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WO2024261290A1 - Procédé de culture de structures de type embryon - Google Patents

Procédé de culture de structures de type embryon Download PDF

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Publication number
WO2024261290A1
WO2024261290A1 PCT/EP2024/067519 EP2024067519W WO2024261290A1 WO 2024261290 A1 WO2024261290 A1 WO 2024261290A1 EP 2024067519 W EP2024067519 W EP 2024067519W WO 2024261290 A1 WO2024261290 A1 WO 2024261290A1
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cell
xen
embryo
epi
aggregates
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Erik Jacob Vrij
Stefan Giselbrecht
Clemens Antoni Van Blitterswijk
Vinidhra SHANKAR
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Universiteit Maastricht
Academisch Ziekenhuis Maastricht
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Universiteit Maastricht
Academisch Ziekenhuis Maastricht
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/44—Thiols, e.g. mercaptoethanol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers

Definitions

  • 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.
  • embryo models exhibit significant morphological heterogeneity, which hinders inferring their relevance to the study of natural embryo development.
  • 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.
  • 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.
  • ECM extracellular matrix
  • embryonic stem cells i.e., pluripotent and/or naive
  • embryonic stem cells i.e., pluripotent and/or naive
  • 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).
  • Epi embryo-like epiblast
  • PAC pro-amniotic-like cavity
  • XEn extraembryonic endoderm-like layer
  • the structures grow and develop in a culture support that is transparent to wavelengths of the visible light spectrum.
  • 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.).
  • 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:
  • 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
  • Epi epiblast
  • XEn extraembryonic endoderm
  • step (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).
  • Epi embryo-like epiblast
  • XEn extraembryonic endoderm-like layer
  • 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).
  • XEn epithelialized extraembryonic endoderm
  • Epi polarized epiblast-like compartment
  • PAC pro-amniotic-like cavity
  • the invention also provides a culture of pre-implantation and post-implantation embryo-like cell three-dimensional aggregates obtained or obtainable from the same.
  • 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.
  • 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.
  • 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;
  • step (c) determining if the contacting of step (b) modulates the embryonic-like development.
  • 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.
  • 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:
  • 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;
  • a source of pluripotent and/or naive embryonic stem-cells preferably mammal and more preferably human embryonic stem cells
  • a source of pluripotent and/or naive embryonic stem-cells preferably mammal and more preferably human embryonic stem cells
  • one or more compounds or compositions for cell aggregate fixation for imaging, and/or one or more compounds or compositions for cell aggregate imaging preferably mammal and more preferably human embryonic stem cells
  • 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);
  • This scalable culture and analysis platform provides a unique opportunity to quantitatively and systematically study effects of pathway modulators on early embryonic development.
  • 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.
  • Epi peri-implantation epiblast
  • XEn extraembryonic endoderm development
  • PAC pro-amniotic cavity
  • 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.
  • 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,
  • 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%.
  • 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.
  • 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.
  • 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.
  • Cell density may be defined as the number of initial seeded cells (i.e., ESC) per volume unit of the container.
  • the units are cells/volumetric measure (e.g., cubic milimeters-mm3).
  • a cell density indicated as from 2 x 10' 5 cell/mm 3 to 2 x 10' 3 cell/mm 3 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' 5 cell per cubic millimetre of the container, to 2 x 10' 3 cells 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.
  • a density expressed as about 18 cells per container of micrometric dimensions supposes about 9 x 10' 5 cell/mm 3 in a microwell of 300 pm of diameter and a depth or volume around 0.5 x 10 8 pm 3 .
  • 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 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.
  • Embryonic stem cells(ESC) 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.
  • endoderm gut, lungs, yolk sac
  • mesoderm muscle, skeleton, blood vascular, urogenital, dermis
  • ectoderm nervous, sensory, epidermis
  • PSC pluripotent stem cells
  • stem cells e.g. ESC
  • stem cells in particular human
  • ESC stem cells
  • 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.
  • iPSC Induced pluripotent stem cells
  • 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.
  • ESCs embryonic stem cells
  • 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).
  • the three germ layers e.g. embryoid bodies-EB, or the aimed embryo-like cell aggregate XEn/EPiC
  • XEn/EPiC embryo-like cell aggregates
  • XEn/EPiC embryo-like cell aggregates
  • XEn/EPiC embryo-like cell aggregates
  • XEn/EPiC embryo-like cell aggregates
  • XEn/EPiC embryo-like cell aggregates
  • 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
  • amorphous XEn/Epi structures with disorganized gata6 expressing XEn).
  • 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).
  • 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.
  • EBs embryoid bodies
  • embryo-like cell aggregates are not able to form or develop into mammal (e.g.
  • 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 10 6 pm 3 to 1.0 x 10 10 pm 3 , more in particular from 1.0 x 10 7 pm 3 to 1.0 x 10 9 pm 3 , even more in particular 0.1 x 10 8 pm 3 to 5.0 x 10 8 pm 3 , and most in particular and preferred from 0.25 x 10 8 pm 3 to 0.75 x 10 8 pm 3 .
  • 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.
  • 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.
  • 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.
  • An induction medium provides signals that change cell behavior, shape, differentiation, mitotic activity, signal cascades and/or gene expression.
  • 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.
  • candidate agent refers to a molecule that may be screened for, or be identified as, modulating the mammalian embryonic development.
  • agent may, for example, be an inhibitor or enhancer (i.e. , promoter) of the development and may find use in a variety of applications, including therapy.
  • the screening methods will typically be assays which provide for qualitative/quantitative measurements of the activity (i.e., modulation of embryonic development) in the presence of a particular candidate agent.
  • (Candidate) agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts or purified compounds are available or may be produced. Additionally, natural or synthetically produced libraries and compounds can be prepared using conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogues or derivates. (Candidate) agents may also be biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogues or combinations thereof.
  • contacting encompasses that a compound or cells, are in touch with compound or compositions which may then interact with each other.
  • 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.
  • 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.
  • the term “providing”, for example, providing a cell includes preparing, isolating, obtaining, and the like, of such cell.
  • 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:
  • 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
  • step (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).
  • Epi embryo-like epiblast
  • XEn extraembryonic endoderm-like layer
  • 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.
  • PAC pro-amniotic-like cavity
  • 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.
  • 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.
  • 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.
  • 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” 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.
  • 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.
  • 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 10 6 pm 3 to 1.0 x 10 10 pm 3 , more in particular from 1.0 x 10 7 pm 3 to 1.0 x 10 9 pm 3 , even more in particular 0.1 x 10 8 pm 3 to 5.0 x 10 8 pm 3 , and most in particular and preferred from 0.25 x 10 8 pm 3 to 0.75 x 10 8 pm 3 .
  • 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.
  • 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).
  • 1 to 100 cells per microwell will suppose an average of cell densities from 2 x 10' 5 cell/mm 3 to 2 x 10' 3 cell mm 3 .
  • Preferred cell densities corresponding from about 14 to about 25 cells per microwell of a depth of 0.5 x 10 8 pm 3 result in a range from 7 x 10' 5 cell/mm 3 to 12.5 x 10 -5 cell/mm 3 .
  • the most preferred density of about 18 cells per container of micrometric dimensions supposes about 9 x 10' 5 cell/mm 3 .
  • the suitable period of time comprises culturing the embryonic stem-cells from 12 to 24 hours, preferably 24 hours
  • 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.
  • 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
  • the presence of p-mercaptoethanol and heparin allowed for a high efficiency in the formation of XEn/EPiCs.
  • 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.
  • 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.
  • 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.
  • 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.
  • the Epi/XEn cell induction culture medium is further supplemented with at least a fibroblast growth factor, in particular with FgF4.
  • 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).
  • Chir99021 Chir
  • RA Retinoic Acid
  • Fgf4 fibroblast growth factor 4
  • 8Br-cAMP ROCK inhibitor
  • 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.
  • 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.
  • 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).
  • FBS fetal bovine serum
  • LIF leukemia inhibitory factor
  • the expansion is carried out on mouse embryonic fibroblasts (MEFs) according to the known for the skilled person conventional techniques.
  • the method is one that comprises:
  • 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
  • step (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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • Epi Epiblast-like
  • XEn extraembryonic endoderm-like
  • PAC pro-amniotic cavity-like
  • 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)
  • Epi Epiblast-like
  • XEn extraembryonic endoderm-like
  • the microscopy technique is any one available and commonly found at the laboratory.
  • 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:
  • 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:
  • 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.
  • 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.
  • modulators can be inhibitors of certain signaling pathways or enhancers.
  • 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 Wnt pathway the Wnt pathway
  • the Fgf/MAPK pathway the Fgf/MAPK pathway
  • the BMP pathway the BMP pathway
  • Tgfp/Nodal pathway the Tgfp/Nodal pathway.
  • 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.
  • the modulators are added from 0 to 120 hours.
  • 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.
  • 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.
  • 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.
  • XEn epithelialized extraembryonic endoderm
  • Epi polarized epiblast-like compartment
  • PAC pro-amniotic-like cavity
  • 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.
  • 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.
  • 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.
  • XEn epithelialized extraembryonic endoderm
  • Epi polarized epiblast-like compartment
  • PAC pro-amniotic-like cavity
  • this culture is obtainable or obtained by the method as defined in the previous aspect and embodiments.
  • 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)
  • the culture is provided on a transparent multiple well cell culture support and that comprises one embryo-like three-dimensional cell aggregate per well.
  • 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.
  • this transparent multiple well cell culture support is a transparent multiple microwell support.
  • 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:
  • step (c) determining if the contacting of step (b) modulates the embryonic-like development.
  • 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.
  • the key is that for all these analysis and studies the culturing is seen in process and in situ.
  • 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.
  • 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.
  • 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.
  • 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.
  • the Epi/XEn cell induction culture medium comprises the basic advanced neural induction medium (N2B27), supplemented with the heparin and the p-mercaptoethanol.
  • 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).
  • the Epi/XEn cell induction culture medium is further supplemented with at least a fibroblast growth factor, in particular with FgF4.
  • 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).
  • a cell culture system or kit for the preparation of pre-implantation and post-implantation embryo-like three-dimensional cell aggregates that comprises:
  • 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;
  • a source of pluripotent and/or naive embryonic stem-cells preferably mammal embryonic stem-cells
  • 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);
  • 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.
  • 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.
  • the system that can optionally include a computer readable software for image analysis, is equally applicable independently from the said analysis.
  • the system can provide an image as an input to a remote or independent software analysis service.
  • 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.
  • 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.
  • 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.
  • the system contemplates paraformaldehyde (PFA) and glutaraldehyde as fixing agents.
  • PFA paraformaldehyde
  • glutaraldehyde glutaraldehyde
  • 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.
  • Anti-Laminin antibodies 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.
  • thermoformed microwell screening arrays Statarrays, 300MICRONS, polystyrene
  • the first step 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.
  • 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).
  • XEn epithelialized extraembryonic endoderm
  • Epi epiblast-like compartment
  • PAC pro- amniotic-like cavity
  • 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).
  • 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).
  • XEn i.e. primitive endoderm
  • Sorting of XEn cells towards the surface sorting of XEn cells towards the surface
  • the formation of a continuous XEn layer engulfing the Epi positioning of Gata6+ cells
  • initiation of Epi polarization F-actin-rich center within Epi
  • formation of the pro-amniotic cavity cavity within the Epi
  • 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).
  • 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).
  • 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.
  • XAV Wnt inhibitor
  • LDN and Noggin BMP inhibitors
  • Fgf4 Fgf activator
  • Nodal Nodal activator
  • 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.
  • 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.
  • 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.
  • PD032 MEK/ERK inhibitor
  • PD17 FGF receptor inhibitor
  • 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.
  • 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.
  • 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.
  • Wnt activation affected the formation of PAC while its inhibition affected XEn patterning with a reduction in the size of XEn/EPiCs.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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
  • Nodal signals through a self-perpetuating loop between the Epi and XEn, are involved in patterning the visceral endoderm.
  • Activin A may be related to its independence of co-factors Cripto/Cryptic to activate the receptor, in contrast to Nodal.
  • BMP pathway inhibitors Dorsomorphin, Noggin
  • Tgfp/Nodal inhibitors SB43, A83
  • Wnt has been reported to play a multitude of roles in development depending on the spatiotemporal expression and activator-inhibitory patterns.
  • 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.
  • 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.
  • 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.
  • other embryo models with increased complexity like ETX embryos, can expectedly also be translated to our novel platform.
  • thermoformed microwells 1. Culture of XEn/EPiC structures within thermoformed microwells a. Preparation of thermoformed microwells
  • STATARRAYs Polystyrene microwell 96-well plate from 300MICRONS GmbH
  • 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. Weibe leopard, A. Welle, Biomed Microdevices 2006, 8, 191). Before usage, the wells were washed 1x with 70% ethanol and 2x with water.
  • Mouse embryonic stem cells 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).
  • 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.
  • 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.
  • 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.
  • the positive control was the XEn- induced medium with DMSO and the negative control was 2i/LIF.
  • 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.
  • 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
  • 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).
  • 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.
  • 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

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Abstract

L'invention concerne un procédé in vitro de préparation d'une culture d'agrégats cellulaires tridimensionnels pré-implantation et post-implantation, le procédé comprenant plusieurs étapes d'induction et de différenciation avec des milieux de culture cellulaire particuliers. L'invention concerne également des milieux de culture cellulaire par induction et un système pour l'analyse et l'imagerie à haut débit d'agrégats cellulaires de type embryon.
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WO2018046929A1 (fr) * 2016-09-09 2018-03-15 Zernicka Goetz Magdalena Procédés et compositions pour la co-culture de cellules pluripotentes et extra-embryonnaires

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WO2018046929A1 (fr) * 2016-09-09 2018-03-15 Zernicka Goetz Magdalena Procédés et compositions pour la co-culture de cellules pluripotentes et extra-embryonnaires

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