Method for the culture of embryo-like structures

Abstract

The invention relates to an in vitro method for the preparation of a culture of pre-implantation and post-implantation embryo-like three-dimensional cell aggregates, the method comprising several stages of induction and differentiation with particular cell culture media. The invention also provides induction cell culture media and a system for the high throughput analysis and imaging of embryo-like cell aggregates.

Description

[0001] Title: Method for the culture of embryo-like structures

[0002] Background of the invention

[0003] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Early mammalian embryonic development is characterized by crucial cell fate decisions of the embryonic and extra-embryonic compartments that ensure developmental progression. The recent advent of stem cell-based embryo models creates new exciting opportunities to study pre- and early post-implantation embryo differentiation and morphogenesis. Typically, embryo models, including blastoids, extraembryonic / embryonic peri-implantation models, and gastruloids, are formed by coaxing stem cells within a non-adhesive (micro-) environment that supports their unrestricted self-organization potential. Embryo models are capable of recapitulating sequences of developmental processes, modeling developmental defects, and providing insights into the molecular regulators driving development. Moreover, they have the potential to reveal hidden complexities in embryo morphogenesis and the assemblage of cell lineages at various stages of development.

[0005] Despite their utility, embryo models exhibit significant morphological heterogeneity, which hinders inferring their relevance to the study of natural embryo development. Moreover, the lack of automated tools to measure and quantify embryonic morphogenesis and associated stages in development limits their potential applications in the screening domain. Part of these challenges may be addressed by forming large numbers of embryo models to determine true variability, as has been done in organoid models. Formulating a better understanding of how different tissues coordinate intricate morphogenetic events, studying the impact of mechanobiological cues, genetic determinants, and drugs, and performing toxicological studies on a large scale would be important in navigating the applications of such embryo models.

[0006] One example of modeling for the study of the embryonic development is disclosed in Vrij et al., A pendulum of induction between the epiblast and extra-embryonic endoderm supports post-implantation progression, Development (2022) 149, dev192310. doi:10.1242 / dev.192310. The authors studied several stages of the embryo development. For this they used particular induction and expansion cell culture media and allowed the structured cell aggregates to grow in Matrigel in microwells. For the visualization using confocal microscopy, the structures were flushed out from the microwells and transferred into non-tissue culture treated polystyrene plates. This is a good method for the study of embryo development, although it needs the transferring of the structures for imaging, which makes it complex. This is in part because, as it is widely known and accepted, for the formation of organoids or in this case embryoids (i.e. , embryo-like structure but non-viable), the structures are let to grow suspended or embedded in a hydrogel, which makes more difficult the imaging of the structures. Matrigel is the trade name of a widely used hydrogel composition, which is an extracellular matrix (ECM) substitute composed of several known and unknown proteins.

[0007] With the aim to develop a high-throughput platform for the analysis of organism development, there are authors that have used a transparent polycarbonate to analyze the elongation of gastruloids, and the cell migration within this process (Samal et al., 2020, A New Microengineered Platform for4D Tracking of Single Cells in a Stem-Cell- Based In Vitro Morphogenesis Model. Adv. Mater, 32, 1907966. DOI: 10.1002 / adma.201907966). This document is silent about the used cell induction culture, since the aim was to study the impact of the shape of the microwells for the analysis of the elongation.

[0008] Thus, there is a need in the art of improved methods for obtaining cultures of preimplantation and post-implantation embryo-like three-dimensional cell aggregates that can provide larger numbers of embryo models, without the need of additional steps of manipulation for imaging.

[0009] Descri ption:

[0010] Summary of the invention

[0011] The inventors have surprisingly found that, under particular culture conditions, embryonic stem cells (i.e., pluripotent and / or naive) can be developed on a ready for imaging support material, to an embryo-like structure or cell aggregate, in which an embryo-like epiblast (Epi) compartment and a pro-amniotic-like cavity (PAC) are encased by an extraembryonic endoderm-like layer (XEn). Unexpectedly, the formation of the embryo-like aggregates or structures could be achieved without the need of any extracellular matrix. The structures grow and develop in a culture support that is transparent to wavelengths of the visible light spectrum. Under these conditions a method for obtaining cultures of pre-implantation and post-implantation embryo-like three-dimensional cell aggregates is provided, which advantageously allows for a high- throughput culture with phenotypes also highly automatically / manually analyzable, in- process, and in-situ. There is no need, moreover, to transfer the embryo-like structures for imaging. This solves the problem of the management of samples in a safe and less complex mode, and it also provides a reproducible system to perform reliable assays on the development of embryos (toxicity, analysis of the key pathways, drug selection, etc.).

[0012] A first aspect of the invention relates to an in vitro method for the preparation of a culture of pre-implantation and post-implantation embryo-like three-dimensional cell aggregates, the method comprising:

[0013] (a) providing a source of pluripotent and / or naive embryonic stem-cells;

[0014] (b) a first differentiation stage in which the pluripotent and / or naive embryonic stemcells are cultured in a container of micrometric dimensions at an initial ratio of about 1 to 100 cells per container, preferably from 5 to 50 cells per container, more preferably from 10 to 25 cells per container, with an epiblast (Epi) / extraembryonic endoderm (XEn) cell induction culture medium, said induction medium comprising heparin and p- mercaptoethanol, for a period of time suitable to provide induced-embryonic stem-cell aggregates; and

[0015] (c) a second differentiation stage, in which in the same container of micrometric dimension the cell induction culture medium of the previous step is replaced by a basic cell culture medium suitable for embryonic stem-cell expansion and differentiation, said medium comprising p-mercaptoethanol; and the induced-cell aggregates of step (b) are cultured for a period of time suitable to provide the formation of the pre- implantation and post-implantation embryo-like three-dimensional cell aggregates, preferably for a period of time suitable to provide embryo-like cell aggregates comprising an embryo-like epiblast (Epi) compartment and an extraembryonic endoderm-like layer (XEn) encasing the said Epi, the embryo-like cell aggregates preferably comprising in addition a pro-amniotic-like cavity (PAC). The method allows for a high yield of embryo-like cell aggregates that comprise an epithelialized extraembryonic endoderm (XEn) layer encasing a polarized epiblast-like compartment (Epi) with a pro-amniotic-like cavity (PAC). With the proposed Epi / XEn cell induction medium and the medium suitable for embryonic stem-cell expansion and differentiation working according to the scheduled stages, the embryo-like cell aggregates surprisingly result in a container without the need of any ECM, which facilitates the visualization and imaging as previously highlighted.

[0016] As a result of the previously disclosed method, the invention also provides a culture of pre-implantation and post-implantation embryo-like cell three-dimensional aggregates obtained or obtainable from the same.

[0017] Thus, in a second aspect the invention also refers to a culture of preimplantation and post-implantation embryo-like cell three-dimensional aggregates, which comprises at least 75 %, preferably from 75 % to 99 %, of embryo-like cell aggregates that comprise an epithelialized extraembryonic endoderm (XEn) layer encasing a polarized epiblast-like compartment (Epi) with a pro-amniotic-like cavity (PAC), the percentages in relation to the total amount of pre-implantation and postimplantation embryo-like cell three-dimensional aggregates. This culture provides embryo-like cell three-dimensional aggregates that are not able to form or develop into mammal (e.g. human) embryos. The culture is used as a model system to conduct research on various aspects of animal, preferably mammal and preferably human developmental biology alleviating any concern regarding actual human embryo manipulation or any concern regarding their industrial applicability.

[0018] With the method and culture previously disclosed, the screening of candidate agents or means to modulate embryo-like development can be performed in a very reproducible and easy mode in an easy and ready to use platform.

[0019] Thus, it is a third aspect of the invention a method for the screening of a candidate agent or of a physical and / or mechanical mean that modulates embryonic development, the method comprising: (a) providing a culture of pre-implantation and post-implantation embryo-like cell three- dimensional aggregates as defined in any the second aspect;

[0020] (b) contacting the culture with a candidate agent or a physical and / or mechanical mean under condition that allow the interaction of the candidate or physical and / or mechanical mean with the culture; and

[0021] (c) determining if the contacting of step (b) modulates the embryonic-like development. As a result of all efforts of the inventors, they surprisingly found a Epi / XEn cell induction culture medium that was particularly useful for the method of the first aspect, in terms of allowing high yields of the pre-implantation and post-implantation embryolike cell three-dimensional aggregates.

[0022] Thus, in a fourth aspect the invention provides an Epi / XEn cell induction culture medium that comprises heparin, p-mercaptoethanol, and fetal bovine serum, and one or more of a fibroblast growth factor, preferably in which the amount of heparin is from 0.7 pg / ml to 1.5 pg / ml, preferably 1.0 pg / ml; the amount of p-mercaptoethanol is from 75 pM to 120 pM, preferably 100 pM; and the amount of fetal bovine serum is from 3.0 % v / v to 7 % v / v, preferably 5 % v / v.

[0023] With the aim of facilitating the carry out of the methods of the invention, a set including all the tools for its performance has also been developed. Thus, the invention also provides, as a fifth aspect a cell culture system or kit for the preparation of pre- implantation and post-implantation embryo-like three-dimensional cell aggregates, that comprises:

[0024] (a) a multiple well cell culture support for cell culturing, or means for preparing it, which is transparent to wavelengths of the visible light spectrum ;

[0025] (b) a cell induction culture medium as defined in the previous (fourth) aspect; and optionally one or more of

[0026] (c) a cell culture medium suitable for embryonic stem-cell expansion and differentiation comprising p-mercaptoethanol at a concentration in the cell culture medium from 75 pM to 120 pM, preferably 100 pM;

[0027] (d) a source of pluripotent and / or naive embryonic stem-cells, preferably mammal and more preferably human embryonic stem cells; (e) one or more compounds or compositions for cell aggregate fixation for imaging, and / or one or more compounds or compositions for cell aggregate imaging;

[0028] (f) a computer readable software for image analysis, the software preferably for performing a method for classifying embryo-like three-dimensional cell aggregates by creating and analyzing a digital image with labeled structures of embryo development stages, said labeled structures preferably selected from Epiblast-like (Epi), Primitive endoderm-like (PrE), extraembryonic endoderm-like (XEn), pro-amniotic cavity-like (PAC);

[0029] (g) instructions for the use of the system or kit, preferably comprising instructions to carry out the method as defined in any one of the first or third aspects.

[0030] This scalable culture and analysis platform provides a unique opportunity to quantitatively and systematically study effects of pathway modulators on early embryonic development.

[0031] Combining high-throughput generation and high-content imaging of embryo models with high fidelity enables large-scale screening assays with an impact on the fields of (embryo) toxicity, drug development, embryogenesis, and reproductive medicine. The invention shows the continuous culture and in situ (i.e., in microwell) imaging-based readout of a 3D stem cell-based model of peri-implantation epiblast (Epi) / extraembryonic endoderm development (XEn) with an expanded pro-amniotic cavity (PAC), namely XEn / EPiCs. Automated image analysis and supervised machine learning permit the identification of embryonic morphogenesis, tissue compartmentalization, cell differentiation, and consecutive classification, respectively. Screens with signaling pathway modulators at different time windows provide information on their phenotypic effect on developmental processes leading to the formation of XEn / EPiCs in space and time.

[0032] FIG. 1. Generation and Characterization of Chemically-Induced XEn / EPiCs: A) Thermoformed microwell screening plate with a zoomed-in inset of a single microwell, B) Schematic depicting the formation of XEn / EPiCs within thermoformed microwells with a top panel indicating the developmental stage and features of a natural embryo they mimic, (ICM: Inner Cell Mass, TE: Trophectoderm, XEn: Extraembryonic Endoderm, Epi: Epiblast, VE: Visceral endoderm, PaE: Parietal Endoderm, PAC: Pro- Amniotic Cavity); Epi / XEn induction: Chir, Fgf4, Retinoic acid, cAMP, Heparin, 5% FBS, C) Immunochemistry image of a XEn / EPiC stained for nuclei (Hoechst) and F- actin (Phalloidin), D) A merged multichannel acquisition image of XEn / EpiCs, E) Immunochemistry characterization of XEn / EPiCs for Oct4, a pluripotency marker, Podocalyxin, a marker for the pro-amniotic cavity lining and XEn lining, and Laminin, lining the epiblast. Scale bars: 100 pm

[0033] FIG. 2. Media optimization for the formation of XEn / EPiCs within thermoformed microwells: mESCs were seeded into thermoformed microwells with (i) advanced N2B27 media, (ii) advanced N2B27 media + 1% LIF, (iii) 33% ESmed+ 67% adv.N2B27, (iv) advN2B27+5% FBS. All the media conditions were tested with XEn- induction factors from 0-24h of ESCs seeding. After that, all conditions were refreshed with advanced N2B27 + 0.2% p-mercaptoethanol + 1 % Penicillin / Streptomycin until 120h. The media condition with advanced N2B27 + 5% FBS-based XEn-induction media gave the highest percentage of XEn / EPiCs with 80%.

[0034] FIG. 3. Imaging and phenotypic classification of XEn / EPiC variants within microwells: A) Nomenclature and classification of different morphologies observed and the features they have (table), B) Percentage of manual vs automated measurement of the different XEn / EPiC variants present. From left to right, first set of columns correspond to XEn / EPiC, second set of columns to XEn / Epi rosettes, third set of columns to XEn / non-polarized Epi, fourth set of columns to EB-like, and fifth set of columns to Amorphous XEn / Epi. Scale bars: 100 pm.

[0035] FIG. 4. Automated phenotypic quantification of XEn / EPiCs exposed to signaling modulators of Wnt and Fgf / MAPK pathways: A) Top: experimental design of the exposure of XEn / EPiCs to different pathway modulators from 0h-72h and 48h-120h of development. Bottom: schematic showing the tissue compartment measurements on XEn / EPiCs, B) Effect of the modulators on the overall size of XEn / EPiCs, Area of Epi, PAC, and XEn in XEn / EPiCs when exposed to Wnt pathway modulators at C) 0-72h, and E) 48-120h, and Fgf / MAPK pathway modulators at D) 0-72h, and F) 48-120h; Data are mean±s.d. obtained from n=3 wells, with each well containing ~ 165 structures. All statistical hypothesis testing was done using Dunnett’s test; * represents P <= 0.05, ** represents P < 0.01 , *** represents P < 0.001 , **** represents P<0.0001 (One-way ANOVA with Dunnet’s post-test).

[0036] FIG. 5. Automated phenotypic quantification of structures exposed to signaling modulators of BMP and Tgffi / Nodal pathway: A) Top: experimental design of exposing the pathway modulators from 0-72h and 48h-120h of development of XEn / EPiCs. Bottom: schematic showing the tissue compartment features measured on XEn / EPiCs, B) Effect of the modulators on the overall size of XEn / EPiCs, Area of Epi, PAC, and XEn in XEn / EPiCs when exposed to BMP pathway modulators at C) 0- 72h, and E) 48-120h, and Tgfp / Nodal pathway modulators at D) 0-72h, and F) 48- 120h; Data are mean±s.d. obtained from n=3 wells, with each well containing ~ 165 structures. All statistical hypothesis testing was done using Dunnett’s test; * represents P<= 0.05, ** represents P < 0.01 , *** represents P < 0.001 , **** represents P<0.0001 (One-way ANOVA with Dunnet’s post-test.

[0037] Definitions

[0038] 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.

[0039] 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.

[0040] “Cell density” may be defined as the number of initial seeded cells (i.e., ESC) per volume unit of the container. Thus, the units are cells / volumetric measure (e.g., cubic milimeters-mm3). As a way of example, a cell density indicated as from 2 x 10'5cell / mm3to 2 x 10'3cell / mm3, means that in the container where the embryonic stem cells are going to be differentiated, the initial number of seeded cells accounts to a value from 2 x 10'5cell per cubic millimetre of the container, to 2 x 10'3cells per cubic millimetre of the container. The volume of the container is to be understood as the total hollow cavity defined by the base and walls of the container. Another equivalent way to express the cell density is by indication of the ratio of number of seeded cells in a micrometer-sized container, such as a microwell (container of micrometric dimensions). This is a very common way to indicate the number of seeded cells, in particular when they are seeded in a microwell of an array as the ones known by the skilled person in the art. Thus, a density expressed as about 18 cells per container of micrometric dimensions supposes about 9 x 10'5cell / mm3in a microwell of 300 pm of diameter and a depth or volume around 0.5 x 108pm3. This description contains both type of indications of the cell density, i.e. , cell per volume unit of the container 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 embryonic stem cells are seeded to form aggregates first, and after the induction to expand and differentiate, they are containers with a shape selected from spherical shaped; hemispherical shaped (i.e., half-spherical shape); 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.

[0041] “Embryonic stem cells(ESC)” (also termed herewith naive embryonic stem cells) are those type of “pluripotent stem cells (PSC)” found in the inner mass of a blastocyst. ESC 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. Mostly, the term PSC is used as an umbrella term for all pluripotent stem cells including embryonic stem cells and “induced pluripotent stem cells”. In any case, when in this description is referred to the provision of stem cells (e.g. ESC), in particular human, they do not proceed from any method that includes the destruction of human embryos. The skilled person in the art with the tenor of this description will understand that the method for the preparation of a culture of pre-implantation and post-implantation embryo-like three-dimensional cell aggregates can either depart from ESC or from induced pluripotent stem cells. “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.

[0042] 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 mammalian cells, in particular of rodent or primate cells, that are adhered forming a defined and discrete structure. It encompasses clusters of ESC; 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, such as the three germ layers (e.g. embryoid bodies-EB, or the aimed embryo-like cell aggregate XEn / EPiC). Among the aggregates comprising the three lineage cells are embryo-like cell aggregates, herewith termed XEn / EPiC, (structures with an epithelialized XEn expressing GATA Binding Protein 6 -Gata6, polarized Epi, and expanded PAC), XEn / Epi rosettes (structures with an epithelialized XEn expressing Gata6, polarized Epi, and with an F-Actin-rich region on the apical side), XEn / Non-polarized Epi (structures with an epithelialized XEn expressing Gata6 and non-polarized Epi), EB-like (ES cell aggregates without XEn specification and negative for Gata6 expression) and amorphous XEn / Epi (structures with disorganized gata6 expressing XEn).

[0043] All along the description the aggregates are defined as “embryo-like” cell aggregates or structures, since they are properly not actual embryos, but in vitro models of the different stages of an embryo development (i.e. embryoids or also called embryoid bodies). As will be illustrated in the examples, the invention proposes a method that models the mammal embryo development comprising the stages from E3.5 to E5.5 days in timeline of a mouse embryonic development. However, it is a model directly transferable to the corresponding embryo-like three-dimensional cell aggregates of other timelines in other mammals. According to the knowledge in the field of embryology, it is widely accepted that embryoid bodies (EBs) also termed in this description embryo-like cell aggregates, are not able to form or develop into mammal (e.g. human) embryos, which alleviates any ethical concern associated with the human embryo manipulation. They are often used as a model system to conduct research on various aspects of developmental biology. They can also contribute to research focused on tissue engineering and regenerative medicine. The container or synonymously well of “micrometric dimensions” refers to a microscale container or well with micrometric longitudinal dimensions and resulting micrometric volume dimensions. Particular volumetric dimensions are from 1.0 x 106pm3to 1.0 x 1010pm3, more in particular from 1.0 x 107pm3to 1.0 x 109pm3, even more in particular 0.1 x 108pm3to 5.0 x 108pm3, and most in particular and preferred from 0.25 x 108pm3to 0.75 x 108pm3. Microwells are well-known cell culture containers of micro-scale size. It is also common that these microwells be arranged in arrays comprising multiple microwells. The arrays are called microwell arrays. Thus, in a particular embodiment, the cells at the indicated density in a microwell are seeded in separated microwells of a microwell array.

[0044] The “epiblast (Epi) / extraembryonic endoderm (XEn) cell induction culture medium” is defined in detail below, but it refers to any a cell culture medium capable to induce a stem cell (i.e. , mammal stem cell) to aggregate to other(s) and to start to differentiate. In particular the induction culture medium triggers induced-embryonic stem-cell aggregates that provide the co-development of both the embryonic Epi compartment and the enveloping XEn layer. There are some commercial and known induction mediums, such as the neural induction medium N2B27 supplemented with particular modulators of the ESC development. An induction medium provides signals that change cell behavior, shape, differentiation, mitotic activity, signal cascades and / or gene expression.

[0045] The “cell culture medium suitable for embryonic stem-cell expansion and differentiation” is a medium that generically promotes cells to change from one type to another, often a less specialized type becoming more specialized in form and function. It is disclosed in more detail below, but the skilled person in the art of cell culturing will know about the commercial and disclosed in bibliography cell culture media with these properties. Again an example is the N2B27 supplemented with other particular modulators of the different stages of the embryo development.

[0046] As used herein, “conventional techniques” or “methods known to the skilled person” refer to a situation wherein the methods of carrying out the conventional techniques used in methods as disclosed herein will be evident to the skilled worker. The practice of conventional techniques in cell culture, molecular biology, biochemistry, genomics, sequencing, and related fields are well-known to those of skill in the art and are discussed, in various handbooks and literature references.

[0047] 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. 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.

[0048] (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.

[0049] The term “contacting” encompasses that a compound or cells, are in touch with compound or compositions which may then interact with each other.

[0050] The expression “physical and / or mechanical mean(s)” refers to any mechanical and / or physical manipulation of the embryo-like structures in vitro, in any of the stages resulting from the method of the invention. 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.

[0051] 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.

[0052] Detailed description of the invention

[0053] As previously indicated, a first aspect of the invention is an in vitro method for the preparation of a culture of pre-implantation and post-implantation embryo-like three- dimensional cell aggregates, the method comprising:

[0054] (a) providing a source of pluripotent and / or naive embryonic stem-cells;

[0055] (b) a first differentiation stage in which the pluripotent and / or naive embryonic stemcells are cultured in a container of micrometric dimensions at an initial ratio of about 1 to 100 cells per container, preferably from about 5 to 50 cells per container, more preferably from 10 to 25 cells per container, with an epiblast (Epi) / extraembryonic endoderm (XEn) cell induction culture medium comprising heparin and p- mercaptoethanol, for a period of time suitable to provide induced-embryonic stem-cell aggregates; and

[0056] (c) a second differentiation stage, in which in the same container of micrometric dimension the cell induction culture medium of the previous step is replaced by a basic cell culture medium suitable for embryonic stem-cell expansion and differentiation, said medium comprising p-mercaptoethanol; and the induced-cell aggregates of step (b) are cultured for a period of time suitable to provide the formation of the preimplantation and post-implantation embryo-like three-dimensional cell aggregates, preferably for a period of time suitable to provide embryo-like cell aggregates comprising an embryo-like epiblast (Epi) compartment and an extraembryonic endoderm-like layer (XEn) encasing the said Epi, the embryo-like cell aggregates preferably comprising in addition a pro-amniotic-like cavity (PAC). Along this description the embryo-like epiblast (Epi) compartment and an extraembryonic endoderm-like layer (XEn) encasing the said Epi, the embryo-like cell aggregates preferably comprise in addition a pro-amniotic-like cavity (PAC) is also abbreviated as XEn / EpiC.

[0057] In a particular embodiment of the first aspect, the source of pluripotent and / or naive embryonic stem cells is a source of mammal embryonic stem cells, preferably selected from rodents, such as mice and rats; or primates, such as human embryonic stem cells. As previously said, the embryonic stem cells are nowadays available from methods that do not suppose or originally come from the destruction of human embryos, in case this specie is selected as source of embryonic stem cells to carry out the method of the invention. 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. This step (a) of the method for the provision of the cells is, thus, carried out without the presence of any human or animal body and any surgical step. The expression “providing a source of pluripotent and / or naive embryonic stemcells” (e.g. in step (a) of the method of the invention) refers to isolated pluripotent and / or naive embryonic stem-cells, which derive from well-established cell lines which do not suppose the destruction of embryos.

[0058] In another particular embodiment, in step (b) the initial ratio of cells in the container is from about 5 to 50 cells per container, more preferably from 10 to 25 cells per container, even preferably from 14 to 25 cells per container of micrometric dimensions, and preferably is about 18 cell per container of micrometric dimensions.

[0059] The container of micrometric dimensions and the initial ratio of cells impairs the adequate spatial constrictions to the cell aggregates that will develop to the preimplantation and post-implantation embryo-like three-dimensional cell aggregates. Particular dimensions (volume) of the containers are from 1.0 x 106pm3to 1.0 x 1010pm3, more in particular from 1.0 x 107pm3to 1.0 x 109pm3, even more in particular 0.1 x 108pm3to 5.0 x 108pm3, and most in particular and preferred from 0.25 x 108pm3to 0.75 x 108pm3. Thus, in a particular embodiment of the in vitro method according to any of the embodiments above or below, the container of micrometric dimensions is a microwell of a multiple well cell culture support (that is, a microarray), and the method comprises in step (b) providing (e.g. seeding) the embryonic stem cells at an initial ratio from about 1 to 100 cells per microwell, preferably from 5 to 50 cells per microwell, more preferably from 10 to 25 cells per microwell, most preferably from about 14 to about 25 cells per microwell, preferably about 18 cells per microwell of the microarray.

[0060] The skilled man in the art knows how to provide an average number of cells per microwell. Indeed, this is a cell density. A particular density of cells (i.e. , average of number of cells per container of micrometric dimension, or microwell) results from a Poison distribution once a suspension with the cells is added to a well plate comprising multiple wells, and for example wells with microwells. The density can in the same way be defined by the number of cells per volume unit of the container of micrometric dimensions (i.e., per microwell). Thus, in the particular case of using a half-spherical microwell with a diameter of 300 pm and a depth or volume around 0.5 x 108pm3, 1 to 100 cells per microwell will suppose an average of cell densities from 2 x 10'5cell / mm3to 2 x 10'3cell mm3. Preferred cell densities corresponding from about 14 to about 25 cells per microwell of a depth of 0.5 x 108pm3, result in a range from 7 x 10'5cell / mm3to 12.5 x 10-5cell / mm3. The most preferred density of about 18 cells per container of micrometric dimensions supposes about 9 x 10'5cell / mm3. Independently of the mode of indicating the cell density, the equivalency is directly derivable from the explanations in this description.

[0061] In another particular embodiment of the first aspect, in the in vitro method in step (b) the suitable period of time comprises culturing the embryonic stem-cells from 12 to 24 hours, preferably 24 hours, and in step (c) the suitable period of time comprises culturing the induced- embryonic stem-cell aggregates to reach a total time of cell culturing including steps (b) and (c) from 120 hours to 150 hours, preferably 120 hours. Also in another particular embodiment of the first aspect, the Epi / XEn cell induction culture medium comprises an amount of heparin from 0.7 pg / ml to 1 .5 pg / ml, preferably 1.0 pg / ml; an amount of p-mercaptoethanol from 75 pM to 120 pM, preferably 100 pM; and preferably an amount of fetal bovine serum from 3.0 % v / v to 7 % v / v, preferably

[0062] 5 % v / v.

[0063] As will be illustrated in the examples, the presence of p-mercaptoethanol and heparin allowed for a high efficiency in the formation of XEn / EPiCs. In addition, the presence of fetal serum promoted an additional increase in efficiency, in particular when its amount was about 5 % v / v in the culture medium.

[0064] The cell induction medium may be prepared from a basic cell culture, in particular for cell induction and / or for the differentiation of stem cells, in particular embryonic stem cells, that the skilled person in the art of the culturing and differentiation of stem cells will know, as well as are the conventional techniques for culturing.

[0065] Thus, in a particular embodiment, the Epi / XEn cell induction culture medium comprises the basic advanced neural induction medium (N2B27), which according to the invention is supplemented with the heparin and the p-mercaptoethanol. In a more particular embodiment, the Epi / XEn cell induction culture medium, for example on the basis of the advanced N2B27, is further supplemented with one or more of Chir99021 (Chir, CAS No. 252917-06-9), Retinoic Acid (RA, CAS No. 302-79-4), fibroblast growth factor, in particular fibroblast growth factor 4 (Fgf4), 8Br-cAMP (CAS No. CAS No. 76939-46-3), ROCK inhibitor (Y27, CAS No. 129830-38-2). In a more particular embodiment, the Epi / XEn cell induction culture medium is further supplemented with at least a fibroblast growth factor, in particular with FgF4. In even a more particular embodiment, the Epi / XEn cell induction culture medium, for example the advanced N2B27, is supplemented with Chir99021 (Chir), Retinoic Acid (RA), fibroblast growth factor, in particular fibroblast growth factor 4 (Fgf4), 8Br-cAMP, ROCK inhibitor (Y27). The particular function of the compounds is disclosed in Table 1 at the Examples section of this description.

[0066] On the other hand and also according to another particular embodiment of the first aspect, optionally in combination with any of the embodiments above or below, the cell culture medium suitable for embryonic stem-cell expansion and differentiation comprises p-mercaptoethanol at a concentration in the cell culture medium from 75 pM to 120 pM, preferably 100 pM. Again, the cell culture medium suitable for embryonic stem-cell expansion and differentiation can be prepared from a basic cell culture medium suitable for embryonic stem-cell expansion and differentiation, said medium comprising, such as the advanced N2B27 medium, and others the skilled person in the art will know.

[0067] For the provision of the pluripotent and / or naive embryonic stem cells, in particular ESC, the method comprises in another particular embodiment a previous step in which the ESC are previously expanded in gelatin and other coated with non-adherent material plates, The expansion medium is in particular one that comprises from 10 % to 20 % of fetal bovine serum (FBS), in particular 15 % of FBS and the leukemia inhibitory factor (LIF). In other particular embodiments, the expansion is carried out on mouse embryonic fibroblasts (MEFs) according to the known for the skilled person conventional techniques.

[0068] In also another embodiment of the in vitro method of the invention, the method is one that comprises:

[0069] (a) providing a source of isolated pluripotent and / or naive embryonic stem-cells;

[0070] (b) a first differentiation stage in which the pluripotent and / or naive embryonic stemcells are cultured in a container of micrometric dimensions at an initial ratio of about 1 to 100 cells per container, preferably from 5 to 50 cells per container, more preferably from 10 to 25 cells per container, with an epiblast (Epi) / extraembryonic endoderm (XEn) cell induction culture medium comprising heparin and p-mercaptoethanol, for a period of time suitable to provide induced-embryonic stem-cell aggregates, wherein the Epi / XEn cell induction culture medium comprises an amount of heparin from 0.7 pg / ml to 1.5 pg / ml, preferably 1.0 pg / ml; an amount of p-mercaptoethanol from 75 pM to 120 pM, preferably 100 pM; and an amount of fetal bovine serum from 3.0 % v / v to 7 % v / v, preferably 5 % v / v; and

[0071] (c) a second differentiation stage, in which in the same container of micrometric dimension the cell induction culture medium of the previous step is replaced by a cell culture medium suitable for embryonic stem-cell expansion and differentiation, said medium comprising p-mercaptoethanol at a concentration in the cell culture medium from 75 pM to 120 pM, preferably 100 pM, and the induced-cell aggregates of step (b) are cultured for a period of time suitable to provide the formation of the preimplantation and post-implantation embryo-like three-dimensional cell aggregates, preferably for a period of time suitable to provide embryo-like cell aggregates comprising an embryo-like epiblast (Epi) compartment and an extraembryonic endoderm-like layer (XEn) encasing the said Epi, the embryo-like cell aggregates preferably comprising in addition a pro-amniotic-like cavity (PAC). Preferred and more preferred suitable times for any of the steps (b) and (c) in this embodiment are the ones previously disclosed.

[0072] In yet another particular embodiment of the first aspect, the in vitro method comprises one or more steps of cell culture refreshment carried out with the cell culture medium suitable for embryonic stem-cell expansion and differentiation that comprises p- mercaptoethanol at a concentration in the cell culture medium from 75 pM to 120 pM, preferably 100 pM.

[0073] In a more particular embodiment, a first step of refreshment is carried out at 24 hours after initiating the culturing of the embryonic stem cells in the container and Epi / XEn cell induction culture medium. The inventors have checked that with this timing the Epi / XEn was already able to induce differentiation. Mainly, these steps of refreshment consist in the washing with cell culture medium suitable for embryonic stem-cell expansion and differentiation that comprises p-mercaptoethanol, and then the addition of the fresh medium, also the cell culture medium suitable for embryonic stem-cell expansion and differentiation that comprises p-mercaptoethanol.

[0074] In another more particular embodiment, the steps of refreshment are carried out each 24 hours after initiation of the culture, more in particular at 24 hours, at 48 hours, at 96 hours and at 120 hours.

[0075] Another particular embodiment of the in vitro method according to the first aspect, is a method in which the container of micrometric dimensions is a container transparent to wavelengths of the visible light spectrum , preferably a container made from a transparent polymer selected from polystyrene, cyclic olefin (co)polymer / polymer, polymethylmethacrylate, fluorinated ethylene propylene and polycarbonate, preferably polystyrene. In the sense of the invention, transparent means that the commonly used wavelengths for imaging in optical microscopes (e.g., optical visible, fluorescence confocal microscope) can be used to visualize the structures in the container. Thus, mainly transparent means transparent in the visible light spectrum, which is to be understood that allows the light to pass through the material of which the container is made. Glass of uniform thickness is understood as the reference to full transparency, but other polymeric compounds as the previously listed do also provide for the transparency for the visualization and imaging by light and fluorescence microscopy. In another particular embodiment of the method of the invention, the container is made of a material with low or no autofluorescence, as a glass with uniform thickness would be. This is in particular of interest when immunofluorescence measurements are done to detect structures in the embryo-like aggregates.

[0076] One of the goals of the method of the invention is that the embryo-like structures are obtained directly upon seeding of embryonic stem cells in the appropriate medium on the container. This is unexpected because for the appropriate induction, expansion and development of stem cells extracellular matrixes are commonly used, which then make difficult any visualization through microscopy and require of the extraction of the structures to be transferred to another support for the visualization. This makes the procedure a complex one. Moreover, the visualized structure is difficult to be used again in any assay.

[0077] With the method of the invention if further a transparent container is used, the developing or developed structures can be visualized and imaged if required all along the culturing time without any additional step of manipulation and without disturbing any in process assay or test.

[0078] Thus, in another particular embodiment of the first aspect, the method is for the continuous high-throughput culture of pre-implantation and post-implantation embryolike three-dimensional cell aggregates and in situ imaging, and it comprises the step of visualizing the aggregates in one or more microwells of a multiple well cell culture support of a transparent polymer with a microscopy technique, and, optionally, the step of obtaining an image (i.e., recorded image) from the visualized aggregates and the computer-processing of the said image to provide a processed image, in which preferably said processed image differentially shows Epiblast-like (Epi) compartment, the extraembryonic endoderm-like (XEn), and the pro-amniotic cavity-like (PAC).

[0079] This embodiment can be reformulated as an in vitro method for the continuous high- throughput culture of pre-implantation and post-implantation embryo-like three- dimensional cell aggregates and in situ imaging, which method comprises carrying out the in vitro method for the preparation of a culture of pre-implantation and postimplantation embryo-like three-dimensional cell aggregates as defined in the previous paragraphs (i.e., aspects and embodimenst), and which further comprises the step of visualizing the aggregates in one or more microwells of a multiple well cell culture support of a polymer which is transparent to the wavelengths of the visible light spectrum with a microscopy technique, and the step of obtaining an image from the visualized aggregates and the computer-processing of the said image to provide a processed image, preferably said processed image differentially showing Epiblast-like (Epi) compartment, the extraembryonic endoderm-like (XEn), and the pro-amniotic cavity-like (PAC)

[0080] The microscopy technique is any one available and commonly found at the laboratory. In particular is a technique for the visualization and imaging by means of fluorescence detection, in particular confocal microscopy.

[0081] The possibility of this in-situ analysis of structures that are developing from the initially embryonic stem cells, do also allow the monitoring of the pre- and post-implantation embryo morphogenesis and differentiation under certain test conditions.

[0082] Therefore, in another particular embodiment, the method is for the analysis of pre- and post-implantation embryo morphogenesis and differentiation, and it comprises the additional step of adding at different times or at different time-windows of the cell culturing:

[0083] (a) one or more modulator compounds of one or more cell signaling pathways; and / or (b) one or more candidate test modulator compounds, physical and / or mechanical means.

[0084] This embodiment can be reformulated as an in vitro method for the analysis of pre- and post-implantation embryo morphogenesis and differentiation, that comprises carrying out the in vitro method for the preparation of a culture of pre-implantation and post-implantation embryo-like three-dimensional cell aggregates and / or the in vitro method for the continuous high-throughput culture of pre-implantation and postimplantation embryo-like three-dimensional cell aggregates and in situ imaging, both methods as defined above in the previous aspects and corresponding embodiments, and which further comprises the additional step of adding at different times or at different time-windows of the cell culturing:

[0085] (a) one or more modulator compounds of one or more cell signaling pathways; and / or

[0086] (b) one or more candidate test modulator compounds, physical and / or mechanical.

[0087] Particular modulator compounds of the one or more cell signaling pathways may be selected from a library of signaling pathway modulators that are known to play an important role in this window of embryo development, thus, from fecundation to an structure comprising in appropriate and functional distribution an Epi, an XEn and a PAC. In particular the one or more modulators are selected from the group consisting of the compounds in Table 1 in the section Materials and methods in the Examples section.

[0088] In the same way, other candidate or test modulator compounds can be assayed and its effects on the development compared with any of the already known modulators. The modulators can be inhibitors of certain signaling pathways or enhancers.

[0089] In a particular embodiment, the modulator is a compound that modulates (i.e., enhances or inhibits), one or more of the following pathways: the Wnt pathway, the Fgf / MAPK pathway, the BMP pathway, and the Tgfp / Nodal pathway. The skilled person in the art knows which are these pathways and in which processes they are involved. In another particular embodiment of the method for the analysis of pre- and postimplantation embryo morphogenesis and differentiation, the modulator compounds of one or more cell signaling pathways, and / or the one or more candidate agents are added to the culture in a time window selected from the initial of the culturing, when the embryonic stem cells are contacted with the XEn / Epi cell induction medium, to a time encompassing all the duration of the culture, in particular to obtain the XEn / EpiCs. In another particular embodiment, the modulators are added from 0 to 120 hours. In another particular embodiment, the effect on the morphogenesis of the structures of the one or more modulators is assayed at one or more time windows comprising from 0 to 72 hours of culturing, and from 48 to 120 hours of culturing, corresponding o hours to the moment in which the embryonic stem-cells are provided in the container and contacted with the induction medium.

[0090] This method for the analysis of pre- and post-implantation embryo morphogenesis and differentiation allows to study the impact or precise role of the one or more modulators, as well as the time window role in which they are crucial.

[0091] Based on morphology and tissue types, the different stages of the development can be characterized. The aim is to analyze the path to the obtention of XEn / EPiC structures. The XEn / EPiC structures are defined by self-organized 3D, spherical structures consisting of an epithelialized extraembryonic endoderm (XEn) layer (expressing Gata6 / Pdgfra), encasing a polarized epiblast-like compartment (Epi) (expressing Otx2 / Oct4), with a pro-amniotic-like cavity (PAC) (lined with sialomucins such as Podocalyxin), namely XEn / EPiCs. Epi expressing Otx2 would indicate their transition from naive to primed pluripotency. Basement membrane deposition by the epiblast, secreting laminin in between Epi and XEn compartment can also be observed.

[0092] In a canonical or expected development process (developmental timeline) from embryonic stem cells to XEn / EPiC structures, some intermediate stages are recognized. Thus, to consider an appropriate development process has taken place the cell aggregates should undergo embryonic development similar to the timeline of natural development, namely, (1) specification of XEn in salt-and-pepper arrangement, which means that the distribution is quite random and stochastic, with the XEn cells and Epi cells randomly arranged throughout the aggregate; (2) epithelialization of XEn around Epi; (3) Polarization of Epi into rosette-like shape; (4) Formation of pro- amniotic cavity; and (5) Expansion of pro-amniotic cavity.

[0093] Thus, the role of a particular known modulator or candidate within a particular pipeline can be elucidated from the analysis of at least these stages, which in each mammal can take place at different times, but that the skilled person in the art will be able to recognize for each case.

[0094] The same is applicable to test the effect of any physical and / or mechanical means applied to the cell aggregates all along the culturing time or at certain time windows.

[0095] As previously indicated and as direct result of the method of the first aspect, another aspect of the invention corresponds to a culture of pre-implantation and postimplantation embryo-like cell three-dimensional aggregates, which comprises at least 75 %, preferably from 75 % to 99 %, of embryo-like cell aggregates that comprise an epithelialized extraembryonic endoderm (XEn) layer encasing a polarized epiblast-like compartment (Epi) with a pro-amniotic-like cavity (PAC), the percentages in relation to the total amount of pre-implantation and post-implantation embryo-like cell three- dimensional aggregates.

[0096] In a particular embodiment, this culture is obtainable or obtained by the method as defined in the previous aspect and embodiments.

[0097] Thus, it can be defined as a culture of pre-implantation and post-implantation embryolike cell three-dimensional aggregates, which comprises at least 75 %, preferably from 75 % to 99 %, of embryo-like cell aggregates that comprise an epithelialized extraembryonic endoderm (XEn) layer encasing a polarized epiblast-like compartment (Epi) with a pro-amniotic-like cavity (PAC), the percentages in relation to the total amount of pre-implantation and post-implantation embryo-like cell three-dimensional aggregates, and obtainable or obtained by a method comprising: a) providing a source of pluripotent and / or naive embryonic stem-cells; (b) a first differentiation stage in which the pluripotent and / or naive embryonic stemcells are cultured in a container of micrometric dimensions at an initial ratio of about 1 to 100 cells per container, preferably from about 5 to 50 cells per container, more preferably from 10 to 25 cells per container, with an epiblast (Epi) / extraembryonic endoderm (XEn) cell induction culture medium comprising heparin and p- mercaptoethanol, for a period of time suitable to provide induced-embryonic stem-cell aggregates, preferably wherein the epiblast (Epi) / extraembryonic endoderm (XEn) cell induction culture medium comprises an amount of heparin from 0.7 pg / ml to 1.5 pg / ml, preferably 1.0 pg / ml; an amount of p-mercaptoethanol from 75 pM to 120 pM, preferably 100 pM; and preferably an amount of fetal bovine serum from 3.0 % v / v to 7 % v / v, preferably 5 % v / v; and c) a second differentiation stage, in which in the same container of micrometric dimension the cell induction culture medium of the previous step is replaced by a basic cell culture medium suitable for embryonic stem-cell expansion and differentiation, said medium comprising p-mercaptoethanol, preferably at a concentration in the cell culture medium from 75 pM to 120 pM, preferably 100 pM; and the induced-cell aggregates of step (b) are cultured for a period of time suitable to provide the formation of the pre-implantation and post-implantation embryo-like three-dimensional cell aggregates, preferably for a period of time suitable to provide embryo-like cell aggregates comprising an embryo-like epiblast (Epi) compartment and an extraembryonic endoderm-like layer (XEn) encasing the said Epi, the embryo-like cell aggregates preferably comprising in addition a pro-amniotic-like cavity (PAC).

[0098] In another particular embodiment of the second aspect, the culture is provided on a transparent multiple well cell culture support and that comprises one embryo-like three-dimensional cell aggregate per well. Particular features and advantages of the transparent (i.e., transparent to wavelengths of the visible light spectrum) multiple well cell culture support have been previously disclosed in relation to the first aspect and apply also to this second aspect.

[0099] Therefore, with the culture of the invention what is provided is a ready to use for the analysis or imaging culture of pre-implantation and post-implantation embryo-like cell three-dimensional aggregates, which culture is provided in a transparent multiple well cell culture support and that comprises one embryo-like three-dimensional cell aggregate per well, wherein at least 75 %, preferably from 75 % to 99 %, of embryo- like cell aggregates in this ready to use culture on the indicated support comprise an epithelialized extraembryonic endoderm (XEn) layer encasing a polarized epiblast-like compartment (Epi) with a pro-amniotic-like cavity (PAC), the percentages in relation to the total amount of pre-implantation and post-implantation embryo-like cell three- dimensional aggregates within the total wells of the support. As previously indicated, this transparent multiple well cell culture support is a transparent multiple microwell support.

[0100] Also another aspect of the invention is a method for the screening of a candidate agent or of a physical and / or mechanical mean that modulates embryonic development, the method comprising:

[0101] (a) providing a culture of pre-implantation and post-implantation embryo-like cell three- dimensional aggregates as defined in any one of the second aspect and its embodiments;

[0102] (b) contacting the culture with a candidate agent or a physical and / or mechanical mean under condition that allow the interaction of the candidate or physical and / or mechanical mean with the culture; and

[0103] (c) determining if the contacting of step (b) modulates the embryonic-like development.

[0104] As previously disclosed in detail for the method for the analysis of pre- and postimplantation embryo morphogenesis and differentiation, the modulation of the embryonic-like development caused by a candidate agent or a physical and / or mechanical mean can be elucidated from the analysis of the morphology and tissue types seen at the different stages of the development. The key structures, namely XEn, Epi and PAC can be visualized by detecting the expression of certain specific markers. If a candidate stops, blocks or slows the evolution to a certain stage, it can be classified as an inhibitor modulator. On the contrary, if it promotes the correct or expected development (tissue type and morphogenesis) and evolution from one stage to another, it is called a an enhancer of the development.

[0105] From all this, the key is that for all these analysis and studies the culturing is seen in process and in situ. For the carry out of all the previously disclosed methods, the inventors developed a particularly preferred Epi / XEn cell induction culture medium, which surprisingly induced the differentiation of embryonic stem cells in an efficient mode.

[0106] Thus, another aspect of the invention is a Epi / XEn cell induction culture medium that comprises heparin, p-mercaptoethanol, and fetal bovine serum, and one or more of a fibroblast growth factor, preferably in which the amount of heparin is from 0.7 pg / ml to 1.5 pg / ml, preferably 1.0 pg / ml; the amount of p-mercaptoethanol is from 75 pM to 120 pM, preferably 100 pM; and the amount of fetal bovine serum is from 3.0 % v / v to 7 % v / v, preferably 5 % v / v.

[0107] In an embodiment thus, the Epi / XEn cell induction culture medium is one in which the amount of heparin is from 0.7 pg / ml to 1.5 pg / ml, preferably 1.0 pg / ml; the amount of P-mercaptoethanol is from 75 pM to 120 pM, preferably 100 pM; and the amount of fetal bovine serum is from 3.0 % v / v to 7 % v / v, preferably 5 % v / v.

[0108] In a particular embodiment, this cell induction medium is prepared from a basic cell culture, in particular for cell induction and / or for the differentiation of stem cells, in particular embryonic stem cells, that the skilled person in the art of the culturing and differentiation of stem cells will know.

[0109] Thus, in a particular embodiment, the Epi / XEn cell induction culture medium comprises the basic advanced neural induction medium (N2B27), supplemented with the heparin and the p-mercaptoethanol. In a more particular embodiment, the Epi / XEn cell induction culture medium, for example on the basis of the advanced N2B27, is further supplemented with one or more of Chir99021 (Chir, CAS No. 252917-06-9), Retinoic Acid (RA, CAS No. 302-79-4), fibroblast growth factor, in particular fibroblast growth factor 4 (Fgf4), 8Br-cAMP (CAS No. CAS No. 76939-46-3), ROCK inhibitor (Y27, CAS No. 129830-38-2). In a more particular embodiment, the Epi / XEn cell induction culture medium is further supplemented with at least a fibroblast growth factor, in particular with FgF4. In even a more particular embodiment, the Epi / XEn cell induction culture medium, for example the advanced N2B27, is supplemented with Chir99021 (Chir), Retinoic Acid (RA), fibroblast growth factor, in particular fibroblast growth factor 4 (Fgf4), 8Br-cAMP, ROCK inhibitor (Y27).

[0110] Finally, another aspect of the invention is, as said, a cell culture system or kit for the preparation of pre-implantation and post-implantation embryo-like three-dimensional cell aggregates, that comprises:

[0111] (a) a multiple well cell culture support for cell culturing or means for preparing it, which is transparent to the wavelengths of the visible light spectrum;

[0112] (b) an Epi / XEn cell induction culture medium as defined in the fourth aspect and its embodiments; and optionally one or more of

[0113] (c) a cell culture medium suitable for embryonic stem-cell expansion and differentiation comprising p-mercaptoethanol at a concentration in the cell culture medium from 75 pM to 120 pM, preferably 100 pM;

[0114] (d) a source of pluripotent and / or naive embryonic stem-cells, preferably mammal embryonic stem-cells;

[0115] (e) one or more compounds or compositions for cell aggregate fixation for visualization and imaging, and / or one or more compounds or compositions for cell aggregate imaging;

[0116] (f) a computer readable software for image analysis, the software preferably for performing a method for classifying embryo-like three-dimensional cell aggregates by creating and analyzing a digital image with labeled structures of embryo development stages, said labeled structures preferably selected from Epiblast-like (Epi), extraembryonic endoderm-like (XEn), pro-amniotic cavity-like (PAC);

[0117] (g) instructions for the use of the system or kit, preferably comprising instructions to carry out the method as defined in the first aspect and its embodiments and / or in the third aspect and its embodiments.

[0118] Thus, the invention also provides simplified kits to carry out the method of the inventions and after or during to optionally automatically analyze in situ the results of the method at a particular time.

[0119] The system, that can optionally include a computer readable software for image analysis, is equally applicable independently from the said analysis. Thus, the system can provide an image as an input to a remote or independent software analysis service.

[0120] In the same way, the software can be provided independent from the system parts, on condition that the required image input data is provided to a method performed by executing the software.

[0121] In a particular embodiment of the system, the multiple well cell culture support for cell culturing which is transparent to wavelengths of the visible light spectrum, is an in-situ thermoformed multiple well cell culture support transparent to wavelengths of the visible light spectrum. In this embodiment, the kit provides the material to prepare by thermoforming the array of wells, commonly the microarray of microwells. Particular materials have been disclosed above and fully apply to this aspect of the invention.

[0122] The skilled person in the art will know which are the possible one or more compounds or compositions for cell aggregate fixation for visualization and imaging, and / or one or more compounds or compositions for cell aggregate imaging. Among those, the system contemplates paraformaldehyde (PFA) and glutaraldehyde as fixing agents. Among the compounds for aiding the imaging of cell structures are 4',6-diamidino-2- phenylindole (DAPI), a fluorescent compound that binds to DNA and thus show the nucleus of the cells; Wheat germ agglutinin (WGA), which binds to glycoproteins of the cell membrane; phalloidin derivatives that binds F-actin in the cells.

[0123] Other compounds for the in situ visualization of structures include antibodies specific for certain antigens in these structures, such as Anti-Laminin antibodies, anti- Podocalyxin antibodies or anti-Oct4, and others the skilled person will know and consider depending on the structure that wants to be visualized.

[0124] Next section includes examples to illustrate the invention and the associated advantages and applications. The materials and methods for the performance of the assays disclosed herewith are at disclosed at the end of this section Example 1. Generation and characterization of XEn / EPiCs within thermoformed microwell arrays to study pre- to post-implantation development.

[0125] A novel in situ imaging and analysis set-up was created using thermoformed microwell screening arrays (Statarrays, 300MICRONS, polystyrene) which allowed the generation of XEn / EPiCs in large numbers and with sufficient control over cell numbers. The first step (FIG. 1 A) involved, seeding an average of 18 mouse embryonic stem (ES) cells per microwell in a previously reported induction medium consisting of Chir99021 (Chir), Retinoic Acid (RA), fibroblast growth factor 4 (Fgf4) with heparin, and 8Br-cAMP (FIG. 1 B). The induction medium triggered the co-development of both the embryonic Epi compartment and the enveloping XEn layer. We chose an ES cell line containing a fluorescent reporter for the gene Gata6 (Gata6:H2B-Venus) to visualize the formation of the extraembryonic endoderm (XEn). The ES cells were cultured in basic serum-free medium containing advanced N2B27, which was daily refreshed. After 120 hours of culture, in -75-80% of microwells, ES cells had selforganized into 3D, spherical structures consisting of an epithelialized extraembryonic endoderm (XEn) layer encasing a polarized epiblast-like compartment (Epi) with a pro- amniotic-like cavity (PAC), namely XEn / EPiCs (FIG. 10, D). These structures could be directly imaged within the microwells and resemble the Epi and XEn compartments of an E5.5 stage embryo (FIG. 1 B). The structures were further characterized for the expression of podocalyxin, which lines the PAC ; Oct4, which marks the pluripotent epiblast; and Laminin, which defines the basement membrane that is deposited by the XEn (FIG. 1 E). The addition of 5% of fetal bovine serum to the induction cocktail from 0- 24 hours improved the yield of XEn / EPiCs to 81% (FIG. 2).

[0126] Of note is that with other methods which also aim for obtaining XEn / EPiCs among other stages of embryo development from ESCs, the herewith indicated yields of XEn / EPiCs could not be achieved. This was the case when the primitive endoderm induction (PrE-ind) medium and the conditions disclosed in Vrij et al. were followed (Vrij et al. 2022. Development (2022) 149, dev192310. doi:10.1242 / dev.192310). The efficiency of formation of XEn / EPiCs were quite low in comparison to the original setting (data not shown). Imaging and phenotypic classification of XEn / EPiC variants within microwells

[0127] In vitro cultures of organoids and stem cell-based embryo models are associated with phenotypic heterogeneity, reflecting variable stages in development or in vitro artifacts. Accordingly, aside from the XEN / EpiCs that we aimed to form, we observed the formation of multiple other phenotypes reflecting earlier stages along the developmental timeline (FIG. 3A). To classify these phenotypes, we imaged all embryo-like structures within the microwells and analyzed them using the open-source software package CellProfiler (CP). First, embryo-like structures were fixed within the microwells at 120 h of culture and stained with Hoechst (nuclei), Phalloidin (F-actin), and WGA (cell membrane). In addition, the Gata6:H2B-Venus fluorescent expression depicted the XEn layer. Montage images containing all structures within the entire wells were acquired using a fluorescence spinning disk microscope (Nikon Ti-E spinning disk). Secondly, a CellProfiler pipeline was generated to identify all embryolike structures and extract their phenotypic features, including area, texture, size, shape, intensity, and radial intensity distribution, among others (e.g., Zernicke features- refer to CellProfiler manual for details on this feature). These phenotypic features can be used to pinpoint the morphogenetic processes occurring during the E3.5 - E5.5 window of mouse embryo development, and thus in which phenotypic class to place the embryo-like structures. The morphogenetic processes that we used to specify these classes include XEn (i.e. primitive endoderm) differentiation (Gata6 expression), sorting of XEn cells towards the surface, and the formation of a continuous XEn layer engulfing the Epi (positioning of Gata6+ cells), initiation of Epi polarization (F-actin-rich center within Epi), and formation of the pro-amniotic cavity (cavity within the Epi). To obtain an automated classification process for the different phenotypes observed, the object measurements from CP were used to train a classifier - a set of rules for each phenotypic class - in CellProfiler Analyst (CPA), an open- source CP extension for supervised machined learning. The classification included five different phenotypic classes, namely, XEn / EPiCs (structures with an epithelialized XEn expressing Gata6, polarized Epi, and expanded PAC), XEn / Epi rosettes (structures with an epithelialized XEn expressing Gata6, polarized Epi, and with an F- Actin-rich region on the apical side), XEn / Non-polarized Epi (structures with an epithelialized XEn expressing Gata6 and non-polarized Epi), EB-like (ES cell aggregates without XEn specification and negative for Gata6 expression) and amorphous XEn / Epi (structures with disorganized gata6 expressing XEn) (FIG. 3A). The XEn / non-polarized Epi, XEn / Epi rosettes, and XEn / EPiCs chronologically resemble the XEn and Epi compartment of the E4.0, E4.5, and E5.5 embryo, respectively, along the peri-implantation developmental timeline (FIG. 1 B).

[0128] We used a supervised machine learning approach to find the linear combinations of phenotypic features that describe each of these phenotypic classes. The object scores that were generated for the automated object classification by CPA were validated by manual scoring, which showed a comparable yield of the different phenotypes. Overall, we show the creation of a microwell-based, high-throughput culture and imaging pipeline to automatically detect and quantify the morphological diversity of XEn / EPiCs embryo models including developmental steps towards the E5.5 stage in mice. We also observe a sufficient overlap between manual and automated measurements, making it a suitable tool to perform high-content analysis of XEn / EPiCs. High-content screening of XEn / EPiC variants with signaling pathway modulators

[0129] To identify essential signaling pathways during pre- to early post-implantation development, we performed a high-content screening using a custom library of signaling pathway modulators that are known to play an important role in this window of embryo development*. This library contains pathway modulators targeting the following pathways: Wnt, Fgf / MAPK, Tgfp / BMP, Activin / Nodal, ROCK, PKC, JAK / STAT, PKA / cAMP, Retinoid, and PI3K / Akt pathway (Table 1 in materials and methods at the end of this section). Since the addition of PD032, Chir, and LIF (2i / LIF) maintains ES cells in a naive pluripotent state and promotes their self-renewal, this condition was included as a negative control for differentiation. Structures were exposed to these modulators for the entire duration of the experiment (0 - 120 hours), after which they were stained, imaged, and the numbers and ratios of XEn / EPiCs and other phenotypic classes were quantified in each condition using the CPA classifier pipeline described before (Example 2). The pathway modulators were then grouped based on the proportion of identified phenotype classes (as described in FIG. 3). The percentages mentioned here are relative to the total of five phenotypes (100%) in each condition. The modulators BMP4, Activin A, Tgfpi (BMP / Tgfp pathway agonists), and Rspondin (Wnt agonist) showed a XEn / EPiCs proportion of 68%, 64%, 70%, and 70%, respectively, which was slightly lower but not significant compared to the 77% found in the control. This showed that the addition of these modulators from 0-120h had minimal to no effect on the differentiation and organization of XEn, as well as on the efficiency of XEn / EPiCs formation. Interestingly, XAV (Wnt inhibitor), LDN and Noggin (BMP inhibitors), Fgf4 (Fgf activator), and Nodal (Nodal activator) showed an overall reduction of 40%, 55%, 57%, 57%, and 30% respectively, for the proportion of XEn / EPiCs. Together, these observations show that activation of Tgfp / Nodal and BMP signaling and amplified canonical Wnt signaling by Rspondin do not interfere with the XEn and Epi morphogenesis. However, their inhibition significantly affects the overall proportion of XEn / EPiCs.

[0130] The modulators Chir, SB43, PD98, A83, Dorsomorphin, WP1066, and DL-adrenaline showed a significant reduction of XEn / EPiCs formation and a significantly higher proportion of XEn / Epi rosettes with an increase from 6% in the control to 9%, 18%, 20%, 16%, 21 %, 37%, 25%, respectively. In addition, there was a higher proportion of XEn / non-polarized Epi in Chir (17%), SB43 (19%), PD98 (58%), Dorsomorphin (28%), and cAMP (39%). This was an interesting observation because the above-mentioned phenotypes represent an earlier stage of epiblast morphogenesis. This suggests that these modulators either incited a roadblock on cavitation, thereby hindering its progression towards forming a PAC, or they slowed down developmental progression. Among these modulators, Dorsomorphin, cAMP, and WP1066 showed more dispersed XEn in comparison to the single epithelialized XEn in control, indicating the role of BMP, PKA, and JAK / STAT pathway in XEn specification and patterning. Together, these results indicate that the inhibition of pathways such as, Tgfp, BMP, Nodal, and JAK signaling, play coordinated roles in the epithelialization of XEn, while Wnt signaling affects the timing of developmental progression to form PAC.

[0131] The inhibitors of the Fgf / MAPK pathway, namely, PD032 (MEK / ERK inhibitor) and PD17 (FGF receptor inhibitor) resulted in 100% of EB-like structures, showing their failure to specify XEn cells. These findings are in line with the established role of Fgf signaling in the specification of XEn in the blastocyst, provided by the inductions from the Epi. Signaling pathway modulators that showed a high proportion of amorphous XEn / Epi included LIF (100%), Indolactam (100%), LY36 (100%), IWR-endo (100%), AGN (100%), UVI3003 (61%), RA (78%), and ML347 (84%). Together, this shows that the prolonged exposure to these signaling pathway modulators affected the correct spatial organization and sorting of XEn lineage cells. Overall, this primary screen confirmed the role of BMP / Tgfp, Activin / Nodal, Fgf / MAPK, Wnt, and JAK / STAT signaling pathways in early embryo development. Partitioning signaling pathway modulation by morphogenetic time windows

[0132] The specification and patterning (i.e. , sorting out) of XEn and Epi in mouse blastocysts occurs between 48 and 96 h after fertilization through the Grb2-MAPK pathway, which corresponds to the window of 0-72 h in the XEn / EPiC model. Exit from naive pluripotency and establishment of a polarized post-implantation epiblast epithelium occurs in natural embryos between 96 and 120 h, which is followed by the formation and expansion of the pro-amniotic cavity within 24 h after. This stage of development corresponds with the culture of XEn / EPiCs between 48 and 120 h. Morphometric analysis of XEn / EPiC cultures exposed to different pathway modulators at this time window could indicate their potential role in this process.

[0133] To increase the temporal resolution and gauge the effect of signaling pathways at specific chronological stages during the development of XEn / EPiCs, we chose a subset of modulators from the library to perform a secondary screen, namely Chir, XAV, Rspondin, Fgf4+Heparin, PD032, BMP4, Dorsomorphin, Tgfpi , Nodal, Activin A, SB43, and A83. These modulators showed distinct phenotypes under the different classifications in the primary screen and significantly affected one or more of the morphological events such as XEn specification and patterning, Epi polarization, and / or PAC formation, along with those that had minimal to no effect on the XEn / EPiCs. The XEn / EPiCs were exposed to the chosen modulators for two distinct windows of development, namely 0-72h and 48-120h, to further delineate the role of these pathways in specific morphogenetic processes during development, and study the plasticity of the cells to catch up with the developmental program when perturbed. The effect of activators and inhibitors of different pathways on the XEn / EPiCs at the two time windows, namely 0-72h and 48-120h are schematically shown in FIG. 4A and FIG. 5A. In addition to quantifying the ratio of different phenotypes, the data obtained from CP were fed-back into the CPA classifier tool to systematically filter XEn / EPiCs from the images and measure the area of individual tissue compartments (Data not shown . This method of quantifying individual cellular compartments in an automated set-up is important because it would be informative to not only identify the effect of different pathways on the developmental progression of XEn / EPiCs but also on how much these pathways are involved in regulating tissue morphogenesis.

[0134] 4.1 Wnt pathway

[0135] Wnt activation affected the formation of PAC while its inhibition affected XEn patterning with a reduction in the size of XEn / EPiCs.

[0136] From the primary screen, it was observed that modulation of the Wnt pathway with Chir, XAV, or Rspondin affected different aspects of XEn / EPiCs development. To observe and systematically quantify the effect of this pathway on Epi differentiation and polarization, XEn / EPiCs were exposed to signaling pathway modulators for two time windows. The exposure of structures to Chir from 0-72h showed a higher number of XEn / Epi rosettes (23% vs 10% in control) than XEn / EPiCs (15% vs 70% in control) (data not shown) with 30% of the structures resembling an EB-like phenotype (data not shown). Similarly, exposure from 48-120h showed a higher occurrence of XEn / Epi rosettes (26% vs 9% in control) and amorphous XEn / Epi (60% vs 10% in control) and no occurrence of XEn / EPiCs (0% vs 70% in control) (data not shown). Quantification of the overall size of Chir-treated XEn / EPiCs showed no significant change in comparison to the control (FIG. 4B).

[0137] By further analyzing the areas of the different compartments in the XEn / EPiCs, namely the Epi size, PAC area, and XEn thickness, in 0-72h treatment, there was a significant decrease in the size of Epi from 11 ,000 pm2 in control to 10,000 pm2 (FIG. 4C). However, the size of PAC and XEn showed no significant effect upon Chir treatment (FIG. 4C, FIG. 4E). Overall, the higher occurrence of structures presumably resembling an earlier time-point of the embryo, namely XEn / Epi rosettes, XEn / non- polarized Epi, and EB-like structures, taken together, could indicate that there could be a slight delay in the developmental progression of XEn / EPiCs after Chir exposure from 0-72h as well as from 0- 48h.

[0138] The inhibition of Wnt with XAV treatment from 0-72h, showed a reduced occurrence of XEn / EPiCs with 42% and a higher proportion of amorphous XEn / Epi (38% vs 10% in control) (Data not shown) with a significantly reduced overall size of XEn / EpiCs (28,000 pm2 vs. 32,000 pm2 in the control) (P < 0.001 )(FIG. 4B), likely contributed by the significant reduction in the size of Epi (10,000 pm2 vs. 11 ,000 pm2 in the control; P<0.0001) and XEn (17,000 pm2 vs 20,000 pm2 in the control; P < 0.01) (FIG. 4G). This observation was also validated by manual quantification of the cellular compartments. 48-120h exposure to XAV showed a similar occurrence of XEn / EPiCs, however, there was no significant effect on the size of different compartments, albeit with a dispersed XEn layer (FIG. 4 E). This suggests that prolonged Wnt inhibition from 0-72h altered the timing of the developmental progression of these structures causing a smaller phenotype, while its exposure from 48-120h affected the epithelialization of XEn.

[0139] Rspondin has been identified to increase the visibility of Wnt receptors and indirectly activate the Wnt pathway. In this screen, Rspondins showed a similar yield of XEn / EPiCs with 81 % at 0-72h compared to the control (70%). The sizes of individual compartments showed a similar ratio as in the control, although the size of PAG showed a marginal increase (11 ,000 pm2 vs. 10,000 pm2 in the control) (FIG. 4G). A similar effect was observed with 48-120h treatment (FIG. 4E). Together, the analysis of the effect of Wnt pathway modulators validated the findings from the literature that show the effect of Wnt in XEn maintenance and sorting by regulating the catenin- cadherin interactions via a feedback loop. Interestingly, the above data also showed that the inhibition of Wnt affected the timeline of the developmental progression of XEn / EPiCs thereby delaying the formation of an epithelialized XEn and expanded PAG.

[0140] 4.2 Fgf / MAPK pathway

[0141] Activation of the Fgf / MAPK pathway reduced the occurrence of XEn / EPiCs while its inhibition from 0-72h showed an EB-like phenotype

[0142] The Fgf / MAPK pathway has been widely studied for its key role in the specification and reinforcement of the XEn lineage in naive pluripotent cells. In addition, maintenance of the post-implantation Epi cells relies on Fgf proteins, which imputes a rapid molecular switch in response to these signals. The exposure of ES cells to MEK inhibitor PD032 or FGF receptor inhibitor PD17 from 0-72h led to the complete failure of structures to specify XEn (data not shown), which is similar to the phenotype observed with Fgf / MAPK signaling inhibition in mouse blastocysts. Interestingly, treatment with PD032 after the specification of XEn, from 48-120h, allowed XEn maintenance but led to a drastically reduced yield of XEn / EPiCs (10%). Possibly, prolonged Fgf inhibition at the later stage of development presumably slows down the transition of primed epiblast rosette into XEn / EPiCs, thereby resulting in a higher occurrence of XEn / Epi rosettes (35%) in PD032 treatment (data not shown). Hyperactivating Fgf / MAPK by exposure to Fgf4 + Heparin showed 60% and 50% of structures forming XEn / EPiCs, at 0-72h and 48-120h windows, respectively. This suggests a larger sensitivity to Fgf / MAPK dosing in pre- than in peri-implantation development (not shown).

[0143] Quantification of the overall size of XEn / EPiCs in these conditions did not show a significant difference when compared to the control. A comparison of the area of Epi, PAC, and XEn in the XEn / EPiCs also did not show a significant difference compared to the control in both time windows (FIG. 4F). The algorithm did not perform these measurements in the case of treatment with PD032 and PD17 for 0-72h because the majority of structures showed an EB-like phenotype without XEn cells (FIG. 4D). Overall, the Fgf / MAPK pathway inhibition from 0-72h displayed a higher occurrence of EB-like structures than the treatment from 48-120h, thus emphasizing the 0-72h crucial window for the specification of XEn.

[0144] 4.3 BMP pathway

[0145] Activation of BMP promoted the developmental progression of XEn / EPiCs and its inhibition affected the sorting and epithelialization of XEn

[0146] BMP signaling plays a role in the establishment and sorting of XEn. Exposure of ES cells to BMP4, which binds to the type-1 receptors activating BMP signaling, from 0- 72h showed a comparable percentage of structures forming XEn / EPiCs (75%) to that of the control (70%) (data not shown). In contrast, treatment with BMP inhibitor 0.5 pM Dorsomorphin (Type-1 receptor) (not shown) reduced the yield of XEn / EPiCs to 50% at the expense of amorphous XEn / Epi structures (25% vs. 10% in control) which contain dispersed XEn. A similar effect was observed in the 48-120h treatment. Quantification of the sizes of individual tissue compartments in XEn / EPiCs upon treatment with BMP4 showed that there was a significant increase in the size of Epi (15,000 pm2), and PAC (12,000 pm2) compared to control (11 ,000 pm2 and 10,000 pm2 respectively) (FIG. 5C). BMP4 inhibition with Dorsomorphin at both time windows of treatment showed comparable sizes of Epi, PAC, and XEn as the control (FIG. 5C, FIG. 5E). Interestingly, the XEn in XEn / EPiCs (both 0-72h and 48-120h) showed a more dispersed organization upon Dorsomorphin treatment (FIG. 5C, FIG. 5E), a phenotype also observed in the primary screen (0-120h) (Example 3). Together, the observations in both the time windows of exposure indicated that suppression of BMP signaling affected XEn sorting, aligning with the literature showing similar effects. The marginally increased sizes of Epi, XEn, and PAC in the BMP4 and DM-treated structures should be further evaluated to validate the findings.

[0147] 4.4 Tgfp / Nodal pathway

[0148] Tgfp / Nodal pathway activation did not show a significant effect on the formation of XEn / EPiCs, although XEn sizes showed large variability upon their inhibition

[0149] Nodal signals, through a self-perpetuating loop between the Epi and XEn, are involved in patterning the visceral endoderm. Here, the treatment of ES cells with Nodal pathway activators, Activin A, and Nodal, from 0-72h had minimal impact on the yield of XEn / EPiCs with 68% and 65%, respectively, but was reduced to 45% and 60%, respectively, in 48-120h treatment, in comparison to the control (70%) (not shown). This was also seen with the reduced size of XEn / EPiCs in the 48-120h treatment with Activin A (31 ,400 pm2 vs 32,000 pm2 in the control) (FIG. 5B). The more pronounced activity of Activin A may be related to its independence of co-factors Cripto / Cryptic to activate the receptor, in contrast to Nodal. However, blocking the Nodal and Tgfp pathway with SB43 and A83, respectively, at both time windows, drastically reduced the yield of XEn / EPiCs and displayed a higher yield of amorphous XEn / Epi and XEn / Epi rosettes, respectively (not shown). This suggests a role for Activin / nodal signaling in XEn establishment, in agreement with findings in ESCs that can be differentiated to XEn through a combination of Activin-A and Wnt activation.

[0150] The overall size of XEn / EPiCs upon Nodal treatment from 0-72h was slightly but significantly reduced by 400 pm2 (P<0.05) in comparison to the control (32,000 pm2) (FIG. 5B). On the contrary, inhibition of Tgfb / Nodal signaling by SB43 also led to a reduction in the size of XEn / EPiCs (31 ,500 pm2 vs. 32,000 pm2 in the control, FIG. 5B), which is in line with findings in Nodal- / - embryos that are of smaller size. Together, this suggests a balanced Nodal activity as a requirement for size regulation, in agreement with its graded activity in regulating target genes. There was no significant difference in the size of XEn / EPiCs in other conditions. Quantification of the sizes of individual tissue compartments in XEn / EPiCs treated with Tgfpi , Nodal, and Activin A from 0-72h, showed an increase in Epi with 19,000 pm2, 17,000 pm2, and 17,000 pm2, PAC with 14,000 pm2, 14,500 pm2, and 13,000 pm2, and XEn with 25,000 pm2, 24,500 pm2, and 25,000 pm2, respectively, compared to the control (11 ,000 pm2, 10,000 pm2, 20,000 pm2, respectively) (FIG. 5D). The treatment from 48-120h also showed a similar increasing trend in the sizes of Epi and PAC (FIG. 5F). While there was no significant change in the sizes of Epi, PAC, and XEn in SB43 and A83 treatment, the XEn layer was highly dispersed (FIG. 5F). Together, these findings indicate the important role of Tgfp / Nodal signaling in XEn patterning and PAC formation where inhibition led to a drastic reduction in XEn / EpiCs formation efficiency.

[0151] As a conclusion of all the examples 1-4 above, automated detection and quantification holds high potential for the future since it provides an efficient means to streamline the focus on finding the most relevant and interesting phenotypes in 3D cell structures like embryo models. High-content phenotypic screening of suspension-based 3D complex cell structures, such as some organoids and embryo models, requires a versatile platform that simultaneously allows reproducible and long-term culture, and in situ staining and image-based readouts. The vast amount of image data should be efficiently and (semi-)automatically analyzed and be interpretable for cell biologists. Therefore, we chose a machine-learning-assisted approach to classify the multiple embryo-like phenotypes in our cultures using unbiased feature measurements. Then, in the phenotype of interest, we developed an additional automated analysis pipeline to identify and measure the relevant tissue compartments. Using this approach, screens of different pathway modulators were conducted on a 3D stem cell-derived embryo-like model capturing the patterning (cell differentiation and sorting) and morphogenesis of the Epi and XEn around peri-implantation development. The structures observed in this developmental landscape were classified into five phenotypes based on the developmental features they display. The different phenotypes, namely XEn / EPiCs, XEn / Epi rosettes, XEn / non-polarized Epi, amorphous XEn / Epi, and EB-like, represented (in descending order) the embryonic stages occurring during E3.5 to E5.5 mouse embryo development.

[0152] It was observed that exposure to BMP pathway inhibitors (Dorsomorphin, Noggin) from 0-72h as well as 48-120h, and Tgfp / Nodal inhibitors (SB43, A83) from 0-72h, affected the XEn specification, patterning, and PAC formation. Similarly, exposure to Fgf / MAPK inhibitors (PD032, PD17) from 0-72h, during which the Epi / XEn specification occurs in XEn / EPiCs, completely interrupted the differentiation of XEn, leading to an EB-like phenotype. This is in alignment with previous reports indicating the role of BMP, Fgf / MAPK, and Tgfp / Nodal pathways on the specification and patterning of extra- embryonic lineages in mouse embryos. Further, it opens up the discussion on how incorrect XEn organization restricts the formation and expansion of the pro-amniotic cavity. Some interesting findings from the analysis pipeline were the effects of Rspondin (0-72h), Tgfpi (0-72h), BMP4 (48-120h), Nodal (0-72h, 48-120h), and SB43 (48-120h) displaying a significant increase in Epi thickness. These findings should be further explored for their biology, for example, whether the Epi has an enriched pseudo-stratified alignment of cells, as in vivo embryos, and if the Epi cells change shape or size with the exposure to some factors.

[0153] Wnt has been reported to play a multitude of roles in development depending on the spatiotemporal expression and activator-inhibitory patterns. During the 0-72h window, the Epi transitions towards a state of rosette-stage pluripotency and becomes a polarized epithelium. Activating the Wnt pathway using Chir led to reduced yields of XEn / EpiCs and instead higher yields of XEn / Epi rosettes and XEn / non-polarized Epi. This could indicate a delay in the formation of PAC, possibly due to the blockage of the naive to rosette-stage epiblast transition, which is necessary for cavity formation. One of the speculations is that continued Wnt activation, prolongs the naive pluripotency, and hence, shows a reduced yield of XEn / EPiCs as well as a reduced size of the epiblast. Wnt activation sustains pluripotency in mouse ES cells and subsequent Wnt inhibition is required to break the pluripotency into a formative stage epiblast for the post-implantation progression. Inhibition of Wnt by XAV from 0-72h or 48-120h resulted in smaller structures with relatively smaller Epi and sometimes showed a dispersed organization of XEn. This indicates that prolonged Wnt inhibition may affect the developmental progression of these structures causing a higher proportion of amorphous XEn / Epi. Another speculation is that there is a minimum cell number required for the formative stage epiblast to proceed development of the PAC, which, when blocked by the inhibition of Wnt, leads to pre-mature differentiation and thereby, smaller XEn / EPiCs.

[0154] In summary, Wnt, Fgf / MAPK, BMP, and Tgfp / Nodal pathways together were found to play a significant role in the pre- to the post-implantation progression of XEn / EPiCs as well as in the development of distinct embryonic tissue types. Overall, the culture of 3D embryo-like structures in this platform and the quantification of different features of the structures using CP and CPA show considerable overlap with literature findings on embryo development. Therefore, these findings in stem cell-based embryo models validate this automated screening platform for efficiently decoding morphogenesis through partitioning signaling pathway activity. Importantly, the screens on XEn / EpiC embryo models provide cues for new hypotheses on the molecular underpinnings of embryo morphogenesis that may be tested in other embryo models or mouse embryos. Moreover, other embryo models with increased complexity, like ETX embryos, can expectedly also be translated to our novel platform. In conclusion, we provide a culture and screening platform that is highly scalable and versatile, which can be applied to different kinds of screening experiments, such as testing the toxicity of drugs, testing the teratogenicity of factors, and identifying disease phenotypes.

[0155] Materials and methods for the examples

[0156] 1. Culture of XEn / EPiC structures within thermoformed microwells a. Preparation of thermoformed microwells

[0157] STATARRAYs (Polystyrene microwell 96-well plate from 300MICRONS GmbH) was used for the 3D culture of mESCs (R. Truckenmuller, S. Giselbrecht, N. Rivron, E. Gottwald, V. Saile, A. van den Berg, M. Wessling, C. van Blitterswijk, Adv Mater 2011 , 23, 1311 , and S. Giselbrecht, T. Gietzelt, E. Gottwald, C. Trautmann, R. Truckenmuller, K. F. Weibezahn, A. Welle, Biomed Microdevices 2006, 8, 191). Before usage, the wells were washed 1x with 70% ethanol and 2x with water. The wells were then incubated with an Anti-adherence solution (StemCell Technologies) for ~20 min at RT inside the laminar flow hood and then washed 3x with PBS and then incubated with fresh PBS until further use. b. Cell culture and reagents

[0158] Mouse embryonic stem cells (mESCs) were expanded on a feeder layer of mouse embryonic fibroblasts (MEFs) on 0.15% gelatin in ES medium containing, Dulbecco's Modified Eagle's Medium High Glucose (Life Technologies) supplemented with 10% fetal bovine serum (FBS, Greiner), 4 mM Glutamax (Life Technologies), 100 U mL-1 penicillin (Life Technologies), 100 mg mL-1 streptomycin (Life Technologies), 10 mM non-essential amino acids (Life Technologies), and freshly supplemented with 0.1 mM 2-mercaptoethanol (Life Technologies), 1000 U mL-1 leukemia inhibitory factor (LIF, Life Technologies), 3 pM CHIR99021 (GSK3P inhibitor, Axon Medchem) and 1 pM PD0325901 (MEK / ERK inhibitor, Sigma Aldrich). The cells were refreshed every 2 days, passaged on the 3rd day with 0.5mL Accutase for 3 min, and seeded at a density of 10,000 cells / cm2 along with 0.5 uM Y27632. For all the experiments in this article, mES:: Gata6: H2B: Venus reporter line was used (mESCs comprising an H2B-Venus reporter under the regulatory elements of Gata6). c. Culture of XEn / EPiCs

[0159] XEn-ind medium containing advanced N2B27 medium was prepared as follows: 46.3% Advanced DMEM / F12 (Invitrogen), 46.3% Neurobasal (Invitrogen), 1 % N2 supplement (Invitrogen), 2% B27 supplement (Invitrogen), 1% Glutamax, 1% Non-Essential Amino Acids, 1.5% BSA (Sigma), 0.5% HEPES, 0.4% Sodium Pyruvate), 3 pM CHIR99021 , 0.1 mM 2-mercaptoethanol, 1mM 8Br-cAMP, 25ug / mL Fgf4, 1 ug / mL Heparin, 10 nM Retinoic acid, 1 uM Y27632, 5% FBS, and 1% Pen / Strep. 25 pL of the medium was added to all the wells and placed inside the incubator. mESCs were washed 2x with PBS to remove the dead cells, treated with 0.5 mL Accutase for 3 min, centrifuged at 200g for 5 min, and the pellet was suspended in 7 mL MEFs medium containing DMEM (high glucose, Sodium pyruvate, and Glutamax) with 15% FBS. MEFs depletion involved seeding the cell suspension first onto a noncoated T75 flask, to allow MEFs to adhere to the plate, for 20-30 min. The cell suspension was then collected from the flask without mixing, centrifuged again, and the pellet re-suspended in 1 mL of adv. N2B27 medium. After counting, the cell suspension was made with XEn-ind medium at a concentration of 160,000 cells / mL and 50 pL of it was added to a tube containing 150 pl of the XEn-ind medium. Finally, 200 pl of cell suspension was added to each well and placed back in the incubator for the cells to settle. This gave an average of about 18 cells per microwell in the STATARRAY. In all the screening experiments, the positive control was the XEn- induced medium with DMSO and the negative control was 2i / LIF. At 24h, the wells were washed 2x with advanced N2B27 medium (+0.2% P-ME+ 1 % Pen / Strep) to remove the XEn-induction medium, and every 24h later, the medium was refreshed. 120h after seeding, XEn / EPiCs structures were formed. d. Fixing and staining

[0160] At 120h, the structures were washed 3x with PBS and fixed with a fixing solution containing ice-cold 2% PFA and 0.1 % Glutaraldehyde for 30 min at RT. The wells were then washed 3x with PBS and continued for further staining or stored at 4 °C.

[0161] Staining was performed by permeabilizing the structures with 0.1 % Triton-x 100 or 1% Tween-20 for 30 mins at RT. Then, the wells were treated with DAPI (1 :300), Phalloidin (1 :300), and WGA (1 :300) in 0.1% Triton-x 100 and incubated for 30 mins at RT. The wells were then washed 3x with PBS and stored at 4 °C or imaged under the microscope. e. Imaging

[0162] The structures were imaged using a Live cell confocal imaging microscope, an inverted Nikon Ti-E microscope, equipped with environmental control, and a CrestOptics X- Light V2 spinning disk unit. The ‘large image’ module was used to achieve a montage of the entire well with all the microwells stitched together. Manual image analysis was performed using NIS software and Imaged (yield and area measurement). f. List of Antibodies:

[0163] 2. CellProfiler pipeline creation and setup a. Measurement of features

[0164] The analysis of 3D structures was performed using CellProfiler (CP) v4.1.3 and

[0165] CellProfiler Analyst (CPA) v3.0.4 (Broad Institute). Each structure within a montage image of a well was identified as an individual object and each object was then quantified for its area, size, shape, texture, intensity, and intensity distribution including Zernicke features. The obtained measurements were then exported into a database file for use with CPA and a spreadsheet for further analysis. b. Yield quantification using CellProfiler Analyst

[0166] The output from the CP pipeline was imported into CPA and the classifier module was used to perform a supervised machine-learning algorithm based on the ‘fast-gentle boosting' scoring method to classify the different phenotypes of objects observed. There was an additional ‘others' bin, where the experimental artifacts and out-of-focus images were sorted out. Most of the images did not have a lot of structures in the ‘others' bin. The software was then trained to generate a set of rules based on the measurements from CP. After a satisfactory training set, the images were scored based on the number of objects falling into each bin. c. Quantification of the area using CellProfiler

[0167] The rules generated after the classifier module training were imported back into the CP pipeline under the “Filter objects” module and the objects falling into each category can be filtered out in each image. Further measurements were performed on the resulting objects.

[0168] 3. Identification and quantification of individual compartments within structures a. Area of epiblast (Epi) with cavity: The area of Epi with pro-amniotic cavity was obtained by using the ‘Image Math’ module to subtract the Gata6 channel from the DAPI channel. b. Area of pro-amniotic cavity (PAC): The PAC compartment was obtained by using the ‘Image Math’ module to multiply the area of the Epi with cavity with F-Actin channel. c. Area of Extraembryonic endoderm (XEn) layer: The area of XEn was determined by subtracting the area of Epi with cavity from the overall area of the DAPI channel.

[0169] 4. Library of signaling pathway modulators

[0170] Table 1 : Library of signaling pathway modulators

[0171]

Claims

CLAIMS1. An in vitro method for the preparation of a culture of pre-implantation and postimplantation embryo-like three-dimensional cell aggregates, the method comprising:(a) providing a source of pluripotent and / or naive embryonic stem-cells;(b) a first differentiation stage in which the pluripotent and / or naive embryonic stemcells are cultured in a container of micrometric dimensions at an initial ratio of about 1 to 100 cells per container, preferably from 5 to 50 cells per container, more preferably from 10 to 25 cells per container, with an epiblast (Epi) / extraembryonic endoderm (XEn) cell induction culture medium comprising heparin and p-mercaptoethanol, for a period of time suitable to provide induced-embryonic stem-cell aggregates; and(c) a second differentiation stage, in which in the same container of micrometric dimension the cell induction culture medium of the previous step is replaced by a cell culture medium suitable for embryonic stem-cell expansion and differentiation, said medium comprising p-mercaptoethanol, and the induced-cell aggregates of step (b) are cultured for a period of time suitable to provide the formation of the preimplantation and post-implantation embryo-like three-dimensional cell aggregates, preferably for a period of time suitable to provide embryo-like cell aggregates comprising an embryo-like epiblast (Epi) compartment and an extraembryonic endoderm-like layer (XEn) encasing the said Epi, the embryo-like cell aggregates preferably comprising in addition a pro-amniotic-like cavity (PAC).

2. The in vitro method according to claim 1 , wherein in step (b) the suitable period of time comprises culturing with the pluripotent and / or naive embryonic stem-cells from 12 to 24 hours, preferably 24 hours, and in step (c) the suitable period of time comprises culturing the induced- embryonic stem-cell aggregates to reach a total time of cell culturing including steps (b) and (c) from 120 hours to 150 hours, preferably 120 hours.

3. The in vitro method according to any one of claims 1-2, in which the Epi / XEn cell induction culture medium comprises an amount of heparin from 0.7 pg / ml to 1.5 pg / ml, preferably 1.0 pg / ml; an amount of p-mercaptoethanol from 75 pM to 120 pM,preferably 100 pM; and preferably an amount of fetal bovine serum from 3.0 % v / v to 7 % v / v, preferably 5 % v / v.

4. The in vitro method according to any one of claims 1-3, in which the Epi / XEn cell induction culture medium comprises an amount of heparin from 0.7 pg / ml to 1.5 pg / ml, preferably 1.0 pg / ml; an amount of p-mercaptoethanol from 75 pM to 120 pM, preferably 100 pM; and an amount of fetal bovine serum from 3.0 % v / v to 7 % v / v, preferably 5 % v / v.

5. The in vitro method according to any one of claims 1-4, in which the cell culture medium suitable for embryonic stem-cell expansion and differentiation comprises p- mercaptoethanol at a concentration in the cell culture medium from 75 pM to 120 pM, preferably 100 pM.

6. The in vitro method according to any one of claims 1-5, which comprises:(a) providing a source of isolated pluripotent and / or naive embryonic stem-cells;(b) a first differentiation stage in which the pluripotent and / or naive embryonic stemcells are cultured in a container of micrometric dimensions at an initial ratio of about 1 to 100 cells per container, preferably from 5 to 50 cells per container, more preferably from 10 to 25 cells per container, with an epiblast (Epi) / extraembryonic endoderm (XEn) cell induction culture medium comprising heparin and p-mercaptoethanol, for a period of time suitable to provide induced-embryonic stem-cell aggregates, wherein the Epi / XEn cell induction culture medium comprises an amount of heparin from 0.7 pg / ml to 1.5 pg / ml, preferably 1.0 pg / ml; an amount of p-mercaptoethanol from 75 pM to 120 pM, preferably 100 pM; and an amount of fetal bovine serum from 3.0 % v / v to 7 % v / v, preferably 5 % v / v; and(c) a second differentiation stage, in which in the same container of micrometric dimension the cell induction culture medium of the previous step is replaced by a cell culture medium suitable for embryonic stem-cell expansion and differentiation, said medium comprising p-mercaptoethanol at a concentration in the cell culture medium from 75 pM to 120 pM, preferably 100 pM, and the induced-cell aggregates of step (b) are cultured for a period of time suitable to provide the formation of the preimplantation and post-implantation embryo-like three-dimensional cell aggregates,preferably for a period of time suitable to provide embryo-like cell aggregates comprising an embryo-like epiblast (Epi) compartment and an extraembryonic endoderm-like layer (XEn) encasing the said Epi, the embryo-like cell aggregates preferably comprising in addition a pro-amniotic-like cavity (PAC).

7. The in vitro method according to any one claims 1-6, which comprises one or more steps of cell culture refreshment carried out with a cell culture medium suitable for embryonic stem-cell expansion and differentiation that comprises p-mercaptoethanol at a concentration in the cell culture medium from 75 pM to 120 pM, preferably 100 pM.

8. The in vitro method according to any one of claims 1-7, wherein the container of micrometric dimensions is a container transparent to the wavelengths of the visible light spectrum , preferably a container made from a transparent polymer selected from polystyrene, cyclic olefin (co)polymer / polymer, polymethylmethacrylate, fluorinated ethylene propylene and polycarbonate, preferably polystyrene.

9. The in vitro method according to any one of claims 1-8, wherein the container of micrometric dimensions is a microwell of a multiple well cell culture support, and the method comprises seeding the pluripotent and / or naive embryonic stem cells at a ratio from about 1 to 100 cells per microwell, preferably from 5 to 50 cells per microwell, more preferably from 10 to 25 cells per microwell.

10. The in vitro method according to claim 1-9, which is a method for the continuous high-throughput culture of pre-implantation and post-implantation embryo-like three- dimensional cell aggregates and in situ imaging, and which comprises the step of visualizing the aggregates in one or more microwells of a multiple well cell culture support of a polymer which is transparent to the wavelengths of the visible light spectrum with a microscopy technique, and the step of obtaining an image from the visualized aggregates and the computer-processing of the said image to provide a processed image, preferably said processed image differentially showing Epiblast-like (Epi) compartment, the extraembryonic endoderm-like (XEn), and the pro-amniotic cavity-like (PAC).

11. The in vitro method according to any one of claims 1-10, which is an in vitro method for the analysis of pre- and post-implantation embryo morphogenesis and differentiation, that comprises the additional step of adding at different times or at different time-windows of the cell culturing:(a) one or more modulator compounds of one or more cell signaling pathways; and / or(b) one or more candidate test modulator compounds, physical and / or mechanical means.

12. A culture of pre-implantation and post-implantation embryo-like three-dimensional cell aggregates, which comprises at least 75 %, preferably from 75 % to 99 %, of embryo-like cell aggregates that comprise an epithelialized extraembryonic endoderm (XEn) layer encasing a polarized epiblast-like compartment (Epi) with a pro-amniotic- like cavity (PAC), the percentages in relation to the total amount of pre-implantation and post-implantation embryo-like cell three-dimensional aggregates.

13. The culture according to claim 12, which is obtainable by the method as defined in any one of claims 1 to 10.

14. The culture according to any one of claims 12-13, which is provided on a multiple well cell culture support, which is transparent to the wavelengths of the visible light spectrum, and that comprises one embryo-like three-dimensional cell aggregate per well.

15. A method for the screening of a candidate agent or of a physical and / or mechanical mean that modulates embryonic development, the method comprising:(a) providing a culture of pre-implantation and post-implantation embryo-like cell three- dimensional aggregates as defined in any one of claims 12-14;(b) contacting the culture with a candidate agent or a physical and / or mechanical mean under condition that allow the interaction of the candidate or physical and / or mechanical mean with the culture; and(c) determining if the contacting of step (b) modulates the embryonic-like development.

16. Mammalian epiblast (Epi) / extraembryonic endoderm (XEn) cell induction culture medium that comprises heparin, p-mercaptoethanol, and fetal bovine serum, and one or more of a fibroblast growth factor, preferably in which the amount of heparin is from 0.7 pg / ml to 1.5 pg / ml, preferably 1 .0 pg / ml; the amount of p-mercaptoethanol is from 75 pM to 120 pM, preferably 100 pM; and the amount of fetal bovine serum is from 3.0 % v / v to 7 % v / v, preferably 5 % v / v.

17. The mammalian epiblast (Epi) / extraembryonic endoderm (XEn) cell induction culture medium according to claim 16, in which the amount of heparin is from 0.7 pg / ml to 1.5 pg / ml, preferably 1.0 pg / ml; the amount of p-mercaptoethanol is from 75 pM to 120 pM, preferably 100 pM; and the amount of fetal bovine serum is from 3.0 % v / v to 7 % v / v, preferably 5 % v / v.

18. A cell culture system or kit for the preparation of pre-implantation and postimplantation embryo-like three-dimensional cell aggregates, that comprises:(a) a multiple well cell culture support for cell culturing or means for preparing it, which is transparent to the wavelengths of the visible light spectrum;(b) a cell induction culture medium as defined in any one of claims 16-17; and optionally one or more of(c) a cell culture medium suitable for embryonic stem-cell expansion and differentiation comprising p-mercaptoethanol at a concentration in the cell culture medium from 75 pM to 120 pM, preferably 100 pM;(d) a source of pluripotent and / or naive embryonic stem-cells;(e) one or more compounds or compositions for cell aggregate fixation for imaging, and / or one or more compounds or compositions for cell aggregate imaging;(f) a computer readable software for image analysis, the software preferably for performing a method for classifying embryo-like three-dimensional cell aggregates by creating and analyzing a digital image with labeled structures of embryo development stages, said labeled structures preferably selected from Epiblast-like (Epi), extraembryonic endoderm-like (XEn), pro-amniotic cavity-like (PAC);(g) instructions for the use of the system or kit, preferably comprising instructions to carry out the method as defined in any one of claims 1-11 and / or 15.

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