced pluripotent stem cells (iPSC) derived from human fibroblasts; Figure 4 is an image from optical microscopy with an example of each of the multiple cysts, trophospheres, embryoid bodies (EB), blastoids with oversized ICM (oversized ICM), blastoids with multiple ICM (multiple ICM) and blastoids with a single ICM (blastoid); and Figure 5 shows images taken with fluorescence microscopy, wherein: Figure 5A shows a blastoid with two ICM obtained with the method of the disclosure; Figure 5B shows the two ICM, detected by the expression of OCT4 (OCT4+) in green; Figure 5C shows the trophectoderm, detected by the expression of GATA3 (GATA3+) in red. Definitions All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition. As used herein, the indefinite articles “a” and “an” are synonymous with “at least one” or “one or more.” Unless indicated otherwise, definite articles used herein, such as “the” also include the plural of the noun. “Cell density” is defined as the number of cells (i.e., PSC) per volume unit of culture medium contained in the confined culture space in which the cells are cultured. The units are cells / volumetric measure (e.g., cubic milimeters-mm3). As a way of example, a cell density indicated as from 1.0x103cells / mm3 to 1.0x104cells / mm3 of culture medium, means that in the confined culture space where the PSC are going to be differentiated, the number of cells accounts to a value between 1.0x103cells per cubic millimetre of culture medium in which the cells are, to 1.0x104cells per cubic millimetre of the container. Another equivalent way to express the cell density is by indication of the ratio of number of cells in a micrometer-sized container, such as a microwell. This is a very common used way to indicated the number of cells, in particular when the cells are seeded in a microwell from a microwell array as the ones known by the skilled person in the art. Thus, a density expressed as 1.2 x 103cells / mm3 can also be expressed as 25 cells per microwell, said microwell with a diameter of 300 µm and a depth of 300 µm (i.e., the volume of the cylindric microwell is approximately 0.02 mm3). This description contains both type of indications of the cell density, i.e., cell per volume unit of culture medium in the confined culture space or cell number per container (i.e., ratio cell / container), the later in the particular cases of using microwells as containers. Regarding the shape of the container in which the PSC are seeded to form aggregates, or in which the already PSC aggregates are provided to differentiate, they are containers with a shape selected from spherical shaped; hemispherical shaped; elongated cylindrical shaped; conical shaped, more in particular selected from conical shaped with hemispherical (i.e., rounded) base; cylindrical shaped, in particular selected from cylindrical shaped with hemispherical (i.e., rounded) base, and pyramidal shaped. Particularly preferred to facilitate aggregation of the seeded PSC or to maintain aggregation in case of providing a PSC aggregate are the hemispherical, conical shaped and pyramidal shaped containers. “Pluripotent stem cells (PSC)” are cells that can differentiate into nearly all cells. They are stem cells that have the potential to differentiate into any of the three germ or founding layers: endoderm (gut, lungs, yolk sac), mesoderm (muscle, skeleton, blood vascular, urogenital, dermis), or ectoderm (nervous, sensory, epidermis), but not into extra-embryonic tissues like the placenta. There are several grades of pluripotency, in terms that some PSC can form every cell of the embryo proper, e.g., embryonic stem cells and iPSCs, meanwhile others are partially pluripotent cells that can form cells of all three germ layers but that may not exhibit all the characteristics of completely pluripotent cells. The term “naïve pluripotent stem cell” (nPSC, or inPSC) relates to cells that are derived from the inner cell mass (ICM) of preimplantation embryos or from parthenogenetically activated oocytes. These PSC have an unlimited self‐renewal capacity when grown under appropriate conditions and are able to differentiate into tissues of all three germ layers in vitro. This pluripotency state is captured in vitro in form of the embryonic stem cells (ESCs). Mostly, the term PSC is used as an umbrella term for all pluripotent stem cells including embryonic stem cells and induced pluripotent stem cells. The PSC for the performance of the disclosure do not suppose the destruction of human embryos. “Induced pluripotent stem cells (iPSC)” are a type of pluripotent stem cell artificially derived from a non-pluripotent cell. These non- pluripotent stem cells are typically adult somatic cells, which are reprogrammed to express genes and the transcription factors Oct4, Sox2, Klf4 and c-Myc. iPSCs exhibit similar traits to those of embryonic stem cells (ESCs), such as the cell morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity, but they do not require the use of embryos. The terms “aggregate” or “cell aggregate” or “three-dimensional cell aggregate” (used in the description as exchangeable synonymous terms) refer to three- dimensional clusters of mammal cells that are adhered forming a defined and discrete structure. It encompasses clusters of PSC; structures or cell systems comprising only one cell type of the originating three germ layers or lineage cells; and structures or cell systems comprising at least two of the three lineage cells (e.g. embryoid bodies- EB), such as the three germ layers. Among the aggregates comprising the at least two of the three lineage cells are those aggregates or cell systems, in which the cells are spatially structured comprising at least a fluid-filled cavity surrounded by a layer of cells, and wherein said at least one fluid-filled cavity comprises one or more clusters of cells different from the cells that surrounds the cavity. The term “differentiated three- dimensional cell aggregate” encompasses, more specifically, the cell clusters obtained after the performance of the method, and they are selected from the group consisting of multiple cysts, embryoid bodies, blastoids with one or more ICM or with oversized ICM, and trophospheres. They are differentiated because departing from a PSC they have changed to one or more specialized cell type. The term “multiple cysts” are defined, according to this description, as structures comprising two or more fluid-filled cavities surrounded by an outer layer of trophoblasts, whose cavities partially share this outer layer. The fluid-filled cavities comprise ICM that can also be shared or be conformed as separated ICM. An image is depicted in Figure 4. “Embryoid bodies” (EBs) are differentiated three-dimensional aggregates of pluripotent stem cells comprising the three embryonic germ layers. An image is depicted in Figure 4. “Blastoids” are EBs spatially structured as a blastocyst stage of mammalian embryonic development. They are also referred in this description as blastocyst-like cell structure or blastocyst-like structure. They comprise an outer-layer of trophoblasts, that surrounds a fluid-filled cavity known as blastocoel, this cavity also including the group of cells called the inner cell mass (ICM). The ICM in the blastoids comprises around 50–150 cells, as in the actual mammal blastocyst. Blastoids with “oversized ICM” encompass blastoids with a single ICM comprising more than 150 cells. Blastoids with multiple ICM refer to blastoids with at least two ICM, each one comprising from 50 to 150 cells. An image of a blastoid with two ICM, and of a blastoid with an ICM with more than 150 cells are depicted in Figure 4. “Trophospheres” are three-dimensional aggregate spheroids of trophoblasts that result from trophoblast cells grown in suspension (in non- adherent surface plate). The trophoblasts cells derive, in the case of the present disclosure, from the differentiation of the cultured pluripotent stem cells in suspension. An image is depicted in Figure 4. The “aggregation medium” as referred in this description is to be understood as a cell medium culture that promotes pluripotent stem cells to form clusters (i.e., generally three-dimensional cell groupings). The skilled person in the art know the composition of different aggregation media useful to allow PSC to aggregate. The “differentiation medium” as referred in this description is to be understood as a medium that promotes pluripotent stem cells to change from a less specialized type or stage to a more specialized in form and function. In the particular case of the disclosure, it is a medium that comprises one or more compounds that allow the cells to propagate and then to capacitate (i.e., to start differentiation towards the three founding lineages of the early embryonic development). The skilled person in the art know these differentiation culture media from the prior art. In this description, particular useful examples to promote the production of blastoids comprising at least two ICM are disclosed. As used herein “candidate agent” or “agent” refers to a molecule that may be screened for, or be identified as, modulating the mammalian embryonic development (e.g. evolution of blastocyst-like stage to an implantation-stage). Such agent may, for example, be an inhibitor or enhancer (i.e., promoter) of the development and may find use in a variety of applications, including therapy. The screening methods will typically be assays which provide for qualitative / quantitative measurements of the activity (i.e., modulation of embryonic development) in the presence of a particular candidate agent. (Candidate) agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts or purified compounds are available or may be produced. Additionally, natural or synthetically produced libraries and compounds can be prepared using conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogues or derivates. (Candidate) agents may also be biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogues or combinations thereof. The expression “physical and / or mechanical mean(s)” refers to any mechanical and / or physical manipulation of the blastoid in vitro, as well as of any of the cell aggregates and differentiated cell aggregates listed in the disclosure, such as the PSC aggregates / clusters. Examples of physical and / or mechanical means are selected from the group consisting of light, temperature, pH, pressure, application of forces, and combinations thereof. The skilled person in the art of embryo and cell manipulation knows the one or more physical techniques and the way to implement them for the alteration of certain parameters in the structured cell aggregates. As used herein, the term “determining”, for example determining activity, and / or amounts of cell-surface markers, of secreted proteins or of transcription factors, includes measuring, analyzing, estimating, following, and the like of such activity, and / or amounts, for example, using conventional means and / or techniques. Likewise, the term “providing”, for example, providing a cell includes preparing, isolating, obtaining, and the like, of such cell. Detailed description of the disclosure The present disclosure relates to an in vitro method of producing a blastoid comprising at least two ICM, the method comprising the step of: to provide one or more PSC aggregates comprising from 150 to 300 cells per aggregate (i.e., cells / aggregate) in a container with a cell differentiation culture medium that comprises an inhibitor of the Hippo pathway, at least one inhibitor of the TGFβ pathway and at least one inhibitor of the ERK pathway, and allow to differentiate to one or more blastoids comprising at least two ICM. In a particular embodiment of the in vitro method of producing a blastoid comprising at least two ICM, the method comprises the steps of: (a) providing a culture of pluripotent stem cells; (b) to allow the pluripotent stem cells to proliferate in a container and form three- dimensional pluripotent stem cell aggregates comprising from 150 to 300 cells per aggregate; (c) to allow one or more pluripotent stem cell aggregates comprising from 150 to 300 cells / aggregate obtained in step (b) to differentiate in a container, which in particular is the container of step (b), said container comprising a cell differentiation culture medium as defined above, to obtain one or more blastoids comprising at least two ICM. The PSC aggregates from 150 to 300 cells are, in a particular embodiment, produced by seeding PSC and letting them grow, and optionally differentiate, in suspension culture. Alternatively, the PSC aggregates from 150 to 300 cells are, in a particular embodiment, produced by seeding PSC adhered on a support and, once the aggregates comprise from 150 to 300 cells, they are optionally transferred to a container to grow and differentiate in suspension. The most common way to obtain PSC aggregates is by suspension in a culture medium allowing or promoting aggregation. In a particular embodiment, the PSC aggregates from 150 to 300 cells / aggregate are obtained by seeding the pluripotent stem cells in a container at a cell density from 0.5x103 cells / mm3 of container to 2.0x104 cells / mm3 of container. Thus, in even a more in particular embodiment the method of the first aspect comprises: (a) providing a culture of pluripotent stem cells; (b) seeding the pluripotent stem cells in a container at a cell density from 0.5x103 cells / mm3 of container to 2.0x104 cells / mm3 of container in a medium (i.e., aggregation medium) to obtain PSC aggregates comprising from 150 to 300 cells / aggregate; (c) to allow the pluripotent stem cells of step (b) to proliferate in a cell differentiation culture medium in the container, which differentiation culture medium comprises an inhibitor of the Hippo pathway, at least one inhibitor of the TGFβ pathway and at least one inhibitor of the ERK pathway, to obtain a culture of differentiated three-dimensional cell aggregates, which culture comprises blastoids comprising at least two ICM; and (d) to optionally isolate the blastoids comprising at least two ICM. Considering the weight of the spatial constraints to the ratio of formation of blastoids with at least two ICM, the present disclosure relates, as previously indicated, to an in vitro method of producing a blastoid comprising at least two ICM, the method comprising the steps of: (a) providing a culture of pluripotent stem cells; (b) seeding the pluripotent stem cells in a container at a cell density from 0.5x103 cells / mm3 of container to 2.0x104 cells / mm3 of container; (c) to allow the pluripotent stem cells to proliferate in a cell differentiation culture medium in the container, which differentiation culture medium comprises an inhibitor of the Hippo pathway, at least one inhibitor of the TGFβ pathway and at least one inhibitor of the ERK pathway, to obtain a culture of cell aggregates that comprise blastoids comprising at least two ICM; and (d) optionally to isolate the blastoids with at least two ICM. All the particular embodiments list below apply to the in vitro method of producing a blastoid comprising at least two ICM in any of its formulations. That is, the method defined including as a starting point the reference to a PSC aggregate with a particular number of cells per aggregate, or alternatively, defined as including as a starting point the reference to the seeding in a container at a particular cell density. Thus, in a particular embodiment of the in vitro method of the first aspect the pluripotent stem cell is selected from one or more of a naïve pluripotent stem cell, an induced pluripotent stem cell, a naïve induced pluripotent stem cell, an embryonic stem cell or a naïve embryonic stem cell. In a more particular embodiment, the pluripotent stem cell is selected from one or more of a naïve pluripotent stem cells and induced pluripotent stem cells. Culture of PSC are commercially available. In another particular embodiment, the PSC or culture of PSC is a PSC obtained by reprogramming an adult somatic cell, in particular a mammalian somatic cell, more in particular, the somatic cell selected from a human fibroblast and peripheral blood mononuclear cell (PBMCs). A way of reprogramming of fibroblasts into in vitro three- dimensional models of blastocysts (i.e., blastoids) is disclosed in the following reference, Liu, X., et al. Modelling human blastocysts by reprogramming fibroblasts into iBlastoids. Nature 591, 627–632 (2021). https: / / doi.org / 10.1038 / s41586-021- 03372-y. In another particular embodiment, the PSC or culture of PSC is a mammalian PSC or culture of mammalian PSC and the blastoid is a mammalian blastoid, more in particular it is a human PSC (hPSC), or culture of hPSC, and the blastoid is a human blastoid. Even more in particular, the PSC or culture of PSC is selected from a human induced PSC or a naïve human PSC, which results from non-human embryo destruction, in particular from parthenogenetically activated human oocytes. In another particular embodiment of the in vitro method of first aspect, optionally in combination with any of the embodiments above or below, the proliferation and differentiation of the PSC is carried out in cell culture suspension. In another particular embodiment of the in vitro method of the first aspect, optionally in combination with any of the embodiments above or below, one PSC aggregate comprising from 150 to 300 cells per aggregate (i.e., cells / aggregate) is provided in a container with a cell differentiation culture medium, and allowed to be differentiated to one blastoid comprising at least two ICM. In another particular embodiment of the in vitro method of the first aspect according to any of its definitions and embodiments, the differentiation of the PSC aggregates to obtain the one or more blastoids comprising at least two ICM is allowed with the culturing of the cells in the cell differentiation culture medium for a time of at least 3 days in the said medium. More in particular, for a time from at least 5 days, and even more in particular for a time from 3 to 7 days, even more in particular from 3 to 5 days. In another particular embodiment of the in vitro method of the first aspect, optionally in combination with any of the embodiments above or below, the container where the PSC are allowed to differentiate comprises a non-cell adherent surface or consists of a non-cell adherent surface material, in particular at least on the side of the container where the cells are cultured. This way, the seeded PSC can proliferate and do not adhere to the surface of the container, which allows or promotes the proliferation in the form of three-dimensional cell aggregates (i.e., adhesion is favoured between cells in relation to between cells and the surface of the container). The skilled person in the art will know which are the materials most commonly used to avoid adherence of cells on the surface (walls and bottom) of the widely used containers for cell culturing. More in particular, the said non- cell adherent material is selected from agarose hydrogels, ethylene oxide, polyethylene glycol and copolymers with polyethylene glycol, such as copolymers of poly-L-lactide acid or poly- caprolactone and PEG, polyethylene oxide (PEO), Poly(N- isopropylacrylamide)), hydrophobic materials, such as olefin polymers, cell-repellent micro- and nanotopographies, among others. A non-adherent surface container can be formed from a non- adhering material, or can be formed from another material coated with a non-adherent material. Particular non-adherent microwell (containers) arrays made from olefin polymers are disclosed, for example, in Vrij, Erik & Espinoza, Sara & Heilig, Markus & Kolew, Alexander & Schneider, Marc & Blitterswijk, Clemens & Truckenmuller, Roman & Rivron, Nicolas. (2016).3D High Throughput Screening and profiling of Embryoid Bodies in thermoformed microwell plates. Lab Chip. 16.10.1039 / C5LC01499A. Other microwell arrays of agarose hydrogel are disclosed in Rivron NC, Vrij EJ, Rouwkema J, Le Gac S, van den Berg A, Truckenmiiller RK, van Blitterswijk CA, Tissue deformation spatially modulates VEGF signalling and angiogenesis, Proc Natl Acad Sci U S A. 2012 May 1; 109(18):6886-91 and is incorporated herein by reference. Both are herewith incorporated by reference. In another particular embodiment of the in vitro method of the first aspect, the container is a microwell. Microwells are well-known cell culture containers of micro- scale size, which means that their dimensions, such as at least the depth and / or the dimensions of the bottom of the microwell are in the micrometric range. It is also common that these microwells be arranged in arrays comprising multiple microwells. The arrays are called microwell arrays. Thus, in another particular embodiment, the cells at the indicated density in a microwell are seeded in separated microwells of a microwell array. In another particular embodiment, optionally in combination with any of the embodiments above or below, the microwells have a length, breadth and depth that are of approximately similar order of magnitude. In a more particular embodiment, the non-cell adherent microwell is a cavity with a length which is up to about 5 times, more in particular 3 times and more in particular equal to its breadth, and the depth is no more than 10 times, more in particular no more than 5 times, and more in particular up to 3 times its breadth. Particular dimensions of the microwells are 20-5000 μm in diameter, more in particular 100-2000 μm, and more in particular are cavities of 150 - 1000 μm, preferably 200 - 800 μm in diameter, or even more in particular from 200 to 500 μm in diameter. The diameter is defined as the longest possible straight-line distance between any two opposite points on the circumference circumscribing the opening of the microwell, independently of the shape (e.g. square, round, oval, or rectangular). In another particular embodiment of the in vitro method of the first aspect, the container is a microwell, and in each microwell a PSC aggregate comprising from 150 to 300 cells / aggregate is provided; or alternatively, the container is a microwell and the pluripotent stem cells are seeded in the container at a cell density (a ratio) from 1 to 500 cell per microwell, more in particular from 100 to 300 cells per microwell. In another more particular embodiment, when the container is a microwell in a microwell array, each microwell has a volume from 0.02 mm3 to 0.08 mm3, and the pluripotent stem cells are seeded in the container at a cell density (a ratio) from 100 to 300 cells per microwell. In another more particular embodiment, the container is a spherical or ellipsoid, free-floating object, e.g. a hollow, core / shell microdroplets, that has a diameter from 100 to 1000 µm, preferably between 100 and 500 µm and even more preferably between 100 and 300 µm; and in step (b) the pluripotent stem cells are seeded at a cell density from 100 to 300 cells per container. The “free-floating” refers to the fact that the container is not attached to anything and is able to move freely, for example floating in or on a liquid (e.g. in a microfluidic channel) or on a support but free of movement (e.g., on a plate that can optionally be an stirred plate). Even more in particular, the pluripotent stem cells are seeded at a ratio or cell density from 100 to 300 cells per microwell. Even more in particular the amount of seeded cells is selected from 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, and 300 cells per microwell. This cell density in the microwell with the indicated dimensions - a volume from 0.02 mm3 to 0.08 mm3 - corresponds to the previously densities from 0.5x103 to 2.0x104 cells per mm3 of the seeded container. In another particular embodiment of the in vitro method of the first aspect, optionally in combination with any of the embodiments above or below, the container, in particular a microwell is of a non-cell adherent material and / or comprises a non-cell adherent surface. Thus, it is, in a more particular embodiment a microwell of non-cell adherent material as previously disclosed. In even a more particular embodiment the container is a microwell made of a non-cell adherent material and / or comprising a non- cell adherent surface. More in particular, each microwell of a non-cell adherent material and / or comprising a non-cell adherent surface has a volume from 0.02 mm3 to 0.08 mm3. In another particular embodiment of the in vitro method of the first aspect, the differentiation of the PSC aggregates is carried out in a medium for PSC differentiation comprising at least one inhibitor of the TGFβ pathway, at least one inhibitor of the ERK pathway, and optionally at least one inhibitor of the Hippo pathway. In another more particular embodiment of the in vitro method of the first aspect, the differentiation of the PSC aggregates is carried out in a medium for PSC differentiation comprising at least one inhibitor of the Hippo pathway, at least one inhibitor of the TGFβ pathway, and at least one inhibitor of the ERK pathway. The Hippo signalling pathway, also known as the Salvador-Warts-Hippo (SWH) pathway, is a signalling pathway that controls organ size in animals through the regulation of cell proliferation and apoptosis. Examples of inhibitors of this pathway include lysophosphatidic acid (LPA, CAS Number.22002-87-5), a ligand of the lysophosphatidic acid receptor (LPAR). Other are NAEPA or OEA-P (oleoyl ethanolamide phosphate), N-[2-(phosphonooxy)ethyl]-9Z-octadecenamide, which three later are ligands of the lysophosphatidic acid receptor. Other inhibitors are disclosed in European patent application with publication number EP4029932 (incorporated herein by reference). Inventors have surprisingly realized that the amount of the inhibitor of the Hippo pathway has also an effect on the rate of the obtained blastoids with at least two ICM. Hence, it is also herewith disclosed the use of an inhibitor of the Hippo pathway for the obtention of blastoids with at least two ICM. In a particular embodiment, the inhibitor of the Hippo pathway is, or comprises, LPA. In another particular embodiment, the inhibitor of the Hippo pathway in the medium for PSC differentiation is in an amount from 1 µM to 20.0 µM, and even in a more particular embodiment in an amount from 1 µM to 10.0 µM. In yet another more particular embodiment, the inhibitor of the Hippo pathway is, or comprises, LPA in an amount in the medium for PSC differentiation from 1 µM to 20.0 µM, and even in a more particular embodiment in an amount from 1 µM to 10.0 µM. In another particular embodiment, the Hippo pathway in the medium for PSC differentiation is LPA, and it is in an amount selected from 1.0, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.6, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 µM. Furthermore, a synergistic effect was also observed between the number of initial cells in the PSC aggregates added in the container (i.e., the initial cell density) and the amount of the inhibitor of the Hippo pathway in the medium for PSC differentiation. Therefore, in another particular embodiment of the in vitro method for the obtention of blastoids comprising at least two ICM, the number of seeded PSC cells to form aggregates, and further differentiate, is from 100 to 300 cells per microwell of a volume from 0.02 mm3 to 0.08 mm3, and the medium for PSC differentiation comprises at least one inhibitor of the Hippo pathway, at least one inhibitor of the TGFβ pathway, and at least one inhibitor of the ERK pathway, wherein the inhibitor of the Hippo pathway in the medium for PSC differentiation is in an amount from 1 µM to 20.0 µM, more in particular in an amount from 1 µM to 10.0 µM. In yet another more particular embodiment, the inhibitor of the Hippo pathway is, or comprises, LPA. In yet another more particular embodiment, the number of seeded PSC cells to form aggregates and further differentiate, is 200 cell per microwell of a volume from 0.02 mm3 to 0.08 mm3 , and the medium for PSC differentiation comprises at least one inhibitor of the Hippo pathway, at least one inhibitor of the TGFβ pathway, and at least one inhibitor of the ERK pathway, wherein the inhibitor of the Hippo pathway in the medium for PSC differentiation is in an amount from 1 µM to 5.0 µM. In yet another more particular embodiment, the inhibitor of the Hippo pathway is, or comprises, LPA. Transforming growth factor beta (TGFβ or TGFB) signalling pathway is involved in many cellular processes in both the adult organism and the developing embryo including cell growth, cell differentiation, cell migration, apoptosis, cellular homeostasis and other cellular functions. The TGFB signalling pathways are conserved. In spite of the wide range of cellular processes that the TGFβ signalling pathway regulates, the process is relatively simple. TGFβ superfamily ligands bind to a type II receptor, which recruits and phosphorylates a type I receptor. The type I receptor then phosphorylates receptor-regulated SMADs (R-SMADs) which can now bind the coSMAD SMAD4. R-SMAD / coSMAD complexes accumulate in the nucleus where they act as transcription factors and participate in the regulation of target gene expression. Among the inhibitors of this pathway are the compound known as A83-01 (CAS No. : 909910-43- 6). A 83-01 is a potent inhibitor of TGF-β type I receptor ALK5 kinase, type I nodal receptor ALK4 and type I nodal receptor ALK7, with IC50s of 12 nM, 45 nM and 7.5 nM against the transcription induced by ALK5, ALK4 and ALK7, respectively Other inhibitors are disclosed in European patent application with publication number EP4029932 (incorporated herein by reference). Some of the inhibitors are referred there as SD-208, GW788388, SRI-011381, TP0427736, RepSox (E-616452, SJN 2511), LY2109761, SB505124, BIBF-0775, LY 3200882, Galunisertib (LY2157299), Vactosertib (TEW-7197, EW-7197), LY364947 (HTS 466284), and SB525334, ITD-1. In a particular embodiment of the in vitro method of the first aspect, the step of differentiation is carried out in a medium for PSC differentiation comprising A 83-01 as inhibitor of the TGFβ pathway. The MAPK / ERK pathway (also known as the Ras-Raf-MEK-ERK pathway) is a chain of proteins in the cell that communicates a signal from a receptor on the surface of the cell to the DNA in the nucleus of the cell. The signal that starts the MAPK / ERK pathway is the binding of extracellular mitogen to a cell surface receptor. This allows a Ras protein (a Small GTPase) to swap a GDP molecule for a GTP molecule, flipping the "on / off switch" of the pathway. The Ras protein can then activate MAP3K (e.g., Raf), which activates MAP2K, which activates MAPK. Finally, MAPK can activate a transcription factor, such as Myc. Mirdametinib (PD0325901, CAS No. : 391210-10-9) is one of the known inhibitors of this pathway. It is a selective and non-ATP-competitive MEK inhibitor with an IC50 of 0.33 nM. Mirdametinib ultimately suppresses the expression of phosphorilated- ERK1 / 2 and induces apoptosis. Other inhibitors are disclosed in European patent application with publication number EP4029932 (incorporated herein by reference). Examples of such other inhibitors are selected from one or more of Selumetinib, Trametinib, U0126-EtOH, PD184352 (CI-1040), PD98059, Pimasertib, TAK-733, AZD8330 (ARRY704), Binimetinib, PD318088, SL327, Refametinib, GDC-0623 (G-868), Cobimetinib. In a particular embodiment of the in vitro method of the first aspect, the step of differentiation is carried out in a medium for PSC differentiation comprising PD0325901 as inhibitor of the ERK pathway. The differentiation medium further comprises, in a particular embodiment, other compounds that allow the aggregation and / or differentiation of the cells. In a particular embodiment, these compounds are selected from the list consisting of leukaemia inhibitory factor (LIF, function in differentiation, UniProtKb database P42702-1), Y- 27632 (CAS No. : 146986-50-7, it is a ROCK inhibitor involved in differentiation an aggregation). Other ROCK inhibitors are also disclosed in European patent application with publication number EP4029932 (incorporated herein by reference), and in a particular embodiment they areselected from one or more of Thiazovivin, Fasudil(HA- 1077), ZINC00881524, Azaindole 1 (TC-S 7001), GSK429286A (RHO-15), RKI-1447, GSK269962A HCl (GSK269962B, GSK269962), Hydroxyfasudil (HA-1100), Netarsudil (AR-13324), Ripasudil (K-115), Y-39983 (Y-33075), and KD025 (SLx-2119). In another particular embodiment of the in vitro method of the disclosure, a step of isolation of a bastoid comprising at least two ICM is included, which means that from the culture of differentiated three- dimensional cell aggregates obtained in the container in which differentiation takes place, those that are blastoids comprising at least two ICM are separated from the rest of three-dimensional cell aggregate types. The isolation is done by commonly known techniques of cell manipulation, widely known or familiar by the skilled person in the art. Indeed, the disclosure refers for the first time to an in vitro generated blastoid comprising at least two ICM. Thus, another aspect of the disclosure is a blastoid comprising at least two ICM. In a particular embodiment of the blastoid comprising at least two ICM (i.e., comprising at least two ICM), it comprises a number of ICM selected from 2, 3, 4, and 5. In particular, it comprises two (2) ICM. Independently of the number of ICM in the blastoid comprising at least two, each of the ICM comprises with a number of cells (i.e., ESCs) at approximately 150 cells. The method according to which the PSC have been allowed to grown, as well as the spatial constrictions, may impart to the cells certain structural and functional features. Thus, the blastoids and other differentiated three-dimensional cell aggregates can also be defined by the method of obtention. As previously indicated, is therefore another (second) aspect of the disclosure a blastoid comprising at least two ICM, and which is obtained or obtainable by a method as disclosed in the first aspect or any of its embodiments and combinations of embodiments. Thus, the disclosure also refers, in more detail, to a blastoid comprising at least two ICM obtainable or obtained by an in vitro method comprising the steps of: (a) providing a culture of pluripotent stem cells; (b) seeding the pluripotent stem cells in a container at a cell density from 0.5x103 cells / mm3 of container to 2.0x104 cells / mm3 of container; (c) to allow the pluripotent stem cells to proliferate in a cell differentiation culture medium in the container, which differentiation culture medium comprises at least one inhibitor of the TGFβ pathway and at least one inhibitor of the ERK pathway, to obtain a culture of differentiated three- dimensional cell aggregates that comprise blastoids comprising at least two ICM; and (d) to optionally isolate the blastoids with at least two ICM. Or alternatively, the disclosure also refers, in more detail, to a blastoid comprising at least two ICM obtainable or obtained by an in vitro method comprising the step of: to provide one or more PSC aggregates comprising from 150 to 300 cells / aggregate in a container with a cell differentiation culture medium, which differentiation culture medium comprises at least one inhibitor of the TGFβ pathway and at least one inhibitor of the ERK pathway, and allow the one or more aggregates to differentiate to one or more blastoids comprising at least two ICM. One aspect of the disclosure relates to cultures of mammalian three-dimensional cell aggregates comprising: - from 2 % to 25 % of blastoids comprising at least two ICM; - from 5 % to 60 % of blastoids comprising one ICM; all the percentages in relation to the total amount / number (i.e., 100 %) of three-dimensional cell aggregates in the culture. This previously disclosed culture of cell aggregates finds direct applications not only as a source of the aggregates of interest, in particular of blastoids comprising at least two ICM, but also to study the differential effects of compounds over all of them. In a particular embodiment, the culture of three-dimensional cell aggregates further comprises three-dimensional cell aggregates selected from the group consisting of embryoid bodies (EB), trophospheres, multiple cysts, and blastoids comprising a single ICM with a number of cells over 150 (also called in this description as blastoids with oversized ICM). In even a more particular embodiment, the culture of differentiated three- dimensional cell aggregates according to the disclosure comprises: - from 2 % to 25 % of blastoids comprising at least two ICM; - from 5 % to 60 % of blastoids comprising one ICM; - from 2 % to 90 % of embryoid bodies; - from 8 % to 30 % of blastoids with oversized ICM (i.e., over 150 cells in ICM); - from 0.5 % to 2 % of trophospheres; and - from 1% to 20 % of multiple cysts, all the percentages up to 100 % of the number of differentiated cell aggregates in the culture. This culture of differentiated three-dimensional cell aggregates is provided in a scaffold, or support, or container, with non-cell adherent properties. Therefore, also herewith disclosed is a container made of a non-cell adherent material and comprising the culture of differentiated three-dimensional cell aggregates defined in the previous aspect and corresponding embodiments. In a more particular embodiment the scaffold, support or container comprising the culture of differentiated three-dimensional cell aggregates is a microwell array comprising one or more, in particular one, differentiated three-dimensional cell aggregate per well of the microwell. As indicated, the present disclosure also relates to the use of an in vitro method of culturing and differentiating of a mammal pluripotent stem cell, in which the pluripotent stem cell is cultured, particularly in suspension, in a differentiation culture medium that comprises at least one inhibitor of the TGFβ pathway and at least one inhibitor of the ERK pathway, to allow the formation of three-dimensional cell aggregates, for the preparation and optional isolation of a blastoid comprising at least two ICM. Indeed, another aspect of the disclosure is the use of a method as defined in any one of the first aspect and its embodiments, for the preparation and isolation of blastoids comprising at least two ICM. More specifically, this aspect is defined as the use of an in vitro method of culturing and differentiating of a mammal pluripotent stem cell, for the preparation and optional isolation of a blastoid comprising at least two ICM, in which the in vitro method comprises the steps of: (a) providing a culture of pluripotent stem cells; (b) seeding the pluripotent stem cells in a container at a cell density from 0.5x103 cells / mm3 of container to 2.0x104 cells / mm3 of container; (c) to allow the pluripotent stem cells to proliferate in a cell differentiation culture medium in the container, which differentiation culture medium comprises at least one inhibitor of the TGFβ pathway and at least one inhibitor of the ERK pathway, to obtain a culture of three-dimensional cell aggregates that comprise blastoids comprising at least two ICM; and (d) to optionally isolate the blastoids comprising at least two ICM. Or, in other words, and also more specifically, this aspect is defined as the use of an in vitro method of culturing and differentiating of a mammal pluripotent stem cell, for the preparation and optional isolation of a blastoid comprising at least two ICM, in which the in vitro method comprises the step of: to provide one or more PSC aggregates comprising from 150 to 300 cells / aggregate in a container with a cell differentiation culture medium, which differentiation culture medium comprises at least one inhibitor of the TGFβ pathway and at least one inhibitor of the ERK pathway, and allow the one or more aggregates to differentiate to one or more blastoids comprising at least two ICM. The embodiments defined for the in vitro method of the first aspect, for example regarding the type of container (microwell), the material (i.e., non-cell adherent), the number of seeded PSCs, number of cells per PSC aggregate, as well as the differentiation culture medium do also apply to the use of any in vitro method of culturing and differentiating of a mammal pluripotent stem cell referred to in this description for the preparation (i.e., obtention) and optional isolation of a blastoid comprising at least two ICM. As indicated, the blastoid with at least two ICM, or the culture of three- dimensional cell aggregates as defined above, are good in vitro models of the blastocyst stage of the mammal, in particular of the human, embryonic development, because they resemble the structural (i.e., spatial distribution) and cell type composition of the blastocyst, although they cannot finally develop a complete mammal organism. The blastoid with at least two ICM is, indeed, a good model for the study of the twinning phenomena, in particular of the monozygotic twins, and more in particular the monochorionic twins (i.e., those that share the placenta). With an appropriate model of implantation, other key pathways and aspects of the development and formation of extra embryonic tissues, such as the placenta, as well as other events that take place for and after implantation of the embryo can be determined. The skilled person in the art will know the commonly accepted models for the study of implantation including, among others, the use of confluent open face endometrial layer (OFEL) comprising endometrial cells that are previously stimulated with oestrogen (E2) and progesterone (E4) to resemble the window in which uterus is receptive to the blastocyst implantation. The model of blastocyst-like proposed by the inventors allows, for example, the screening of a candidate agent, or of a physical and / or mechanical mean, with the capacity to modulate the mammal, in particular the human, embryonic development. Thus, also as previously indicated, the disclosure also relates to the use of the blastoid as defined in any of the previous aspects (i.e. comprising at least two ICM), or the culture of three- dimensional cell aggregates as defined above, for the screening of candidate agents, or physical and / or mechanical means, with the capacity to modulate the mammal, in particular the human, embryonic development. Also as indicated, another aspect of the disclosure is an in vitro screening method for identifying an agent or physical and / or mechanical mean that modulates the mammal embryonic development, the method comprising: (a) providing a blastoid comprising at least two ICM, or a culture of three- dimensional cell aggregates comprising blastoids with at least two ICM, both as defined in the previous aspects; (b) to contact the blastoid or culture of the previous step with a candidate agent, or alternatively, to subject the blastoid or culture to a physical and / or mechanical mean; (c) detecting a change in mammal embryonic development compared to a control to determine the candidate agent’s modulatory activity, or the modulatory activity of the physical and / or mechanical mean. In all the methods and uses to screen if a candidate agent, or a physical and / or mechanical means, has the capacity to modulate the mammal embryonic development, the step of detecting a change in mammal embryonic development comprises, in particular embodiments, to determine if the blastoid comprising at least two ICM evolves to stages that are reminiscent of stages later than the blastocyst stage in the mammal embryonic development, or if the blastoid comprising at least two ICM does not evolve to any later stage and / or involutes to a previous stage, or if the at least two ICM merge in one single ICM. Blastocyst-like stage can evolve in a later stage to mimic an implantation- like stage when brought in contact with the candidate agent, or subjected to the physical and / or mechanical mean. Blastocyst-like stage can involute to a previous stage, in which the cell aggregate is not spatially structured as in the blastoid, such as a three- dimensional aggregate of cells non- phenotypically corresponding to trophoblasts surrounding ICM cells and fluid-filled cavity. If due to the contacting with the candidate agent, or due to the subjection to the physical and / or mechanical mean, the blastoid of the disclosure involutes to a previous than the blastocyst-like stage, the candidate, or physical and / or mechanical mean, is identified as an inhibitor of embryonic development. On the contrary, the candidate agents or physical and / or mechanical means are identified as enhancers or promoters of embryonic development, if due to the contacting with the candidate agent or physical and / or mechanical mean, the blastoid of the disclosure with at least two ICM evolves to a further stage. In all these methods and uses, common techniques known by the person skilled in the art are used to analyze or determine the stage-like of the embryonic development. For example and in a particular embodiment, there can be analyzed one or more of the type and amounts of a cell-surface marker(s), the type and amounts of a cell secreted protein(s), and the type and amounts of a transcription factor(s). The skilled person in the art will know that all these are particular phenotypic traits associated to the diverse differentiation states and functionalities of the cells involved in embryonic development. As a way of example, a transcriptomic analysis can be used, in a particular embodiment, to determine if the three founding lineages are present or not in the blastoid of the disclosure after the contacting with the candidate agent. More in particular if one or more of the genes GATA- binding factor 2 (GATA2) and GATA- binding factor 3 (GATA3), specific for trophoblast are transcribed; one or more of the genes POU domain, class 5, transcription factor 1 (POU5F1, also known as Oct-4 or OCT4) and Krueppel-like factor 17 (KLF17), specific for epiblast (ICM) are transcribed; and one or more of the genes GATA-binding factor 4 (GATA4) and SRY-box 17 (SOX17), specific for the primitive endoderm are transcribed. In another particular embodiment, the embryonic-like development stage achieved after the application of the physical and / or mechanical mean or after contacting the blastoid or culture of the disclosure with the candidate modulator agent, can be determined by means of visual inspection, more in particular using a microscope (such as an optical or confocal microscope). In particular embodiments, the control to determine the candidate agent’s modulatory activity, thus the candidate agent’s capacity to modulate embryonic development, is selected from a blank (no candidate), a known enhancer or promoter of the mammal embryonic development and / or a known inhibitor of the mammal embryonic development. Known enhancers or promoters of the mammal embryonic development include prostaglandin I2, and anastrazole. Known inhibitors of the mammal embryonic development include calmodulin inhibitors, such as 2-chloro-10-aminopropyl phenothiazine and 10-aminopropyl phenothiazine, as well as most of the compounds previously commented when disclosing in more detail the differentiation medium. In another particular embodiments, the physical and / or mechanical mean is any mean that modulates embryonic development in particular by modulating or affecting the ICM merging, more in particular that prevents the formation of at least two ICM in the blastoids. Particular physical and / or mechanical means are selected from the group consisting of light, temperature, pH, pressure, application of forces, and combinations thereof. For the performance of the methods of screening several high- throughput procedures can be applied, as the skilled person in the art will recognize. As a way of example, the screening is performed in known test kits (e.g., in a multiple well plate like a Thermofisher cell assay kit). This way a screening device is provided, in which multiple twinning conditions are tested in an array of different conditions in a kit. As directly derivable from the aspects and embodiments of the disclosure, it is herewith provided for the first time a model for the study of monozygotic twinning that does not imply the working with actual embryos neither with stem cells derived from the destruction of embryos. This opens a myriad of possibilities to determine the causes of the phenomena, but also to screen for candidate agents or physical and / or mechanical means with a therapeutic effect in a model of twinning, such as candidates to avoid the connection of blood supplies between the two embryos and further fetuses. As previously indicated, one of the major complications in monozygotic twins that are monochorionic is the twin-to-twin transfusion syndrome (TTTS). In this syndrome there is disproportionate blood supply between the fetuses that leads to death of the undersupplied twin and, without treatment, and usually death or a range of birth defects or disabilities for a surviving twin. Current treatments include the adjustment of amniotic fluid by serial amniocentesis, or the disruption of the dividing membrane. Also employed are other surgical interventions, aiming to interrupt the vessels that allow the exchange of blood between fetuses. The provision of therapeutic agents for preventing TTTS in case of early detected monochorionic twins is derivable from the blastoids comprising at least two ICM according to the disclosure. The possibility to study the mammal monochorionic twinning phenomena for example in non-human animals (bovine, ovine, porcine, cats, dogs, rabbits, etc.) has also a great impact on veterinary and livestock economy, allowing to avoid complications and loses due to monochorionic gestations in animals. Last but not least, the provision of these three-dimensional cell aggregates resembling a blastocyst stage comprising at least two ICM, opens the possibilities of a more efficient in vitro growth of specialized tissues and organs applicable to regenerative medicine. Particularly useful for this purpose are the blastoids comprising at least two ICM, but also other embryoid bodies, as well as the blastoids with a single ICM comprising more than 150 cells (oversized ICM). The skilled person will be aware of the differentiation, stimulation and culturing techniques available to obtain a specialized tissue of interest (e.g., cardiomyocytes, osteocytes, epithelia, etc.). So that, once the blastoids with at least two ICM or the blastoids with one ICM, oversized or not, are obtained, they can be subjected to particular stimulation conditions that will promote the development of particular tissues of interest, such as neural organoids, hepatocytes, erythroid cells, osteocytes, etc. The combination of this differentiation in particular 3D- scaffolds allows the formation of in vivo resembling microenvironment for inducing stem cell derived tissue formation. All the organoids and tissues obtainable from the differentiated three-dimensional cell aggregates obtained with the method and uses of the disclosure are entities for use in the prevention and / or treatment of disorders or diseases by means of regenerative medicine. Thus, they are, more in particular, for use as tissue and / or organ replacers. Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprise” encompasses the case of “consisting of”. Additional objects, advantages and features of the disclosure will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the disclosure. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present disclosure. Furthermore, the present disclosure covers all possible combinations of particular and preferred embodiments described herein. Examples Formation of monochorionic twin structures in blastoid cultures Human naïve pluripotent stem cells are capable of self-organizing into blastocyst-like structures when placed in a non-adhesive microenvironment. Here, human naïve embryonic and induced pluripotent stem cell lines (HNES1 and niPSC HDF75) were used to generate blastoids using previously reported PAY conditions and within a custom high- throughput thermoformed microwell platform (target seeding number of 100 cells per microwell). Within these cultures, the rare occurrence (1 to 5%) of blastoids is observed, with the blastoids having formed a single cavity containing multiple ICM-like clusters between 72 and 96 hours after cell seeding (Figure 1A). Since inhibition of the Hippo signaling pathway and starting cell number can affect the dynamics of single trophectoderm cavity (TE ) versus ICM specification, an increase of the rate of spontaneous twinning was sought by adding lysophosphatidic acid (LPA) in increasing concentrations in combination with higher cell seeding numbers. Using niPSCs, it was found that a pulse of LPA from 0 to 48 hours of culture (Figure 1B) led to a significant increase in the number of singleton blastoids. In addition, increasing LPA concentrations in combination with increasing cell densities led to a relative increase of blastoids containing two ICMs in a single cavity (termed twin blastoids, Figure 1C-E and S1A, B). Seeding 200 and 300 cells with 2.5 µM LPA (conditions hereafter referred to as 200c and 300c, respectively) resulted in the highest percentage of twin blastoids (16.4±3.2% and 18.2±7.1% of all aggregates, respectively; averaged across three independent experiments, ± indicates standard deviation (SD); Figure 1E). The vast majority of multiple ICM- containing blastoids contained two separate ICMs, although blastoids with three or, even less frequently, four separate ICM clusters were observed as well (Figure S1B, C). The 200c twin blastoids had a cyst diameter of 228±32 µm, which is in the upper range of the cyst diameter of the blastocyst. The 300c twin blastoids were significantly larger than those generated from 200 cells (diameter of 266±50 µm; Welch ANOVA, P=0.003). It is noticed that in both 200c and 300c conditions twins had a similar diameter to their singleton counterparts that formed within the same cultures (Figure S1D, Welch ANOVA, P>0.8 for both 200c and 300c blastoids). However, it is observed that twin blastoids are not fully circular, therefore it was decided to measure the projected areas of the TE cysts as a more rigorous and accurate metric to compare between singleton and twin blastoids. The 200c twin blastoids showed a marginally larger projected cyst area compared to their singleton counterparts within the same culture (on average 32.888±11.758 versus 27.872±8.147 µm2, so 18% larger), yet this difference was not statistically significant (Figure 1F; Kruskal-Wallis test, P>0.9). The same was true for 300c twin blastoids compared to the 300c singletons, though the difference was smaller (59.185 versus 57.849 µm2, 2% larger, Kruskal-Wallis test, P>0.9). Unsurprisingly, 300c twin blastoids had a larger projected cyst area than the 200c twin blastoids (Kruskal-Wallis test, P=0.0001). Twin blastoids are bi-polar containing a double pluripotent ICM with respective hypoblast and polar trophectoderm Using immunofluorescent staining, it was observed that both ICM- like clusters in twin blastoids were KLF17+ and OCT4+, indicating that pluripotency is similarly preserved in both clusters (Figure 1G). Furthermore, most twin blastoids contained a small population of SOX17+ cells in both ICM clusters (84%, n=50, Figure 1H, I), indicative of the hypoblast lineage. Confocal imaging verified that both clusters were enveloped by a single cyst consisting of a continuous single layer of GATA3+ cells, indicative of the TE epithelium (Figure 1J). In human, the polar TE that surrounds the ICM facilitates the initial adhesion of the blastocyst to the endometrium during implantation. Since in human blastocysts, polar expression of NR2F2 identifies TE maturation adjacent to the ICM, it was hypothesized that twin blastoids could possess a bi-polar TE maturation identity. Indeed, it was found in 60% of all twins (n=20) that the regional TE-like epithelium surrounding each ICM expressed the polar TE marker NR2F2, indicating that both ICM clusters maintain their local polar TE-like populations (Figure 1K, L). Since epiblast signals appear to drive polar TE maturation, this finding suggests the presence of a bi-polar TE identity in monochorionic twin embryos, which may have implications for the implantation kinetics and post-implantation development. TE expansion drives ICM division Initially, it was hypothesized that splitting of the ICM may be due to insufficient proliferation of the ICM cells as the cyst expands. Insulin growth factor 1 (IGF1), which was previously reported to boost proliferation of the ICM cells in human embryos, was added but no effect was found on the yield of twin blastoids (Figure S1E-G, all Welch ANOVAs, P>0.4). Then the fluorescent imaging capability of the thin polymer film-based thermoformed microwell platform was used to further investigate the morphokinetics of twin blastoid formation through time-lapse imaging. The twin blastoid cultures were imaged from 24 to 72 hours post seeding (hps), during which cavitation and cyst expansion occurred. Using a niPSC OCT4- GFP reporter, the division of the ICM in two was visually captured. It was observed that twinning typically occurred during TE cyst expansion in which the unified ICM cluster elongated into a line or smeared configuration of cells that successively thinned out in the middle and reconsolidated into separate clusters (Figure 2A). Twin blastoids started cavitation rather consistently between 36 and 48 hps (Figure 2B, average at 41±4 hps). On average the ICM split 12 hours after the start of cavitation, however with considerable variability (12±7 hours, Figure 2B, C). As a result, between 48 and 72 hps, a variety of TE cyst-like structures were observed at various stages of ICM division along the transition from singleton to twin. These included TE cysts enveloping a single OCT4+ clump, OCT4+ cell populations being extended along the TE lining, the emergence of two separately condensing OCT4+ populations bridged by a thin line of OCT4+ cells, and finally, a TE cyst with two distinctly condensed OCT4+ clusters. Additionally, data derived from the time lapse experiments show that within the same culture wells TE cysts expand faster in twin blastoids than in singleton blastoids (linear regression, P<0.0001, Figure S1H). Given that LPA has been reported to accelerate in vitro blastocyst formation in bovines and an increase in cyst diameter is seen in response to increasing LPA concentrations (Kruskal-Wallis test, P<0.0001, Figure S1I), it is suspected that accelerated TE expansion may drive the ICM splitting in the system. This is further supported by the finding that delaying TE induction by 24 hours decreased twin blastoid formation (Student’s t-test, P=0.019, Figure S1J). Taken together, these findings suggest that monochorionic twins result from TE expansion, loss of coherence of the pluripotent cell cluster and re-condensation of the respective ICMs. Both sibling ICM-like clusters preserve similar epiblast and hypoblast cell proportions The morphological features of 200c and 300c twin blastoids were then assessed. The first feature of note was the consistent positioning of the ICMs nearly 150° apart at 72 hps, both in 200c and 300c conditions (146.6±26 and 152.2±18°, respectively; Figure 2D). These 2-dimensional image projections were validated with measurements on 3D reconstructions (n = 5), which showed a similar average angular distance of 147.5±19°. This suggests a minimal angular distance for establishing two separate, condensed ICMs within this culture system for twin blastoids. Expanding on the projected TE cyst area measurements, the projected area of the corresponding ICM(s) was also investigated in the blastoids to have an approximation of the (relative) sizes of both compartments. The total ICM area of 200c twin blastoids was slightly larger than that of their singleton counterparts, though again not significantly (9.815±4.541 and 13.040±5.554 µm2, Kruskal-Wallis test, P=0.25; Figure S2A). Remarkably, the total ICM areas were not significantly different between 200c and 300c twin blastoids (10.834±4.840 µm2, Kruskal-Wallis test, P= 0.47; Figure S2A). The ICM area to cyst area ratio was then calculated for each blastoid using paired measurements of cyst and total ICM area. Both 200c singleton and twin blastoids had similar ICM / cyst ratios (0.35±0.11 and 0.41±0.13, respectively; Kruskal-Wallis test, P>0.9). Similarly, the ICM / cyst ratios for 300c singleton and twin blastoids were comparable at 0.16±0.06 and 0.19±0.06, respectively (Kruskal-Wallis test, P>0.9). However, the 300c blastoids displayed strikingly smaller ratios than the 200c blastoids (Kruskal-Wallis, P<0.0001; Figure 2E). Since the total projected area of the ICMs was found to be not significantly different for all tested conditions, we wondered if the total ICM cell number was similarly consistent in our culture system, which would be in line with findings in human blastocysts. While both 200c and 300c conditions depend on a high starting cell number, 200 cells are a closer approximation to the number of cells present in the blastocyst, as the comparable diameter of 200c blastoids already suggests. Although the 2D measurements provided a rough indication of the relative sizes of the cyst and ICM compartments, these measurements do not volumetrically scale in a similar degree between singleton (with a single ICM projected area) and twin blastoids (with the sum of two ICM projected areas). For a more accurate comparison the cell numbers per structure for both singleton and twin 200c blastoids at 72 hps were then counted by dissociating individual, manually picked structures in TrypLE Express (Figure 2F, G). These cell counts revealed that, despite the similar projected areas, twin blastoids on average contained more cells than their singleton counterparts (564±159 and 421±131, respectively, Welch's t-test P=0.0017, Figure 2F), which was also reflected in the higher OCT4+ and SOX17+ cell numbers in twin blastoids (Welch’s t-test, P=0.0005 and Mann-Whitney test, P=0.0004, respectively; Figure S2B, C). While the absolute number of cells differed between twins and singletons, the proportion of the epiblast-like (OCT4+) and hypoblast-like (SOX17+) cell numbers to the total cell number per blastoid was similar (Welch ANOVA, P>0.2; Figure 2F, G). This indicates that the number of ICM cells was higher in twins than singletons in 200c conditions. However, it appears that, independent of the total cell number and ICM splitting, an equilibrium between the three lineages is established. The sibling ICMs within individual twin blastoids were then compared. An apparent dichotomy was observed in the ratio of the projected areas of ICM within twin blastoids of 200c, while no such pattern emerged in 300c twin blastoids.200c twin ICMs were observed that either were similar in size (ratio 0.76 at 75% percentile) or were of distinct sizes, where one was 4-5 times larger than the other (ratio 0.22 at 25% percentile). The 300c twins on the other hand did not show a predisposition for a particular size ratio between ICMs (Figure S2D). When the number of OCT4+ cells per ICM-like cluster in our 200c twin blastoids were counted based on high resolution confocal image stacks, again a higher number of OCT4+ cells were observed in twins compared to singleton blastoids, however the dichotomy in ICM size was less pronounced (Figure 2H, I and S2E). Furthermore, consistent with the bulk count performed on dissociated blastoids, the ratio between OCT4+ and SOX17+ cells in 200c singleton and twin blastoids was comparable. Moreover, this ratio was maintained within the individual ICM-like cluster of twin blastoids (Kruskal-Wallis test, P=0.21; Figure 2J), which further supports the establishment of a similar equilibrium in both singleton and twin blastoids for the trophectoderm, epiblast and hypoblast lineages. Materials and Methods Cell culture Naïve pluripotent stem cells (niPSCs and HNES1) were cultured on irradiated DR4 mouse embryonic fibroblasts (MEF; iPS core Erasmus Rotterdam) in PXGL medium (Bredenkamp et al., 2019b). PXGL medium is N2B27 (DMEM / F12:Neurobasal 1:1, 1% N2 (made in-house), 2% B27 (Gibco), 2mM L-glutamine (Gibco), 1 mM beta-mercaptoethanol (Gibco)medium supplemented with 1 μM PD0325901, 2 μM XAV939, 2 μM Gö6983and 10 ng / ml human LIF. Every 3-4 days, cells were dissociated withAccutase for 3-5 minutes, resuspended in PXGL medium and 1:2-1:8 of the cell suspension was reseeded on new MEF-covered wells. For the first 24 hours of culture, 10 μM Y-27632 was included in the medium. Cells were kept at 37°C in 5% O2, 5% CO2. Cells were cultured without antibiotics and periodically tested for mycoplasma. Microwell preparation Microwell arrays were fabricated via microthermoforming. Briefly, thin polycarbonate films (50 μm thickness) were clamped between a counter-plate and a brass mold with arrays of 288 micromilled dead-end holes. The plates and film were heated up to 153°C and a differential gas pressure of 10 bar was applied via a pressure reservoir connected to the counter-plate. In this manner the softened polymer film was stretched into the holes of the mold, thus forming thin-walled microwells. The film was cooled down to below 80°C and the gas pressure released. The film could be demolded and the arrays were cut to chips that fit into 24 wells plate wells. Microwell arrays were kept in place in the well plate by placing O-rings (ERIKS) on top. Alternatively, commercially available microwell arrays in 96 wells plate format (300MICRONS GmbH, STATARRAY MCA96-8.736- PS) were used. Both in-house produced and commercial microwells were rinsed with 100% ethanol and sterilized for 30-60 minutes in 70% ethanol. After three washes with PBS to remove the ethanol, the microwells were incubated for at least 60 minutes in 1% (w / v) pluronic F108 (Sigma-Aldrich) to prevent cell adhesion to the film. Wells were rinsed with PBS and could be stored at 4 degrees in N2B27 or DMEM medium for several days before use. Generation of blastoids Prior to blastoid culture, the niPSCs were grown for 3 days on wells coated with 1% Geltrex (Gibco). Cells were dissociated with TrypLE Express (Gibco) for 3-5 minutes and collected in DMEM / F12 (Sigma-Aldrich) + 0.15% BSA V fraction (Gibco). Cells were spun down for 3 minutes at 200xg and resuspended in PAY medium, which is N2B27 medium supplemented with 2 μM PD0325901, 2 μM A83-01 and 10 μM Y-27632. LPA was also added in concentrations mentioned per experiment. Cells were then manually counted using Neubauer counting chambers. Microwells were prepared by removing the media inside and replacing it with 50 µL (in 96wp) or 100 µL (in 24wp) to aid even distribution of cells. Cell seeding densities varied per experiment. After seeding, plates were moved to an incubator (37°C, 5% CO2) for 10-15 minutes to let cells settle in the microwells. After the cells had settled, the medium was topped up to the final volume (250 µL / well in 96wp, 1 mL / well in 24wp). Half of the medium was refreshed after 24 hours. PAY medium (with / without LPA) was replaced with N2B27 supplemented with 0.5 µM A83-01 after 48 hours. At 72 hours, the medium was changed to plain N2B27. Structures were analysed at 72-96 hours after cell seeding. Blastoids were categorized as ‘twin blastoids’ if they met all of the following criteria: 1) the structure comprises only a single TE epithelium-like cyst, which consists of OCT4- negative cells, 2) the cyst does not contain protruding clumps of OCT+ cells outside of the TE cyst, 3) the cyst contains two foci of OCT4+ cell clumps, with a minimum of 5 cells or 600 µm2 projected surface area per OCT4+ clump, 4) to qualify as separate ICM clumps, the closest OCT4+ cells of the two clumps should be at least 3 OCT4+ cell lengths removed from each other. REFERENCES CITED IN THE APPLICATION - Kagawa et al. (2021) in Human blastoids model blastocyst development and implantation, Nature, Vol.601, 600-605 - Yanagida et al., in Naive stem cell blastocyst model captures human embryo lineage segregation, Cell Stem Cell.2021 Jun 3; 28(6): 1016–1022.e4. - EP2986711 (University of Maastricht et al.) - Vrij, Erik & Espinoza, Sara & Heilig, Markus & Kolew, Alexander & Schneider, Marc & Blitterswijk, Clemens & Truckenmuller, Roman & Rivron, Nicolas. (2016).3D High Throughput Screening and profiling of Embryoid Bodies in thermoformed microwell plates. Lab Chip.16.10.1039 / C5LC01499A. - Rivron NC, Vrij EJ, Rouwkema J, Le Gac S, van den Berg A, Truckenmiiller RK, van Blitterswijk CA, Tissue deformation spatially modulates VEGF signalling and angiogenesis, Proc Natl Acad Sci U S A.2012 May 1; 109(18):6886-91 - Liu, X., et al. Modelling human blastocysts by reprogramming fibroblasts into iBlastoids. Nature 591, 627–632 (2021). https: / / doi.org / 10.1038 / s41586-021-03372-y

Claims

Claims 1. An in vitro method of producing a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures, the method comprising: providing at least 150 of pluripotent stem cells (PSC’s) in a confined culture space in which the PSC’s are kept spatially constrained in a microvolume of culture medium at a cell density between 0.5x103cells to 2.0x104cells per mm3culture medium, allowing the PSC’s to aggregate into an aggregate structure comprising at least 150 cells, adding an inhibitor of the Hippo pathway in the culture medium containing the aggregate structure in a concentration of at least 1µM, and differentiating the PSC’s in the aggregate structure to form the blastocyst-like 3D structure of cells; and to optionally isolate the blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures.

2. The in vitro method according to claim 1, wherein the pluripotent stem cell is selected from one or more of naïve pluripotent stem cells and induced pluripotent stem cells.

3. The in vitro method according to any one of claims 1-2, wherein the confined culture space is provided in a cell culture container comprising a non-cell adherent surface at least at the location of the confined culture space.

4. The in vitro method according to claim 3, wherein the cell culture container is a microwell with a diameter from 20 to 5000μm, more in particular from 200 to 800μm, the said diameter defined as the longest possible straight-line distance between any two opposite points on the circumference circumscribing the opening of the microwell.

5. The in vitro method according to any one of claims 1-4, wherein the cell culture container is a microwell that has a diameter from 150 to 300 µm and a depth from 200 to 300µm, and wherein the pluripotent stem cells are seeded at a cell density from 50 to 300 cells per microwell.

6. The in vitro method according to any one of claims 1-4, wherein the cell culture container is a spherical or ellipsoid, free-floating object, in particular a hollow, core / shell microdroplet, that has a diameter from 100 to 1000 µm, in particular from 100 to 500 µm, and even more in particular from 100 to 300 µm, and wherein the pluripotent stem cells are seeded at a cell density from 50 to 300 cells per free-floating object.

7. The in vitro method according to any one of claims 1-6, wherein the inhibitor of the Hippo pathway is lysophosphatidic acid (LPA).

8. The in vitro method according to any one of claims 1-7, wherein at least 200 pluripotent stem cells (PSC’s) are provided in the confined culture space in which the PSC’s are kept spatially constrained in a microvolume of culture medium at a cell density between 0.5x103cells to 2.0x104cells per mm3culture medium, and wherein the inhibitor of the Hippo pathway in the culture medium containing the aggregate structure is added in a concentration of at least 2.5µM.

9. The in vitro method according to any one of claims 1-8, wherein the cell differentiation culture medium comprises at least one inhibitor of the TGFβ pathway and at least one inhibitor of the ERK pathway.

10. A blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures obtained or obtainable by a method as disclosed in any one of claims 1-9.

11. A culture of differentiated three-dimensional cell aggregate structures comprising: - from 2 % to 25 % of a blastocyst-like 3D structure of cells having cells of embryonic lineage forming at least two distinguishable epiblast-like structures and cells of extraembryonic lineage forming at least two distinguishable hypoblast-like structures; - from 5 % to 60 % of a blastocyst-like 3D structure of cells having cells of embryonic lineage forming one distinguishable epiblast-like structure and cells of extraembryonic lineage forming one distinguishable hypoblast-like structure; all the percentages in relation to the total number (100 %) of three-dimensional cell aggregates in the culture.

12. Use of the blastocyst-like 3D structure of cells as defined in claim 10, or the culture of differentiated three-dimensional cell aggregate structures as defined in claim 11, for the screening of a candidate agent or a physical and / or mechanical mean with the capacity to modulate the mammal embryonic development.

13. Use of the blastocyst-like 3D structure of cells as defined in claim 10, or the culture of differentiated three-dimensional cell aggregate structures asdefined in claim 11, as an in vitro model of the mammal embryonic development of a blastocyst stage comprising at least two inner cell masses.

14. An in vitro screening method for identifying an agent or a physical and / or mechanical mean that modulates the mammal embryonic development, the method comprising: providing a blastocyst-like 3D structure of cells as defined in claim 10 or a culture as defined in claim 9; contacting the blastocyst-like 3D structure of cells or culture with a candidate agent or alternatively, to subject the blastocyst-like 3D structure of cells to a physical and / or mechanical mean; and detecting a change in mammal embryonic development compared to a control to determine the candidate agent’s modulatory activity or the modulatory activity of the physical and / or mechanical mean.

15. The method according to claim 14, wherein change in mammal embryonic development is detected by determining if the blastocyst-like 3D structure of cells evolves to stages that are reminiscent of stages later than the blastocyst stage in the mammal embryonic development, or if the blastocyst-like 3D structure of cells does not evolve to any later stage, and / or involutes to a previous stage in the mammal embryonic development, or if the at least two distinguishable epiblast-like structures and at least two distinguishable hypoblast-like structures merge in one single epiblast / hypoblast-like structure.

Images