Methods for producing synthetic embryos

Abstract

A method for producing a synthetic embryo is provided. Thus, the method for producing a synthetic embryo is provided, comprising inducing the expression of a factor that induces differentiation into trophectoderm cells in a subpopulation of naive pluripotent stem cells (PSCs) to obtain trophectoderm primed cells, inducing the expression of a factor that induces differentiation into extraembryonic primitive endoderm cells in a second subpopulation of naive PSCs to obtain extraembryonic primitive endoderm primed cells, and mixing the trophectoderm primed cells and the extraembryonic primitive endoderm primed cells with naive PSCs under conditions that allow the formation of aggregated cells. Also provided are cell products, mixtures, and aggregates, and methods of using them.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 63 / 317,036, filed March 6, 2022, and U.S. Provisional Application No. 63 / 390,695, filed July 20, 2022, the contents of which are incorporated herein by reference in their entireties.

[0002] Sequence Listing An XML file entitled 95053.XML, created on March 6, 2023, consisting of 327,680 bytes, was submitted concurrently with the filing of this application and is incorporated herein by reference.

[0003] FIELD OF THE INVENTION The present invention, in some embodiments thereof, relates to methods for producing synthetic embryos. [Background technology]

[0004] Advanced artificial embryogenesis utilizing in vitro established embryonic stem cell lines and induced pluripotent stem cell lines (iPSCs) while avoiding the use of oocytes and zygotes or blastocysts has become a desired goal in the fields of developmental biology and reproductive medicine. Establishing methods and systems for generating such synthetic embryos (abbreviated as sEmbryo or sEmbryoid, also sometimes referred to as stem cell-derived embryos or embryoid body models (SEMs) or stem cell-derived whole embryoid body models (SWEMs)) outside the uterus can be a powerful tool in basic research, for example, as a framework for investigating the emergence of cell diversity, cell fate determination, and how tissues and organs emerge from single pluripotent cells. Furthermore, they can serve as a source of cells, tissues, embryos, and organs for transplantation, for testing the effects of drugs on embryonic development, and potentially for embryo bioengineering.

[0005] The blastocyst (early mammalian embryo) develops all of the embryonic and extraembryonic tissues, including the placenta. This structure arises at the 64-cell stage, shortly after the embryo loses its "ball of cells" shape and forms a cavity. In this structure, the inner cell mass (ICM) develops into the embryo proper and is positioned above the blastocoel, a hallmark of the blastocyst stage. The cells surrounding the ICM are trophectoderm (TE) cells, which later contribute to the trophoblast extraembryonic placental tissue (the fetal placenta). A portion of the ICM further differentiates into primitive endoderm (PRE), which later gives rise to the extraembryonic yolk sac and membranes during preparation for implantation [Zhang et al. (2020) Front. Bioeng. Biotechnol. 8:781].

[0006] Embryonic stem cell lines (ESCs) and iPSCs in various pluripotent states can be propagated in vitro and can contribute to somatic and / or extraembryonic tissues when injected into a host embryo. For example, mouse naive ESCs or iPSCs (Bayerl et al. Cell Stem Cell (2021) 28(9): 1549-1565.e12, Weinberger et al. Nat Rev Mol Cell Biol (2016) 17(3): 155-69, Manor et al. Curr Opin Genet Dev (2015) 34: 35-45, Shakiba et al. Nat Commun (2015) 16 6:7329, Gafni et al. Nature (2013) 504(7479): 282-6, Geula et al. Science (2015) 347(6225): 1002-6) can function like resident pluripotent cells and contribute to the developing embryo when microinjected into mouse blastocysts (Nicols and Smith Cell Stem Cell (2009) 4(6): 487-92). Conversely, mouse primed pluripotent stem cells (also known as epiblast stem cells (EpiSCs)) grown in N2B27 FGF2 / activin conditions are highly inefficient at generating chimeric embryos and fail to significantly contribute to such embryos when injected into host blastocysts or E7.5 embryos (Alejandro-Castrejon et al. Nature (2021) 593(7857): 119-124).

[0007] Mouse trophoblast stem cells (TSCs) can be established from early mouse embryos (postimplantation E5.5–E7.5) or directly from mouse ESCs in vitro (through a process requiring several passages of growth and selection to establish stable mouse TSC lines, typically involving overexpression of a transgene such as Cdx2). Conventional stabilized mouse TSC lines can be microinjected into blastocysts and contribute slightly to placental development in advanced embryos, but they have not previously been shown to be able to reconstitute the entire placenta and trophectoderm progeny (Posfai et al. Nat Cell Biol (2021) 23(1): 49–60). By injecting mouse naive ESCs or iPSCs using the tetraploid embryo complementation microinjection method, it is possible to generate unique "all-ESC / iPSC" chimeras in which all embryonic somatic cells are derived exclusively from the injected mouse stem cells and all extraembryonic cells are derived exclusively from electrofused cells of the host embryo (Choi et al. Nature (2017) 548(7666): 219-223).

[0008] However, the goal of artificial synthetic embryos is to establish embryo-like structures without any contribution of germ cells, zygotes, or blastocysts. Some studies have attempted to create synthetic embryos by coaggregation of two or three different cell types [see, e.g., Zhang et al. (2020) Front. Bioeng. Biotechnol. 8:781]. For example, Harrison et al. [Science (2017), DOI:10.1126 / science.aa1810] used in vitro-expanded mouse PSCs, non-pluripotent conventional trophoblast stem cells (TSCs), Sozen et al. [Nature Cell Biology volume 20, pages 979-989 (2018)] used in vitro-expanded mouse PSCs, non-pluripotent conventional trophoblast stem cells (TSCs), and non-pluripotent extraembryonic endoderm cells (termed XEN cells), whereas Amadei et al. [Developmental Cell (2021) 56, 366-382] used naive iPSCs, non-pluripotent conventional TSC lines, and mouse ES cells transiently overexpressing the Gata4 transcription factor, which helps promote primitive endoderm fate (instead of using a stable mouse XEN line). However, the resulting embryos were grossly abnormal, failing to progress through or complete gastrulation or initiate organogenesis, and had morphological abnormalities, lacking key features that define an embryo (DOI: 10.1126 / sciadv.add2913). Summary of the Invention

[0009] According to an aspect of some embodiments of the present invention there is provided a method of producing a synthetic embryo, the method comprising: (a) inducing the expression of factors that induce differentiation into trophectoderm cells in a subpopulation of naive pluripotent stem cells (PSCs) to obtain trophectoderm-primed cells; and (b) mixing the trophectoderm-primed cells with naive PSCs under conditions that allow for the formation of aggregate cells; When the factor that induces differentiation into trophectoderm cells is an exogenous factor, the induction of expression includes inducing transient expression that begins within 0 hours to 14 days before mixing and ends within 120 hours after mixing; When the factor that induces differentiation into trophectoderm cells is an endogenous factor, inducing expression includes culturing the subpopulation of naive PSCs under conditions that allow expression of the factor, starting within a period of 14 days to 6 hours before mixing; A method is provided whereby a synthetic embryo is produced.

[0010] According to some embodiments of the invention, the method further comprises inducing expression of a factor that induces differentiation into extraembryonic primitive endoderm cells in a second subpopulation of naive PSCs to obtain extraembryonic primitive endoderm primed cells in (a), and mixing the extraembryonic primitive endoderm primed cells with the cells of (b).

[0011] According to some embodiments of the present invention, when the factor that induces differentiation into extraembryonic primitive endoderm cells is an exogenous factor, the induction of expression comprises induction of transient expression that begins within 0 hours to 14 days before mixing and ends within 120 hours after mixing; The factors that induce differentiation into extraembryonic primitive endoderm cells are endogenous factors, and induction of expression involves culturing a subpopulation of naive PSCs under conditions that allow expression of the factors, starting within 6 hours to 14 days before mixing.

[0012] According to an aspect of some embodiments of the present invention there is provided a method of producing a synthetic embryo, the method comprising: (a) inducing transient expression of an exogenous factor that induces differentiation into trophectoderm cells in a subpopulation of naive pluripotent stem cells (PSCs) to obtain trophectoderm-primed cells; and (b) mixing the trophectoderm-primed cells with naive PSCs under conditions that allow for the formation of aggregate cells; Induction of transient expression begins within the period from 14 days to 0 hours before mixing and ends within 120 hours after mixing. A method is provided whereby a synthetic embryo is produced.

[0013] According to some embodiments of the invention, the method further comprises (a) inducing transient expression of an exogenous factor that induces differentiation into extraembryonic primitive endoderm cells in a second subpopulation of naive PSCs to obtain cells primed towards extraembryonic primitive endoderm, and (b) mixing the cells primed towards extraembryonic primitive endoderm with the cells.

[0014] According to some embodiments of the present invention, naive PSCs, trophectoderm-primed cells, and extraembryonic primitive endoderm-primed cells are mixed simultaneously.

[0015] According to some embodiments of the invention, naive PSCs are mixed with cells primed to extraembryonic primitive endoderm before being mixed with cells primed to trophectoderm.

[0016] According to some embodiments of the invention, trophectoderm cells and / or cells primed to extraembryonic primitive endoderm express pluripotency markers at 0 hours.

[0017] According to some embodiments of the present invention, trophectoderm cells and / or cells primed to extraembryonic primitive endoderm express pluripotency markers upon mixing.

[0018] According to some embodiments of the invention, cells primed towards trophectoderm express trophectoderm markers at 0 hours.

[0019] According to some embodiments of the present invention, cells primed towards trophectoderm express trophectoderm markers upon mixing.

[0020] According to some embodiments of the invention, cells primed to extraembryonic endoderm express extraembryonic primitive endoderm markers at 0 hours.

[0021] According to some embodiments of the present invention, cells primed to extraembryonic endoderm express extraembryonic primitive endoderm markers upon mixing.

[0022] According to an aspect of some embodiments of the present invention, (i) trophectoderm-primed cells, including naive pluripotent stem cells (PSCs), containing an exogenous polynucleotide suitable for transient expression of a factor that induces differentiation into trophectoderm cells; and (ii) a naive PSC.

[0023] According to some embodiments of the present invention, the article of manufacture comprises: (iii) Cells primed to extraembryonic primitive endoderm are further included, including naive pluripotent stem cells (PSCs) containing an exogenous polynucleotide suitable for transient expression of factors that induce differentiation into extraembryonic primitive endoderm cells.

[0024] According to an aspect of some embodiments of the present invention, (i) trophectoderm-primed cells, including naive pluripotent stem cells (PSCs), containing an exogenous polynucleotide suitable for transient expression of a factor that induces differentiation into trophectoderm cells; and (ii) An article of manufacture is provided that includes cells primed to extraembryonic primitive endoderm, including naive pluripotent stem cells (PSCs) that include an exogenous polynucleotide suitable for transient expression of a factor that induces differentiation into extraembryonic primitive endoderm cells.

[0025] According to some embodiments of the present invention, (i), (ii), and (iii) are in a single container.

[0026] According to some embodiments of the present invention, (i), (ii), and (iii) are in separate containers.

[0027] According to an aspect of some embodiments of the present invention, there is provided a method for ex utero culturing of a synthetic embryo, comprising mixing cells of an article of manufacture under conditions that allow for the formation of aggregated cells, the method comprising inducing transient expression of a factor beginning within a period of 14 days to 0 hours before mixing and ending within 120 hours of mixing, thereby producing a synthetic embryo.

[0028] According to an aspect of some embodiments of the present invention there is provided a mixture or aggregate obtainable by the method.

[0029] According to an aspect of some embodiments of the present invention, (i) naive pluripotent stem cells (PSCs), (ii) cells primed to trophectoderm, including stem cells that express endogenous trophectoderm markers and endogenous pluripotency markers; and (iii) a mixture or aggregate comprising cells primed to extraembryonic primitive endoderm, the cells comprising stem cells expressing endogenous extraembryonic primitive endoderm markers and endogenous pluripotency markers, is provided; The mixture or aggregate is not structurally organized like a natural embryo at any stage of development, and The mixture or aggregate is capable of forming a synthetic embryo that is structurally organized like a natural embryo at least at the post-implantation egg cylinder or bilayered scutellum stage of development.

[0030] According to an aspect of some embodiments of the present invention, (i) trophectoderm-primed cells, including stem cells that express exogenous factors that induce differentiation into trophectoderm cells, endogenous trophectoderm markers, and endogenous pluripotency markers; and (ii) A mixture or aggregate containing naive pluripotent stem cells (PSCs) is provided.

[0031] According to some embodiments of the present invention, the mixture or aggregate is (iii) The cells further include cells primed to extraembryonic primitive endoderm, including stem cells that express exogenous factors that induce differentiation into extraembryonic primitive endoderm cells, endogenous extraembryonic primitive endoderm markers, and endogenous pluripotency markers.

[0032] According to some embodiments of the invention, factors that induce differentiation into extraembryonic primitive endoderm cells comprise Gata4, Gata6, and / or SOX17.

[0033] According to some embodiments of the invention, factors that induce differentiation into extraembryonic primitive endoderm cells include Gata4 and / or Gata6.

[0034] According to some embodiments of the invention, inducing expression of a factor that induces differentiation into extraembryonic primitive endoderm cells comprises culturing the second subpopulation of naive PSCs with a factor that induces differentiation into extraembryonic primitive endoderm cells, starting within 14 days to 6 hours before mixing.

[0035] According to some embodiments of the invention, the method further comprises contacting the second subpopulation of naive PSCs with a factor that induces differentiation into extraembryonic primitive endoderm cells within 0 hours to 14 days prior to mixing.

[0036] According to some embodiments of the present invention, the factor that induces differentiation into extraembryonic primitive endoderm cells is selected from the group consisting of a GSK-3 inhibitor, a WNT ligand, heparin, FGF2, FGF4, PDGF, leukemia inhibitory factor (LIF), and insulin.

[0037] According to some embodiments of the invention, the factor that induces differentiation into extraembryonic primitive endoderm cells is selected from the group consisting of a GSK-3 inhibitor, a WNT ligand, heparin, FGF2, and FGF4.

[0038] According to some embodiments of the invention, the factor that induces differentiation into trophectoderm cells is selected from the group consisting of Cdx2, Gata3, and Gata2.

[0039] According to some embodiments of the invention, inducing expression of a factor that induces differentiation into trophectoderm cells comprises culturing a second subpopulation of the naive PSCs with a factor that induces differentiation into extraembryonic primitive endoderm cells, starting within a period of 14 days to 6 hours before mixing.

[0040] According to some embodiments of the invention, the method further comprises contacting the subpopulation of naive PSCs with a factor that induces differentiation into trophectoderm cells within a period of 14 days to 0 hours before mixing.

[0041] According to some embodiments of the present invention, the factor that induces differentiation into trophectoderm cells is selected from the group consisting of a TGFR inhibitor, an FGFR inhibitor, a MEK / ERK inhibitor, BMP4, a JAK inhibitor, a ROCK pathway inhibitor, FGF4, FGF2, heparin, a sumoylation inhibitor, a histone deacetylase inhibitor, a HIPPO signaling pathway inhibitor, and a factor that induces YAP nuclear translocation.

[0042] According to some embodiments of the present invention, the factor that induces differentiation into trophectoderm cells is selected from the group consisting of FGF4, FGF2, heparin, a sumoylation inhibitor, a histone deacetylase inhibitor, a HIPPO signaling pathway inhibitor, and a factor that induces YAP nuclear translocation.

[0043] According to some embodiments of the invention, transient expression is achieved by introduction of a polynucleotide encoding the factor.

[0044] According to some embodiments of the invention, the polynucleotide is DNA.

[0045] According to some embodiments of the invention, expression of the polynucleotide is regulated by an inducible promoter.

[0046] According to some embodiments of the invention, the introducing is by transient transfection.

[0047] According to some embodiments of the invention, the polynucleotide is RNA.

[0048] According to some embodiments of the present invention, induction of transient expression is initiated within a period of 120 hours to 0 hours before mixing.

[0049] According to some embodiments of the present invention, induction of transient expression is initiated within a period of 48 hours to 0 hours before mixing.

[0050] According to some embodiments of the present invention, induction of transient expression is initiated within a period of 24 hours to 0 hours before mixing.

[0051] According to some embodiments of the present invention, induction of transient expression is initiated within a period of 48 hours to 12 hours before mixing.

[0052] According to some embodiments of the present invention, induction of transient expression is completed within 48 hours of mixing.

[0053] According to some embodiments of the invention, induction of transient expression is carried out for 96 hours or less.

[0054] According to some embodiments of the present invention, induction of expression of endogenous factors is initiated within a period of 10 days to 1 day before mixing.

[0055] According to some embodiments of the present invention, the ratio of naive PSCs to cells primed to trophectoderm is 1:1 to 1:5.

[0056] According to some embodiments of the invention, the ratio of naive PSCs to trophectoderm-primed cells is 1:3.

[0057] According to some embodiments of the present invention, the ratio of naive PSCs to cells primed to primitive endoderm is 1:1 to 2:1.

[0058] According to some embodiments of the invention, the ratio of naive PSCs to cells primed towards primitive endoderm is 1:1.

[0059] According to some embodiments of the invention, the method further comprises, after (b), culturing the aggregated cells ex utero.

[0060] According to an aspect of some embodiments of the present invention there is provided a method of ex utero culturing a synthetic embryo, the method comprising ex utero culturing a mixture or aggregate.

[0061] According to some embodiments of the present invention, the culturing is carried out by static culturing followed by dynamic culturing.

[0062] According to some embodiments of the present invention, static culture is carried out until the embryo reaches at least the early post-implantation egg cylinder stage or the bilayered blastocyst stage.

[0063] According to some embodiments of the present invention, dynamic culture begins at the latest when the embryo reaches the early somite stage.

[0064] According to an aspect of some embodiments of the present invention, there is provided a method for culturing a synthetic embryo ex utero, comprising culturing an aggregate-stage synthetic embryo ex utero by static culture followed by dynamic culture, wherein the static culture is performed until the embryo reaches at least the early post-implantation egg cylinder stage or the bilayered blastocyst stage, and the dynamic culture is initiated at the latest when the embryo reaches the early somite stage.

[0065] According to some embodiments of the present invention, dynamic culturing comprises shaking culturing, roller culturing, or a sequential combination thereof.

[0066] According to some embodiments of the present invention, the dynamic cultivation is carried out under high pressure greater than 0.1 pounds per square inch (psi) and less than 10.2 psi.

[0067] According to some embodiments of the present invention, the dynamic cultivation is carried out under high pressure greater than 5 pounds per square inch (psi) and less than 10.2 psi.

[0068] According to some embodiments of the present invention, the culture is carried out in an atmosphere containing 10 to 40% oxygen.

[0069] According to some embodiments of the invention, the culture is carried out in a medium containing serum or serum replacement in increasing concentrations throughout the culture.

[0070] According to some embodiments of the invention, culture is carried out in a medium containing at least 15% serum or serum replacement from the start until the embryo reaches the early post-implantation egg cylinder stage or bilayered blastocyst stage, and thereafter in a medium containing at least 30% serum or serum replacement.

[0071] According to some embodiments of the invention, the culturing is carried out in a medium comprising human serum for at least a portion of the culture.

[0072] According to some embodiments of the invention, the method further comprises implanting the synthetic embryo into a uterus.

[0073] According to some embodiments of the invention, the cell is a mammalian cell.

[0074] According to some embodiments of the invention, the mammalian cell is a mouse cell.

[0075] According to some embodiments of the invention, the mammalian cell is a human cell.

[0076] According to some embodiments of the invention, the mammalian cell is a non-human primate cell.

[0077] According to some embodiments of the invention, the mammalian cell is a livestock cell.

[0078] According to an aspect of some embodiments of the present invention there is provided a synthetic embryo obtainable by the method.

[0079] According to one aspect of some embodiments of the present invention, there is provided a method for obtaining cells primed to extraembryonic primitive endoderm, the method comprising: + GATA6 + OCT4 + Nanog - A method is provided that includes culturing naive PSCs in a basal medium lacking activin A under conditions that allow differentiation into cells, thereby obtaining cells primed towards extraembryonic primitive endoderm.

[0080] According to some embodiments of the invention, the culturing is carried out for at least 1 day.

[0081] According to some embodiments of the invention, the culturing is carried out for up to 10 days.

[0082] According to some embodiments of the invention, the cells are + GATA6 + OCT4 + Nanog - They can be propagated in culture for at least five passages while maintaining their phenotype.

[0083] According to some embodiments of the present invention, the basal medium is supplemented with at least one of a GSK-3 inhibitor, leukemia inhibitory factor (LIF), and insulin.

[0084] According to some embodiments of the present invention, the basal medium is supplemented with at least one of a GSK-3 inhibitor, heparin, FGF4, PDGF, and BSA.

[0085] According to some embodiments of the invention, the cells are human cells.

[0086] According to some embodiments of the invention, the cell is a mouse cell.

[0087] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.Although methods and materials similar or equivalent to those described herein can be used to practice or test embodiments of the present invention, exemplary methods and / or materials are described below.In case of conflict, the present patent specification, including definitions, will prevail.In addition, the materials, methods, and examples are merely illustrative and are not necessarily intended to be limiting.

[0088] Some embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings. With specific reference now made in detail to the drawings, it is emphasized that the details shown are by way of example only and for the purpose of illustratively discussing embodiments of the invention. In this regard, it will become apparent to those skilled in the art how embodiments of the present invention may be practiced, upon reading the description in conjunction with the drawings.

[0089] The drawings are as follows: [Brief explanation of the drawings]

[0090] [Figure 1] Figure 1 shows immunostained images of mouse TSC line #1, established from E6.5 mouse embryos at passage 3 (day 14 after establishment), demonstrating the expression of the TSC markers Cdx2 and Gata3. DAPI was used for counterstaining. [Figure 2] Figure 2 shows immunostained images of mouse TSC line #1 at passage 5 (day 25 after establishment), demonstrating expression of the TSC markers Cdx2, TBR2, Gata3, and TFAP2C, but not Oct4 or Nanog. DAPI was used for counterstaining. [Figure 3]Figure 3A-B shows immunostained images of the parental ESC line (Figure 3A) and the mouse TSC line #2 established after transient knockdown of Oct4 in the parental ES line (Figure 3B) at passage 3 (approximately day 12 from the start of the protocol), demonstrating that the established TSC lines express the TSC markers Cdx2 and Gata3 but not Oct4. DAPI was used for counterstaining. [Figure 4] Figure 4 shows immunostained images of mouse TSC line #2 at passage 5 (day 30 from the start of the protocol), demonstrating expression of the TSC markers Cdx2, TBR2, Gata3, and TFAP2C, but not Oct4 or Nanog. DAPI was used for counterstaining. [Figure 5] Figure 5 shows immunostained images of mouse TSC line #3 at passage 4 (approximately 24 days after transfection) demonstrating expression of the TSC markers Cdx2, Gata3, and TFAP2C, but not Oct4. DAPI was used for counterstaining. [Figure 6A] Figure 6A shows immunostained images of mouse XEN strain #7 at passage 5, demonstrating expression of the XEN markers Foxa2 and Gata4, but not Oct4 or Nanog. DAPI was used for counterstaining. [Figure 6B] Figure 6B shows the RNA expression levels of XEN markers Gata6, PDGFRA, Sox17, and Gata4 in the mouse XEN line, XEN-ICR clone #7 and XEN-Prep1, as determined by real-time PCR analysis. Mouse V6.5 naive ESCs grown under 2i-LIF conditions were used as a reference control (value set to 1). [Figure 7-1]Figure 7A-C demonstrates that coaggregation of naive mouse PSCs (V6.5-GFP), mouse XEN cells (mouse XEN line #7 cells), and mouse TSCs (mouse TSC line #1 cells) followed by ex utero culture does not result in the generation of organized embryos. Figure 7A is a schematic representation of the protocol. Aggregation Medium (AM): 1:1 DMEM-F12 (Thermo, 11320033) and Neurobasal (Thermo, 21103049), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin, 1x NEAA, 1x N2 supplement (Thermo, 17502048), 1x B27 supplement (Thermo, 17504044), 0.1mM beta-mercaptoethanol (Thermo, 31350010), and 0.45% BSA (Sigma-Aldrich, A7979) (500ml total). IVC1 medium: Modified DMEM / F12 (Gibco, 12634010), 1x Glutamax, 1x penicillin / streptomycin, 1x ITS-X (Gibco, 51500056), 8 nM beta-estradiol, 20 ng / ml progesterone, 25 μM N-acetyl-L-cysteine, and serum as described in the scheme [total 500 ml].EUCM: 25% DMEM (Gibco, 11880), 25% HAS (human adult serum or human umbilical cord serum), 50% rat serum (RAS), 1x Glutamax (Gibco, 35050038), 2 mM HEPES (Gibco, 15630056), 1x penicillin / streptomycin. Figure 7B shows an image of a representative embryoid body aggregate on day 4 of the protocol, immunostained for TBR2 (red), which characterizes the trophectoderm lineage, Gata4, which characterizes the primitive endoderm lineage, and GFP signal, which characterizes ESCs contributing to the epiblast. Figure 7C shows an image or representative structure on day 7 of the protocol, demonstrating that there is no organized embryo within the yolk sac, and no signs of the emergence of neural folds and heart field. [Figure 7-2] Same as above [Figure 7-3]Figure 7D-F demonstrates that coaggregation of naive mouse PSCs (V6.5-GFP), mouse KH2-Gata4 Dox-inducible ES cells, and mouse TSCs (mouse TSC line #1 cells) followed by ex utero culture does not result in the generation of organized embryos. Figure 7D is a schematic representation of the protocol. Aggregation Medium (AM): 1:1 DMEM-F12 (Thermo, 11320033) and Neurobasal (Thermo, 21103049), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin, 1x NEAA, 1x N2 supplement (Thermo, 17502048), 1x B27 supplement (Thermo, 17504044), 0.1mM beta-mercaptoethanol (Thermo, 31350010), 0.45% BSA (Sigma-Aldrich, A7979) (500ml total). IVC1 medium: Improved DMEM / F12 (Gibco, 12634010), 1x Glutamax, 1x penicillin / streptomycin, 1x ITS-X (Gibco, 51500056), 8 nM beta-estradiol, 20 ng / ml progesterone, 25 μM N-acetyl-L-cysteine, and serum or KSR as specified in the scheme (total 500 ml).EUCM: 25% DMEM (Gibco, 11880), 25% HAS (human adult serum or human umbilical cord serum), 50% rat serum (RAS), 1x Glutamax (Gibco, 35050038), 2 mM HEPES (Gibco, 15630056), 1x penicillin / streptomycin. Figure 7E shows images of representative embryoid body aggregates on days 5 and 6 of the protocol, immunostained for TBR2 (red), which characterizes the trophectoderm lineage, Gata4, which characterizes the primitive endoderm lineage, and GFP signal, which characterizes ESCs that contribute to the epiblast. Figure 7F shows images or representative structures on days 7 and 8 of the protocol, demonstrating the absence of organized embryos within the yolk sac and no signs of the emergence of neural folds and heart regions. [Figure 7-4] Same as above [Figure 8]FIG. 8 shows a schematic representation of the generation and validation of KH2-Gata4 Dox-inducible naive ES cells. [Figure 9] Figure 9 shows real-time PCR analysis of exogenous GATA4 expression in various KH2-Gata4 mouse ES clones targeted with TetOn-Gata4 within the collagen locus after treatment with DOX. KH2-wild-type and irradiated mouse fibroblasts (MEFs) were used as negative controls. [Figure 10] Figure 10 shows immunostaining images of various KH2 mouse naive ES clones targeted with TetOn-Gata4 (KH2-Gata4) within the collagen locus before and after treatment with DOX under 2i / LIF naive conditions. Gata4 was strongly expressed in the tested clones only after DOX treatment. DAPI was used for counterstaining. [Figure 11] FIG. 11 shows a schematic representation of the generation and validation of KH2-Cdx2 Dox-inducible cells. [Figure 12] Figure 12 shows real-time PCR analysis of exogenous Cdx2 expression in various KH2-Cdx2 mouse ES clones targeted with TetOn-Cdx2 within the collagen locus after treatment with DOX. KH2-wild-type cells were used as a negative control. [Figure 13] Figure 13 shows immunostaining of clone #3 of KH2 mouse naive ES cells targeted with TetOn-Cdx2 (KH2-Cdx2) within the collagen locus before and after treatment with DOX under 2i / LIF naive conditions. Cdx2 was strongly expressed in the tested clones only after DOX treatment. DAPI was used for counterstaining. [Figure 14] Figure 14 shows immunostaining of KH2 mouse naive ES clone #7 targeted with TetOn-Cdx2 (KH2-Cdx2) within the collagen locus before and after treatment with DOX under 2i / LIF naive conditions. Cdx2 was strongly expressed in the tested clones only after DOX treatment. DAPI was used for counterstaining. [Figure 15-1]Figure 15A-F demonstrates that coaggregation of naive KH2-wild-type ES cells with DOX-pretreated KH2-Gata4 naive ES cells and DOX-pretreated KH2-Cdx2 naive ES cells, followed by ex utero culture, leads to the generation of organized embryos. Figure 15A is a schematic representation of the protocol. Aggregation Medium (AM): 1:1 DMEM-F12 (Thermo, 21331-020) and Neurobasal (Thermo, 21103049), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin, 1x NEAA, 1x N2 supplement (Thermo, 17502048), 1x B27 supplement (Thermo, 17504044), 0.1mM beta-mercaptoethanol (Thermo, 31350010), 0.45% BSA (Sigma-Aldrich, A7979) (500ml total). EUCM2-CMRL: CMRL (Gibco, 11530037), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin + 1x NEAA, 4mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 20% FBS (500ml total). EUCM3-CMRL: CMRL (Gibco, 11530037), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin + 1x NEAA, 4mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 30% FBS (500ml total). EUCM4-CMRL: CMRL (Gibco, 11530037), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin + 1x NEAA, 4mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 30% HAS (500ml total).EUCM: 25% DMEM (GIBCO 11880), 25% HAS, 50% rat serum, 1x Glutamax (GIBCO 35050038), 2mM HEPES (GIBCO 15630056), 1x penicillin / streptomycin. Figure 15B shows phase-contrast images of the resulting synthetic embryos at the indicated time points. On days 4 and 5, a distinct egg-cylinder-shaped embryo was evident, with extraembryonic ectoderm (EXE) appearing on the opposite side. On day 6.5, the yolk sac and ectoplacental cone were evident, with an internally enclosed embryo-like structure. On day 7, a gastrulated embryo with distinct neural folds, somitogenesis, and a beating heart was evident. Figure 15C-F show immunostained images of the resulting synthetic embryos. Embryos obtained on day 7 of the protocol correspond to E8.5 embryos (Figure 15C-D). In Figure 15C, the embryo shows neural folds staining positive for the Sox2 marker (red) and Brachyury (green) staining for the mesoderm. In Figure 15D, the embryo shows neural folds staining positive for the Pax6 marker (green) and the Sox17 marker (red), which characterizes the developing endodermal gut. Embryos obtained on day 8 of the protocol correspond to E9.5 embryos (Figure 15E-F). In Figure 15E, the embryo shows neural tube closure and somitogenesis (positively stained red for Sox9). Otx2 (magenta) characterizes the forebrain region. In Figure 15F, the embryo shows advanced organogenesis and normal morphology with properly distributed brain neurons staining positive for the Tuj1 marker (green). [Figure 15-2] Same as above [Figure 15-3] Same as above [Figure 16-1]Figure 16A-B demonstrates that coaggregation of KH2-wildtype naive ES cells with naive KH2-Gata4 and KH2-Cdx2 naive cells in the presence of Dox, followed by ex utero culture, leads to the generation of organized embryos. Figure 16A is a schematic representation of the protocol. Aggregation Medium (AM): 1:1 DMEM-F12 (Thermo, 11320033) and Neurobasal (Thermo, 21103049), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin, 1x NEAA, 1x N2 supplement (Thermo, 17502048), 1x B27 supplement (Thermo, 17504044), 0.1mM beta-mercaptoethanol (Thermo, 31350010), 0.45% BSA (Sigma-Aldrich, A7979) (500ml total). EUCM2-CMRL: CMRL (Gibco, 11530037), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin + 1x NEAA, 4mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 20% FBS (500ml total). EUCM3-CMRL: CMRL (Gibco, 11530037), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin + 1x NEAA, 4mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 30% FBS (500ml total). EUCM4-CMRL: CMRL (Gibco, 11530037), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin + 1x NEAA, 4mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 30% HAS (500ml total).EUCM: 25% DMEM (GIBCO 11880), 25% HAS, 50% rat serum (RAS), 1x Glutamax (GIBCO 35050038), 2mM HEPES (GIBCO 15630056), 1x penicillin / streptomycin. Figure 16B shows phase-contrast images of the synthetic embryos obtained at the indicated time points. At days 4 and 6, a distinct egg-cylinder-shaped embryo was evident, with extraembryonic ectoderm (EXE) on the opposite side. At day 6.5, the yolk sac and ectoplacental cone were evident, with an internally enclosed embryo-like structure. At day 7, a fully gastrulated embryo with a distinct neural fold, early somitogenesis, and a beating heart was evident. [Figure 16-2] Same as above [Figure 17]Figure 17A-B demonstrates that coaggregation of KH2-wild-type naive ES cells with Dox-pretreated naive KH2-Gata4 ES cells and Dox-pretreated KH2-Cdx2 ES cells, followed by ex utero culture, leads to the generation of organized embryos. Figure 17A is a schematic representation of the protocol. Aggregation Medium (AM): 1:1 DMEM-F12 (Thermo, 11320033) and Neurobasal (Thermo, 21103049), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin, 1x NEAA, 1x N2 supplement (Thermo, 17502048), 1x B27 supplement (Thermo, 17504044), 0.1mM beta-mercaptoethanol (Thermo, 31350010), 0.45% BSA (Sigma-Aldrich, A7979) (500ml total). EUCM2-CMRL: CMRL (Gibco, 11530037), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin + 1x NEAA, 4mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 20% FBS (500ml total). EUCM3-CMRL: CMRL (Gibco, 11530037), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin + 1x NEAA, 4mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 30% FBS (500ml total). EUCM4-CMRL: CMRL (Gibco, 11530037), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin + 1x NEAA, 4mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 30% HAS (500ml total).EUCM: 25% DMEM (GIBCO 11880), 25% HAS, 50% RAS, 1x Glutamax (GIBCO 35050038), 10 mM HEPES (GIBCO 15630056), 1x penicillin / streptomycin. Figure 17B shows phase-contrast images of synthetic embryos obtained at the indicated time points. At day 6, a distinct egg-cylinder-shaped embryo was evident, with extraembryonic ectoderm (EXE) on the opposite side. At day 6.5, the yolk sac and ectoplacental cone were evident, with an internally enclosed embryo-like structure. At day 7, a completely gastrulated embryo with distinct neural folds, early somitogenesis, and a beating heart was evident, equivalent to E8.5. [Figure 18-1]Figure 18A-B demonstrates that coaggregation of KH2-wildtype naive ES cells with KH2-Gata4 and KH2-Cdx2 naive ES cells in the presence of Dox, followed by ex utero culture, leads to the generation of organized embryos. Figure 18A is a schematic representation of the protocol. Aggregation Medium (AM): 1:1 DMEM-F12 (Thermo, 11320033) and Neurobasal (Thermo, 21103049), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin, 1x NEAA, 1x N2 supplement (Thermo, 17502048), 1x B27 supplement (Thermo, 17504044), 0.1mM beta-mercaptoethanol (Thermo, 31350010), 0.45% BSA (Sigma-Aldrich, A7979) (500ml total). EUCM2-CMRL-Improved DMEM / F12: 1:1 mixture of CMRL (Gibco, 11530037) and Improved DMEM / F12 (Thermo, 12634010), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin + 1x NEAA, 1mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 20% FBS (500ml total). EUCM3-CMRL-Improved DMEM / F12: 1:1 mixture of CMRL (Gibco, 11530037) and Improved DMEM / F12 (Thermo, 12634010), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin + 1x NEAA, 4mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 30% FBS (500ml total).EUCM4-CMRL-Improved DMEM / F12: 1:1 mixture of CMRL (Gibco, 11530037) and Improved DMEM / F12 (Thermo, 12634010), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin + 1x NEAA, 4mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 30% HAS (500ml total). EUCM: 25% DMEM (GIBCO 11880), 25% HAS, 50% RAS, 1x Glutamax (GIBCO 35050038), 10 mM HEPES (GIBCO 15630056), 1x penicillin / streptomycin. Figure 18B shows phase-contrast images of the synthetic embryos obtained at the indicated time points. On days 4, 5, and 6, a distinct egg-cylinder-shaped embryo was evident, with extraembryonic ectoderm (EXE) on the opposite side. On day 6.5, the yolk sac, ectoplacental cone, and umbilical invagination were evident, with an internally enclosed embryo-like structure. On day 7, a completely gastrulated embryo with a distinct neural fold, early somitogenesis, and a beating heart was evident. [Figure 18-2] Same as above [Figure 19]Figure 19A-B demonstrates that coaggregation of KH2-wild-type naive ES cells with Dox-pretreated KH2-Gata4 naive ES cells and Dox-pretreated KH2-Cdx2 naive ES cells, followed by ex utero culture, leads to the generation of organized embryos. Figure 19A is a schematic representation of the protocol. Aggregation Medium (AM): 1:1 DMEM-F12 (Thermo, 11320033) and Neurobasal (Thermo, 21103049), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin, 1x NEAA, 1x N2 supplement (Thermo, 17502048), 1x B27 supplement (Thermo, 17504044), 0.1mM beta-mercaptoethanol (Thermo, 31350010), 0.45% BSA (Sigma-Aldrich, A7979) (500ml total). IVC1: Modified DMEM / F12 (Gibco, 12634010), 1x Glutamax, 1x penicillin / streptomycin, 1x ITS-X (Gibco, 51500056), 8 nM beta-estradiol, 20 ng / ml progesterone, 25 μM N-acetyl-L-cysteine, and FBS or HAS as indicated (500 ml total). EUCM: 25% DMEM (Gibco, 11880), 25% HAS, 50% RAS, 1x Glutamax (Gibco, 35050038), 2 mM HEPES (Gibco, 15630056), 1x penicillin / streptomycin. Figure 19B shows phase-contrast images of synthetic embryos obtained at the indicated time points. On day 6, a distinct egg cylinder-shaped embryo was evident, with the extraembryonic ectoderm (EXE) on the opposite side. On day 6.5, the yolk sac and ectoplacental cone were evident, with an embryo-like structure enclosed within. [Figure 20-1]Figure 20A-B demonstrates that coaggregation of KH2-wild-type naive ES cells with Dox-pretreated KH2-Gata4 naive ES cells and Dox+FGF4+LPA-pretreated KH2-Cdx2 naive ES cells, followed by ex utero culture, leads to the generation of organized embryos. Figure 20A is a schematic representation of the protocol. Aggregation Medium (AM): 1:1 DMEM-F12 (Thermo, 11320033) and Neurobasal (Thermo, 21103049), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin, 1x NEAA, 1x N2 supplement (Thermo, 17502048), 1x B27 supplement (Thermo, 17504044), 0.1mM beta-mercaptoethanol (Thermo, 31350010), 0.45% BSA (Sigma-Aldrich, A7979) (500ml total). EUCM2-CMRL-Improved DMEM / F12: 1:1 mixture of CMRL (Gibco, 11530037) and Improved DMEM / F12 (Thermo, 12634010), 1x Glutamax, 0.5mM sodium pyruvate, 1x penicillin / streptomycin + 0.5x NEAA, 4mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 20% FBS (500ml total). EUCM3-CMRL-Improved DMEM / F12: 1:1 mixture of CMRL (Gibco, 11530037) and Improved DMEM / F12 (Thermo, 12634010), 1x Glutamax, 0.5mM sodium pyruvate, 1x penicillin / streptomycin + 0.5x NEAA, 4mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 30% FBS (500ml total).EUCM4-CMRL-Improved DMEM / F12: 1:1 mixture of CMRL (Gibco, 11530037) and Improved DMEM / F12 (Thermo, 12634010), 1x Glutamax, 0.5mM sodium pyruvate, 1x penicillin / streptomycin + 0.5x NEAA, 4mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 30% HAS (500ml total). EUCM: 25% DMEM (GIBCO 11880), 25% HAS, 50% RAS, 1x Glutamax (GIBCO 35050038), 2mM HEPES (GIBCO 15630056), 1x penicillin / streptomycin. Figure 20B shows phase-contrast images of the synthetic embryos obtained at the indicated time points. At day 6, embryos were evident with a distinct egg cylinder shape and amnion, and extraembryonic ectoderm (EXE) appeared on the opposite side. At day 6.5, the yolk sac, ectoplacental cone, and umbilical cord invagination were evident, with an internally enclosed embryo-like structure. At day 7, embryos that had completed gastrulation with distinct neural folds, early somitogenesis, and a beating heart were evident. [Figure 20-2] Same as above [Figure 21]FIG. 21 shows a schematic protocol for coaggregation of KH2-wild type naive cells with KH2-Gata4 naive cells pretreated with Dox+CHIR99021 and KH2-Cdx2 naive cells pretreated with Dox+LPA, followed by ex utero culture, leading to the generation of organized embryos. Aggregation Medium (AM): 1:1 DMEM-F12 (Thermo, 11320033) and Neurobasal (Thermo, 21103049), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin, 1x NEAA, 1x N2 supplement (Thermo, 17502048), 1x B27 supplement (Thermo, 17504044), 0.1mM beta-mercaptoethanol (Thermo, 31350010), 0.45% BSA (Sigma-Aldrich, A7979) (500ml total). EUCM2-CMRL-Improved DMEM / F12: 1:4 mixture of CMRL (Gibco, 11530037) and Improved DMEM / F12 (Thermo, 12634010), 1x Glutamax, 0.5mM sodium pyruvate, 1x penicillin / streptomycin + 0.5x NEAA, additional 2mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 20% FBS (500ml total). EUCM3-CMRL-Improved DMEM / F12: 4:1 mixture of CMRL (Gibco, 11530037) and Improved DMEM / F12 (Thermo, 12634010), 1x Glutamax, 0.5mM sodium pyruvate, 1x penicillin / streptomycin + 0.5x NEAA + additional 2mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 30% FBS (500ml total).EUCM4*-CMRL-Improved DMEM / F12: 4:1 mixture of CMRL (Gibco, 11530037) and Improved DMEM / F12 (Thermo, 12634010), 1x Glutamax, 0.5mM sodium pyruvate, 1x penicillin / streptomycin + 0.5x NEAA, additional 3mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 20% RAS, 10% HAS, 10% KSR, 2mM HEPES (Gibco, 15630056) (500ml total). EUCM5-CMRL-Improved DMEM / F12: 4:1 mixture of CMRL (Gibco, 11530037) and Improved DMEM / F12 (Thermo, 12634010), 1x Glutamax, 0.5mM sodium pyruvate, 1x penicillin / streptomycin + 0.5x NEAA + additional 3mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 30% RAS, 15% HAS, 5% KSR, 2mM HEPES (Gibco, 15630056) (500ml total). EUCM: 25% improved DMEM / F12 (Thermo, 12634010), 25% HAS, 50% RAS, 1x Glutamax (GIBCO, 35050038), 2mM HEPES (GIBCO, 15630056), 1x penicillin / streptomycin. [Figure 22]FIG. 22 shows a schematic protocol for coaggregation of KH2-wild-type naive ES cells with KH2-Gata4 naive ES cells pretreated with Dox+CHIR99021 and KH2-Cdx2 naive cells pretreated with Dox+LPA, followed by ex utero culture, leading to the generation of organized embryos. Aggregation Medium (AM): 1:1 DMEM-F12 (Thermo, 11320033) and Neurobasal (Thermo, 21103049), 1x Glutamax, 1mM sodium pyruvate, 1x penicillin / streptomycin, 1x NEAA, 1x N2 supplement (Thermo, 17502048), 1x B27 supplement (Thermo, 17504044), 0.1mM beta-mercaptoethanol (Thermo, 31350010), 0.45% BSA (Sigma-Aldrich, A7979) (500ml total). EUCM2-CMRL-Improved DMEM / F12: 1:4 mixture of CMRL (Gibco, 11530037) and Improved DMEM / F12 (Thermo, 12634010), 1x Glutamax, 0.5mM sodium pyruvate, 1x penicillin / streptomycin + 0.5x NEAA, additional 2mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 20% FBS (500ml total). EUCM3-CMRL-Improved DMEM / F12: 4:1 mixture of CMRL (Gibco, 11530037) and Improved DMEM / F12 (Thermo, 12634010), 1x Glutamax, 0.5mM sodium pyruvate, 1x penicillin / streptomycin + 0.5x NEAA + additional 2mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 30% FBS (500ml total).EUCM4*-CMRL-Improved DMEM / F12: 4:1 mixture of CMRL (Gibco, 11530037) and Improved DMEM / F12 (Thermo, 12634010), 1x Glutamax, 0.5mM sodium pyruvate, 1x penicillin / streptomycin + 0.5x NEAA, additional 3mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 20% RAS, 10% HAS + 2mM HEPES (Gibco, 15630056) (500ml total). EUCM5-CMRL-Improved DMEM / F12: 4:1 mixture of CMRL (Gibco, 11530037) and Improved DMEM / F12 (Thermo, 12634010), 1x Glutamax, 0.5mM sodium pyruvate, 1x penicillin / streptomycin + 0.5x NEAA + additional 3mg / mL D-glucose, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / mL progesterone, 30% RAS, 15% HAS, 2mM HEPES (Gibco, 15630056) (500ml total). EUCM: 25% improved DMEM / F12 (Thermo, 12634010), 25% HAS, 50% RAS, 1x Glutamax (GIBCO, 35050038), 2mM HEPES (GIBCO, 15630056), 1x penicillin / streptomycin. [Figure 23-1]Figure 23A-D shows immunostained images of representative synthetic embryos obtained according to the protocol described in Example 2, demonstrating the generation of organized synthetic embryos. Figure 23A shows immunostained images of a representative day 4 mouse synthetic embryo obtained using the described regimen. OTX2 (gray) was used to stain primed pluripotent epiblast, Gata4 (red) was used to stain extraembryonic primitive endoderm progeny, and TFAP2C (green) was used to characterize the trophoblast lineage. The images show normal embryonic structure with egg cylinder morphology, with trophoblast EXE and pluripotent epiblast self-organizing on opposite sides of the embryo as expected, and Gata4 primitive endoderm cells surrounding the embryo. Figure 23B shows immunostained images of a representative day 4 mouse synthetic embryo obtained using the described regimen. Oct4 (gray) was used to stain primed pluripotent epiblasts within the egg cylinder epiblast, Sox17 (red) was used to stain extraembryonic primitive endoderm progeny, and Cdx2 was used to characterize the trophoblast lineage. The image shows normal embryonic structure with egg cylinder morphology, with trophoblast EXEs and pluripotent epiblasts (Oct4+ regions) self-organizing on the opposite side of the Cdx2+ region, and Sox17+ primitive endoderm cells surrounding the embryo. Figure 23C shows immunostaining of a representative day 4 or 5 mouse composite embryo using the described regimen. Oct4 (gray) was used to stain primed pluripotent epiblasts within the egg cylinder epiblast, Sox17 (red) was used to stain extraembryonic primitive endoderm progeny, and Cdx2 was used to characterize the trophoblast lineage. The image shows normal embryonic structure with egg cylinder morphology, with the trophoblast EXE and pluripotent epiblast (Oct4+ region) self-organizing on the opposite side of the Cdx2+ region, and Sox17+ primitive endoderm cells surrounding the embryo. Figure 23D shows immunostaining of a representative mouse synthetic embryo using the regimen shown in Figure 21. Oct4 (magenta) was used to stain the primed pluripotent epiblast within the egg cylinder-shaped epiblast. T / Brachyury stains green, marking the emergence of the primitive streak. DAPI is shown in blue as a counterstain. Day 6 egg cylinder-shaped mouse embryoid bodies demonstrate mimicking primitive streak formation and gastrulation on day 7 of the protocol, as evidenced by specific immunostaining. [Figure 23-2] Same as above [Figure 23-3] Same as above [Figure 23-4] Same as above [Figure 24] Figure 24 demonstrates the endogenous expression of the indicated markers in KH2-Gata4 naive ES cells before and after Dox administration, as determined by real-time PCR, and in day 4 synthetic embryos obtained according to the protocol described in Figure 17A. [Figure 25] Figure 25 demonstrates the endogenous expression of the indicated markers in KH2-Cdx2 naive ES cells before and after Dox administration, as well as in day 4 synthetic embryos obtained according to the protocol described in Figure 17A, as determined by real-time PCR. [Figure 26] Figure 26 demonstrates the endogenous expression of the indicated markers in KH2-Cdx2 naive cells before and after administration of Dox and LPA, as well as in day 4 synthetic embryos obtained according to the protocol described in Figure 19A, as determined by real-time PCR. [Figure 27] FIG. 27 demonstrates the endogenous expression of the indicated markers in KH2-Cdx2 naive ES cells before and after treatment with Dox and the indicated media and factors, as determined by real-time PCR. [Figure 28] FIG. 28 shows a schematic representation of targeting vectors designed to generate inserts that overexpress human TetON-inducible Gata4, or Cdx2, or Gata3 in Gata3-2A-Cdx2. [Figure 29] Figure 29 demonstrates clones of human naive PSCs that were correctly targeted for either GATA4 or CDX2 overexpression. The targeting scheme and pre- and post-DOX Western blot analysis demonstrate the specific overexpression of each TF after DOX. [Figure 30]Figure 30 shows the plasmid design for generating a lentivirus that allows TetOn-induced overexpression of Gata4 after treating lentivirus-infected mouse or human PSCs with Dox. The green reporter is also DOX-responsive. This lentivirus can be used with PSCs that already express M2RTTA or by co-infecting them with a lentivirus encoding FUW-CAGGS-M2Rtta. [Figure 31] Figure 31 shows the plasmid design for generating a lentivirus that allows TetOn-induced overexpression of CDX2 after treating lentivirally infected mouse or human PSCs with Dox. The green reporter is also DOX-responsive. This lentivirus can be used with PSCs that already express M2RTTA or by co-infecting them with a lentivirus encoding FUW-CAGGS-M2Rtta. [Figure 32] Figure 32 shows the plasmid design of the pcDNA3 plasmid used for transient transfection of mouse or human naive PSCs, which encodes GATA4-IRES-green fluorescent under the CMV promoter. [Figure 33] Figure 33 shows the plasmid design used for transient transfection of human naive PSCs, which encodes human CDX2-IRES-green fluorescent under the CAGGS promoter. [Figure 34]Figure 34 shows the plasmid design for generating a human lentivirus that allows TetOn-inducible overexpression of YAP*mut (carrying a mutation that forces nuclear localization of YAP) after treating lentivirus-infected mouse or human PSCs with Dox. The green GFP reporter is also DOX-responsive. This lentivirus can be used with PSCs that already express M2RTTA or by co-infecting them with a lentivirus encoding FUW-CAGGS-M2Rtta. For this construct, we used constitutively active (mut) human YAP (serines at positions 61, 109, 127, 128, 131, 163, 164, and 381 changed to alanines (phosphorylation sites). See addgene entry #27371 (Yap-mut) from which the insert was obtained). [Figure 35] Figure 35 shows the plasmid design for generating a lentivirus that allows TetOn-induced overexpression of Gata3 after treating lentivirally infected mouse or human PSCs with Dox. The CFP reporter is also DOX-responsive. This lentivirus can be used with PSCs that already express M2RTTA or by co-infecting them with a lentivirus encoding FUW-CAGGS-M2Rtta. [Figure 36] Figure 36 shows the plasmid design for use in transient transfection of mouse or human naive PSCs, which encodes GATA3-IRES-CFP fluorescent under the CAGGS promoter. [Figure 37]Figure 37 shows the plasmid design of the pcDNA3 plasmid used for transient transfection of mouse or human naive PSCs. It encodes YAP*mut-IRES-CFP fluorescent under the CMV promoter. Yap-mut: Carries a mutation that forces nuclear localization of YAP. For this construct, constitutively active (mut) human YAP was used (serines at positions 61, 109, 127, 128, 131, 163, 164, and 381 changed to alanines (phosphorylation sites). See addgene entry #27371 (Yap-mut) from which the insert was obtained). [Figure 38] Figures 38A-B show images of representative synthetic embryoid bodies obtained according to the protocol described in Example 3, demonstrating the generation of day 4.5 human synthetic embryos. Figure 38A shows a phase-contrast image of a day 4.5 synthetic human embryoid body. Figure 38B shows an image of a representative day 4.5 synthetic human embryoid body immunostained for the Oct4 pluripotency marker, the Sox17 primitive endoderm (hypoblast) marker, and the CDX2 trophectoderm marker. The image shows a typical human embryo in which aggregates of OCT4+ pluripotent cells are surrounded by spheres of trophectoderm CDX2+ cells, and the SOX17+ primitive endoderm (hypoblast region) is immediately beneath the OCT4+ cell cluster. [Figure 39-1]Figures 39A-G demonstrate that naive ESC-derived synthetic embryos complete gastrulation and reach the organogenesis stage within the extraembryonic membranes. Figure 39A shows a schematic depiction of the synthetic embryo production and culture protocol. Pre-induced with DOX (2i Self-organized synthetic embryos ["sEmbryo(iCdx2)] that grow to early organogenesis are generated by aggregating populations derived from three naive ESC types (day -1 for Gata4 in LIF, and day -14 to day -1 for Cdx2 in TSC-LPA medium) and subsequently culturing for 8 days in AM (containing DOX for the first 2 days), EUCM2 (20-30% FBS), EUCM2 (30% FBS), and EUCM. Figure 39B shows brightfield images of representative embryos at each day of the culture protocol compared to stage-matched natural embryos. Figure 39C shows the configuration of the electronically controlled roller bottle ex utero culture platform used for sEmbryo expansion. Figure 39D shows the roller culture bottles. Images of day 7 and day 8 sEmbryos cultured ex utero are shown. Figure 39E shows a brightfield image of day 8 sEmbryos growing ex utero within the extraembryonic membranes (yolk sac and amnion). Figure 39F shows images of day 8 sEmbryo (iCdx2) and E8.5 native embryos after dissection and removal of the extraembryonic membranes. The insets are enlarged views of the dashed boxes. Figure 39G shows a representative image of an empty yolk sac obtained after continuous culture in an IVC-static culture-based protocol. All images are representative of a minimum of three biological replicates. Scale bars are 100 μm in Figure 39B-C and 500 μm in Figure 39G. A-anterior, AC-amniotic cavity, Am-amnion, A-allantois, AB-allantoic bud. bud), BI-blood island, Epi-epiblast, EC-exocoelomic cavity, EPC-exocene placental cone, ExE-extraembryonic ectoderm, Fg-foregut pocket, H-heart, NF-neural fold, NT-neural tube, P-posterior, PAC-preamniotic cavity, PS-primitive streak, S-somites, VE-visceral endoderm, YS-yolk sac. [Figure 39-2] Same as above [Figure 39-3] Same as above [Figure 40-1]Figure 40A-G demonstrates that the expression patterns of lineage markers in egg cylinder-type sEmbryo(iCdx2) are similar to those in native embryos. Figure 40A shows confocal images of midsections of day 4 egg cylinder sEmbryo(iCdx2) and native E5.5 embryos immunostained for extraembryonic ectoderm markers, visceral endoderm markers, and epiblast markers. Figure 40B shows the migration of the anterior visceral endoderm (AVE) from the distal to the future anterior portion, as revealed by CER1 staining (magenta) in day 4 sEmbryo(iCdx2). Figure 40C shows representative immunofluorescence images (midsections) of day 5 sEmbryo(iCdx2) for visceral endoderm markers, trophoblast markers, epiblast markers, and gastrulation markers (Brachyury, yellow arrows) compared with matched E6.5 native embryos. Figure 40D-E demonstrates primitive streak migration and definitive endoderm establishment in day 6 sEmbryo(iCdx2) and control E7.5 native embryos. Figure 40D shows immunofluorescence images demonstrating Brachyury+ cell migration and the presence of Brachyury / Foxa2 double-positive cells. Figure 40E shows SOX17 / FOXA2 immunostaining revealing invaginating definitive endoderm cells. FOXA2+ / SOX17- cells along the epiblast reveal nodal-like organizers in sEmbryo(iCdx2). The inset is a magnified view of the dashed box. Figure 40F shows a mid-section image of Blimp1-mVenus fluorescence detected by live imaging in day 5 sEmbryo(iCdx2) (top panel), characterizing PGC specification. Sox2+ PGCs are shown to be properly positioned in the anterior ventral aspect of sEmbryo(iCdx2) at day 8 (bottom panel). Figure 40G shows flow cytometry plots of Blimp1-Venus / Stella-CFP in sEmbryo(iCdx2) at days 4 and 5, characterizing their appearance at day 5 (equivalent to E6.5) as expected. Scale bars are indicated above each panel. [Figure 40-2] Same as above [Figure 40-3] Same as above [Figure 41-1]Figures 41A-E demonstrate that day 8 postgastrulation sEmbryo(iCdx2) appropriately recapitulate the spatial expression patterns of tissues derived from all three germ layers. Figure 41A shows a representative whole-mount immunofluorescence image of day 8 sEmbryo(iCdx2) demonstrating appropriate expression of neural lineage markers (Sox2), cardiac lineage markers (MHC-II), and notochord lineage markers (Brachyury). Scale bar: 200 μM. Figure 41B shows maximum intensity projection confocal images of day 8 sEmbryo(iCdx2) for Sox2 and Brachyury compared to native E8.5 embryos grown in utero. Scale bar: 200 μM. Figure 41C shows maximum intensity projection confocal images of day 8 sEmbryo(iCdx2) for OTX2 and FOXA2 compared to native E8.5 embryos grown in utero. Scale bar: 200 μM. Figure 41D shows a schematic representation of a section of a neural tube (upper panel). Representative immunosection confocal images (mid-section, transverse section) of the closed neural tube in day 8 sEmbryo(iCdx2) compared to a native E8.5 embryo in utero (lower panel). Scale bar: 20 μM. Figure 41E shows a schematic representation of a section of a heart (upper panel). Representative immunohistochemical images (mid-section, transverse section) of the heart tube in day 8 sEmbryo(iCdx2) (lower panel). Scale bar: 50 μM. Images are representative of at least three biological replicates. FB - forebrain, FP - floor plate, H - heart, LV - left ventricle, Nc - notochord, NF - neural fold, NT - neural tube, RV - right ventricle, TB - tail bud. [Figure 41-2] Same as above [Figure 42-1]Figures 42A-F demonstrate that sEmbryo(iCdx2) develop within the extraembryonic membranes and develop the allantois, blood islands, and ectoplacental cones. Figure 42A shows representative images of day 7 and day 8 sEmbryo(iCdx2) demonstrating the presence of the amnion, yolk sac, allantois, ectoplacental cones, and blood islands. Scale bar: 200 μM. Figure 42B shows a schematic diagram of the extraembryonic compartments present in day 8 sEmbryo(iCdx2). Figure 42C shows representative whole-mount immunofluorescence images of standard markers in day 8 sEmbryo(iCdx2) and the yolk sac isolated from E8.5 native embryos in utero. Scale bar: 500 μM. Figure 42D shows representative images of RUNX1 immunostaining in yolk sacs obtained from day 8 sEmbryo(iCdx2) and equivalent to E8.5 native embryos. Scale bar: 50 μM. Figure 42E shows representative flow cytometry contour plots of CD45 and CD34 expression in Kit+CD41+ progenitor cells derived from E8.5 (upper panel) and sEmbryo(iCdx2) (lower panel) within the yolk sac, as well as Kit and CD41 expression in the lineage-negative population. Gates were set based on fluorescence minus 1 (FMO) for each cell surface marker antibody. Figure 42F shows the methylcellulose-based in vitro culture, colony-forming ability, and morphology of erythroid progenitor cells derived from day 8 sEmbryo(iCDX2) along with erythroid progenitor cells derived from E8.5 native controls in utero. Data are representative of a minimum of three biological replicates. Scale bars are indicated above each panel. [Figure 42-2] Same as above [Figure 43-1]Figures 43A-D show scRNA-seq analysis of natural versus synthetic embryos. Figure 43A shows a UMAP plot representing individual cells (n=15,284 natural E8.5 embryos, n=31,151 day 8 synthetic embryos). Points are colored according to their assigned cell cluster. Cell lineage annotation of clusters based on marker genes for the major cell types identified in E8.5 mouse embryos. EN = total embryos, CN = total cells. Figure 43B shows a UMAP plot representing individual cells from the various samples shown. Colors are the same as in Figure 43A. Figure 43C shows natural embryo cells (black) and synthetic embryo cells (red) projected onto the same UMAP plot. Figure 43D shows a pie chart depicting the proportional abundance of each cell cluster in both natural and synthetic embryos. Asterisks indicate clusters with statistically significant differences between the two groups. *t-test, p<0.05. [Figure 43-2] Same as above [Figure 44-1]Figures 44A-M show an optimized gas and pressure regulation controller for a roller culture incubator. Figure 44A is a schematic representation of an embryonic incubation system. Figure 44B is an image of an exemplary embryonic incubation system. Figure 44C shows the configuration of the gas and pressure regulation electronic module. O2, N2, and CO2 are introduced into the system at pressures of 0.1-0.5 psi and mixed by a centrifugal blower. The gases are then pumped into water bottles inside the incubator (high pressure) by a pump that allows for control of the gas pressure. Lph = liters per hour. Figure 44D is a schematic representation of an exemplary gas and pressure controller. Figure 44E shows a perspective view of an exemplary gas and pressure controller configured to monitor and manipulate CO2 and O2 levels by supplying CO2 and / or N2. Figure 44F shows a top view of the gas and pressure controller with the controller opened to reveal the internal components. Figure 44G shows a front view of the gas and pressure controller. Figure 44H is a schematic representation of an exemplary gas mixing box. Figure 44I is an image of an exemplary gas mixing box. Figure 44J is a schematic representation of an exemplary incubator. Figure 44K is an image of the interior of a precision incubator system (manufactured by BTC Engineering) showing the direction of gas flow (indicated by white arrowheads). Figure 44L is an image of an embryo cultured in a roller bottle (yellow arrowhead). Figure 44M is a flowchart of an exemplary method for the embryonic incubation system. [Figure 44-2] Same as above [Figure 44-3] Same as above [Figure 44-4] Same as above [Figure 44-5] Same as above [Figure 44-6] Same as above [Figure 44-7] Same as above [Figure 44-8] Same as above [Figure 44-9] Same as above [Figure 44-10] Same as above [Figure 44-11] Same as above [Figure 44-12] Same as above [Figure 45-1] Figures 45A-E demonstrate the coaggregation of human naive PSC cells with DOX-pretreated iGata4 human naive PSCs and DOX-pretreated iCdx2 human naive PSCs, followed by ex utero culture, leading to the generation of organized embryos. Figure 45A is a schematic representation of the protocol. Figure 45E shows phase-contrast and immunostained images of synthetic embryos obtained at the indicated time points after aggregation. Figure 45B shows the appearance of a bilayered germinal disc on day +4. Figure 45C demonstrates the formation of a primary yolk sac on day +5. Figure 45D demonstrates the formation of a secondary yolk sac on day +6 after coaggregation. Figure 45E demonstrates the establishment of a bilayered germinal disc in a human synthetic embryo on day +7. [Figure 45-2] Same as above [Figure 45-3] Same as above [Figure 45-4] Same as above [Figure 45-5] Same as above [Figure 45-6] Figure 45F shows a schematic representation of an inducible plasmid for transient expression of human CDX2, GATA3, GATA4, or GATA6. Shown is a PiggyBac plasmid expressing a selected cDNA insert under the control of a doxycycline-inducible promoter. The PiggyBac vector used (www(dot)addgene(dot)org / 104454 / ) carries M2Rtta and a site for inserting the cDNA of a transcription factor of interest. This vector was used to generate four different plasmids: human CDX2 or human Gata3 (to promote differentiation of human PSCs toward trophectoderm), and human GATA4 or human GATA6 (to promote priming of human PSCs toward human primitive endoderm). [Figure 46-1]Figure 46A-C shows immunostained images of human PSC lines expressing the inducible plasmids shown in Figure 45. Figure 46A shows JH33 iPS (a genetically unmodified human iPSC line generated from a healthy Caucasian adult male) and WIBR3 wild-type human female ESCs, which were used to generate a CDX2-inducible line (iCDX2) using Piggybac plasmids containing a CDX2 insert. Immunostained analysis of CDX2 protein expression in human primed PSCs grown in mTESR conditions with and without DOX (2 μg / ml Dox) was shown. CDX2 expression was specifically detected only after DOX addition. Figure 46N shows WIBR3 wild-type human female ESCs, which were used to generate GATA4 or GATA6 DOX-inducible lines (iGATA4 or iGATA6) using Piggybac plasmids containing human GATA4 or GATA6, as shown here. Figure 46C shows immunostaining analysis of GATA6 or GATA4 protein expression in human primed PSCs (iGATA6 or iGATA4, respectively) grown in mTESR conditions with and without DOX (final concentration of 2 μg / ml Dox). As shown, GATA4 or GATA6 expression was specifically detected only after DOX addition in the corresponding lines. Figure 46C shows WIBR3 wild-type human female ESCs used to generate a GATA3-inducible line (iGATA3) using a Piggybac plasmid containing a GATA3 insert. Figure 46C shows immunostaining analysis of GATA3 protein expression in human primed PSCs grown in mTESR conditions with and without DOX (final concentration of 2 μg / ml Dox in the growth medium). GATA3 expression was specifically detected only after DOX addition. [Figure 46-2] Same as above [Figure 46-3] Same as above [Figure 47-1]Figures 47A-C demonstrate the performance of alternative conditions for generating naive PSCs as a starting population for generating organized embryos. Figure 47A shows a schematic representation of the protocol. Figure 47B shows egg cylinder-shaped sEmbryos obtained on day 6 of the protocol from BVSC, KH2-iGata4, and Kh2-iCDX2 cells grown in 2i / LIF and a2i / LIF (alternative to 2i / Lif) conditions (blue arrows highlight normal day 6 sEmbryos morphology). Figure 47C is a graph demonstrating the efficiency of egg cylinder-shaped sEmbryos obtained on day 5 of the protocol from BVSC, KH2-iGata4, and Kh2-iCDX2 cells grown in 2i / LIF and a2i / LIF (alternative to 2i / Lif), EPS (LCDM naive condition), and serum-LIF conditions. Error bars indicate SEM from the mean. [Figure 47-2] Same as above [Figure 47-3] Same as above [Figure 48] Figure 48 demonstrates that a two-step aggregation protocol improves the efficiency of sEmbryo formation. A schematic representation of the protocol and various regimens is shown, as well as a graph demonstrating the efficiency of egg-cylindrical sEmbryo formation obtained on day 5 of the protocol. [Figure 49-1] Figures 49A-D demonstrate the generation of trophectoderm (TE)-primed human cells by inducing endogenous expression of differentiation factors. Figure 49A is a schematic representation of the culture conditions. Figure 49B shows immunostaining characterization of trophectoderm (TE)-primed human cells. Staining was performed on day 3 of the trophectoderm induction regimen (TE). Figure 49C shows immunostaining characterization of trophectoderm-primed human cells with or without transgenic iGATA3 expression. Staining was performed on day 3 of the trophectoderm induction regimen (TE). Figure 49D demonstrates RT PCR expression of various trophectoderm markers in wild-type and iGATA3 WIBR3 cells. [Figure 49-2] Same as above [Figure 49-3] Same as above [Figure 49-4] Same as above [Figure 50-1] Figures 50A-D demonstrate the generation of cells primed for extraembryonic primitive endoderm (PRE) by inducing endogenous expression of differentiation factors. Figure 50A shows FACS analysis of PDGFRa expression 6 days after induction with the indicated media. Figure 50B shows FACS analysis of PDGFRa expression in iGATA6 cells 6 days after induction with the indicated media. Figure 50C demonstrates RT PCR expression of PRE markers (PDGFRa, GATA4, GATA6, Nid2, SOX17) and definitive endoderm markers (Gsc and Foxa2) in wild-type and iGATA6 WIBR3 cells. Figure 50D shows immunostaining characterization of human PRE cells with or without transgenic iGATA3 expression. Staining was performed on day 6 of the PRE induction regimen. [Figure 50-2] Same as above [Figure 50-3] Same as above [Figure 50-4] Same as above [Figure 51-1]Figures 51A-L demonstrate the coaggregation of human naive PSCs with trophectoderm (TE)-primed cells as described in Figures 49A-D and extraembryonic primitive endoderm-primed cells as described in Figures 50A-D, followed by ex utero culture, leading to the generation of organized embryos. Figure 51A shows a schematic representation of the protocol. Figure 51B demonstrates the progression of human sEmbryos from days 4 to 8 of the protocol (top panel) compared to human embryos 9-12 dpf (shown diagrammatically at the bottom). The top panel shows images of human sEmbryos from days 4 to 8 immunostained for OCT4 (cyan), SOX17 (yellow), and CK7 (magenta), which are markers of epiblast, hypoblast, and trophoblast, respectively. Scale bar: 50 μm. Abbreviations: TB: trophoblast, EB: epiblast, HB: hypoblast, L: lacunar, AC: amniotic cavity, PYS: primary yolk sac, ChC: chorionic cavity, ExEM: extraembryonic mesoderm, SK: stem, dpf: days post-fertilization. Figure 51C shows characterization of epiblast pluripotency in human sEmbryo. Images of day 4 sEmbryo immunostained for OCT4, SOX2, KLf17, and DNA (DAPI) are shown. Scale bar: 25 μm. Top panel - 2x magnification of epiblast region. Figure 51D-E shows apical cell polarity and lumen formation in human sEmbryo. Figure 51D shows images of day 6 sEmbryo immunostained for OCT4 (cyan), F-actin (red), and DNA (DAPI, gray). Scale bar: 50 μm. The asterisk indicates the putative chorionic cavity. The upper right shows a 2x magnification of the amniotic cavity. The lower right shows a 2x magnification of the yolk sac cavity. The arrow indicates the apical cell surface with aPKC (green). Scale bar: 12.5 μm. Figure 51E shows an image of a day 6 sEmbryo immunostained for SOX17 (yellow), F-actin (red), and DNA (DAPI, gray). Scale bar: 50 μm. The right shows a magnification of the yolk sac cavity, showing the yolk sac layer, which contains columnar cells (top) and flat cells (bottom). The cell periphery is delineated by a dashed line. Scale bar: 5 μm. OCT4 and SOX17 characterize the epiblast and hypoblast, respectively.Figure 51F-G shows the breaking of anterior-posterior symmetry in human sEmbryos. Figure 51F shows an image of a day 6 sEmbryo immunostained for OCT4 (cyan), CER1 (red), and DNA (DAPI, gray). Scale bar: 50 μm. Bottom: 4x magnification of the hypoblast region, rich in apical vesicles containing CER1 (indicated by arrows), which give rise to the AVE (anterior visceral endoderm). Figure 51G shows an image of a day 6 sEmbryo immunostained for OCT4 (cyan), Brachyury (BRA, red), and DNA (DAPI, gray). Scale bar: 50 μm. Right: 4x magnification of a BRA-positive epiblast cell (indicated by an arrow). OCT4 characterizes the pluripotent epiblast, whereas BRA and CER1 are early markers of epiblast and hypoblast differentiation, respectively, and are associated with the breakdown of anterior-posterior symmetry in early postimplantation human embryos. Figure 51H-J shows characterization of the trophoblast compartment in sEmbryos. Figure 51H shows an image of a day 6 sEmbryo immunostained for epiblast (OCT4, cyan), hypoblast (SOX17, yellow), and three different trophoblast markers (CK7, SDC1, and GATA3, magenta). All three types of trophoblast are present and form a surrounding layer covering the structure. The top panel of Figure 51I shows an image of a day 6 sEmbryo immunostained for F-actin (red) and HCGB (green), showing the membrane and syncytial trophoblast. The enlarged image shows multinucleated cells expressing HCGB, characteristic of syncytial trophoblasts. The bottom panel of Figure 51I shows an image of day 6 sEmbryo immunostained for CK7 (magenta) and HCGB (green), demonstrating partial, but not complete, overlap of the two markers, indicating the presence of cellular trophoblasts (CK7+ only) and syncytial trophoblasts (CK7+ and HCGB+). The top panel of Figure 51J shows an image of day 6 sEmbryo immunostained for F-actin and HCGB. A single stack of the membrane marker F-actin and HCGB indicates the formation of lacunae (dotted lines) by the syncytial trophoblasts.The bottom panel of Figure 51J shows a maximum projection image demonstrating that syncytial trophoblasts surround the entire structure. Scale bar: 50 μm. Figure 51K-L demonstrate the presence and organization of extraembryonic mesoderm (ExEm) in sEmbryo. Figure 51K shows images of day 6 sEmbryo immunostained for epiblast (OCT4, cyan), PYS (SOX17, yellow), and ExEm markers (BST2, GATA6, red). The top panel demonstrates that BST2+ cells have invaded the space between the yolk sac (SOX17+) and outer trophoblast layers (DAPI, SOX17-, OCT4-, BST2-), indicating the presence of ExEm cells. The middle panel demonstrates that ExEm also expresses GATA6, a common marker for HB / PYS, but at lower levels than in PYS. The bottom panel demonstrates that ExEM cells organize to form a cavity between the PYS and trophoblast cells, indicating the formation of a chorionic cavity (asterisk). Figure 51L shows a magnified view of a central section of an immunofluorescence image stained for OCT4 (cyan), SOX17 (green), and BST2 (red). BST2 is expressed between the yolk sac (SOX17+) and outer trophoblast cells (DAPI, SOX17-, OCT4-, BST2-) and in the epiblast (OCT4+). ExEM cells are characterized here by the expression of BST2 and the absence of SOX17 and OCT4 expression. Scale bar: 50 μm. Abbreviations: Max. Proj.: maximum intensity projection of z-stack. [Figure 51-2] Same as above [Figure 51-3] Same as above [Figure 51-4] Same as above [Figure 51-5] Same as above [Figure 51-6] Same as above [Figure 51-7] Same as above [Figure 52-1]Figures 52A-B demonstrate the generation of naive rhesus monkey (RM) PSCs. Figure 52A shows a phase image demonstrating the typical dome-shaped morphology of RM-1 naive hESCs grown in NENSM conditions at the indicated passages. Figure 52B shows an image of naive RM1-ESCs immunostained for several naive pluripotency markers (DPPA3, TFCP2L1, KLF17, and NANOG). DAPI was used for counterstaining. [Figure 52-2] Same as above [Figure 53] FIG. 53 is a schematic representation of the protocol for generating RM synthetic embryos. [Figure 54] Figure 54 demonstrates that coaggregation of RM naive PSC cells with trophectoderm (TE)-primed cells and human extraembryonic primitive endoderm-primed cells as described in Example 12, followed by ex utero culture, leads to the generation of organized embryos. The figure demonstrates the progression of human sEmbryos on day 6 of the protocol (left panel) compared to RM embryos at 9-12 dpf (schematically shown on the right). sEmbryos were immunostained for OCT4 (green), SOX17 (red), and Gata3 (magenta), which are markers of epiblast, hypoblast, and trophoblast, respectively. Scale bar: 50 μm. [Figure 55] Figure 55 demonstrates the formation of stable human TSC lines. Images of cells immunostained for CDX2, cytokeratin 7 (CK7), GATA3, and TFAP2C are shown. DAPI was used for counterstaining. [Figure 56]Figure 56 demonstrates that stable, established human TSC lines (human stable-derived TSC lines) are unable to form normal sEmbryos. Three starting cell types were coaggregated: naive wild-type hESCs cultured in HENSM medium; for the primitive endoderm compartment, naive wild-type cells were plated on HENSM supplemented with 10 μM ROCKi (Axon Medchem, 1683) in mouse embryonic fibroblast conditions, the medium was replaced with RCL medium the next day, and RCLs were maintained for 72 hours with medium changes every 24 hours; and for the trophoblast compartment, a human TSC line (passage 5) established from WIBR3 naive ESCs was used. Human TSC lines were generated by transferring human naive WIBR3 cells grown in HENSM conditions to TSC medium on MEF-coated plates, serially passaged in TSC medium, and validated as TSC lines. We constitutively labeled TSC lines with lentivirus encoding RFP (labeled as RED). Human TSCs were introduced on days 0, 1, and 2 of the sEmbryo protocol. In all cases, cells were visible at day 5 (as recognized by their RED labeling) colocalizing to one side of the aggregate without surrounding the entire sEmbryo. This is inconsistent with the expected morphology, which does not result in normally developing embryos surrounded only by the trophoblast lineage. [Figure 57-1]Figures 57A-C demonstrate the establishment of novel, stable, extraembryonic primitive endoderm-primed mouse cells (mouse PRE) from wild-type mouse naive ESCs / iPSCs or after transient overexpression of Gata4 in mouse ESCs. Figure 57A shows three strategies used to establish mouse PRE cells capable of permanent growth in the described mouse PRE medium. Images were taken at time 0 (2i / LIF stage naive ESCs) and after initiation of differentiation. The initial regimen used DOX for 72 hours as described. Figure 57B shows FACS analysis of cells using an anti-PDGFRa antibody. The parental ESC line is shown as a control. An unstained sample is shown as a negative control. Note that MEFs are also known to express PDGFRA, but at lower levels than mouse PRE cells. Figure 57C shows representative images of cells immunostained for the indicated markers. Immunostaining was performed on mouse PRE cells established from unmodified CD1 iPSCs grown in PRE medium. DAPI was used as a counterstain. [Figure 57-2] Same as above [Figure 57-3] Same as above [Figure 58] Figure 58 demonstrates that organized embryos could be generated by coaggregating naive mouse PSCs with mouse PRE cells and trophectoderm-primed mouse iCDX2 cells described in Figure 57A-C, followed by ex utero culture. The figure shows images of day 8 mouse sEmbryos generated according to the protocol shown in the figure. [Figure 59] Figures 59A-B demonstrate that coaggregation of human naive PSC cells with trophectoderm (TE)-primed cells and extraembryonic primitive endoderm-primed cells, as described in Example 15 below, followed by ex utero culture, leads to the generation of organized embryos. Figure 59A shows a schematic representation of the protocol. Figure 59B is a representative phase image showing the formation of a bilayered germinal disc, amniotic cavity, and yolk sac cavity, all surrounded by a trophoblast compartment. [Figure 60]Figures 60A-B demonstrate that co-aggregation of human naive PSC cells with cells primed to the trophectoderm (TE), as described in Example 16 below, followed by ex utero culture, leads to the generation of organized embryos. Figure 60A shows a schematic representation of the protocol. Figure 60B is a representative phase image showing the formation of a bilayered germinal disc, amniotic cavity, and yolk sac cavity, all surrounded by a trophoblast compartment. DETAILED DESCRIPTION OF THE INVENTION

[0091] The present invention, in some embodiments thereof, relates to methods for producing synthetic embryos.

[0092] Before describing at least one embodiment of the present invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by the examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0093] Advanced artificial embryogenesis utilizing in vitro established embryonic stem cell lines (ESCs) and induced pluripotent stem cell lines (iPSCs) while avoiding the use of oocytes and zygotes or blastocysts has become a desired goal in the fields of developmental biology and reproductive medicine. Although various pluripotent states of stem cells can be expanded in vitro and can contribute to embryonic and / or extraembryonic tissues when injected into a host embryo, there has thus far been failure to obtain a gastrulated whole embryo-like structure with both embryonic and extraembryonic compartments without relying on microinjected host embryos (see, e.g., Zhang et al. (2020) Front. Bioeng. Biotechnol. 8:781; Harrison et al. Science (2017), DOI:10.1126 / science.aa1810; Amadei et al. Developmental Cell (2021) 56, 366-382; and as illustrated in Example 1 in the Examples section below).

[0094] In putting into practice certain embodiments of the present invention, the inventors have now developed a method for generating synthetic embryos with both embryonic and extraembryonic compartments by starting only with naive ESCs, followed by culturing them in a controlled ex utero embryo culture platform.

[0095] As illustrated hereinafter and in the Examples section below, we demonstrate that coaggregating three populations of mouse stem cells—naive, untransduced PSCs, PSCs transiently overexpressing a master regulatory transcription factor for trophectoderm cells (Cdx2), and ESCs transiently overexpressing a master regulatory transcription factor for extraembryonic primitive endoderm cells (Gata4)—followed by ex utero culture of the aggregates in a recently described controlled platform and growth conditions (Aguilera-Castrejon et al., 2021a) results in the construction of fully synthetic mouse embryos that complete gastrulation and develop progenitors for the brain, neural folds, neural tube, beating heart, somites, and other organs (Examples 2-3 and 7-8 in the Examples section below). The synthetic embryos developed within extraembryonic membranes similar to natural embryos. Furthermore, thorough analysis of both the embryonic and extraembryonic compartments demonstrated that these synthetic embryos reached a level of lineage complexity comparable to that seen in natural intrauterine embryos, with all annotated cell types represented in the synthetic embryos at a developmental stage equivalent to E8.5. Furthermore, a very high level of correlation in gene expression was observed for each tissue between natural intrauterine embryos and synthetic mouse embryos. In a similar manner, we demonstrate that coaggregation of three identical but human-derived stem cell populations, followed by ex utero culture of the aggregates, results in the construction of fully synthetic human embryos with both embryonic and extraembryonic compartments (Examples 4-6 in the Examples section below). Subsequently, we show that various culture conditions can support the differentiation of naive PSCs (from mouse, human, or rhesus monkey) into trophectoderm (TE)-primed or extraembryonic primitive endoderm (PRE)-primed cells, and that coaggregation of these stem cell populations followed by ex utero culture of the aggregates results in the construction of complete synthetic embryos (Examples 9-12 and 14-16 in the Examples section below).

[0096] Establishing methods and systems for generating such synthetic embryos ex utero could not only be a powerful tool in basic research, for example, as a framework for investigating the emergence of cellular diversity, cell fate determination, and how tissues and organs arise from single pluripotent cells, but could also serve as a source of cells, tissues, embryos, and organs for transplantation, testing the effects of drugs on embryonic development, and potentially embryo bioengineering.

[0097] Thus, according to one aspect of the present invention, there is provided a method for producing a synthetic embryo, comprising the steps of: (a) inducing the expression of factors that induce differentiation into trophectoderm cells in a subpopulation of naive pluripotent stem cells (PSCs) to obtain trophectoderm-primed cells; and (b) mixing the trophectoderm-primed cells with naive PSCs under conditions that allow for the formation of aggregate cells; When the factor that induces differentiation into trophectoderm cells is an exogenous factor, the induction of expression includes induction of transient expression that begins within a period of 14 days to 0 hours before mixing and ends within 120 hours after mixing; the factor that induces differentiation into trophectoderm cells is an endogenous factor, and the induction of expression begins within a period of 14 days to 6 hours before mixing, comprising culturing a subpopulation of naive PSCs under conditions that allow expression of the factor; A method is provided whereby a synthetic embryo is produced.

[0098] According to a further or alternative aspect of the present invention, there is provided a method of producing a synthetic embryo, comprising: (a) inducing transient expression of an exogenous factor that induces differentiation into trophectoderm cells in a subpopulation of naive pluripotent stem cells (PSCs) to obtain trophectoderm-primed cells; and (b) mixing the trophectoderm-primed cells with naive PSCs under conditions that allow for the formation of aggregate cells; Induction of transient expression begins within the period from 14 days to 0 hours before mixing and ends within 120 hours after mixing. A method is provided whereby a synthetic embryo is produced.

[0099] According to certain embodiments, the method further comprises (a) inducing expression of a factor that induces differentiation into extraembryonic primitive endoderm cells in a second subpopulation of naive PSCs to obtain cells primed towards extraembryonic primitive endoderm, and (b) mixing the cells primed towards extraembryonic primitive endoderm with the cells.

[0100] According to certain embodiments, the method further comprises (a) inducing transient expression of an exogenous factor that induces differentiation into extraembryonic primitive endoderm cells in a second subpopulation of naive PSCs to obtain cells primed towards extraembryonic primitive endoderm, and (b) mixing the cells primed towards extraembryonic primitive endoderm with the cells.

[0101] According to additional or alternative aspects of the present invention there is provided a mixture or aggregate of cells obtainable by the methods disclosed herein, as further described below.

[0102] According to further or alternative aspects of the present invention, there is provided a synthetic embryo obtainable by the methods disclosed herein.

[0103] As used herein, the term "synthetic embryo" refers to a collection of at least different cells produced ex vivo using pluripotent stem cells without any germ cell, zygote, or blastocyst donation. When cultured, the synthetic embryo undergoes self-organization and differentiation to give rise to multiple tissue types that are organized like a natural embryo or exhibit embryonic-like properties.

[0104] According to certain embodiments, the synthetic embryo structure mimics the developmental stages of a natural or naturally occurring embryo, at least during the early stages of embryonic development after the blastocyst stage.

[0105] According to certain embodiments, the synthetic embryo is not a blastocyst (eg, a structure having characteristics of E3.5 / 4-5 of a natural mouse embryo in utero or PFD6 / PFD7 of a natural human embryo in utero).

[0106] According to certain embodiments, the synthetic embryo at least mimics (with respect to humans) Carnegie stage 5, Carnegie stage 6, Carnegie stage 8, or later developmental stages, an embryo with a primary and secondary yolk sac, a bilayered blastocyst stage surrounded by trophoblast, and formation of the amniotic membrane, yolk, and chorionic cavity, including formation of extraembryonic mesodermal cells.

[0107] According to certain embodiments, the synthetic mouse embryo at least mimics the formation of the egg cylinder stage, the completion of gastrulation (E7.5), and the onset of organogenesis (E8.0 or later in the mouse embryo).

[0108] According to certain embodiments, the synthetic embryo is produced exclusively outside the uterus.

[0109] As used herein, the phrase "pluripotent stem cells (PSCs)" refers to undifferentiated cells (e.g., mammalian cells, primate cells) that can differentiate into all three embryonic germ cell layers, namely, into the embryonic germ layers mesoderm, ectoderm, and endoderm, or can remain in an undifferentiated state.

[0110] Methods for determining the ability of stem cells to differentiate into the embryonic germ layers of endoderm, mesoderm, and ectoderm are known in the art (e.g., as disclosed in WO 2014 / 174470, the contents of which are fully incorporated herein by reference in their entirety) and include, for example, the generation of embryoid bodies (in vitro) or teratomas (in vivo).

[0111] According to some embodiments of the invention, the pluripotent stem cells are selected from the group consisting of embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and embryonic germ cells (EGCs).

[0112] According to certain embodiments, the cells are mammalian cells, e.g., rodent (e.g., mouse, rat), human (homo sapiens), monkey (e.g., chimpanzee, gorilla, rhesus monkey, and / or baboon), livestock animal (e.g., cow, sheep, pig, goat, horse, donkey, mule).

[0113] According to a particular embodiment, the cells are mouse cells.

[0114] According to certain embodiments, the cells are human cells.

[0115] According to other particular embodiments, the cells are non-human primate (e.g., macaque) cells.

[0116] The term "embryonic stem cells" refers to embryonic cells that can differentiate into cells of all three embryonic germ layers (i.e., endoderm, ectoderm, and mesoderm) or can remain undifferentiated. The term "embryonic stem cells" can include cells obtained before implantation of an embryo (i.e., preimplantation blastocysts) from embryonic body tissues (e.g., blastocysts) formed after conception, expanded blastocyst cells (EBCs) obtained from blastocysts at the postimplantation / pregastrulation stage (see WO 2006 / 040763), and embryonic germ (EG) cells obtained from fetal (offspring) reproductive tissue at any time during pregnancy, preferably before 10 weeks of gestation.

[0117] Embryonic stem cells in some embodiments of the present invention can be obtained using well-known cell culture methods. For example, human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo preimplantation embryos or in vitro fertilized (IVF) embryos. Alternatively, single-cell human embryos can be grown to the blastocyst stage. To isolate human ES cells, the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by immunosurgery. Trophectoderm cells are then lysed and removed from the intact ICM by gentle pipetting.

[0118] The ICM is then plated into tissue culture flasks containing the appropriate medium to allow for ICM growth. After 9–15 days, the ICM-derived outgrowths are dissociated into clumps by either mechanical dissociation or enzymatic digestion, and the cells are then replated in fresh tissue culture medium. Colonies exhibiting undifferentiated morphology are individually selected with a micropipette, mechanically dissociated into clumps, and replated. The resulting ES cells are then split regularly every 4–7 days. For further details on methods for preparing human ES cells, see Thomson et al., [U.S. Patent No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133, 1998; Proc. Natl. Acad. Sci. USA 92: 7844, 1995], Bongso et al., [Hum Reprod 4: 706, 1989], and Gardner et al., [Fertil. Steril. 69: 84, 1998].

[0119] Another method for preparing ES cells is described in Chung et al., Cell Stem Cell, Volume 2, Issue 2, 113-117, 7 February 2008. This method involves removing a single cell from an embryo during an in vitro fertilization process, in which the embryo is not destroyed.

[0120] It will be appreciated that commercially available stem cells can also be used in accordance with some embodiments of the present invention. Human ES cells can be purchased from the NIH Human Embryonic Stem Cell Registry [Hypertext Transfer Protocol: / / grants(dot)nih(dot)gov / stem_cells / registry / current(dot)htm]. Non-limiting examples of commercially available embryonic stem cell lines include BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE32, CHB-4, CHB-5, CHB-6, CHB-8, CHB-9, CHB-10, CHB-11, CHB-12, HUES 1, HUES 2, HUES 3, HUES 4, HUES 5, HUES 6, HUES 7, HUES 8, HUES 9, HUES 10, HUES 11, HUES 12, HUES 13, HUES 14, HUES 15, HUES 16, HUES 17, HUES 18, HUES 19, HUES 20, HUES 21, HUES 22, HUES 23, HUES 24, HUES 25, HUES 26, HUES 27, HUES 28, HUES 29, HUES 30, HUES 31, HUES 32, HUES 33, HUES 34, HUES 35, HUES 36, HUES 37, HUES 38, HUES 39, HUES 40, HUES 41, HUES 42, HUES 43, HUES 44, HUES 45, HUES 46, HUES 47, HUES 48, HUES 49, HUES 50, HUES 51, HUES 52, HUES 53, HUES 54, HUES 55, HUES 56, HUES 57, HUES 58, HUES 59, 26, HUES 27, HUES 28, CyT49, RUES3, WA01, UCSF4, NYUES1, NYUES2, NYUES3, NYUES4, NYUES5, NYUES6, NYUES7, UCLA 1, UCLA 2, UCLA 3, WA077(H7), WA09(H9), WA13(H13), WA14(H14), HUES 62, HUES 63, HUES 64, CT1, CT2, CT3, CT4, MA135, Eneavour-2, WIBR1, WIBR2, WIBR3, WIBR4, WIBR5, WIBR6, HUES 45, Shef 3, Shef 6, BJNhem19, BJNhem20, SA001, SA001.

[0121] In addition, ES cells have been used to generate embryonic stem cells from mice (Mills and Bradley, 2001), golden hamsters (Doetschman et al., 1988, Dev Biol. 127: 224-7), rats (Iannaccone et al., 1994, Dev Biol. 163: 288-92), rabbits (Giles et al. 1993, Mol Reprod Dev. 36: 130-8; Graves & Moreadith, 1993, Mol Reprod Dev. 1993, 36: 424-33), and several livestock species (Notarianni et al., 1991, J Reprod Fertil Suppl. 43: 255-60; Wheeler 1994, Reprod Fertil Dev. 6: 563-8; Mitalipova et al., 2001, Cloning. 3: 59-67], as well as from other species, including non-human primate species (rhesus monkeys and marmosets) [Thomson et al., 1995, Proc Natl Acad Sci U S A. 92: 7844-8; Thomson et al., 1996, Biol Reprod. 55: 254-9].

[0122] Non-limiting examples of commercially available mouse embryonic stem cell lines are v6.5, KH2, BVSC ES line, NGFP1 iPSC line, NGFP2 iPSC line.

[0123] Expanded blastocyst cells (EBCs) can be obtained from blastocysts at least 9 days post-fertilization, at the pre-gastrulation stage. Prior to culturing the blastocysts, the zona pellucida is digested (e.g., with Tyrode's acid solution (Sigma-Aldrich, St. Louis, MO, USA)) to expose the inner cell mass. The blastocysts are then cultured in vitro as whole embryos for at least 9 days post-fertilization and no more than 14 days (i.e., before the gastrulation event) using standard embryonic stem cell culture methods.

[0124] EG cells are prepared from primordial germ cells obtained from fetuses (human fetuses) approximately 8-11 weeks gestation using laboratory techniques known to those skilled in the art. The genital ridges are dissociated and cut into small clumps, which are then disaggregated into cells by mechanical dissociation. EG cells are then grown in tissue culture flasks with appropriate medium. The cells are cultured with daily medium changes until a cell morphology consistent with EG cells is observed (usually after 7-30 days or 1-4 passages). For further details on the preparation of human EG cells, see Shamblott et al., [Proc. Natl. Acad. Sci. USA 95: 13726, 1998] and U.S. Patent No. 6,090,622.

[0125] Induced pluripotent stem cells (iPS, embryonic-like stem cells) are cells obtained by dedifferentiation of adult somatic cells that have been endowed with pluripotency (i.e., the ability to differentiate into the three embryonic germ cell layers: endoderm, ectoderm, and mesoderm). According to some embodiments of the present invention, such cells are obtained from differentiated tissues (e.g., somatic tissues such as skin) and dedifferentiated by genetic manipulations that reprogram the cells to acquire embryonic stem cell characteristics. According to some embodiments of the present invention, induced pluripotent stem cells are formed by inducing the expression of Oct-4, Sox2, Kfl4, and c-Myc in somatic stem cells.

[0126] Induced pluripotent stem cells (iPS) (embryonic-like stem cells) can be generated from somatic cells by genetic manipulation, for example, by retroviral transduction of transcription factors such as Oct-3 / 4, Sox2, c-Myc, and KLF4 into somatic cells such as fibroblasts, hepatocytes, and gastric epithelial cells [Yamanaka S, Cell Stem Cell. 2007, 1(1):39-49; Aoi T, et al., Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach Cells. Science. 2008 Feb 14. (Epub ahead of print); IH Park, Zhao R, West JA, et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 2008;451:141-146; K Takahashi, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861-872]. Other combinations of iPSCs generated by overexpression of pluripotency factors are also included (Buganim et al. Cell (2012) 150(6): 1209-1222 www(dot)doi(dot)org / 10.1016 / j.cell.2012.08.023). Other embryonic-like stem cells can be generated by nuclear transfer into oocytes, fusion with embryonic stem cells, or by nuclear transfer into zygotes if the recipient cell is arrested in mitosis.

[0127] The methods of some embodiments of the present invention utilize a naive PSC.

[0128] The phrase "naive pluripotent stem cells (PSCs)" refers to PSCs that express pluripotency markers (such as Oct4, Nanog, and Klf4) and lack markers specific to extraembryonic endoderm, trophectoderm, and early somatic lineages. These cells retain unlimited developmental potential, as they can reliably differentiate into all cell types in vitro and, when injected into blastocysts, efficiently contribute to the three germ layers and the germline of chimeric animals.

[0129] Naive PSCs in humans and non-human primates are also in a state prior to X-chromosome inactivation.

[0130] X-chromosome inactivation is an early developmental process in mammalian females that results in equal gene dosage between males and females by transcriptionally silencing one of the paired X chromosomes. This process is regulated by several factors, including a region of the X chromosome called the X-inactivation center (XIC). The XIC contains several non-coding and protein-coding genes, and the XIST gene was the first non-coding gene identified within the XIC.

[0131] When XIST bodies are present, they are expressed exclusively from the XIC of the inactive X chromosome and are essential for the propagation of X chromosome inactivation.

[0132] The pre-X chromosome inactivation state according to some embodiments of the invention is characterized by the presence of two unmethylated alleles in the promoter of the X chromosome inactivation-specific transcription (XIST) gene in female cells and the presence of an unmethylated allele in the promoter of the XIST gene in male cells.

[0133] This is in contrast to "primed" or "conventional PSCs" in humans and non-human primates, which are characterized by one methylated allele of XIST and one unmethylated allele of XIST in female cells and one methylated allele in male cells.

[0134] The XIST gene is located on the human chromosome Xq13.2 and has the sequence shown in clone NC_000023.10 (73040486.73072588, complementary strand, based on GenBank version GRCh37.p10). The XIST gene has a non-coding RNA shown in GenBank accession number NR_001564.2.

[0135] Methods for determining the methylation status of the XIST gene are known in the art (e.g., Lengner Cell 141, 872-883 (2010); Hanna et al., Cell 143, 508-525; (2010); TAKASHI SADO et al., DEVELOPMENTAL DYNAMICS 205: 421-434 (1996); and WO 2016 / 016894 and WO 2014 / 174470, each of which is fully incorporated by reference in its entirety), and include, for example, bisulfite sequencing and Southern blot analysis.

[0136] According to some embodiments of the present invention, the presence of two unmethylated alleles of the XIST gene in female cells refers to less than about 20% of CpG methylated reads sequenced in the XIST promoter, e.g., less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, e.g., 0% (e.g., completely absent) of CpG methylated reads sequenced in the XIST promoter.

[0137] According to some embodiments of the present invention, the presence of one unmethylated allele of the XIST gene in male cells refers to less than about 20% of CpG methylated reads sequenced in the XIST promoter, for example, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, e.g., 0% of CpG methylated reads sequenced in the XIST promoter.

[0138] A non-limiting example of an XIST promoter containing a CpG island, which can be either methylated or unmethylated, is shown in the XIST promoter amplicon represented by SEQ ID NO:65.

[0139] According to some embodiments of the invention, naive PSCs exhibit a pre-X chromosome inactivation state similar to that of the human inner cell mass (ICM).

[0140] According to some embodiments of the present invention, naive PSCs are characterized by reduced CpG island methylation compared to the CpG island methylation level of primed PSCs.

[0141] According to some embodiments of the present invention, naive PSCs are characterized by significantly low levels of total methylated cytosines (e.g., 1-2%) of total guanine nucleotides in each cell, as determined by liquid chromatography-mass spectrometry (LC-MS) quantitative analysis.

[0142] According to some embodiments of the present invention, naive PSCs are characterized by having 0-3% total methylated cytosines of the total guanine nucleotides in the naive PSC cells, compared to primed PSCs or somatic cells, which have 3.5-5% total methylated cytosines of the total guanine nucleotides in the primed PSC cells.

[0143] According to some embodiments of the invention, human naive PSCs express XIST.

[0144] Methods for detecting XIST expression are known in the art and include, for example, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis using XIST-specific PCR primers, such as forward primer: 5'-AGGGAGCAGTTTGCCCTACT (SEQ ID NO: 66) and reverse primer: 5'-CACATGCAGCGTGGTATCTT (SEQ ID NO: 67).

[0145] According to some embodiments of the invention, naive PSCs lack XIST bodies.

[0146] As used herein, the phrase "XIST body" refers to an inactive X chromosome coated with XIST.

[0147] Methods for detecting XIST bodies are known in the art, including, for example, RNA fluorescence in situ hybridization. RNA fluorescence in situ hybridization (FISH) is performed as previously described [Hanna J., et al., Cell 143, 508-525]. Briefly, human pluripotent stem cells are harvested, MEFs are depleted, cytospun onto glass slides, and then fixed. cDNA probes were generated for XIST exon 1 (GenBank accession no. U80460:61251-69449, SEQ ID NO: 26) and exon 6 (GenBank accession no. U80460:75081-78658, SEQ ID NO: 27) and labeled with Cy3-dUTP (Amersham) by nick translation (Roche), and Cot-1 DNA was labeled with fluorescein-12-dUTP using the Prime-It Fluor Labeling Kit (Stratagene).

[0148] According to some embodiments of the invention, naive PSCs lack H3K27me3 / polycomb foci.

[0149] As used herein, the phrase "H3K27me3 / Polycomb foci" refers to nuclear foci obtained after immunostaining that correspond to the condensed inactive X chromosome.

[0150] Methods for detecting H3K27me3 / polycomb foci are known in the art and include, for example, the use of immunofluorescence analysis using an anti-H3K27me3 antibody (e.g., rabbit anti-H3K27me3, Millipore, CA, USA, Catalog No. 07-449), as shown, for example, in Figure 23C-D.

[0151] According to some embodiments of the invention, naive PSCs have low levels of XIST expression while in the naive state, none of the X chromosomes are inactivated, and XIST bodies are absent.

[0152] It should be noted that naive PSCs (before X-chromosome inactivation and in a naive state) of some embodiments of the present invention are capable of inactivating one of the X-chromosome alleles and methylating one of the XIST genes upon differentiation.

[0153] According to certain embodiments, the expression level of transcription factor E3 (TFE3) in naive PSCs is characterized by a nuclear to cytoplasmic expression ratio of 1 or greater, as determined, for example, by an immunostaining assay.

[0154] According to certain embodiments, naive cells express c-Kit on the cell surface, as determined, for example, by flow cytometry or immunostaining assays.

[0155] In contrast to known primed PSCs, naive PSCs of some embodiments of the present invention are "resistant" to induction of differentiation by FGFR / MEK / ERK inhibitors, JNK inhibitors, and / or P38 inhibitors. Thus, according to some embodiments of the present invention, when isolated naive PSCs are incubated in the presence of an agent selected from the group consisting of an FGFR / MEK / ERK inhibitor, a JNK inhibitor, and a P38 inhibitor, the naive PSCs remain in a pluripotent state.

[0156] According to some embodiments of the present invention, the p38 pathway is inhibited in naive PSCs compared to primed PSCs, e.g., p38 activity is inhibited in naive PSCs.

[0157] According to some embodiments of the invention, the level of p38 RNA and / or phosphorylated p38 protein in naive PSCs is less than about 30%, such as less than about 20%, such as less than about 5%, such as less than about 0.5%, such as less than about 0.1%, compared to the level of p38 RNA and / or phosphorylated p38 protein, respectively, in non-naive PSCs incubated and / or cultured under the same conditions but not subjected to p38 inhibition.

[0158] According to some embodiments of the present invention, the JNK pathway is inhibited in naive PSCs compared to primed PSCs, e.g., JNK activity is inhibited in naive PSCs.

[0159] According to some embodiments of the invention, the level of JNK RNA and / or phosphorylated JNK protein in naive PSCs is less than about 30%, such as less than about 20%, such as less than about 5%, such as less than about 0.5%, such as less than about 0.1%, compared to the level of JNK RNA and / or phosphorylated JNK protein, respectively, in non-naive PSCs incubated and / or cultured under the same conditions but not subjected to JNK inhibition.

[0160] According to some embodiments of the present invention, the ROCK pathway is inhibited in naive PSCs compared to primed PSCs, e.g., ROCK activity is inhibited in naive PSCs.

[0161] According to some embodiments of the invention, the level of ROCK RNA and / or phosphorylated ROCK protein in naive PSCs is less than about 30%, such as less than about 20%, such as less than about 5%, such as less than about 0.5%, such as less than about 0.1%, compared to the level of ROCK RNA and / or phosphorylated ROCK protein, respectively, in non-naive PSCs incubated and / or cultured under the same conditions but not subjected to ROCK inhibition.

[0162] According to some embodiments of the invention, naive PSCs maintained in an undifferentiated, pluripotent, and naive state (as defined above) express significantly lower levels of SOX1 compared to the expression levels present in primed PSCs (e.g., primed ESCs) under identical SOX1 assay conditions, and primed PSCs exhibit one methylated and one unmethylated allele of XIST (in female cells) or one methylated allele of XIST (in male cells), express XIST, exhibit XIST bodies, and exhibit H3K27me3 / Polycomb foci.

[0163] According to some embodiments of the present invention, naive PSCs express lower levels of MHC class I compared to primed PSCs under identical detection assay conditions, and primed PSCs exhibit one methylated and one unmethylated allele of XIST (in female cells) or one methylated allele of XIST (in male cells), express XIST, exhibit XIST bodies, and exhibit H3K27me3 / Polycomb foci.

[0164] The level of MHC class I can be determined by various methods known in the art, such as FACS analysis using specific antibodies to detect surface expression of MHC class I molecules, and FACS analysis using fluorescently labeled anti-HLA-A, anti-HLA-B, and anti-HLA-C antibodies (BD Biosciences).

[0165] According to some embodiments of the invention, naive PSCs are characterized by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% more RNA polymerase II pausing on chromosomes compared to primed PSCs under identical assay conditions, and primed PSCs exhibit one methylated and one unmethylated allele of XIST (in female cells) or one methylated allele of XIST (in male cells), express XIST, exhibit XIST bodies, and exhibit H3K27me3 / Polycomb foci.

[0166] According to certain embodiments, naive PSCs express pluripotency markers, i.e., are positive for one or more pluripotency markers.

[0167] According to certain embodiments, the expression is of an endogenous protein.

[0168] Positive is also abbreviated as (+). Positive for a marker means that at least about 70%, 80%, 85%, 90%, 95%, or 100% of the cells in a population show detectable levels of the marker assayed by methods known to those skilled in the art. Developmental markers can be detected using immunological techniques well known in the art (e.g., as described in Thomson JA et al., (1998). Science 282: 1145-7). Examples include, but are not limited to, immunostaining, microscopy, flow cytometry, Western blot, and enzyme immunoassay. Other non-limiting methods include PCR analysis, RNA fluorescence in situ hybridization (FISH), Northern blot, and single-cell RNA sequencing.

[0169] Pluripotency markers are well known in the art and include, but are not limited to, Oct4, NANOG, KLF4, KLF17, SUSD2, TFCP2L1.

[0170] Naive PSCs of some embodiments of the present invention are characterized by positive expression of C-KIT protein (also known as CD117) on the cell surface.

[0171] According to certain embodiments, naive PSCs can be obtained during culture in a naive state without the need for exogenous expression of Oct4, Sox2, Klf4, and / or c-Myc factors.

[0172] According to certain embodiments, naive PSCs are negative for differentiation markers.

[0173] Negative is also abbreviated as (-). Negative for a marker means that about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less of the cells in the population show detectable levels of the marker as assayed by methods known to those skilled in the art, such as FACS, immunofluorescence, immunohistochemistry, Western blot analysis, RT-PCR, etc. Non-limiting examples of differentiation markers include Gata3, Gata2, tbr2, tfap2c, Cdx2, Elf5, Hand1, EOMES, ETS2, Foxa2, Gata4, Gata6, PDGFRA, and Sox17.

[0174] PSC differentiation can also be assessed by measuring alkaline phosphatase activity, which is present in undifferentiated human ES cells and can be detected by fixing the cells with 4% paraformaldehyde and developing with the Vector Red substrate kit according to the manufacturer's instructions (Vector Laboratories, Burlingame, CA, USA).

[0175] According to certain embodiments, naive PSCs can be obtained during culture in a naive state under feeder-layer-free conditions (e.g., on a surface coated with Matrigel, laminin, and / or fibronectin, but not on feeder cells, e.g., fibroblast feeder cells, e.g., mouse embryonic fibroblasts (MEFs)).

[0176] According to certain embodiments, naive PSCs can be obtained during culture in a naive pluripotent state in the absence of exogenous L-glutamine in the culture medium.

[0177] According to certain embodiments, naive PSCs can be obtained during culture in a naive pluripotent state in the absence of DNA methyltransferase 1 (DNMT1) expression. Methods for obtaining naive PSCs and maintaining them in a naive and pluripotent state are known in the art and are described in the Examples section below, which form an integral part of this specification, as well as, for example, Choi, J., Huebner, A., Clement, K et al. Nature 548, 219-223 (2017), www(dot)doi(dot)org / 10.1038 / nature23274; Yang et al. Cell (2017) 169(2): 243-257, www(dot)cell(dot)com / fulltext / S0092-8674%2817%2930183-6; Bayerl et al. 2021, Cell Stem Cell 28, 1549-1565, and WO 2016 / 016894 and WO 2014 / 174470, the contents of which are incorporated herein by reference in their entireties.

[0178] Several naive PSC cell lines are also known and commercially available from public cell line repositories, for example, the V6.5 mouse ESC line, the BVSc ES line, the NGFP1 iPSC line, the WIBR3 line, the WIS1 ES line, and the WIS ES line.

[0179] As used herein, the term "trophectoderm (TE)-primed cells" refers to stem cells derived from naive PSCs that contain an exogenous nucleic acid sequence encoding a factor that induces differentiation into a trophectoderm cell, and / or cells derived from naive PSCs that have committed to the trophectoderm lineage (e.g., trophectoderm cells). It will be understood that prior to the induction of expression of the factor (i.e., the exogenous nucleic acid sequence or the protein product expressed from the endogenous factor), the cell is in a naive and undifferentiated state (i.e., a naive PSC), and that after the induction of expression of the factor, the cell acquires the characteristics of a trophectoderm cell.

[0180] According to certain embodiments, the trophectoderm-primed cells comprise an exogenous polynucleotide suitable for the transient expression of factors that induce differentiation into trophectoderm cells, as further described below.

[0181] According to certain embodiments, the cells primed towards trophectoderm comprise a protein product expressed from an exogenous nucleic acid sequence encoding a factor.

[0182] According to other particular embodiments, the cells primed towards trophectoderm do not comprise a protein product expressed from an exogenous nucleic acid sequence encoding a factor.

[0183] As used herein, "trophectoderm cells" refers to cells with restricted potential that give rise to all placental lineages but no other cell lineages.

[0184] According to some embodiments, the trophectoderm cells give rise to the ectoplacental cone, and / or extraembryonic ectoderm (ExE), and / or the umbilical cord, and / or the chorion.

[0185] Non-limiting examples of trophectoderm cell markers include Gata3, Gata2, tbr2, tfap2c, Cdx2, Elf5, Hand1, EOMES, KLF5, and ETS2.

[0186] According to certain embodiments, the trophectoderm cells express Cdx2, ie are positive for Cdx2.

[0187] According to certain embodiments, cells primed to trophectoderm express trophectoderm markers. According to certain embodiments, cells primed to trophectoderm express at least one trophectoderm marker selected from the group consisting of Gata3, Gata2, Cdx2, and Elf5.

[0188] According to certain embodiments, cells primed to trophectoderm express pluripotency markers. Such markers are known in the art and are further described above and below. According to certain embodiments, cells primed to trophectoderm express (are positive for) at least one pluripotency marker selected from the group consisting of Oct4, Tfap2c, Susd2, Sox2, SSEA1, SSEA3, SSEA4, and TRA1-60. According to certain embodiments, cells primed to trophectoderm express Oct4. According to certain embodiments, cells primed to trophectoderm are negative for pluripotency markers selected from the group consisting of Klf17, Klf4, and Nanog.

[0189] According to certain embodiments, cells primed towards trophectoderm express trophectoderm markers and pluripotency markers.

[0190] According to certain embodiments, the marker is endogenous to the cell.

[0191] Thus, according to certain embodiments, cells primed towards trophectoderm express exogenous factors that induce differentiation into trophectoderm cells, endogenous trophectoderm markers, and endogenous pluripotency markers.

[0192] According to certain embodiments, cells primed towards trophectoderm express trophectoderm markers immediately after mixing (ie, at time 0), in the mixture, or in the aggregates.

[0193] According to certain embodiments, cells primed towards trophectoderm express pluripotency markers immediately after mixing (ie, at time 0), in the mixture, or in the aggregates.

[0194] According to certain embodiments, cells primed towards trophectoderm do not express Oct4 and Nanog immediately after mixing (ie, at time 0), in the mixture, or in the aggregates.

[0195] According to certain embodiments, the cells primed to trophectoderm (e.g., immediately after mixing, in the mixture, in the aggregate) are not at the differentiation stage of trophoblast stem cells (TSCs). In other words, the cells primed to trophectoderm do not have the phenotypic signature of TSCs (derived from trophectoderm, artificial TSCs, or obtained by cell dedifferentiation or reprogramming).

[0196] According to certain embodiments, the TSCs (e.g., human TSCs) express CDX2 - TFAP2C + GATA3 + SIGLE6 + have a phenotype.

[0197] In comparison, human cells primed to trophectoderm or some embodiments of the present invention contain CDX2 + TFAP2c + GATA3 + SIGLEC6 - have a phenotype.

[0198] According to certain embodiments, the TSCs (e.g., mouse TSCs) express CDX2 + TFAP2C + MHC class 1 + OCT4 - have a phenotype.

[0199] In comparison, mouse cells primed to trophectoderm or some embodiments of the present invention contain CDX2 + TFAP2C + MHC class 1 - Oct4 +(低) have a phenotype.

[0200] The factors whose expression induces differentiation of naive PSCs in some embodiments of the present invention can be endogenous and / or exogenous to the cells.

[0201] According to certain embodiments, the method comprises inducing expression of an exogenous factor that induces differentiation into (towards) trophectoderm cells.

[0202] As used herein, the term "exogenous" refers to a heterologous polynucleotide or a heterologous polypeptide that is not naturally expressed in a cell or whose overexpression in a cell is desired. It should be noted that an exogenous polynucleotide and / or exogenous polypeptide may contain nucleic acid sequences and / or amino acid sequences, respectively, that are identical or partially homologous to endogenous nucleic acid sequences and / or endogenous amino acid sequences of the cell.

[0203] According to additional or alternative embodiments, the method includes inducing the expression of endogenous factors that induce differentiation into (toward) trophectoderm cells. Thus, according to certain embodiments, the cells are cultured under conditions that allow the expression of endogenous factors that induce differentiation into trophectoderm-primed cells. Such conditions include, for example, appropriate temperature (e.g., 37°C), atmosphere (e.g., O2% and CO2%), pressure, pH, light, medium, additives (e.g., differentiation factors disclosed herein), etc. Further description of culture conditions is provided below. Thus, according to certain embodiments, naive PSC cells or trophectoderm-primed cells are contacted with factors. Under this culture scenario, the cells may further comprise or lack exogenous polynucleotides suitable for expressing factors that induce differentiation into trophectoderm cells. According to certain embodiments, the cells do not comprise exogenous polynucleotides suitable for expressing factors that induce differentiation into trophectoderm cells. In this case, induction of expression of, culturing, or contacting of endogenous factors is typically performed before mixing the cell subpopulations (e.g., within the period 14 days to 0 hours before mixing, within the period 13 days to 0 hours before mixing, within the period 12 days to 0 hours before mixing, within the period 11 days to 0 hours before mixing, within the period 10 days to 0 hours before mixing, within the period 9 days to 0 hours before mixing, within the period 8 days to 0 hours before mixing, within the period 7 days to 0 hours before mixing, within the period 6 days to 0 hours before mixing, within the period 5 days to 0 hours before mixing, within the period 120 hours to 0 hours before mixing, within the period 100 hours to 0 hours before mixing, within the period 80 hours to 0 hours before mixing). Within the period, within the period 60 hours to 0 hours before mixing, within the period 48 hours to 0 hours before mixing, within the period 36 hours to 0 hours before mixing, within the period 24 hours to 0 hours before mixing, within the period 12 hours to 0 hours before mixing, within the period 14 days to 1 day before mixing, within the period 13 days to 1 day before mixing, within the period 12 days to 1 day before mixing, within the period 11 days to 1 day before mixing, within the period 10 days to 1 day before mixing, within the period 9 days to 1 day before mixing, within the period 8 days to 1 day before mixing, within the period 7 days to 1 day before mixing, within the period 6 days to 1 day before mixing, within the period 5 days to 1 day before mixing, within the period 120 hours to 20 hours before mixing,Within the period of 100 hours to 20 hours before mixing, within the period of 80 hours to 20 hours before mixing, within the period of 60 hours to 20 hours before mixing, within the period of 48 hours to 20 hours before mixing, within the period of 14 days to 12 hours before mixing, within the period of 13 days to 12 hours before mixing, within the period of 12 days to 12 hours before mixing, within the period of 11 days to 12 hours before mixing, within the period of 10 days to 12 hours before mixing, within the period of 9 days to 12 hours before mixing Within the period from 1 day to 12 hours before mixing, within the period from 8 days to 12 hours before mixing, within the period from 7 days to 12 hours before mixing, within the period from 6 days to 12 hours before mixing, within the period from 5 days to 12 hours before mixing, within the period from 120 hours to 12 hours before mixing, within the period from 100 hours to 12 hours before mixing, within the period from 80 hours to 12 hours before mixing, within the period from 60 hours to 12 hours before mixing, within the period from 48 hours to 12 hours before mixing Within, within the period 14 days to 6 hours before mixing, within the period 13 days to 6 hours before mixing, within the period 12 days to 6 hours before mixing, within the period 11 days to 6 hours before mixing, within the period 10 days to 6 hours before mixing, within the period 9 days to 6 hours before mixing, within the period 8 days to 6 hours before mixing, within the period 7 days to 6 hours before mixing, within the period 6 days to 6 hours before mixing, within the period 5 days to 6 hours before mixing, within the period 1 within a period of 20 hours to 6 hours before mixing, within a period of 100 hours to 6 hours before mixing, within a period of 80 hours to 6 hours before mixing, within a period of 60 hours to 6 hours before mixing, within a period of 48 hours to 6 hours before mixing, within a period of 36 hours to 6 hours before mixing, within a period of 24 hours to 6 hours before mixing, or within a period of 12 hours to 6 hours before mixing, each possibility representing a separate embodiment of the present invention.

[0204] According to certain embodiments, the culturing or contacting is initiated at least 6 hours, at least 10 hours, at least 12 hours, at least 16 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, or at least 96 hours prior to mixing.

[0205] According to certain embodiments, the culturing or contacting is initiated within 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 14 days, 16 days, or 20 days prior to mixing.

[0206] According to certain embodiments, the culturing or contacting is initiated within about 6 to 3 days prior to mixing.

[0207] According to certain embodiments, the culturing or contacting is initiated about 3 days prior to mixing.

[0208] Factors that induce differentiation toward trophectoderm cells are well known in the art, including factors expressed in cells and factors that come into contact with cells. Non-limiting examples of such factors include Cdx2, Gata3, Gata2, TGFR inhibitors, FGFR inhibitors, MEK / ERK inhibitors, BMP4, JAK inhibitors, FGF4, FGF2, heparin, SUMOylation inhibitors, histone deacetylase inhibitors, HIPPO signaling pathway inhibitors, and factors that induce YAP nuclear translocation.

[0209] According to a particular embodiment, the factor that induces differentiation into trophectoderm cells is Cdx2.

[0210] As used herein, the term "Cdx2" refers to polynucleotides and expression products, e.g., polypeptides, of the CDX2 gene (corresponding to human Gene ID No. 1045) or its homologs or orthologs. According to certain embodiments, Cdx2 refers to human Cdx2, such as that set forth in the following GeneBank Nos. NP_001256, NP_001341629, NM_001265, and NM_001354700 (SEQ ID NOs: 68-70). According to certain embodiments, Cdx2 refers to mouse Cdx2, such as that set forth in the following GeneBank Nos. NP_031699 and NM_007673 and (SEQ ID NOs: 72-73). Homologues (as defined in more detail below) refer to functional expression products of Cdx2 capable of inducing differentiation of naive PSCs into trophectoderm cells.

[0211] Thus, as noted above, any of the polynucleotides and expression products (e.g., Cdx2, Gata4, etc.) described herein also refer to homologs that exhibit the desired activity defined herein. Such homologs can be, for example, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide or polynucleotide sequences provided herein.

[0212] Homologues may also refer to orthologs, deletion, insertion, or substitution variants containing amino acid substitutions, so long as they retain activity.

[0213] Sequence identity or homology can be determined using any protein or nucleic acid sequence alignment algorithm, such as Blast, ClustalW, and MUSCLE.

[0214] According to a particular embodiment, the factor that induces differentiation into trophectoderm cells is Gata3.

[0215] As used herein, the term "Gata3," also known as GATA binding protein 3 and HDRS, refers to polynucleotides and expression products, e.g., polypeptides, of the GATA3 gene (corresponding to human Gene ID No. 2625) or its homologs or orthologs. According to certain embodiments, Gata3 refers to human Gata3, such as that set forth in the following GeneBank Nos. NP_001002295 and NM_001002295 (SEQ ID NO: 74). According to certain embodiments, Gata3 refers to mouse Gata3, such as that set forth in the following GeneBank Nos. NP_032117, NP_001342039, NP_001342040, NP_001342041, NM_008091, NM_001355110, NM_001355111, and NM_001355112 (SEQ ID NOs: 76-83). Homologue (as defined in more detail above) refers to a functional expression product of Gata3 that is capable of inducing differentiation of naive PSCs into trophectoderm cells.

[0216] According to a particular embodiment, the factor that induces differentiation into trophectoderm cells is Gata2.

[0217] As used herein, the term "Gata2," also known as GATA-binding protein 2, refers to the polynucleotides and expression products, e.g., polypeptides, of the GATA2 gene (corresponding to human Gene ID No. 2624) or its homologs or orthologs. According to certain embodiments, Gata2 refers to human Gata2, such as that set forth in the following GeneBank Nos. NP_001139133, NP_001139134, NP_116027, NM_032638, NM_001145661, and NM_001145662 (SEQ ID NOs: 84-89). According to certain embodiments, Gata2 refers to mouse Gata2, such as that set forth in the following GeneBank Nos. NP_032116, NP_001342182, NM_008090, and NM_001355253 (SEQ ID NOs: 90-93). Homologue (as defined in more detail above) refers to a functional expression product of Gata2 that is capable of inducing differentiation of naive PSCs into trophectoderm cells.

[0218] According to a particular embodiment, the factor that induces differentiation into trophectoderm cells is a TGFR inhibitor.

[0219] As used herein, the term "transforming growth factor receptor (TGFR)" (also known as "TGFβR") refers to ALK5, a TGF-β type I receptor, ALK4, a type I activin / nodal receptor, and ALK7, a type I nodal receptor.

[0220] As used herein, the term "TGFR inhibitor (or TGFRi)" refers to a molecule capable of inhibiting the expression and / or activity level of TGFR, as determined by phosphorylated ALK4, ALK5, and ALK7. Non-limiting examples of TGFR inhibitors include the small molecule compounds SB431542 and A 83-01.

[0221] According to a particular embodiment, the factor that induces differentiation into trophectoderm cells is an FGFR inhibitor.

[0222] As used herein, the term "fibroblast growth factor receptor (FGFR)" refers to FGFR1, FGFR2, and FGFR3.

[0223] As used herein, the term "FGFR inhibitor (or FGFRi)" refers to a molecule capable of inhibiting the expression and / or activity levels of FGFRs as determined by the levels of phosphorylated FGFR1, FGFR2, and FGFR3. Non-limiting examples of FGFR inhibitors include PD173074, SU5401, and PD0325901.

[0224] According to a particular embodiment, the factor that induces differentiation into trophectoderm cells is a ROCK inhibitor.

[0225] As used herein, the term "ROCK" includes ROCK1 and ROCK2, which are also known as Rho-associated protein kinases and have serine / threonine kinase activity.

[0226] As used herein, the term "ROCK inhibitor" refers to any molecule capable of inhibiting the activity of ROCK as determined by inhibition of ROCK phosphorylation levels (e.g., as detected by Western blot analysis).

[0227] Non-limiting examples of ROCK inhibitors include Y27632 (TOCRIS, Catalog No. 1254), blebbistatin (TOCRIS, Catalog No. 1760), and thiazovivin (Axon Medchem-Axon 1535). Blebbistatin is a selective inhibitor of myosin II ATPase, a downstream effector of the ROCK pathway, and therefore effectively inhibits the ROCK pathway (Chen G1, Hou Z et al. Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells. Cell Stem Cell. 2010; 7(2):240-8, the entire contents of which are fully incorporated herein by reference).

[0228] According to certain embodiments, the factor that induces differentiation into trophectoderm cells is a MEK / ERK inhibitor (as defined in more detail below).

[0229] According to a particular embodiment, the factor that induces differentiation into trophectoderm cells is BMP4.

[0230] As used herein, the term "BMP4," also known as bone morphogenetic protein 4, refers to polynucleotides and expression products, e.g., polypeptides, of the BMP4 gene (corresponding to human gene ID number 652) or homologs or orthologs thereof. According to certain embodiments, BMP4 refers to human BMP4, such as that set forth in the following UniProt number: P12644 (SEQ ID NO: 195). According to certain embodiments, BMP4 refers to mouse BMP4, such as that set forth in the following UniProt number: P21275 (SEQ ID NO: 196). A homolog (as defined in more detail above) refers to a functional expression product of BMP4 that is capable of inducing differentiation of naive PSCs into trophectoderm cells.

[0231] According to certain embodiments, the factor that induces differentiation into trophectoderm cells is a JAK inhibitor.

[0232] As used herein, the term "JAK" refers to Janus kinases and includes JAK1, JAK2, JAK3, and TYK2.

[0233] As used herein, the term "JAK inhibitor (or JAKi)" refers to a molecule capable of inhibiting the expression and / or activity level of JAK, as determined by the level of phosphorylated STAT (signal transducer and activator of transcription). Non-limiting examples of JAK inhibitors include Calbiochem 420099, tofacitinib, baricitinib, ruxolitinib, fedratinib, upadacitinib, cedulatinib, gandotinib, lesraurtinib, momelotinib, pacritinib, curuevitacin I, CHZ868, and the like.

[0234] According to a particular embodiment, the factor that induces differentiation into trophectoderm cells is FGF4.

[0235] As used herein, the term "FGF4," also known as fibroblast growth factor 4, refers to polynucleotides and expression products, e.g., polypeptides, of the FGF4 gene (corresponding to human Gene ID No. 2249) or homologs or orthologs thereof. According to certain embodiments, FGF4 refers to human FGF4, such as that set forth in the following GeneBank Nos. NP_001998 and NM_002007 (SEQ ID NOs: 94-95). According to certain embodiments, FGF4 refers to mouse FGF4, such as that set forth in the following GeneBank Nos. NP_034332 and NM_010202 (SEQ ID NOs: 96-97). Homologues (as defined in more detail above) refer to functional expression products of FGF4 capable of inducing differentiation of naive PSCs into trophectoderm cells and / or extraembryonic primitive endoderm cells.

[0236] According to a particular embodiment, the factor that induces differentiation into trophectoderm cells is FGF2.

[0237] As used herein, the term "FGF2," also known as basic fibroblast growth factor (bFGF) and FGF-β, refers to the polynucleotides and expression products, e.g., polypeptides, of the FGF2 gene (corresponding to human Gene ID No. 2247) or its homologs or orthologs. According to certain embodiments, FGF2 refers to human FGF2, such as that set forth in the following GeneBank Nos. NP_001997, NP_001348594, NM_002006, and NM_001361665 (SEQ ID NOs: 98-101). According to certain embodiments, FGF2 refers to mouse FGF2, such as that set forth in the following GeneBank Nos. NP_032032 and NM_008006 (SEQ ID NOs: 102-103). Homologues (as defined in more detail above) refer to functional expression products of FGF2 that are capable of inducing differentiation of naive PSCs into trophectoderm cells and / or extraembryonic primitive endoderm cells.

[0238] According to certain embodiments, the factor that induces differentiation into trophectoderm cells is a sumoylation inhibitor, such as, but not limited to, DC 2-D08 (2',3',4'-trihydroxyflavone).

[0239] According to certain embodiments, the factor that induces differentiation into trophectoderm cells is a histone deacetylase inhibitor (HDACi), such as, but not limited to, VPA (valproic acid), trichostatin A, sodium butyrate, or the small molecule inhibitor UF010.

[0240] According to certain embodiments, the factor that induces differentiation into trophectoderm cells is an inhibitor of the HIPPO signaling pathway or a factor that induces YAP nuclear translocation.

[0241] The HIPPO signaling pathway, also known as the Salvador / Warts / Hippo (SWH) pathway, involves a complex cascade of serine / threonine protein kinases that ultimately regulate the phosphorylation of Yap / Taz. Specifically, when the Hippo pathway is "off," phosphorylated YAP / TAZ are retained in the cytoplasm and may undergo proteolysis. When the Hippo pathway is "on," unphosphorylated YAP / TAZ translocate to the nucleus and bind to transcription factors called TEA DNA-binding proteins (TEAD1-4). Non-limiting examples of components of the HIPPO signaling pathway include Hpo, serine threonine kinase 3 (STK3) and STK4 (also known as MST1 and MST2), salvador homolog 1 (SAV1), large tumor suppressor 1 / 2 (LATS1 / 2), MAP4K, TAOK, MOB kinase activator 1A / B (MOB1A / B), yes-associated protein (YAP1), transcriptional coactivator with PDZ-binding motifs (TAZ or WWTR1), and neurofibromin 2 (NF2).

[0242] The HIPPO signaling pathway inhibitors disclosed herein bind to components of the HIPPO signaling pathway and inhibit their expression and / or activity. Such inhibitors are known in the art and include, but are not limited to, LPA and XMU-MP-1.

[0243] As used herein, the term "cells primed towards extraembryonic primitive endoderm (PRE)" refers to stem cells derived from naive PSCs that contain exogenous nucleic acid sequences encoding factors that induce differentiation into extraembryonic primitive endoderm cells, and / or cells derived from naive PSCs that have committed to the extraembryonic primitive endoderm lineage (e.g., extraembryonic primitive endoderm cells).

[0244] It will be understood that prior to the induction of expression of exogenous factors (i.e., exogenous nucleic acid sequences or protein products expressed from endogenous factors), the cells are in a naive and undifferentiated state (i.e., naive PSCs), and following the induction of expression of exogenous factors, the cells acquire characteristics of extraembryonic primitive endoderm cells.

[0245] According to certain embodiments, cells primed to extraembryonic primitive endoderm comprise an exogenous polynucleotide suitable for transient expression of factors that induce differentiation into extraembryonic primitive endoderm cells, as further described below.

[0246] According to certain embodiments, cells primed towards extraembryonic primitive endoderm comprise a protein product expressed from an exogenous nucleic acid sequence encoding a factor.

[0247] According to other specific embodiments, the cells primed to extraembryonic primitive endoderm do not comprise a protein product expressed from an exogenous nucleic acid sequence encoding a factor.

[0248] As used herein, "extraembryonic primitive endoderm cells" refer to cells with restricted potential to give rise to all primitive endoderm lineages but not other cell lineages.

[0249] Non-limiting examples of extraembryonic primitive endoderm cell markers include Foxa2, Gata4, Gata6, PDGFRA, and Sox17.

[0250] According to certain embodiments, the extraembryonic primitive endoderm cells express Gata4, ie, are positive for Gata4.

[0251] According to certain embodiments, cells primed to extraembryonic primitive endoderm express extraembryonic primitive endoderm markers. According to certain embodiments, cells primed to extraembryonic primitive endoderm express at least one extraembryonic primitive endoderm marker selected from the group consisting of PDGFRA, GATA4, SOX17, NID2, and GATA6.

[0252] According to certain embodiments, cells primed to extraembryonic primitive endoderm express pluripotency markers. Such markers are known in the art and are further described above and below. According to certain embodiments, cells primed to extraembryonic primitive endoderm express (are positive for) at least one pluripotency marker selected from the group consisting of OCT4, SSEA1, and SSEA4. According to certain embodiments, cells primed to extraembryonic primitive endoderm are negative for pluripotency markers selected from the group consisting of NANOG, KLF17, NANOG-NB, and TFCP2L1.

[0253] According to certain embodiments, cells primed towards extraembryonic primitive endoderm express extraembryonic primitive endoderm markers and pluripotency markers.

[0254] According to certain embodiments, the marker is endogenous to the cell.

[0255] Thus, according to certain embodiments, cells primed to extraembryonic primitive endoderm express exogenous factors that induce differentiation into extraembryonic primitive endoderm cells, endogenous extraembryonic primitive endoderm markers, and endogenous pluripotency markers.

[0256] According to certain embodiments, cells primed to extraembryonic primitive endoderm express extraembryonic primitive endoderm markers immediately after mixing (i.e., at time 0), in the mixture, or in the aggregates.

[0257] According to certain embodiments, cells primed to extraembryonic primitive endoderm express pluripotency markers immediately after mixing (i.e., at time 0), in the mixture, or in the aggregates.

[0258] According to certain embodiments, cells primed towards extraembryonic primitive endoderm express Oct4 immediately after mixing (ie, at time 0), in the mixture, or in the aggregates.

[0259] According to other specific embodiments, cells primed to extraembryonic primitive endoderm do not express Oct4 immediately after mixing (i.e., at time 0), in the mixture, or in the aggregates.

[0260] According to certain embodiments, cells primed to extraembryonic primitive endoderm (e.g., immediately after mixing, in the mixture, in the aggregate) are not at the differentiation stage of XEN cells. In other words, cells primed to extraembryonic primitive endoderm do not have the phenotypic signature of XEN (which can be obtained from blastocysts, early postimplantation embryos, or artificial XEN obtained by cell dedifferentiation or reprogramming).

[0261] According to a particular embodiment, XEN is OCT4 - GATA6 + GATA4 + SOX17 + PDGFRa + have a phenotype.

[0262] In comparison, cells primed to extraembryonic primitive endoderm in some embodiments of the present invention are OCT4 + GATA6 + GATA4 + SOX17 + PDGFRa + have a phenotype.

[0263] As previously mentioned, factors whose expression induces differentiation of naive PSCs in some embodiments of the present invention can be endogenous and / or exogenous to the cells.

[0264] According to certain embodiments, the method comprises inducing expression of an exogenous factor that induces differentiation into (towards) extraembryonic primitive endoderm cells.

[0265] According to additional or alternative embodiments, the method includes inducing expression of an endogenous factor that induces differentiation toward extraembryonic primitive endoderm cells. Thus, according to certain embodiments, the cells are cultured under conditions that allow expression of an endogenous factor that induces differentiation toward extraembryonic primitive endoderm-primed cells. Such conditions include, for example, appropriate temperature (e.g., 37°C), atmosphere (e.g., O2% and CO2%), pressure, pH, light, medium, additives (e.g., differentiation factors disclosed herein), etc. Culture conditions are further described below. Thus, according to certain embodiments, naive PSC cells or extraembryonic primitive endoderm-primed cells are contacted with a factor. Under this culture scenario, the cells may further comprise or lack an exogenous polynucleotide suitable for expression of a factor that induces differentiation toward extraembryonic primitive endoderm cells. According to certain embodiments, the cells do not comprise an exogenous polynucleotide suitable for expression of a factor that induces differentiation into extraembryonic primitive endoderm. In this case, the induction of expression of, culturing, or contacting of endogenous factors is typically performed before mixing the subpopulations of cells (e.g., within a period of 14 days to 0 hours before mixing, within a period of 13 days to 0 hours before mixing, within a period of 12 days to 0 hours before mixing, within a period of 11 days to 0 hours before mixing, within a period of 10 days to 0 hours before mixing, within a period of 9 days to 0 hours before mixing, within a period of 8 days to 0 hours before mixing, within a period of 7 days to 0 hours before mixing, within a period of 6 days to 0 hours before mixing, within a period of 5 days to 0 hours before mixing, within a period of 120 hours to 0 hours before mixing, within a period of 100 hours to 0 hours before mixing, within a period of 8 days to 0 hours before mixing, within a period of 120 hours ... Within the period from 0 hours to 0 hours before mixing, within the period from 60 hours to 0 hours before mixing, within the period from 48 hours to 0 hours before mixing, within the period from 36 hours to 0 hours before mixing, within the period from 24 hours to 0 hours before mixing, within the period from 12 hours to 0 hours before mixing, within the period from 14 days to 1 day before mixing, within the period from 13 days to 1 day before mixing, within the period from 12 days to 1 day before mixing, within the period from 11 days to 1 day before mixing, within the period from 10 days to 1 day before mixing, within the period from 9 days to 1 day before mixing, within the period from 8 days to 1 day before mixing, within the period from 7 days to 1 day before mixing, within the period from 6 days to 1 day before mixing, within the period from 5 days to 1 day before mixing,Within the period of 120 hours to 20 hours before mixing, within the period of 100 hours to 20 hours before mixing, within the period of 80 hours to 20 hours before mixing, within the period of 60 hours to 20 hours before mixing, within the period of 48 hours to 20 hours before mixing, within the period of 14 days to 12 hours before mixing, within the period of 13 days to 12 hours before mixing, within the period of 12 days to 12 hours before mixing, within the period of 11 days to 12 hours before mixing, within the period of 10 days to 12 hours before mixing Within the period 2 hours before mixing, within the period 9 days to 12 hours before mixing, within the period 8 days to 12 hours before mixing, within the period 7 days to 12 hours before mixing, within the period 6 days to 12 hours before mixing, within the period 5 days to 12 hours before mixing, within the period 120 hours to 12 hours before mixing, within the period 100 hours to 12 hours before mixing, within the period 80 hours to 12 hours before mixing, within the period 60 hours to 12 hours before mixing, or within the period Within the period of 48 hours to 12 hours before mixing, within the period of 14 days to 6 hours before mixing, within the period of 13 days to 6 hours before mixing, within the period of 12 days to 6 hours before mixing, within the period of 11 days to 6 hours before mixing, within the period of 10 days to 6 hours before mixing, within the period of 9 days to 6 hours before mixing, within the period of 8 days to 6 hours before mixing, within the period of 7 days to 6 hours before mixing, within the period of 6 days to 6 hours before mixing, within the period of 5 days to 6 hours before mixing (e.g., within a period of 120 hours to 6 hours before mixing, within a period of 100 hours to 6 hours before mixing, within a period of 80 hours to 6 hours before mixing, within a period of 60 hours to 6 hours before mixing, within a period of 48 hours to 6 hours before mixing, within a period of 36 hours to 6 hours before mixing, within a period of 24 hours to 6 hours before mixing, or within a period of 12 hours to 6 hours before mixing, each possibility representing a separate embodiment of the present invention.)

[0266] According to certain embodiments, the culturing or contacting is initiated at least 6 hours, at least 10 hours, at least 12 hours, at least 16 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 72 hours, or at least 96 hours prior to mixing.

[0267] According to certain embodiments, the culturing or contacting is initiated within 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 14 days, 16 days, or 20 days prior to mixing.

[0268] According to certain embodiments, the culturing or contacting is initiated within about 6 to 3 days prior to mixing.

[0269] According to certain embodiments, the culturing or contacting is initiated about 3 days prior to mixing.

[0270] Factors that induce differentiation into extraembryonic primitive endoderm cells are well known in the art, including factors expressed in cells and factors that come into contact with cells. Non-limiting examples of such factors include Gata4, Gata6, GSK-3 inhibitors, WNT ligands, heparin, FGF2, FGF4, PDGFRA, leukemia inhibitory factor (LIF), and insulin.

[0271] According to a particular embodiment, the factor that induces differentiation into extraembryonic primitive endoderm cells is Gata4.

[0272] As used herein, the term "Gata4," also known as GATA-binding protein 4 and transcription factor Gata4, refers to the polynucleotides and expression products, e.g., polypeptides, of the GATA4 gene (corresponding to human Gene ID No. 2626) or its homologs or orthologs. According to certain embodiments, Gata4 refers to human Gata4, such as that set forth in the following GeneBank Nos.: NP_001295022, NP_001295023, NP_002043, NP_001361202, NP_001361203, NM_001308093, NM_001308094, NM_002052, NM_001374273, and NM_001374274 (SEQ ID NOs: 104-113). According to certain embodiments, Gata4 refers to mouse Gata4, such as that set forth in the following GeneBank numbers: NP_001297539, NP_032118, NM_008092, and NM_001310610 (SEQ ID NOs: 114-117). Homologues (as defined in more detail above) refer to functional expression products of Gata4 that are capable of inducing differentiation of naive PSCs into extraembryonic primitive endoderm cells.

[0273] According to a particular embodiment, the factor that induces differentiation into extraembryonic primitive endoderm cells is Gata6.

[0274] As used herein, the term "Gata6," also known as GATA-binding protein 6 and transcription factor Gata6, refers to polynucleotides and expression products, e.g., polypeptides, of the GATA6 gene (corresponding to human gene ID number 2627) or its homologs or orthologs. According to certain embodiments, Gata6 refers to human Gata6, such as that set forth in the following GeneBank numbers: NP_005248 and NP_005257 (SEQ ID NOs: 118-119). According to certain embodiments, Gata6 refers to mouse Gata6, such as that set forth in the following GeneBank numbers: NP_034388 and NM_010258 (SEQ ID NOs: 120-121). Homologues (as defined in more detail above) refer to functional expression products of Gata6 capable of inducing differentiation of naive PSCs into extraembryonic primitive endoderm cells.

[0275] According to a particular embodiment, the factor that induces differentiation into extraembryonic primitive endoderm cells is SOX17.

[0276] As used herein, the term "SOX17," also known as SRY-box 17, refers to polynucleotides and expression products, e.g., polypeptides, of the SOX17 gene (corresponding to human gene ID number 64321) or its homologs or orthologs. According to certain embodiments, SOX17 refers to human SOX17, such as that set forth in the following GeneBank numbers: NP_071899 and NM_022454 (SEQ ID NOs: 197-198). According to certain embodiments, SOX17 refers to mouse SOX17, such as that set forth in the following UniProt number: Q61473 (SEQ ID NO: 199). Homologues (as defined in more detail above) refer to functional expression products of SOX17 that are capable of inducing differentiation of naive PSCs into extraembryonic primitive endoderm cells.

[0277] According to certain embodiments, the factor that induces differentiation into extraembryonic primitive endoderm cells is a GSK-3 inhibitor.

[0278] As used herein, the term "GSK-3" refers to glycogen synthase kinase 3 beta protein, designated by Genbank accession number NP_002084 (SEQ ID NO: 122) and / or NP_001139628 (SEQ ID NO: 123), which has WNT signaling modulating activity via kinase activity.

[0279] As used herein, the term "GSK-3 inhibitor" refers to any molecule capable of inhibiting the activity of GSK-3 as determined by specifically inhibiting the level of phosphorylated GSK3b (out of the total GSK3b present in the cell).

[0280] Non-limiting examples of GSK-3 inhibitors include CHIR99021 (AXON MEDCHEM - AXON 1386), BIO (AXON MEDCHEM - Axon 1693), and Kenpaullone (TOCRIS - Catalog No. 1398).

[0281] According to certain embodiments, the factor that induces differentiation into extraembryonic primitive endoderm cells is a WNT ligand.

[0282] Wnt ligands are a large family of secreted glycoproteins that are rich in cysteine ​​and highly hydrophobic. Non-limiting examples of Wnt ligands that can be used in certain embodiments of the present invention include Wnt-1, Wnt-2, Wnt-2b, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt8a, Wnt-8b, Wnt-9a, Wnt-9b, Wnt-10a, Wnt-10b, Wnt-11, and Wnt16b.

[0283] According to a particular embodiment, the factor that induces differentiation into extraembryonic primitive endoderm cells is FGF2.

[0284] According to a particular embodiment, the factor that induces differentiation into extraembryonic primitive endoderm cells is FGF4.

[0285] According to certain embodiments, the factor that induces differentiation into extraembryonic primitive endoderm cells is PDGF.

[0286] As used herein, the term "PDGF" refers to platelet-derived growth factor, which includes various subtypes of PDGF, including PDGF-B (e.g., GenBank Accession Nos. X02811 and CAA26579), PDGF-A (e.g., GenBank Accession Nos. X06374 and CAA29677), PDGF-C (e.g., GenBank Accession Nos. NM 016205 and NP 057289), PDGF-D, variant 1 and variant 2 (e.g., GenBank Accession Nos. NM 025208, NP 079484, NM 033135, NP 149126), and dimerized forms thereof, including PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD. Typically, platelet-derived growth factors comprise homodimers or heterodimers of A and B chains, which bind to and dimerize two related receptor tyrosine kinases, the platelet-derived growth factor cell surface receptors, PDGFR-α (also known as PDGFRA, e.g., GenBank Accession Nos. NM 006206 and NP 006197) and PDGFR-β (e.g., GenBank Accession Nos. NM 002609 and NP 002600). For PDGF sequences, see also International Publication No. WO 2010 / 127029 and U.S. Patent Application Publication No. 20150182623, which are incorporated herein by reference in their entireties. According to certain embodiments, PDGF can induce the differentiation of naive PSCs into extraembryonic primitive endoderm cells.

[0287] According to certain embodiments, the factor that induces differentiation into extraembryonic primitive endoderm cells is LIF (as defined in more detail below).

[0288] According to certain embodiments, the factor that induces differentiation into extraembryonic primitive endoderm cells is insulin.

[0289] Having developed culture conditions that allow for the generation of cells primed towards extraembryonic primitive endoderm from naive PSCs (Examples 10 and 14 in the Examples section below), which can be used, for example, to generate synthetic embryos, embodiments of the present invention also contemplate methods for obtaining these cells.

[0290] Therefore, according to one aspect of the present invention, there is provided a method for obtaining cells primed to extraembryonic primitive endoderm, the method comprising: converting naive PSCs into extraembryonic primitive endoderm-primed GATA4 + GATA6 + OCT4 + Nanog - In one embodiment, a method is provided that comprises culturing the cells in a basal medium lacking activin A under conditions that allow differentiation into extraembryonic primitive endoderm-primed cells, thereby obtaining cells primed towards extraembryonic primitive endoderm.

[0291] According to a particular embodiment, GATA4 + GATA6 + OCT4 + Nanog - The cells are also positive for PDGFRA and NID2.

[0292] According to certain embodiments of this aspect of the invention, the method further comprises inducing expression (eg, transient expression) of an exogenous factor that induces differentiation into extraembryonic primitive endoderm cells.

[0293] According to other particular embodiments of this aspect of the invention, the method does not include inducing expression (e.g., transient expression) of an exogenous factor that induces differentiation into extraembryonic primitive endoderm cells.

[0294] According to particular embodiments, culturing is carried out for at least 6 hours, at least 12 hours, at least 16 hours, at least 1 day (ie at least 24 hours), at least 2 days, or at least 3 days.

[0295] According to certain embodiments, culturing is carried out for up to 14 days, up to 13 days, up to 12 days, up to 11 days, up to 10 days, up to 9 days, up to 8 days, up to 7 days, or up to 6 days.

[0296] According to a particular embodiment, the culturing is carried out for 1 to 10 days.

[0297] According to a particular embodiment, the culturing is carried out for about 3 to 6 days.

[0298] According to a particular embodiment, the culturing is carried out for about 3 days or more.

[0299] According to certain embodiments, the resulting cells retain the phenotype of cells primed towards extraembryonic primitive endoderm (e.g., GATA4 + GATA6 + OCT4 + Nanog - Thus, according to certain embodiments, the cells are stable lines that maintain the phenotype of cells primed to extraembryonic primitive endoderm (e.g., GATA4 + GATA6 + OCT4 + Nanog - The cells can be expanded in culture for at least 3 passages, at least 4 passages, at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, at least 10 passages, at least 20 passages, or at least 30 passages while maintaining a specific phenotype.

[0300] The exogenous factors of some embodiments of the present invention are transiently expressed in the cells.

[0301] Methods for expressing exogenous nucleic acid sequences and / or exogenous amino acid sequences are known in the art and include, for example, the materials and methods in the Examples section below, as well as those described in Mansour et al. 2012, Warren et al. 2010 and Hongyan Zhou et al. Cell Stem Cell (2009) 4(6): 581, Rabinovich and Weissman (2013) Methods Mol Biol. 969:3-28, WO 2013049389, and U.S. Pat. No. 8,557,972 (which are hereby fully incorporated by reference in their entireties).

[0302] Additionally, methods of transient expression are well known in the art and include, but are not limited to, expression under the control of an inducible promoter, by introducing mRNA into the cell (wherein the mRNA is translated in the cell and degraded after a relatively short period of time), or by transfection of a protein, as further described below.

[0303] The cells that express exogenous polynucleotides and / or exogenous polypeptides can be distinguished from the cells that do not express exogenous polynucleotides and / or exogenous polypeptides by, for example, determining the level and / or distribution of RNA and / or protein molecules in cells, the location of DNA integration in the genome of cells, and / or the number of gene copies.Methods for determining the presence of exogenous polynucleotides and / or exogenous polypeptides in cells are well known in the art, and include, for example, PCR, DNA sequencing and RNA sequencing, Southern blot, Western blot, immunoprecipitation, immunocytochemistry, flow cytometry, and imaging.

[0304] According to certain embodiments, expression is achieved by introducing a polynucleotide encoding the factor into the cell.

[0305] According to certain embodiments, the expression is not at the natural location (ie, locus) and / or expression level (eg, copy number and / or cellular localization) of the factor's native gene.

[0306] Alternatively or additionally, exogenous expression of factors can be promoted by activation of the endogenous locus of these genes such that the factors are overexpressed in the cells. Methods for activating and overexpressing endogenous genes are well known in the art [see, e.g., Menke D. Genesis (2013) 51: -618; Capecchi, Science (1989) 244:1288-1292; Santiago et al. Proc Natl Acad Sci USA (2008) 105:5809-5814; International Publication Nos. 2014085593, 2009071334, and 2011146121; U.S. Patent Nos. 8,771,945, 8,586,526, and 6,774,279; and U.S. Patent Application Publication Nos. 20030232410, 20050026157, and 20060014264. [The contents of which are incorporated by reference in their entireties], which include, but are not limited to, targeted homologous recombination (e.g., "hit-and-run," "double replacement"), site-specific recombinases (e.g., Cre recombinase and Flp recombinase), PB transposases (e.g., Sleeping Beauty, piggyBac, Tol2, or Frog Prince), genome editing with engineered nucleases (e.g., meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR / Cas systems), genome editing using recombinant adeno-associated virus (rAAV) platforms, and small molecules. Agents for introducing nucleic acid modifications into genes of interest can be designed from publicly available sources or are commercially available from Transposagen, Addgene, and Sangamo Biosciences.

[0307] As used herein, the term "polynucleotide" refers to a single- or double-stranded nucleic acid sequence in the form of an RNA sequence (e.g., mRNA), a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence (e.g., a sequence isolated from a chromosome), a composite polynucleotide sequence (e.g., a combination of the above polynucleotides), or a mimetic or analog thereof. The term includes polynucleotides and / or oligonucleotides derived from naturally occurring nucleic acid molecules (e.g., RNA or DNA), synthetic polynucleotide and / or oligonucleotide molecules composed of naturally occurring bases, sugars, and covalent internucleoside linkages (e.g., backbones), and synthetic polynucleotides and / or oligonucleotides having non-naturally occurring portions that function similarly to the respective naturally occurring portions.

[0308] According to certain embodiments, the polynucleotide is a modified polynucleotide, such as a modified RNA.

[0309] Such modified polynucleotides may contain modifications in the backbone, internucleoside linkage, or base. Modified polynucleotides may contain naturally modified nucleotides or synthetic nucleoside analogs. In certain embodiments, modified polynucleotides may be preferred over natural forms due to desirable properties such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, and reduced immunogenicity.

[0310] Such modifications include, but are not limited to, 5-methoxyuridine, pseudouridine, 5-methylcytidine, N6-methyladenosine, 2'-O-methyl, 2'-O-methyl 3' phosphorothioate, 2'-O-methyl 3' thiophosphonoacetate, locked nucleic acid (LNA).

[0311] Methods for stabilizing mRNA are known in the art and include adjusting the length of the polyadenine tail found at the 3' end of mRNA transcripts. Alternatively, or in addition, the RNA cap found at the 5' end of the molecule can be modified. The naturally occurring cap structure typical of mammalian cells tends to be improperly incorporated into in vitro synthesized RNA, reducing the effectiveness of the RNA. Synthetic "anti-reverse cap analogs" (e.g., those commercially available from Thermo Fisher Scientific) can prevent this misincorporation, resulting in more stable RNA and improved translation efficiency. To reduce immunogenicity, certain nucleotide substitutions can be replaced with chemically modified alternatives such as 5-methylcytosine or pseudouridine. Such substitutions can also suppress immune responses while simultaneously increasing mRNA stability and translation efficiency. Other exemplary chemically modified nucleotides are described herein above.

[0312] Alternatively or additionally, mRNA can be encapsulated in lipid-based particles to enhance fusion with lipid cell membranes.

[0313] Polynucleotides designed according to the teachings of some embodiments of the present invention can be made according to any method of polynucleotide synthesis known in the art, such as enzymatic synthesis or solid-phase synthesis. Equipment and reagents for performing solid-phase synthesis are commercially available, for example, from Applied Biosystems. Any other means for such synthesis can also be used. The actual synthesis of polynucleotides is well within the capabilities of one of ordinary skill in the art and can be achieved using solid-phase chemistry, e.g., cyanoethyl phosphoramidites, followed by deprotection, desalting, and purification (e.g., by automated trityl-ion or HPLC), according to established methodologies detailed, for example, in "Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R.M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); and "Oligonucleotide Synthesis" Gait, M.J., ed. (1984).

[0314] The term "polypeptide" or "protein" as used herein encompasses native peptides (either degradation products, synthetically synthesized peptides, or recombinant peptides) and peptidomimetics (typically synthetically synthesized peptides), as well as peptide analogs such as peptoids and semipeptoids, which may have modifications that make the peptide more stable in the body or more permeable to cells. Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, peptide bond modifications, backbone modifications, and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are described, for example, in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein.

[0315] The peptide bond (-CO-NH-) in a peptide can be substituted with, for example, an N-methylated amide bond (-N(CH3)-CO-), an ester bond (-C(=O)-O-), a ketomethylene bond (-CO-CH2-), a sulfinylmethylene bond (-S(=O)-CH2-), an α-aza bond (-NH-NNAive-CO-) (wherein R is any alkyl (e.g., methyl)), an amine bond (-CH2-NH-), a sulfide bond (-CH2 -S-), an ethylene bond (-CH2-CH2-), a hydroxyethylene bond (-CH(OH)-CH2-), a thioamide bond (-CS-NH-), an olefinic double bond (-CH=CH-), a fluorinated olefinic double bond (-CF=CH-), an inverted amide bond (-NH-CO-), a peptide derivative Naive(R)-CH2-CO- (where R is a "normal" side chain naturally occurring on a carbon atom).

[0316] These modifications can be made at any bond along the polypeptide chain, and can also be made at multiple (2-3) bonds simultaneously.

[0317] The natural aromatic amino acids Trp, Tyr, and Phe can be substituted with unnatural aromatic amino acids such as 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine, ring-methylated derivatives of Phe, halogenated derivatives of Phe, or O-methyl-Tyr.

[0318] Polypeptides of some embodiments of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (eg, fatty acids, complex carbohydrates, etc.).

[0319] The term "amino acid" or "amino acids" should be understood to include the 20 naturally occurring amino acids, as well as amino acids that are often post-translationally modified in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine, and other less common amino acids, including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine, and ornithine. Furthermore, the term "amino acid" includes both D- and L-amino acids.

[0320] The polypeptides of some embodiments of the present invention can be synthesized by any technique known to those skilled in the art of peptide synthesis, such as, but not limited to, recombinant DNA techniques or solid phase peptide synthesis.

[0321] The following is a non-limiting description of expression vectors that can be used to express a polypeptide of interest (e.g., any of the factors described above and below, e.g., Cdx2, Gata4) in cells and ways to administer them to cells.

[0322] According to certain embodiments, expression comprises introducing into the cell a polynucleotide encoding the polypeptide of interest (eg, a factor).

[0323] According to a particular embodiment, the polynucleotide is DNA.

[0324] According to certain embodiments, polynucleotide is RNA.Usually, mRNA introduced into cell only exists in cytoplasm, does not cause genome disturbance, and is essentially transient.Unless mRNA expression causes cell epigenetic changes, transient transfection is limited by the time that mRNA and corresponding cognate protein remain in cell, and does not persist after corresponding protein is degraded.

[0325] To express exogenous proteins in mammalian cells, it is preferred that a polynucleotide sequence encoding the polypeptide of interest be ligated into a nucleic acid construct suitable for expression in mammalian cells.

[0326] It will be understood that overexpression of a gene can be achieved using a knock-in construct and / or a knock-out construct [see, for example, Fukushige, S. and Ikeda, JE: Trapping of mammalian promoters by Cre-lox site-specific recombination. DNA Res 3 (1996) 73-50; Bedell, MA, Jerkins, NA and Copeland, NG: Mouse models of human disease. Part I: Techniques and resources for genetic analysis in mice. Genes and Development 11 (1997) 1-11; Bermingham, JJ, Scherer, SS, O'Connell, S., Arroyo, E., Kalla, KA, Powell, FL and Rosenfeld, MG: Tst-1 / Oct-6 / SCIP regulates a unique step in peripheral myelination and is required for normal respiration. Genes Dev 10 (1996)]. See 1751-62].

[0327] Such a nucleic acid construct or system comprises at least one cis-acting regulatory element for directing expression of a nucleic acid sequence, including those that direct constitutive expression of a nucleotide sequence and those that direct inducible expression of a nucleotide sequence only under certain conditions.

[0328] According to certain embodiments, the nucleic acid construct comprises an inducible promoter sequence for inducibly directing the transcription of the polynucleotide sequence in cells.Mammalian inducible promoters are known to those skilled in the art (see, for example, Bitter et al. (1987) Methods in Enzymology 153: 516-544).Inducible promoters can be activated by external signals or agents (i.e., inducers).Inducers can directly activate promoters or inactivate the repressors of the promoters. For example, inducible systems endogenous to mammalian cells include promoters inducible by heavy metals (Brinster et al. Nature (1982) 296:39-42, Mayo et al. Cell (1982) 29:99-108, and Searle et al. Molecular and Cellular Biology (1985) 5:1480-1489), promoters inducible by steroid hormones (Hynes et al. Proc. Natl. Acad. Sci. USA (1981) 78:2038-2042, Lee et al. Nature (1981) 294:228-232, and Klock et al. Nature (1987) 329:734-736), and promoters inducible by heat shock (Nouer, Heat Shock Response. Boca Raton, FL, Ed. CRC, 1991) (Mullick, A. and B. Massie, Encyclopedia of Cell Technology, pp. 1140-1164, 2000), and these promoters have been well characterized. International Publication No. 2002 / 088346 discloses a cumate-inducible promoter. Additional inducible promoters are known in the art, including, but not limited to, promoters induced by inflammation and hypoxia. Prokaryotic and insect inducible promoter systems have been adapted for regulated expression in mammalian cells.For example, see Gossen et al. (1993) TIBS 18:471-475 and No et al. (1996) Proc. Natl. Acad. Sci. USA 93:3346-3351. Insect ecdysone-inducible promoters are tightly regulated, and there is no detectable background expression in the absence of inducer. Ecdysone is a naturally occurring lipophilic steroid that can penetrate tissues, is inactive in mammals, and exhibits rapid clearance kinetics, making it suitable for in vivo use (No et al.). Gupta et al. (PNAS (2004) 101: 1927-1932) discloses the retroviral delivery of an ecdysone-inducible gene expression system under the control of a modified RNA polymerase III-specific U6 promoter.

[0329] Prokaryotic repressors derived from the lac and tet operons have been incorporated into eukaryotic inducible expression systems. Repression of expression is mediated by the repressor binding to an operator site located downstream of a minimal promoter in the absence of an inducer, and repression is relieved by the addition of an inducer (Brown et al. (1987) Cell 49:603-612; Hu and Davidson (1987) Cell 48:555-566; Blau and Rossi, Proc. Natl. Acad. Sci. USA (1999) 96:797-799; and Gossen et al. (1995) Science 268:1766-1769).

[0330] According to a particular embodiment, the inducible promoter is a Tet-on promoter that is induced by tetracycline or doxycycline.

[0331] Methods for constructing nucleic acid constructs or systems containing an inducible promoter operably linked to the coding sequence of any polypeptide are known to those skilled in the art, as are methods for introducing such systems and vector constructs containing such expression cassettes into cells.

[0332] In the case of mRNA, gene expression from an RNA source does not require transcription, and therefore no promoter sequence or additional sequences involved in transcription, as described below, are required.

[0333] The nucleic acid constructs or nucleic acid systems (also referred to herein as "expression vectors") of some embodiments of the present invention contain additional sequences that make the vector suitable for replication and integration in prokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). In addition, typical cloning vectors may also contain transcription and / or translation initiation sequences, transcription and / or translation terminators, and polyadenylation signals. By way of example, such constructs usually include a 5'LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3'LTR or a portion thereof.

[0334] Eukaryotic promoters typically contain two types of recognition sequences: the TATA box and upstream promoter elements. The TATA box, located 25–30 base pairs upstream of the transcription start site, is thought to be involved in directing RNA polymerase to begin RNA synthesis. Other upstream promoter elements determine the rate at which transcription is initiated.

[0335] Enhancer elements can stimulate transcription from linked homologous or heterologous promoters up to 1,000-fold. Enhancers are active when placed downstream or upstream of the transcription start site. Many enhancer elements derived from viruses have a broad host range and are active in a variety of tissues. For example, the SV40 early gene enhancer is suitable for many cell types. Other enhancer / promoter combinations suitable for some embodiments of the present invention include those derived from polyomavirus, human or mouse cytomegalovirus (CMV), murine leukemia virus, mouse or Rous sarcoma virus, and long-term repeats of various retroviruses, such as HIV. See Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor, NY 1983, incorporated herein by reference.

[0336] In constructing expression vectors, the promoter is preferably positioned approximately the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting, although, as is known in the art, some variation in this distance can be tolerated without loss of promoter function.

[0337] Polyadenylation sequences can also be added to expression vectors to increase the translation efficiency of mRNA. Accurate and efficient polyadenylation requires two distinct sequence elements: a GU- or U-rich sequence located downstream of the polyadenylation site and a highly conserved six-nucleotide sequence, AAUAAA, located 11 to 30 nucleotides upstream. Suitable termination and polyadenylation signals for some embodiments of the present invention include those derived from SV40.

[0338] In addition to the elements already described, the expression vectors of some embodiments of the present invention may contain other specialized elements, typically intended to increase the level of expression of the cloned nucleic acid or to facilitate identification of cells harboring the recombinant DNA. For example, some animal viruses contain DNA sequences that promote extrachromosomal replication of the viral genome in permissive cell types. Plasmids carrying these viral replicons will replicate episomally as long as the appropriate factors are provided, either by genes carried on the plasmid or by genes present in the host cell genome.

[0339] The vector may or may not contain a eukaryotic replicon.If a eukaryotic replicon is present, the vector can be amplified in eukaryotic cells using an appropriate selection marker.If the vector does not contain a eukaryotic replicon, episomal amplification is not possible.Instead, recombinant DNA is integrated into the genome of engineered cells, where the promoter directs the expression of desired nucleic acid.

[0340] The expression vectors of some embodiments of the present invention may further comprise additional polynucleotide sequences that allow for the translation of multiple proteins from a single mRNA, such as, for example, an internal ribosome entry site (IRES) and sequences for genomic integration of the promoter-chimeric polypeptide.

[0341] It will be understood that the individual elements contained within an expression vector can be arranged in a variety of configurations. For example, enhancer elements, promoters, etc., as well as polynucleotide sequences encoding proteins of interest, can be arranged in a "head-to-tail" configuration, can exist as reverse complements, or can exist in complementary configurations as antiparallel strands. While such configurational variations are likely to occur with non-coding elements of expression vectors, alternative configurations of coding sequences within expression vectors are also contemplated.

[0342] According to certain embodiments, the expression construct comprises a label for imaging in cells, such as a fluorescent label.

[0343] Examples of mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1(+ / -), pGL3, pZeoSV2(+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 available from Invitrogen, pCI available from Promega, pMbac, pPbac, pBK-RSV, and pBK-CMV available from Strategene, pTRES available from Clontech, and derivatives thereof.

[0344] Expression vectors containing regulatory elements derived from eukaryotic viruses, such as retroviruses, can also be used. SV40 vectors include pSVT7 and pMT2. Bovine papillomavirus-derived vectors include pBV-1MTHA, and Epstein-Barr virus-derived vectors include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A. + , pMTO10 / A + , pMAMneo-5, baculovirus pDSVE, and any other vector that allows expression of proteins under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells.

[0345] As mentioned above, viruses are often highly specialized infectious pathogens that have evolved to evade host defense mechanisms. Viruses typically infect and propagate in specific cell types. The targeting specificity of viral vectors utilizes their natural specificity to specifically target a given cell type, thereby introducing a recombinant gene into the infected cells. Therefore, the type of vector used in some embodiments of the present invention will depend on the cell type to be transformed. The ability to select a suitable vector depending on the cell type to be transformed is well within the capabilities of those skilled in the art, and therefore, no general explanation of the selection considerations is provided herein.

[0346] A variety of methods can be used to introduce the polynucleotides or polypeptides of some embodiments of the present invention into cells. These methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995); Vega et al., Gene Targeting, CRC Press, Ann Arbor, Mich. (1995); Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988); and Gilboa et al. [Biotechniques 4 (6): 504-512, Methods for the transfer of nucleic acids have been described in

[1986] , including stable or transient transfection, lipofection, electroporation, nucleofection, microinjection, and infection with recombinant viral vectors. Additionally, see U.S. Patent Nos. 5,464,764 and 5,487,992 for positive-negative selection methods. Currently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, herpes simplex virus type I, or adeno-associated virus (AAV), and lipid-based systems.

[0347] According to certain embodiments, the introduction is by transient transfection.

[0348] As is known in the art, naked DNA or RNA, cell membrane-permeable peptides, or viral and non-viral vectors (e.g., but not limited to, liposomes, nanoparticles, mammalian vectors, etc.) can be used as delivery vehicles for polynucleotides or polypeptides. According to certain embodiments of the present invention, the delivery systems used are biocompatible and non-toxic.

[0349] The following are exemplary embodiments suitable for enhancing the penetration of exogenous polynucleotides or exogenous polypeptides into cells.

[0350] According to an exemplary embodiment, naked DNA or RNA (e.g., naked plasmid DNA (pDNA)) is a non-viral vector that can be produced in bacteria and manipulated using standard recombinant DNA techniques. Naked DNA or RNA does not induce an antibody response against itself (i.e., no anti-DNA or anti-RNA antibodies are generated) and allows long-term gene expression without chromosomal integration. Naked DNA or RNA can be introduced by a number of means, including, but not limited to, intravascular and electroporation techniques [Wolff JA, Budker V, 2005, Adv. Genet. 54: 3-20] or jet injection [Walther W, et al., 2004, Mol. Biotechnol. 28: 121-8].

[0351] According to another exemplary embodiment, as further described above, a mammalian vector is used.

[0352] According to certain embodiments, the polynucleotide is contained in a viral vector. The introduction of nucleic acid by viral infection has several advantages over other methods such as lipofection and electroporation, because it can achieve higher transfection efficiency due to the infectious nature of viruses. The viral vector may be a virus with a DNA-based genome or a virus with an RNA-based genome (i.e., a positive single-stranded RNA virus and a negative single-stranded RNA virus). Examples of viral vectors include, but are not limited to, lentivirus, adenovirus, and retrovirus.

[0353] Viral constructs, such as retroviral constructs, contain at least one transcriptional promoter / enhancer or locus-defining element(s), or other elements that control gene expression by other means, such as alternative splicing, nuclear RNA transport, or post-translational modification of messengers. Such vector constructs also contain a packaging signal appropriate for the virus being used, a long terminal repeat (LTR) or a portion thereof, and binding sites for plus-strand and minus-strand primers, unless already present in the viral construct. Protocols for generating recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found, for example, in Ausubel et al., [eds., Current Protocols in Molecular Biology, Greene Publishing Associates, (1989)]. Other suitable expression vectors may be adenovirus, lentivirus, herpes simplex type I virus, or adeno-associated virus (AAV).

[0354] Regulatory elements that restrict expression to particular cell types can also be included. Such features include, for example, promoters and regulatory elements specific for the desired cell type.

[0355] According to certain embodiments, expression involves introducing the polypeptide of interest (eg, factor) into a cell.

[0356] According to certain embodiments, the polypeptide is provided in a formulation suitable for cell penetration, which enhances intracellular delivery of the polypeptide, as further described below.

[0357] Cell-penetrating peptides (CPPs) are short peptides (40 amino acids or less) that have the ability to access the interior of almost any cell. They are highly cationic and typically rich in the amino acids arginine and lysine. They have the extraordinary property of transporting a wide variety of covalently and non-covalently conjugated cargoes into cells, such as proteins, oligonucleotides, and even 200 nm liposomes. Thus, according to additional exemplary embodiments, CPPs can be used to transport polynucleotides or polypeptides into the interior of cells.

[0358] TAT (a transcriptional activator derived from HIV-1), pAntp (an antennapedia homeodomain transcription factor from Drosophila, also named penetratin), and VP22 (derived from herpes simplex virus) are examples of CPPs that can enter cells nontoxicly and efficiently and may be suitable for use in some embodiments of the present invention. Protocols for generating CPP-cargo conjugates and infecting cells with such conjugates can be found, for example, in L Theodore et al. [The Journal of Neuroscience, (1995) 15(11): 7158-7167], Fawell S, et al. [Proc Natl Acad Sci USA, (1994) 91:664-668], and Jing Bian et al. [Circulation Research. (2007) 100: 1626-1633].

[0359] The expression and / or activity levels of exogenous polynucleotides and / or exogenous polypeptides expressed in cells of some embodiments of the present invention can be determined using methods known in the art, such as, but not limited to, Northern blot analysis, PCR analysis, Western blot analysis, immunohistochemistry, and fluorescence-activated cell sorting (FACS).

[0360] As mentioned above, according to certain embodiments, expression of the exogenous factor is transient.

[0361] According to a particular embodiment, induction of transient expression is initiated within a period of 14 days to 0 hours before mixing.

[0362] According to certain embodiments, induction of transient expression is initiated within 13 days to 0 hours before mixing, within 12 days to 0 hours before mixing, within 11 days to 0 hours before mixing, within 10 days to 0 hours before mixing, within 9 days to 0 hours before mixing, within 8 days to 0 hours before mixing, within 7 days to 0 hours before mixing, within 6 days to 0 hours before mixing, within 5 days to 0 hours before mixing, within 120 hours to 0 hours before mixing, within 100 hours to 0 hours before mixing, within 80 hours to 0 hours before mixing, within 60 hours to 0 hours before mixing, within 48 hours to 0 hours before mixing, within 36 hours to 0 hours before mixing, within 24 hours to 0 hours before mixing, or within 12 hours to 0 hours before mixing, each possibility representing a separate embodiment of the present invention.

[0363] According to a particular embodiment, induction of transient expression is initiated within a period of 120 hours to 0 hours before mixing.

[0364] According to certain embodiments, induction of transient expression is initiated within a period of 48 hours to 0 hours before mixing.

[0365] According to certain embodiments, induction of transient expression is initiated within a period of 42 to 0 hours prior to mixing.

[0366] According to certain embodiments, induction of transient expression is initiated within 48 hours, within 24 hours, or within 12 hours of mixing.

[0367] According to certain embodiments, induction of transient expression is initiated within 14 to 1 day before mixing, within 13 to 1 day before mixing, within 12 to 1 day before mixing, within 11 to 1 day before mixing, within 10 to 1 day before mixing, within 9 to 1 day before mixing, within 8 to 1 day before mixing, within 7 to 1 day before mixing, within 6 to 1 day before mixing, within 5 to 1 day before mixing, within 120 to 20 hours before mixing, within 100 to 20 hours before mixing, within 80 to 20 hours before mixing, within 60 hours (60: house) before mixing, or within 48 to 20 hours before mixing, each possibility representing a separate embodiment of the present invention.

[0368] According to certain embodiments, induction of transient expression is initiated within a period of 14 days to 12 hours before mixing, within a period of 13 days to 12 hours before mixing, within a period of 12 days to 12 hours before mixing, within a period of 11 days to 12 hours before mixing, within a period of 10 days to 12 hours before mixing, within a period of 9 days to 12 hours before mixing, within a period of 8 days to 12 hours before mixing, within a period of 7 days to 12 hours before mixing, within a period of 6 days to 12 hours before mixing, within a period of 5 days to 12 hours before mixing, within a period of 120 hours to 12 hours before mixing, within a period of 100 hours to 12 hours before mixing, within a period of 80 hours to 12 hours before mixing, within a period of 60 hours to 12 hours before mixing, or within a period of 48 hours to 12 hours before mixing, each possibility representing a separate embodiment of the present invention.

[0369] According to certain embodiments, induction of transient expression is initiated within a period of 48 hours to 12 hours before mixing.

[0370] According to certain embodiments, induction of transient expression is completed within 120 hours of mixing.

[0371] According to certain embodiments, induction of transient expression is completed within 110 hours, 100 hours, 90 hours, 80 hours, 70 hours, 60 hours, 50 hours, 48 ​​hours, 36 hours, 24 hours, 20 hours, or 12 hours after mixing, with each possibility representing a separate embodiment of the present invention.

[0372] According to certain embodiments, induction of transient expression is completed within 48 hours of mixing.

[0373] According to a particular embodiment, induction of transient expression is carried out for up to 19 days.

[0374] According to certain embodiments, induction of transient expression is performed within 18 days, within 17 days, within 16 days, within 15 days, within 14 days, within 13 days, within 12 days, within 11 days, within 10 days, within 9 days, within 8 days, within 7 days, within 6 days, within 5 days.

[0375] According to a particular embodiment, the induction of transient expression is carried out within 16 days.

[0376] According to a particular embodiment, induction of transient expression is carried out within a period of 96 hours or less.

[0377] According to a particular embodiment, induction of transient expression begins 24 hours before mixing and continues for 96 hours (ie, ending 48 hours after mixing).

[0378] It will be understood that culture conditions suitable for maintaining naive PSCs, as well as trophectoderm-primed cells and extraembryonic primitive endoderm-primed cells before mixing, include various tissue culture medium compositions, feeder cells, oxygen concentrations, culture vessels, etc., and that determining which conditions to apply is within the ability of one skilled in the art. Non-limiting examples of such are described in the Examples section below, which form an integral part of this specification, as well as Choi, J., Huebner, A., Clement, K. et al. Nature 548, 219-223 (2017); Yang et al. Cell (2017) 169(2): 243-257, www(dot)cell(dot)com / fulltext / S0092-8674%2817%2930183-6; www(dot)doi(dot)org / 10.1038 / nature23274; www(dot)doi(dot)org / 10.1242 / dev.180620; DOI:10.1126 / science.aay3325; DOI:10.1016 / j.stem.2017.11.004; Shingo Io et al. (2021) Cell Stem Cell, 28(6): 1023-1039 and Mitsuyoshi Amita et al. (2013) PNAS E1212-1221; and WO 2016 / 016894 and WO 2014 / 174470, the contents of which are incorporated herein by reference in their entireties.

[0379] According to certain embodiments, the culture medium comprises a STAT3 activator.

[0380] As used herein, the term "STAT3" refers to the signal transducer and activator of transcription 3 gene product (acute phase response factor) (gene ID 6774). STAT family members are phosphorylated by receptor-associated kinases in response to cytokines and growth factors, then form homodimers or heterodimers, which translocate to the cell nucleus where they function as transcriptional activators. Known STAT3 activators include, but are not limited to, interferon (IFN), epidermal growth factor (EGF), interleukin 5 (IL5), interleukin 6 (IL6), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), and bone morphogenetic protein 2 (BMP2).

[0381] According to some embodiments of the present invention, the STAT3 activator used in the culture medium of some embodiments of the present invention is selected from the group consisting of LIF, IL6, and EGF.

[0382] According to some embodiments of the present invention, the STAT3 activator used in the culture medium of some embodiments of the present invention is selected from the group consisting of LIF and IL6.

[0383] According to some embodiments of the present invention, the STAT3 activator used in the culture medium of some embodiments of the present invention is LIF.

[0384] As used herein, the term "leukemia inhibitor factor (LIF)" refers to the polynucleotide and expression product, e.g., polypeptide, of the LIF gene (e.g., human gene ID number 3976). A non-limiting example of a human LIF polypeptide is set forth in the following GenBank accession number: NP_001244064 (SEQ ID NO: 124). Preferably, the LIF used by the methods according to some embodiments of the present invention, together with other factors, is capable of supporting the undifferentiated proliferation of naive PSCs while maintaining their pluripotent potential. LIF can be obtained from various manufacturers, such as Millipore, Peprotech, and R&D Systems.

[0385] According to certain embodiments, the medium comprises a GSK-3 inhibitor.

[0386] According to certain embodiments, the medium comprises an ERK1 / 2 inhibitor.

[0387] As used herein, the term "ERK1" refers to mitogen-activated protein kinase 3 (MAPK3) isoform 1, e.g., as represented by GeneBank accession number NP_002737 (SEQ ID NO: 125), MAPK3 isoform 2, e.g., as represented by GenBank accession number NP_001035145 (SEQ ID NO: 126), MAPK3 isoform 3, e.g., as represented by GenBank accession number NP_001103361 (SEQ ID NO: 127), and / or ERK1, as represented by GenBank accession number M84490 (SEQ ID NO: 128), which have MAPK signaling activity.

[0388] As used herein, the term "ERK2" refers to mitogen-activated protein kinase 1 (MAPK1) having MAPK signaling activity, as represented, for example, by Genbank Accession No. NP_002736 (SEQ ID NO: 129) and / or Genbank Accession No. NP_620407 (SEQ ID NO: 130).

[0389] As used herein, the term "ERK1 / 2 inhibitor" refers to any molecule capable of inhibiting the activity of ERK1 / 2 as determined by Western blot protein detection of phosphorylated ERK1 / 2 protein.

[0390] Non-limiting examples of ERK1 / 2 inhibitors (also known as MEK1 / 2 inhibitors) include PD0325901 (AXONMEDCHEM - AXON 1408), PD98059 (AXONMEDCHEM - Axon 1223), and PD184352 (AXONMEDCHEM - AXON 1368).

[0391] According to certain embodiments, the medium comprises at least one of a GSK-3 inhibitor, a WNT ligand, heparin, FGF2, FGF4, PDGF, leukemia inhibitory factor (LIF), and insulin.

[0392] According to certain embodiments, the medium comprises at least one of a GSK-3 inhibitor, leukemia inhibitory factor (LIF), and insulin.

[0393] According to certain embodiments, the medium comprises at least one of a GSK-3 inhibitor, heparin, FGF4, PDGF, and BSA.

[0394] According to certain embodiments, the medium lacks activin A (also known as inhibin beta A, polynucleotides and expression products, eg, polypeptides, of the INHBA gene (corresponding to human gene ID3624) or homologs or orthologs thereof).

[0395] According to certain embodiments, the medium lacks REPROCELL.

[0396] According to certain embodiments, the culture medium comprises at least one of a TGFR inhibitor, an FGFR inhibitor, a MEK / ERK inhibitor, BMP4, a JAK inhibitor, FGF4, FGF2, heparin, a sumoylation inhibitor, a histone deacetylase inhibitor, a HIPPO signaling pathway inhibitor, and a factor that induces YAP nuclear translocation.

[0397] According to certain embodiments, the following components, taken together or individually, may be useful additives for pre-induction of primed cells into TSCs: A-83, a TGFRi; LIF; LPA, a HIPPO inhibitor; Y27632, a ROCK inhibitor; PD0325901, a MEK / ERK inhibitor; EGF; and JAKi.

[0398] Further description of media and additives that can be used in certain embodiments of the present invention is provided below.

[0399] According to certain embodiments, the cell subpopulations are mixed together under conditions that allow the formation of aggregated cells.

[0400] These conditions may include culture time, culture vessel, medium composition, oxygen concentration, amount and ratio of cells, etc. Determining such conditions is well within the ability of one skilled in the art. Non-limiting examples of aggregation protocols that can be used in certain embodiments of the present invention are described in the Examples section below, which forms an integral part of this specification.

[0401] According to certain embodiments, the naive PSCs, the trophectoderm-primed cells, and the extraembryonic primitive endoderm-primed cells are mixed simultaneously.

[0402] According to other specific embodiments, the mixing of naive PSCs, trophectoderm-primed cells, and extraembryonic primitive endoderm-primed cells is performed sequentially.

[0403] Thus, for example, according to certain embodiments, naive PSCs are mixed with cells primed to extraembryonic primitive endoderm before being mixed with cells primed to trophectoderm, according to certain embodiments, naive PSCs are mixed with cells primed to trophectoderm before being mixed with cells primed to extraembryonic primitive endoderm.

[0404] According to certain embodiments, the trophectoderm-primed cells and the extraembryonic primitive endoderm-primed cells are mixed prior to mixing with the naive PSCs.

[0405] According to certain embodiments, this difference in mixing time can be up to 48 hours, up to 40 hours, up to 35 hours, up to 30 hours, up to 24 hours, up to 16 hours, or up to 12 hours, with each possibility representing a separate embodiment of the present invention.

[0406] According to certain embodiments, the ratio of naive PSCs to cells primed towards trophectoderm is 1:1 to 1:5.

[0407] According to certain embodiments, the ratio of naive PSCs to cells primed to trophectoderm is 1:3 to 1:4.

[0408] According to certain embodiments, the ratio of naive PSCs to trophectoderm-primed cells is about 1:3.3.

[0409] According to certain embodiments, the ratio of naive PSCs to cells primed towards primitive endoderm is 1:1 to 2:1.

[0410] According to certain embodiments, the ratio of naive PSCs to cells primed towards primitive endoderm is about 1:1.

[0411] According to certain embodiments, aggregation is carried out in aggregation plates, such plates being known to those skilled in the art and commercially available, for example, from STEMCELL Technologies.

[0412] Non-limiting examples of protocols for culturing naive PSCs, inducing trophectoderm-primed cells and primitive endoderm-primed cells, and aggregation of the three cell types are provided in the Examples section below.

[0413] Thus, for example, naive KH2 ESCs containing a nucleic acid sequence encoding Gata4 under the control of the TetOn promoter can be cultured in 2i / Lif medium and treated with DOX (e.g., 2 μg / ml—Sigma, D9891) for 24 hours in 2i / Lif before the start of the experiment. Gata4 induction can be similarly performed in N2B27 medium, MEF medium, TSC medium, or aggregation medium. Naive KH2 ESCs containing a nucleic acid sequence encoding Cdx2 under the control of the TetOn promoter are cultured in 2i / Lif and treated with DOX (2 μg / ml, Sigma, D9891) for various time points (days -1 to -14, e.g., 24 h) in TSC medium (25 ng / ml FGF4 (Peprotech), 1 μg / ml heparin (Sigma)) supplemented with or without 0.5–1 μM lysophosphatidic acid (LPA), a Hippo pathway inhibitor. TGFR and / or ERK inhibitors can also be optionally added during this pre-induction step. The non-induced ESC fraction is maintained in 2i / Lif or serum-LIF conditions without any special pretreatment. Other naive conditions that can be used instead of the 2i / LIF condition include the a2i condition, which uses basal medium supplemented with N2B27 containing the GSK3 inhibitor CHIR99021 (final concentration 3 μM), Lif (final concentration 20 ng / ml), and the SRCI CGP77675 (final concentration 1 μM). Aggregation is then performed in an AggreWell 24-well plate 400 (STEMCELL Technologies, 34415) or AggreWell 24-well plate 800 (STEMCELL Technologies, 34815). Non-adherent 96-well plates (NUNC or THERMÔ) can also be used. AggreWell plates can be prepared according to the manufacturer's instructions. For example, 500 μl of anti-adherent rinse solution (STEMCELL Technologies, 07010) is added to each well. The plate is centrifuged at 2,000 g for 5 minutes and incubated at room temperature for 30 minutes. After incubation, the rinse solution is removed and the plates are washed twice with PBS.Next, 500 μl of aggregation medium (AM) supplemented with DOX (final concentration 2 μg / ml, Sigma, D9891) and ROCKi Y27632 (final concentration 5 nM-10 μM, Axon Medchem, 1683) is added to each well. On the day of aggregation (day 0), the three cell populations can be trypsinized with 0.05% trypsin-EDTA solution (Biological Industries (Sartorius), 03-053-1B) at 37°C for 4-6 minutes. The trypsin enzyme reaction is stopped by adding aggregation medium (AM). Alternatively, TrypleE or Accutase can be used instead of trypsin. The cells were centrifuged at 1200 rpm for 3 minutes and resuspended in AM containing DOX (2 μg / ml, Sigma, D9891) and ROCKi Y27632 (final concentration 5 nM-10 μM, Axon Medchem, 1683). To deplete mouse embryonic fibroblasts, the cells were plated on gelatin-treated tissue culture plates for 20 minutes at 37°C. The supernatant was then collected, centrifuged, and the cells were resuspended. Three cell fractions were counted and resuspended in AM containing DOX (2 μg / ml, Sigma, D9891) and ROCKi Y27632 (5 nM, Axon Medchem, 1683). In aggregation experiments, an exemplary ratio of 1:1 naive ESC:iGata4 ESC:iCdx2 ESC ratio (3.33) is maintained, depending on the aggregation plate used, with the following exact cell numbers: A) AggreWell 800: Number of microwells per well in a 24-well plate = 300, Number of cells added per well of a 24-well plate = iCdx2: 5,000 cells + iGata4: 1,500 cells + naive ESC: 1,500 cells, Number of cells per single microwell = approximately 27 cells; B) AggreWell 400: Number of microwells per well in a 24-well plate = 1,200, Number of cells added per well of a 24-well plate = iCdx2: 20,000 cells + iGata4: 6,000 cells + naive ESC: 6,000 cells, Number of cells per single microwell = approximately 27 cells.

[0414] As another example, naive PSCs are used to generate the following three cell populations: 1) Naive wild-type PSC cells cultured in HENSM medium. 2) For the primitive endoderm compartment: naive wild-type cells plated in mouse embryonic fibroblast conditions on HENSM supplemented with ROCKi, followed by a change to RCL medium the next day for 72 hours. 3) For trophectoderm lineage: naive wild-type cells plated in feeder-free conditions on HENSM supplemented with ROCKi, followed by a 24-hour medium change the next day with (B) AP medium, then switched to AP (J) for the next 48 hours.

[0415] All three cell types were supplemented with ROCKi before being harvested for coaggregation the following day. On the day of aggregation (day 0), Aggrewell 400, 24-well plates (Stemcell Technologies) were prepared and pre-filled with 500 μL of N2B27 medium and maintained at 37°C for medium equilibration. The three cell populations were disaggregated using TrypLE (Thermo Fisher, 12604054) at 37°C for 3 minutes for wild-type and primitive endoderm cells and 5 minutes for trophectoderm cells. The enzyme was then removed, the cells were incubated at room temperature for 2 minutes, and then collected with PBS. The cells were centrifuged at 1300 rpm for 3 minutes and resuspended in N2B27 ROCKi. To deplete mouse embryonic fibroblasts, the primitive endoderm cells were plated on gelatin-treated tissue culture plates for 30 minutes at 37°C. The supernatant was collected, all three components were centrifuged separately, and the cells were resuspended in N2B27 medium. The three cell fractions were counted and resuspended in N2B27 supplemented with ROCKi. The coaggregation maintained a ratio of 1:1:3 (WT:PRE:TE).

[0416] The present invention also contemplates compositions and articles of manufacture comprising the cells and aggregates disclosed herein.

[0417] Therefore, according to one aspect of the present invention, (i) trophectoderm-primed cells, including naive pluripotent stem cells (PSCs), containing an exogenous polynucleotide suitable for transient expression of a factor that induces differentiation into trophectoderm cells; and (ii) a naive PSC.

[0418] According to certain embodiments, the article of manufacture comprises: (iii) cells primed to extraembryonic primitive endoderm, including naive pluripotent stem cells (PSCs) containing an exogenous polynucleotide suitable for transient expression of factors that induce differentiation into extraembryonic primitive endoderm cells.

[0419] According to a further or alternative aspect of the present invention, (i) trophectoderm-primed cells, including naive pluripotent stem cells (PSCs), containing an exogenous polynucleotide suitable for transient expression of a factor that induces differentiation into trophectoderm cells; and (ii) An article of manufacture is provided that includes cells primed to extraembryonic primitive endoderm, including naive pluripotent stem cells (PSCs) that include an exogenous polynucleotide suitable for transient expression of a factor that induces differentiation into extraembryonic primitive endoderm cells.

[0420] According to certain embodiments, the different cell subpopulations are in a single container.

[0421] According to other particular embodiments, the different cell subpopulations are in separate containers.

[0422] According to further or alternative aspects of the present invention, there is provided a method for ex utero culturing a synthetic embryo, comprising mixing cells of an article of manufacture disclosed herein under conditions that allow for the formation of aggregated cells, the method comprising inducing transient expression of a factor beginning within a period of 14 days to 0 hours before mixing and ending within 120 hours of mixing, thereby producing a synthetic embryo.

[0423] According to additional or alternative aspects of the present invention, there is provided a mixture or aggregate made or obtainable by the methods disclosed herein.

[0424] According to certain embodiments, the mixture or aggregate is not structurally organized like a natural or naturally occurring embryo at any developmental stage. According to certain embodiments, the different cells in the mixture or aggregate are randomly scattered in an unorganized manner within the mixture of the aggregate. According to certain embodiments, the mixture or aggregate does not form a blastocyst at any stage. According to certain embodiments, the mixture or aggregate does not contain a cavity.

[0425] According to certain embodiments, the mixture or aggregate comprises at least 12, at least 16, at least 32, at least 40, at least 50, or at least 64 cells.

[0426] According to certain embodiments, the mixture or aggregate comprises more than 64 cells.

[0427] According to certain embodiments, the mixture or aggregate comprises up to 500 cells, up to 450 cells, up to 400 cells, up to 350 cells, up to 300 cells, up to 250 cells, up to 200 cells, up to 150 cells, or up to 100 cells.

[0428] It will be appreciated that during the cultivation of a mixture or aggregate of unorganized cells, there is a process by which the cells reorganize to form a synthetic embryo having the structure or tissue of a natural embryo.

[0429] According to certain embodiments, the mixture or aggregate is capable of forming a synthetic embryo that is structurally organized like a natural embryo at least at the post-implantation egg cylinder or bilayered scutellum development stage.

[0430] Thus, according to a further or alternative aspect of the present invention, (i) naive pluripotent stem cells (PSCs), (ii) cells primed to trophectoderm, including stem cells that express endogenous trophectoderm markers and endogenous pluripotency markers; and (iii) cells primed to extraembryonic primitive endoderm, including stem cells expressing endogenous extraembryonic primitive endoderm markers and endogenous pluripotency markers; There is provided a mixture or aggregate comprising: The mixture or aggregate is not structurally organized like a natural embryo at any stage of development, and The mixture or aggregate is capable of forming a synthetic embryo that is structurally organized like a natural embryo at least at the post-implantation egg cylinder or bilayered scutellum stage of development.

[0431] According to a further or alternative aspect of the present invention, (i) trophectoderm-primed cells, including stem cells that express exogenous factors that induce differentiation into trophectoderm cells, endogenous trophectoderm markers, and endogenous pluripotency markers; and (ii) naive pluripotent stem cells (PSCs); There is provided a mixture or aggregate comprising:

[0432] According to certain embodiments, the mixture or aggregate comprises: (iii) The cells further include cells primed to extraembryonic primitive endoderm, including stem cells that express exogenous factors that induce differentiation into extraembryonic primitive endoderm cells, endogenous extraembryonic primitive endoderm markers, and endogenous pluripotency markers.

[0433] It will be appreciated that the mixtures or aggregates of some embodiments of the present invention comprise different stem cell populations that all start from naive pluripotent stem cells (PSCs).

[0434] According to some embodiments of the invention, the method further comprises growing the synthetic embryo ex vivo (outside the uterus) or in vivo (inside the uterus).

[0435] Thus, according to one aspect of the present invention, there is provided a method of ex utero culturing a synthetic embryo, the method comprising ex utero culturing a mixture or aggregate as disclosed herein.

[0436] As described in the Examples section below, the inventors were able to generate post-gastrulation synthetic embryos by utilizing controlled ex utero embryo culture platforms and growth conditions that previously enabled them to capture gastrulation and organogenesis of native mouse embryos outside the uterus (Aguilera-Castrejon et al., 2021a), demonstrating that these platforms and growth conditions can be used to culture ex utero synthetic embryos generated by both the methods disclosed herein and any other methods.

[0437] Therefore, according to one aspect of the present invention, there is provided a method for ex utero culture of a synthetic embryo, which comprises carrying out ex utero culture of an aggregate-stage synthetic embryo by static culture followed by dynamic culture, wherein the static culture is carried out until the embryo reaches at least the early post-implantation egg cylinder or bilayered scutellum development stage, and the dynamic culture is initiated at the latest when the embryo reaches the early somite stage.

[0438] According to certain embodiments, the method further comprises determining the development of the embryo before, during, and / or after culture. Methods for assessing development are well known in the art and are further detailed herein.

[0439] As used herein, the term "culturing a synthetic embryo" refers to at least a synthetic embryo and a culture medium being in an in vitro or ex vivo (outside the uterus) environment. The culture is maintained under a condition (or set of conditions) that can induce the development and growth of the synthetic embryo. Such conditions include, for example, an appropriate temperature (e.g., 37°C), atmosphere (e.g., O2%, CO2%), pressure, pH, light, medium, additives, etc.

[0440] The culture may be in a glass, plastic, or metal container that can provide a sterile environment for the culture. According to certain embodiments, the culture container includes a dish, plate, flask, bottle, vial, bag, bioreactor, or any device that can be used to grow cells.

[0441] According to certain embodiments, the culture is maintained under sterile conditions.

[0442] According to a particular embodiment, the culture is maintained at 37-38°C.

[0443] According to a particular embodiment, the culture is maintained at 38°C.

[0444] According to a particular embodiment, the culture is maintained at 37°C.

[0445] Because temperature changes can affect embryo development, according to certain embodiments, care should be taken to avoid keeping embryos at temperatures above 38° C. and below 35° C. for extended periods of time. Thus, for example, opening the culture incubator or keeping embryos at room temperature for extended periods of time should be avoided.

[0446] According to a particular embodiment, the culture is a static culture.

[0447] According to other particular embodiments, the culture is a dynamic culture.

[0448] According to a particular embodiment, the culturing is a static culturing followed by a dynamic culturing.

[0449] As used herein, the term "static culture" refers to cell culture that is carried out without agitating the culture.

[0450] According to a particular embodiment, the static culture is carried out for 3 to 4 days.

[0451] According to a particular embodiment, the static culture is carried out for about 3 days.

[0452] Embryonic stage and development can be assessed in comparison with in vivo embryonic counterparts at the same developmental stage by several methods well known in the art, including, but not limited to, morphology, length, weight, expression of developmental marker genes using specific antibodies or specific primers, transcriptional profiling, etc., as further described in the Examples section below, which forms an integral part of this specification.

[0453] As used herein, the terms "embryonic day (E)," "days post fertilization (PFD)," or "gestational day (GD)" refer to an embryo having the developmental characteristics of its embryonic counterpart in vivo (in the fallopian tube or in the uterus depending on the day) on a specified day after fertilization or mating, where E0 / PFD0 / GD0 is considered a fertilized egg.

[0454] According to a particular embodiment, the static culture is carried out for 2 to 5 days.

[0455] According to a particular embodiment, the static culture is carried out for about 4 days.

[0456] According to a particular embodiment, the static culture is carried out for 10 to 20 days.

[0457] According to a particular embodiment, the static culture is carried out for about 12 days.

[0458] According to certain embodiments, static culture is terminated at the latest when the synthetic embryo reaches the egg cylinder stage (eg, in the case of mice) or the bilayered blastocyst stage (eg, in the case of humans).

[0459] As used herein, the term "egg cylinder stage" refers to the composite embryo after (but not including) the blastocyst stage and before the post-gastrulation stage, characterized by the expansion of the yolk sac surrounding the embryo and the completion of gastrulation and the appearance of somites.

[0460] According to certain embodiments, the egg cylinder stage refers to a synthetic embryo having characteristics of E5-7 of a natural mouse embryo in utero.

[0461] As used herein, the term "bilayered disc stage" refers to a composite embryo after (but not including) the blastocyst stage and before the 15-day stage (Carnegie stage 6), characterized by two dorsally convex cell discs: one disc formed by a thick layer of pseudostratified columnar cells called the epiblast, and the other by a thin layer of cuboidal cells called the hypoblast. The disc-shaped epiblast is slightly dorsally convex and is separated from the amnion by the amniotic cavity in the center of the disc.

[0462] According to certain embodiments, the bilayered disc stage refers to a synthetic embryo having characteristics of PFD8-14 of a natural human embryo in utero.

[0463] According to certain embodiments, to prevent the embryonic epiblast and yolk sac from adhering to the culture vessel during static culture, the cultures are inspected to ensure that only the ectoplacental cone remains attached to the surface of the plate. According to certain embodiments, if necessary, the embryos are carefully pushed away from the surface of the plate, e.g., using forceps.

[0464] As used herein, the term "dynamic culture" refers to cell culture performed while the culture is agitated (e.g., rotated, shaken, inverted). Again, in dynamic culture, the entire culture, including the embryos, is agitated. Non-limiting examples of dynamic culture include roller culture (culture on a rotating device) and shaking culture (culture on a shaker, e.g., an orbital shaker).

[0465] According to a particular embodiment, the dynamic culture is a roller culture.

[0466] According to a particular embodiment, the rotating culture is rotated at 30 rpm.

[0467] Rotator-type culture units are commercially available, for example from Cullum Starr Precision Engineering Ltd, UK, but can be further modified and adapted.

[0468] According to another particular embodiment, the dynamic culture is a shaking culture.

[0469] According to certain embodiments, the shaker rotates at 30-80 rpm, 40-70 rpm, 50-70 rpm, or 55-65 rpm.

[0470] According to a particular embodiment, the shaker rotates at about 60 rpm.

[0471] According to certain embodiments, dynamic culturing comprises shaking culturing, roller culturing, or a sequential combination thereof.

[0472] According to certain embodiments, dynamic culture is initiated when the embryos reach the pre-gastrulation stage.

[0473] As used herein, the term "pre-gastrulation" refers to a composite embryo after the egg cylinder or bilayered blastodisc stage but before early gastrulation, characterized by the presence of OCT4 and OTX2+ epiblast, formation of the pre-amniotic cavity, and formation of an extra ectoderm embryo (ExE).

[0474] According to certain embodiments, the pre-gastrulation stage refers to a synthetic embryo having characteristics of E5-6 of a natural mouse embryo in utero or PFD8-14 of a natural human embryo in utero.

[0475] According to certain embodiments, dynamic culture begins with shaking culture (eg, for 1 to 4 days), followed by roller culture.

[0476] According to certain embodiments, roller culture is initiated when the embryos have reached at least the early gastrulation stage.

[0477] As used herein, the term "early gastrulation" refers to a composite embryo after the pre-gastrulation stage and before the late gastrulation stage, characterized, for example, by an egg cylinder with a posterior primitive streak in the mouse and a bilayered embryonic disc cylinder with a posterior primitive streak in the human.

[0478] According to certain embodiments, the early gastrulation stage refers to a synthetic embryo having characteristics of E6-7 of a natural mouse embryo in utero or PFD12-18 of a natural human embryo in utero.

[0479] According to certain embodiments, roller culture is initiated at the latest when the embryos reach the early somite stage.

[0480] According to certain embodiments, dynamic culture begins at the latest when the embryo reaches the early somite stage.

[0481] As used herein, the term "early somitic" refers to the composite embryo after late gastrulation and before neural tube closure, characterized by the appearance of somites and the formation of the first organs.

[0482] According to certain embodiments, the early somite stage refers to a synthetic embryo having characteristics of E8-9 of a natural mouse embryo in utero or PFD18-24 of a natural human embryo in utero.

[0483] As used herein, the phrase "basal medium" refers to a liquid substance used to support the growth / survival of stem cells and / or synthetic embryos and, optionally, to guide their development.

[0484] Media used in accordance with some embodiments of the present invention (such as cell or embryo culture media, aggregation media, etc.) may be water-based media that contain a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, and / or proteins (e.g., cytokines, growth factors, and hormones), all of which are necessary for cell growth and embryo development.

[0485] Preferably, all components contained in the culture media of the present invention are substantially pure, i.e., tissue culture grade.

[0486] For example, the culture medium may comprise a synthetic tissue culture medium, e.g., a basal medium such as DMEM, DMEM / F12, or modified DMEM / F12 (commercially available, e.g., from GIBCO® or Biological Industries), Neurobasal (Invitrogen, 21103-049 or ThermoFisher, 21103049), KO-DMEM (commercially available, e.g., from GIBCO®), CMRL (commercially available, e.g., from GIBCO®), TCM199 (commercially available, e.g., from Sigma), StemPro® (commercially available, e.g., from ThermoFisher Scientific), RPMI (commercially available, e.g., from Biological Industries), or combinations thereof supplemented with necessary additives as further described herein.

[0487] Non-limiting examples of media that can be used in particular embodiments and at particular stages of the disclosed methods are described in detail in the Examples section below, and in, for example, WO 2016 / 016894 and WO 2014 / 174470, the contents of which are incorporated herein by reference in their entireties.

[0488] Furthermore, since the inventors have identified novel culture media containing specific factors that can be used to enable the generation of trophectoderm-primed cells and extraembryonic primitive endoderm-primed cells and the development of synthetic embryos (see the Examples section below), the present invention also contemplates embodiments relating to the media described in the Examples section below, wherein the components of the media are provided at concentrations of ±20%.

[0489] According to certain embodiments, the basal medium lacks phenol red.

[0490] According to certain embodiments, the basal medium comprises DMEM having the same components as GIBCO® catalog number 11880 DMEM.

[0491] According to certain embodiments, the basal medium comprises DMEM / F12 having the same components as GIBCO® catalog number 12634-010 DMEM / F12.

[0492] According to certain embodiments, the basal medium comprises CMRL having the same components as GIBCO® catalog number 11530037 CMRL.

[0493] According to certain embodiments, the basal medium comprises TCM199 having the same components as TCM199 from Sigma catalog number M4530.

[0494] According to certain embodiments, the culture medium comprises serum or a serum replacement.

[0495] According to certain embodiments, the medium comprises 10-80%, 15-80%, 20-80%, 15-75%, or 20-75% [volume / volume (v / v)] serum or serum substitute.

[0496] According to particular embodiments, the medium comprises 15-60%, 15-40%, or 15-30% (v / v) serum or serum replacement.

[0497] According to particular embodiments, the culture medium comprises 20-40% or 20-30% (v / v) serum or serum replacement.

[0498] According to other particular embodiments, the culture medium comprises at least 20% (v / v) serum or serum replacement.

[0499] According to certain embodiments, the medium comprises about 20% (v / v) serum or serum replacement.

[0500] According to certain embodiments, the medium comprises about 30% (v / v) serum or serum replacement.

[0501] According to other particular embodiments, the culture medium comprises at least 30% (v / v) serum or serum replacement.

[0502] According to other particular embodiments, the culture medium comprises at least 35% (v / v), at least 40% (v / v), at least 45% (v / v), at least 50% (v / v), at least 55% (v / v), at least 60% (v / v), at least 65% (v / v), at least 70% (v / v) of serum or serum replacement.

[0503] According to other particular embodiments, the medium comprises 40-80%, 50-80%, 60-80%, 70-80% (v / v) serum or serum substitute.

[0504] According to other particular embodiments, the medium comprises 70-80% (v / v) serum or serum replacement.

[0505] According to certain embodiments, the medium comprises about 75% (v / v) serum or serum replacement.

[0506] According to certain embodiments, the medium comprises increasing concentrations of serum or serum substitutes throughout the culture of the embryo.

[0507] According to certain embodiments, the medium comprises at least 15% serum or serum substitute from the start until the embryo reaches the early post-implantation egg cylinder stage or bilayered blastocyst stage, and thereafter comprises at least 30% serum or serum substitute.

[0508] Serum can be obtained from rodents (eg, rats, mice) or mammals (eg, cows, humans).

[0509] According to certain embodiments, care should be taken to ensure that the serum (eg, human serum) is free of any traces of hemolysis.

[0510] According to certain embodiments, the serum is obtained from an adult animal.

[0511] According to other particular embodiments, the serum is obtained from a fetal animal.

[0512] According to certain embodiments, the serum comprises cord blood serum. Methods for obtaining cord blood serum (e.g., human cord blood serum) are well known in the art and are further described in the Examples section below.

[0513] According to certain embodiments, the serum comprises bovine serum (eg, FCS).

[0514] According to certain embodiments, the serum comprises rat serum.

[0515] According to certain embodiments, the serum comprises human serum.

[0516] According to certain embodiments, the serum comprises human serum during at least part of the culture.

[0517] According to certain embodiments, the human serum comprises umbilical cord serum (HCS).

[0518] According to other particular embodiments, the human serum comprises human bovine serum (HBS).

[0519] According to certain embodiments, the serum comprises rat serum and human serum.

[0520] According to a particular embodiment, the ratio of rat serum to human serum in the culture medium is 1:1 to 5:1 (v / v).

[0521] According to a particular embodiment, the ratio of rat serum to human serum in the culture medium is 1:1 to 3:1 (v / v).

[0522] According to a particular embodiment, the ratio of rat serum to human serum in the culture medium is about 2:1 (v / v).

[0523] According to a particular embodiment, the ratio of rat serum to human serum in the culture medium is 2:1 (v / v).

[0524] In some embodiments, the medium optionally contains knockout serum replacement (KSR) instead of animal serum.

[0525] In some embodiments, the medium optionally contains knockout serum replacement (KSR) in addition to animal serum, such as rat serum and human serum.

[0526] In some embodiments, optionally, either human serum or rat serum is partially exchanged with KSR, or an amount of both is partially exchanged with KSR.

[0527] According to certain embodiments, the ratio of serum to basal medium in the culture medium is 1:0.5 to 10:1, 1:1 to 10:1, or 1:1 to 8:1 (v / v).

[0528] According to a particular embodiment, the ratio of serum to basal medium in the culture medium is 1:1 to 5:1 (v / v).

[0529] According to a particular embodiment, the ratio of serum to basal medium in the culture medium is about 3:1 (v / v).

[0530] According to a particular embodiment, the ratio of serum to basal medium in the culture medium is 3:1 (v / v).

[0531] According to a particular embodiment, the culture medium comprises 20-30% basal medium, 40-60% rat serum, and 20-30% human serum (v / v).

[0532] According to a particular embodiment, the culture medium comprises 25% basal medium, 50% rat serum, and 25% human serum (v / v).

[0533] According to certain embodiments, the serum is heat inactivated (eg, for 30-45 minutes at 55°C).

[0534] According to certain embodiments, serum is added to the medium immediately prior to use.

[0535] According to some embodiments of the present invention, the medium may further comprise additional additives, including, but not limited to, antibiotics (e.g., PEN-STREP), L-glutamine (e.g., GlutaMAX™), sodium pyruvate, non-essential amino acids (NEAA), insulin-transferrin-selenium-ethanolamine (ITS-X), β-estradiol, progesterone, N-acetyl-L-cysteine, 3,3',5-triiodo-L-thyronine sodium salt (T3), sodium lactate, glucose, serum replacement, and / or HEPES.

[0536] According to some embodiments of the invention, the medium comprises N2 and B27.

[0537] According to certain embodiments, the medium comprises glucose.

[0538] According to certain embodiments, the medium comprises at least 1 mg / ml, at least 2 mg / ml, at least 3 mg / ml, or at least 4 mg / ml glucose.

[0539] According to certain embodiments, the medium comprises at least 4 mg / ml, at least 5 mg / ml, at least 6 mg / ml, at least 7 mg / ml, or at least 8 mg / ml glucose.

[0540] According to certain embodiments, the medium comprises 2 to 12 mg / ml glucose, 3 to 12 mg / ml glucose, 4 to 12 mg / ml glucose, or 4 to 8 mg / ml glucose.

[0541] According to certain embodiments, glucose is supplied in the medium at a constant or increasing concentration throughout the culture.

[0542] Thus, according to certain embodiments, the glucose concentration supplied in the medium throughout the culture does not decrease throughout the culture (e.g., during the transition from one set of conditions to the next set of conditions).

[0543] According to certain embodiments, the culture medium lacks MATRIGEL®.

[0544] According to certain embodiments, the conditions include changing at least half of the medium every 1-2 days of culture (eg, in static culture).

[0545] According to certain embodiments, the conditions include changing all the medium every 1-2 days of culture (eg, in dynamic culture).

[0546] According to certain embodiments, the culture is maintained under high pressure.

[0547] According to certain embodiments, the dynamic culture is maintained under high pressure.

[0548] According to certain embodiments, the roller culture is maintained under high pressure.

[0549] As used herein, the term "high pressure" refers to pressure above atmospheric pressure, which is generally considered to be 14.7 pounds per square inch (psi). Thus, when a specific high pressure is mentioned herein, it refers to the indicated pressure above atmospheric pressure, not the indicated value itself. For example, a high pressure of 5 psi refers to a pressure of 19.7 psi, a high pressure of 6.5 psi refers to a pressure of 21.2 psi, and a high pressure of 10.2 psi refers to a pressure of 24.7 psi.

[0550] According to certain embodiments, the high pressure is greater than 2.5 psi, greater than 4 psi, greater than 5 psi, or greater than 6 psi.

[0551] According to certain embodiments, the high pressure is greater than 5 psi.

[0552] According to certain embodiments, the high pressure is less than 10.2 psi, less than 9 psi, less than 8 psi, or less than 7 psi.

[0553] According to a particular embodiment, the high pressure is less than 10.2 psi.

[0554] According to certain embodiments, the high pressure is greater than 5 psi and less than 10.2 psi.

[0555] According to a particular embodiment, the high pressure is 6-7 psi.

[0556] According to a particular embodiment, the high pressure is 6.5 psi.

[0557] According to a particular embodiment, the high pressure is between 0.1 and 0.5 psi.

[0558] According to other particular embodiments, the culture is maintained at atmospheric pressure.

[0559] According to certain embodiments, the static culture is maintained at atmospheric pressure.

[0560] According to certain embodiments, the cultures are maintained in an atmosphere containing controlled levels of O2, N2, and / or CO2.

[0561] According to a particular embodiment, the culturing is carried out in an atmosphere containing 5% CO2.

[0562] According to a particular embodiment, the culturing is carried out in an atmosphere containing 5 to 40% oxygen.

[0563] According to particular embodiments, the culturing is carried out in an atmosphere containing 5-30%, 5-25%, or 5-21% oxygen.

[0564] According to particular embodiments, the culturing is carried out in an atmosphere containing 10-40%, 10-30%, or 15-30% oxygen.

[0565] According to a particular embodiment, the culturing is carried out in an atmosphere containing 15-30% oxygen.

[0566] According to a particular embodiment, the culturing is carried out in an atmosphere containing 18 to 23% oxygen.

[0567] According to certain embodiments, the culturing is carried out in an atmosphere containing a constant or increasing oxygen concentration throughout the culturing.

[0568] Thus, according to certain embodiments, oxygen concentration does not decrease throughout the culture (eg, during the transition from one set of conditions to the next set of conditions).

[0569] According to certain embodiments, the following embryonic incubation system is used, which includes gas and pressure controllers and static and / or rotary incubators to control pressure and oxygen levels during culture.

[0570] Exemplary Embryonic Incubation System Reference is now made to FIG. 44A, which shows a schematic representation of an embryonic incubation system according to some embodiments of the present invention. In some embodiments, the system includes a gas and pressure controller 502, one or more gas sources 504, 505, and 506 (shown in FIG. 44A are tanks of carbon dioxide (CO2) 506, oxygen (O2) 505, and nitrogen (N2) 504), a gas mixing box 508, and an incubator 510. In some embodiments, gases from gas sources 504, 505, 506 are delivered to gas and pressure controller 502, which delivers the gases to gas mixing box 508. In some embodiments, once the mixture of gases reaches the required concentration, the gases are returned to gas and pressure controller 502 for controlled delivery to incubator 510. In some embodiments, incubator 510 optionally includes an internal rotating incubator module configured to grip one or more vials holding embryos. In some embodiments, the gas mixture is delivered to an internal rotating incubator module where the gas is delivered evenly to each vial (see below for details).

[0571] FIG. 44B shows an image of an exemplary embryonic incubation system including a gas and pressure controller 502, a gas mixing box 508, and an incubator 510, according to some embodiments of the present invention.

[0572] FIG. 44C shows a general schematic representation of an exemplary configuration of the gas electronics module (gas and pressure controller 502 together with gas mixing box 508) and the principles of pressure regulation. In some embodiments, N, O, and / or CO enter the system at a pressure of 0.5 psi and are mixed by a mixing centrifugal blower (see below). In some embodiments, the gas is then optionally injected into a water bottle inside the incubator by a pump that allows control of the gas pressure, thereby enabling high pressure. In some embodiments, the system is equipped with one or more sampling ports (e.g., for O and / or CO) that allow additional monitoring of the levels of gases in the system.

[0573] Exemplary Gas and Pressure Controller Reference is now made to FIG. 44D, which shows a schematic representation of an exemplary gas and pressure controller 502, according to some embodiments of the present invention. In some embodiments, as previously described, one or more gas sources 504, 505, 506 are connected to the gas and pressure controller 502. In some embodiments, each gas source is connected to a dedicated motorized valve 512, 513, and 514 within the gas and pressure controller 502. For example, gas from a CO source (e.g., tank) 506 is connected to a dedicated CO motorized valve 514, gas from an N source (e.g., tank) 504 is connected to a dedicated N motorized valve 512, and gas from an O source (e.g., tank) 505 is connected to a dedicated O motorized valve 513. In some embodiments, the gas and pressure controller 502 includes dedicated "individual gas controllers 516 / 518" for manipulating and monitoring the gases within the system (hereinafter referred to as the CO controller 518 or the O controller 516, which are distinct from the main gas and pressure controller 502 of the embryonic incubation system). For example, when the CO2 level within the embryonic incubation system needs to be manipulated, the user accesses the CO2 controller 518 to set the required CO2 level within the system; when the O2 level within the embryonic incubation system needs to be manipulated, the user accesses the O2 controller 516 to set the required O2 level within the system by adding or not adding N2 gas to the system and / or by adding or not adding O2 gas to the system. It should be noted that oxygen is approximately 21% in normal air, and when a lower level of oxygen (e.g., 5% or 10%) is required within the embryonic incubation system, nitrogen gas is inserted to reduce the oxygen level within the system. In some embodiments, when a higher oxygen concentration is required, the N2 gas is turned off and pure O2 is supplied, for example, such that the O2 / CO2 level within the vial is 95% / 5%.In some embodiments, the system is configured to deliver any combination of gas mixtures, for example, a gas mixture containing 0% O to a gas mixture delivering 100% O, or any combination of O / CO ratios, for example, a ratio of about 1% / 99% to 99% / 1%. In some embodiments, the gas and pressure controller 502 delivers and ensures that any gas delivered to the embryonic incubation system is within a tolerance of about 0.2%. In some embodiments, the tolerances are about 0.1% to about 1% for CO gas, about 0.1% to about 2% for O gas, and about 0.1% to about 2% for N gas. In some embodiments, the tolerance for CO does not exceed 2%. In some embodiments, the tolerance for N does not exceed 5%. In some embodiments, the tolerance for O does not exceed 5%. In some embodiments, each specific gas controller 516 / 518 controls the opening and closing of specific motorized valves 512, 513, and 514 as needed. In some embodiments, the requests set by the user using the individual gas controllers 516 / 518 are monitored by dedicated sensors in the gas mixing box 508 (see information below regarding the gas mixing box 508). Thus, in some embodiments, information from the gas sensors in the gas mixing box 508 is sent to the dedicated gas controllers 516 / 518. In some embodiments, the dedicated gas controllers 516 / 518 use the information from the gas sensors in the gas mixing box 508 to open or close specific motorized valves 512, 513, 514, again according to the predetermined requests set by the user. In some embodiments, the gas and pressure controller 502 includes a vacuum pump 520 and a pressure pump 522. In some embodiments, after the gases are mixed in the gas mixing box 508 (see information below regarding the gas mixing box 508), the vacuum pump 520 is activated to push the mixed gases out of the gas mixing box 508. In some embodiments, the gas is then accumulated in the pressure pump 522 until a predetermined pressure level is reached before being delivered to the incubator 510 .In some embodiments, the pressure pump is configured to deliver the gas mixture at a pressure of about 1 psi to about 6 psi, optionally about 0.5 psi to about 8 psi, and optionally about 0.05 psi to about 12 psi. 6.5 psi is preferred when needed. In some embodiments, a user manually sets the pressure level at which the pressure pump 522 delivers the gas mixture. In some embodiments, the gas and pressure controller 502 includes a power supply unit 524 configured to provide dedicated power to various portions of the gas and pressure controller 502. In some embodiments, the gas and pressure controller 502 optionally includes a pressure transmitter 526 configured to monitor the pressure within the system / gas and pressure controller 502. In some embodiments, the gas and pressure controller 502 optionally includes a check valve 528 configured to ensure that the gas mixture exiting the gas and pressure controller 502 does not return (backflow) to the gas and pressure controller 502. In some embodiments, the gas and pressure controller 502 optionally includes a gas adapter control 530 configured to control the flow rate within the system. In some embodiments, the gas and pressure controller 502 includes one or more filters (e.g., 1 μ filters), shown at E (531) in FIG. 44, attached to the tubing through which the gas flows to ensure gas purity and potentially avoid contamination.

[0574] FIG. 44E, FIG. 44F, and FIG. 44G show various images of an exemplary gas and pressure controller 502, in accordance with some embodiments of the present invention. Specifically, FIG. 44E shows a perspective view of the gas and pressure controller 502, showing the gas lines entering and exiting the gas and pressure controller 502. Also shown are gas controllers 516 / 518 and optional filter 531. FIG. 44F shows the internal layout of the gas and pressure controller 502, showing exemplary electrically operated valves 512, 513, 514, in accordance with some embodiments of the present invention. FIG. 44G shows an exemplary gas and pressure controller 502 configured to monitor and deliver only CO and / or N, in accordance with some embodiments of the present invention.

[0575] Exemplary Gas Mixing Box Reference is now made to FIG. 44H, which shows a schematic representation of an exemplary gas mixing box 508, according to some embodiments of the present invention. In some embodiments, the gas mixing box 508 is used to ensure a thorough and uniform mixing of the different gases required to provide the necessary environment for the vials within the incubator. In some embodiments, the gas mixing box 508 has a volume of approximately 250,000 cm 3 ~Approx. 260,000cm 344H. In some embodiments, the gas mixing box 508 has an internal volume of 1000 psi. In some embodiments, different sizes may be used to provide different amounts of mixed gas as needed. In some embodiments, the gas mixing box 508 is made of plastic, such as Perspex. In some embodiments, the gas mixing box 508 is made of a material other than plastic. In some embodiments, the gas mixing box 508 includes dedicated gas sensors 532 / 534 configured to monitor the gas content inside the gas mixing box 508. Following the previous description, FIG. 44H shows two sensors, an O2 sensor 532 and a CO2 sensor 534. In some embodiments, the sensors have a sensitivity ranging from about 95% accuracy to about 100% accuracy. In some embodiments, the gas mixing box 508 includes a mixer blower 536 configured to thoroughly mix the gases coming from the gas and pressure controller 502. In some embodiments, when the gas levels detected inside the gas mixing box 508 by the sensors 532 / 534 reach a desired level, the mixed gas is sucked out by a vacuum pump 520 in the gas and pressure controller 502. In some embodiments, the gas and pressure controller 502 optionally includes a limit flow 538 configured to maintain a uniform flow rate within the system, thereby potentially avoiding possible flow rate variations. In some embodiments, the gas mixing box 508 includes one or more filters (e.g., 1 μ filters) not shown attached to the tubing through which the gases flow to ensure gas purity and potentially avoid contamination.

[0576] FIG. 44I shows an image of an exemplary gas mixing box 508 according to some embodiments of the present invention.

[0577] Exemplary Incubator In some embodiments, the incubator is a static incubator. In some embodiments, the incubator is a rotary incubator. In some embodiments, the incubator is a static incubator with a rotary module therein. In some embodiments, the static incubator includes one or more temperature regulators configured to maintain a temperature inside the static incubator, including the rotary module disposed therein. In some embodiments, the temperature inside the incubator is regulated between 4°C and about 60°C. In some embodiments, the incubator is, for example, a "Precision" incubator system (BTC01 model with gas bubbler kit - manufactured by BTC Engineering (Cullum Starr Precision Engineering Ltd) in the UK).

[0578] Reference is now made to FIG. 44J, which shows a schematic representation of an exemplary incubator 510, according to some embodiments of the present invention. In some embodiments, a gas mixture is delivered to the incubator 510 from a gas and pressure controller 502. In some embodiments, the incubator includes a one-way valve 540 connected to a tube through which the gas mixture is delivered from the gas and pressure controller 502. In some embodiments, the one-way valve 540 is a manual one-way valve that is manually opened and closed by a user. In some embodiments, the one-way valve 540 is an automatic one-way valve controlled by a master controller (see below). In some embodiments, the gas mixture is optionally delivered to a bubbler bottle 542. In some embodiments, the bubbler bottle 542 is partially filled with a liquid. In some embodiments, the liquid is water. In some embodiments, gas is delivered to the liquid in the bubbler bottle 542, thereby creating bubbles in the liquid. In some embodiments, the bubbler bottle 542 allows the user to verify that the system is delivering the gas mixture by visually assessing 1. whether bubbles are present and 2. the rate at which bubbles are generated. In some embodiments, the bubbler bottle 542 also functions as a humidifier for the gas (see below). In some embodiments, the bubbler bottle 542 provides protection against pressure buildup from the gas mixture being delivered to the incubator 510. For example, in the event of a malfunction, if the gas mixture is delivered at a higher than desired pressure, the excess pressure is trapped within the bubbler bottle 542, where it dissipates. In some embodiments, the gas mixture is then optionally delivered to an additional humidifier 544. In some embodiments, the inventors have found that in certain cases, a dry gas mixture can be harmful to samples in the incubator. Therefore, adding the humidifier 544 overcomes this problem. In some embodiments, the additional humidifier reduces excess humidity from the gas coming from the bubbler bottle 542.In some embodiments, the gas mixture is delivered from the humidifier 544 to a rotating drum 546 of a rotating module that includes all containers (vials) containing samples placed in the incubator. In some embodiments, the rotating drum 546 is configured to deliver the gas mixture evenly among the containers, optionally while rotating the samples. In some embodiments, the containers (vials) in the rotating drum 546 contain culture media necessary for embryo growth and / or maintenance. In some embodiments, the gas is delivered into the containers (vials) and absorbed / used through the culture media. It should be noted that in some embodiments, because the embryos remain suspended in the culture media, continuously delivering new culture media containing already mixed gases is problematic. This is because this type of mechanism requires that old / used culture media be removed from the vials while new culture media containing the new mixed gases is placed in the vials, which may increase the chance of embryo loss during replacement. Therefore, new gas delivery is achieved by delivering the gas mixture to the vials without the need to change the culture media accordingly. In some embodiments, regardless of the need for continuous gas exchange, medium changes can be performed by removing each vial and carefully changing the medium according to known techniques. In some embodiments, the rotating drum 546 is configured to be positioned at an angle, allowing the vials to be angled relative to the base on which the entire rotating module is positioned upright. In some embodiments, the angle is from about 0 degrees (no angle; i.e., the vial is held sideways, as shown, for example, in FIG. 44 at K) to about 45 degrees. In some embodiments, the angle is set so that the top of the vial is always positioned above the bottom of the vial. In some embodiments, the rotation of the rotating drum 546 is independent of the operation of delivering the mixed gas to the vial. In some embodiments, the rotating drum is configured to rotate at a speed of about 1 rpm to about 100 rpm. In some embodiments, gas exiting the rotating drum 546 is delivered to a gas outlet bottle 548.In some embodiments, the outlet bottle 548 acts as a pressure buffer for the system, helping to maintain a constant pressure throughout the system. In some embodiments, the incubator 510 has dark walls, allowing samples to be kept in the dark. In some embodiments, the incubator 510 optionally includes one or more cameras for monitoring the samples, such as a conventional video camera, an IR camera, a night vision camera, etc. In some embodiments, the incubator 510 includes one or more heaters configured to maintain the samples at a constant temperature. In some embodiments, the incubator 510 includes one or more light sources, such as white light, IR light, UV light, and / or black light.

[0579] Figure 44K shows an image of an exemplary rotating incubator module 510 according to some embodiments of the present invention. Figure 44L shows an image of an exemplary container (vial or bottle) containing a sample according to some embodiments of the present invention.

[0580] Exemplary Automated Embryonic Incubation System In some embodiments, the embryonic incubation system disclosed above is connected to a master controller configured to perform automated operations according to a predetermined protocol provided by a user. For example, a user programs the master controller to perform changes in the incubation chamber over a period of time. In some embodiments, the system includes motorized valves throughout the system that are activated and deactivated according to a programmed protocol. In some embodiments, a potential advantage of utilizing an automated system is reduced opportunity for human error during embryo development. In some embodiments, optionally, the master controller provides the user with periodic updates to a PC or mobile electronic device.

[0581] Example general information about the system Generally, since the 1930s, numerous culture techniques have been proposed over the years, from culturing embryos in traditional static conditions, culturing in rotating bottles on drums (referred to as "roller culture systems"), or culturing on circulator platforms. These platforms remain highly inefficient for embryo survival and are limited to short-term use, as embryonic developmental abnormalities begin to appear as early as 24 hours after the initiation of culture. Therefore, a stable and efficient protocol for long-term culture of pre-gastrulation mouse embryos through to advanced organogenesis has been developed and disclosed herein. In some embodiments, several newly established cell culture additives or biomechanical principles in stem cell research were tested to evaluate whether they could help keep embryos alive (e.g., hyperbaric chambers, synthetic serum). In some embodiments, a "roller culture system" on a drum was used, which was integrated with a customized, in-house developed electronic gas regulation module 502, which not only allowed for sensitive and precise control of N2, O2, and CO2 levels, but also allowed for control of atmospheric pressure. In some embodiments, sequential increases in oxygen levels were applied every 24 hours, starting with 5% O2 on E7.5, 13% on E8.5, 18% on E9.5, and ending with 21% O2 on E10.5, and were found to be highly optimal for obtaining the robust results reported herein. Furthermore, increases in oxygen levels reached 95% when necessary. Additionally, in some embodiments, maintaining elevated pressure of approximately 6.5 psi was also found to be essential for normal and efficient embryo development.

[0582] In some embodiments, the sample is held in a static incubator. In some embodiments, the sample is held in a dynamic incubator, e.g., a rotating incubator. In some embodiments, the sample is first held in a static incubator and then moved to a dynamic incubator, or vice versa. In some embodiments, the sample is held in a static incubator, e.g., with a rotating incubator therein. In some embodiments, when held in a dynamic incubator, the sample is held in a rolling bottle on a drum (referred to as a "roller culture system") or on a circulator platform.

[0583] In some embodiments, embryos are maintained on a roller bottle culture unit inside a "precision" incubator system (e.g., BTC01 model with gas bubbler kit - manufactured by BTC Engineering (Cullum Starr Precision Engineering Ltd), UK) for the entire duration of culture. In some embodiments, a "rolling incubator" culture method is used, which provides a continuous flow of oxygenated gas to the cultures in the roller bottles, as disclosed elsewhere herein (e.g., BTC Rolling Bottle Culture Unit BTC02 model, manufactured by BTC Engineering (Cullum Starr Precision Engineering Ltd), UK). In some embodiments, culture bottles (small glass bottle BTC 03 and large glass bottle BTC 04) are inserted into a perforated rotating drum. In some embodiments, gas flows axially and is distributed to the culture bottles by a baffle plate inside the drum. In some embodiments, the system maintains a stable pH compared to other systems with sealed culture bottles. In some embodiments, the roller incubator is supplied by the manufacturer complete with gas filters, bubblers, and leads. In some embodiments, the BTC Precision Incubator uses a thyristor-controlled heater and high-flow fan to provide a very stable, uniform temperature throughout an easily accessible workspace. In some embodiments, the incubator has a 370 mm x 350 mm x 200 mm high workspace accessed through a hinged top lid. In some embodiments, the heater element is rated at 750 watts. In some embodiments, a BTC 06 (hollow) bung is used to seal the bottles, and a BTC 07 (solid) bung is used to seal the drums (manufactured by BTC Engineering (Cullum Starr Precision Engineering Ltd) in the UK).

[0584] In some embodiments, the incubator module is connected to a gas and pressure control unit 502 (model number - HannaLab1, assembled and sold by Arad Technologies LTD, Ashdod, Israel) to achieve constant O2 and CO2 levels in the culture medium throughout the incubation period. In some embodiments, carbon dioxide and oxygen concentrations are regulated by specific controllers located in the gas and pressure control unit 502. In some embodiments, the pressure controller can control the gas pressure at 5-10 psi (a positive pressure above the ambient external atmospheric pressure). In some embodiments, nitrogen, O2, and / or CO2 are then injected into the gas mixer box at a pressure of 6.5 psi, which has been found to be an optimal level. In some embodiments, the gas mixture in the gas box is uniform and mixed by a centrifugal mixer blower. In some embodiments, gas is injected into the incubator by a pump that provides pressure and sufficiency depending on the number of bubbles created in a water bottle under the control of a one-way flow meter. In some embodiments, the bubble rate (which indicates the rate at which gas enters the bottle) is adjusted by the user as needed. In some embodiments, gas enters the water bottle through an inlet, and the rate at which gas enters the bottle is controlled by a valve. In some embodiments, the humidified gas flows into a glass test tube and then into the bottle inside the rotating drum. In some embodiments, the gas flow rate is monitored by the rate at which bubbles are created inside the water-filled test tube at the outlet. In some embodiments, the bottle containing the sample is placed on a rotating bottle incubation system rotating at 30 revolutions per minute at 37°C and continuously gassed with, for example, an atmosphere of 5% O2, 5% CO2 at 6.5 pounds per square inch (psi), or, for example, 5% O2, 5% CO2 at 0.1 pounds per square inch (psi), or a gas mixture of, for example, 13% O2, 5% CO2, or, for example, a gas atmosphere of 18% O2 and 5% CO2, or, for example, a gas supply of 21% O2 and 5% CO2.In some embodiments, for medium exchange, the culture medium is pre-warmed for at least 1 hour by placing the culture medium inside a glass bottle on a roller culture with an appropriate gas atmosphere depending on the stage of the embryo being cultured.

[0585] Exemplary Methods for Embryonic Incubation Systems Reference is now made to FIG. 44M, which illustrates a flowchart of an exemplary method for an embryonic incubation system, according to some embodiments of the present invention. In some embodiments, a user sets the desired levels of gas and pressure inside the incubator (550). For example, 5% CO2 and 10% O2 at 6.1 psi pressure. In some embodiments, the gas and pressure controller activates sensors in the gas mixing box to receive information about the actual levels of gas in the gas mixing box (552). In some embodiments, an electric valve is opened to allow gas from the gas source to flow through the gas and pressure controller and into the gas mixing box (554). In some embodiments, the gases are mixed inside the gas mixing box by activating a mixer blower (556). In some embodiments, sensors in the gas mixing box are activated to monitor the levels of gases inside until they reach their desired levels (558). In some embodiments, the mixed gas is drawn from the gas mixing box by activating a vacuum pump (560). In some embodiments, the pressure of the drawn mixed gas is increased to the desired level by activating a pressure pump (562). In some embodiments, a one-way valve inside the incubator is opened to allow the pressurized gas mixture to enter the incubator (564). In some embodiments, the pressurized gas mixture passes through a humidifier (566). In some embodiments, the humidified pressurized gas mixture is delivered to individual tubes containing samples (568). In some embodiments, the released gas passes through an outlet tube before being released from the incubator (570).

[0586] According to certain embodiments, the method further comprises isolating cells, tissues, or organs from the embryo after culturing.

[0587] Non-limiting examples of such cells include stem cells (e.g., but not limited to, embryonic stem cells, mesenchymal stem cells, neural stem cells, and hematopoietic stem cells), blood cells, liver cells, insulin-secreting pancreatic beta cells, muscle cells, lung epithelial cells, endothelial cells, and glial cells.

[0588] These cells, tissues, or organs can be used, for example, as cell-based therapies, tissue replacements, organ or tissue implants, or for research purposes.

[0589] According to other particular embodiments, the method further comprises implanting the synthetic embryo into a uterus.

[0590] The established methods for producing synthetic embryos described herein, as well as methods and systems for culturing them outside the uterus, can be further combined with, for example, genetic modification, chemical screening, tissue manipulation, and microscopy, and can be powerful tools in basic research, for example, as a framework for investigating the emergence of cellular diversity, cell fate determination, and how tissues and organs emerge from a single totipotent cell, as well as a source of cells, tissues, and organs for transplantation, production of chimeric embryos, testing the effects of drugs on embryonic development (e.g., teratogenic effects), etc. Such methods are known in the art.

[0591] Thus, according to certain embodiments, the method comprises manipulating a synthetic embryo or cell subpopulation as described herein.

[0592] According to certain embodiments, the manipulation comprises introducing a gene of interest into the cell or embryo.

[0593] According to certain embodiments, the manipulation comprises introducing a polynucleotide of interest into the cell or embryo.

[0594] According to certain embodiments, the manipulation comprises introducing a genome editing agent or an RNA silencing agent into the cell or embryo.

[0595] According to certain embodiments, the manipulation involves producing an embryo that is incompatible with life. Thus, for example, the manipulation may involve knocking selected genes and selectively perturbing certain organs, as further described in the Examples section below, to create embryos that are developmentally restricted and unable to retain viability, such as headless embryos (e.g., deletion of Mesp1 or NKX2-5) or heartless embryos (e.g., deletion of Lim1).

[0596] Thus, according to certain embodiments, the manipulation involves introducing into the embryo a polynucleotide that renders the embryo incompatible with life.

[0597] Methods for designing, expressing, and introducing a polynucleotide of interest into a cell of interest so that it is expressed are well known in the art and are further described herein above and below.

[0598] According to certain embodiments, the manipulation comprises microinjecting cells into the embryo, thereby obtaining a chimeric embryo.

[0599] As used herein, the phrase "chimeric embryo" refers to an animal that contains cells from at least two genetically distinct individuals.

[0600] It should be noted that a chimeric embryo may be composed of cells from two different individuals, either belonging to two different species or belonging to the same species.

[0601] According to some embodiments of the invention, the cells are allogeneic to the embryo.

[0602] According to some embodiments of the invention, the cells are xenogeneic to the embryo.

[0603] As used herein, the term "heterogeneous" refers to at least two individuals of different species.

[0604] According to certain embodiments, the cells are stem cells (for example, but not limited to, embryonic stem cells, mesenchymal stem cells, neural stem cells, hematopoietic stem cells, induced pluripotent stem cells (iPS)).

[0605] According to some embodiments of the invention, the introduction of cells is performed ex vivo via direct injection or aggregation with a developing embryo.

[0606] According to certain embodiments, the manipulation comprises introducing a drug of interest into the cell or embryo.

[0607] According to certain embodiments, the method further comprises determining the effect of the manipulation on the development of the embryo.

[0608] According to certain embodiments, any of the genes, polynucleotides, proteins, polypeptides, and / or proteinaceous portions described herein may have the sequence of a human gene, polynucleotide, protein, polypeptide, and / or proteinaceous portion, or a functional fragment or homolog thereof, that exhibits a desired activity as described herein.

[0609] According to particular embodiments, the genes, polynucleotides, proteins, polypeptides and / or proteinaceous portions are of human origin.

[0610] According to other specific embodiments, the gene, polynucleotide, protein, polypeptide, and / or proteinaceous portion is a homolog of a human gene, polynucleotide, protein, polypeptide, and / or proteinaceous portion. Such a homolog can be, for example, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical or homologous to the human sequence.

[0611] As used herein, the term "about" refers to ±10%.

[0612] The terms "comprises," "comprising," "includes," "including," "having," and their conjugations mean "including but not limited to."

[0613] The term "consisting of" means "including and limited to."

[0614] The term "consisting essentially of" means that a composition, method, or structure may include additional ingredients, steps, and / or moieties, but only if the additional ingredients, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0615] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include multiple compounds, including mixtures thereof.

[0616] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an indefinite limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0617] Whenever a range of numerical values ​​is given herein, the range is meant to include any recited number (fractional or integer) within the range given. The phrases "ranging between" a first indicated number and a second indicated number and "ranging from" a first indicated number to a second indicated number are used interchangeably herein and are meant to include the first indicated number and the second indicated number, and all fractional and integer numbers therebetween.

[0618] As used herein, the term "method" refers to manners, means, techniques, and procedures for accomplishing a given task, including, but not limited to, manners, means, techniques, and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry, and medicine, or those readily developed by such practitioners from known manners, means, techniques, and procedures.

[0619] When referring to a particular sequence listing, it should be understood that such reference also encompasses sequences that substantially correspond to the complementary sequence, including minor sequence variations due, for example, to sequencing errors, cloning errors, or other alterations that result in base substitutions, deletions, or additions, provided that the frequency of such variations is less than 1 in 50 nucleotides, or less than 1 in 100 nucleotides, or less than 1 in 200 nucleotides, or less than 1 in 500 nucleotides, or less than 1 in 1000 nucleotides, or less than 1 in 5,000 nucleotides, or less than 1 in 10,000 nucleotides.

[0620] It will be understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately, or in any suitable subcombination, or as preferred in any otherwise described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0621] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. [Example]

[0622] Reference is now made to the following examples, which together with the above description illustrate some embodiments of the present invention in a non-limiting manner. Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques.

[0623] Example 1 Aggregation of mouse naive PSCs, TSCs, and XENs does not produce organized embryos

[0624] material and method Embryonic mouse TSC line #1 (mTSC#1) was established according to a previously published classical protocol (doi: 10.1126 / science.282.5396.2072.) Briefly, TSCs were established from E3.5 blastocysts collected by flushing the uterus of 5-week-old female CD1 mice. TSCs were cultured on mitotically inactivated mouse MEF cells (feeder cells) in TSC culture medium [15–20% (v / v) FBS (Gibco, ES cell-qualified or Biological Industries), 1× GlutaMAX (35050061, Thermo Fisher Scientific or Invitrogen), 0.1 mM β-mercaptoethanol (21985-023, Thermo Fisher Scientific or Sigma), 1 mM sodium pyruvate (11360070, Thermo Fisher Scientific), 1% (v / v) penicillin-streptomycin (15140122, Thermo Fisher Scientific or Biological Industries]. Blastocysts were cultured using modified RPMI 1640 (11875-093, Thermo Fisher Scientific) or RPMI (Invitrogen) supplemented with 25 ng / ml recombinant human FGF4 (235-F4-025, R&D or Peprotech), 1 μg / ml heparin (07980, StemCell or Sigma), and 1× non-essential amino acids (Biological Industries). 25 ng / ml FGF2 (Peprotech) was optionally added. DMEM can also be used instead of RPMI, as has typically been done previously in the field to generate stable TSC lines. Cells were then disaggregated by incubation in 0.1% trypsin-EDTA for 5 minutes in an incubator until outgrowths formed on day 4. The reaction was stopped by adding mouse 70% fetal fibroblast conditioned medium containing FGF4 and heparin, and the cells were returned to the incubator.The medium was changed every other day until TS cell colonies were observed (not before day 14 (passage 3)). The medium was then changed to TSC culture medium to maintain the cells. Immunostaining for TSC markers Cdx2, Gata3, TBR2, and TFAP2C, as well as the absence of Oct4 and Nanog pluripotency markers, confirmed the identity of this representative TSC line as TSCs (Figures 1 and 2). The cells could be passaged more than 50 times while maintaining their morphology and markers.

[0625] Mouse TSC line #2 (mTSC#2) established from mouse ES cells: This was established by inhibiting Oct4 and transferring cells to TSC medium as detailed for mTSC#1 above, as previously established (PMID: 10742100). Briefly, we used ZHBTC4, a mouse ES cell line with DOX-induced inhibition of Oct4 blockade (Tet-off), and passaged it in TSC medium with DOX (PMID: 10742100). Immunostaining revealed that the parental ESC line was positive for the Oct4 pluripotency marker and negative for the Cdx2 and Gata3 TSC markers (Figure 3A). The established mTSC#2 line lacked the Oct4 and Nanog pluripotency markers and stably expressed the TSC markers Cdx2, TBR2, Gata3, and TFAP2C (Figure 3B and Figure 4), confirming the TSC identity of this representative TSC line. Furthermore, the mTSC#2 line was maintained on MEFs and had classic TSC colony morphology (doi: 10.1126 / science.282.5396.2072).

[0626] Mouse TSC line #3 (mTSC#3) established from mouse ES cells: This was established by transiently overexpressing Cdx2 in mouse ES cells grown in semi-primed FBS / LIF conditions, as previously described (www(dot)doi(dot)org / 10(dot)1016 / j(dot)cell(dot)2005(dot)08(dot)040), and then transferring the cells to TSC medium as detailed for mTSC#1. Specifically, we used a previously published ICR mouse ES cell line (PMID: 19427283) transiently transfected with Cdx2 and passaged it in TSC medium. TSC colonies were subsequently selected, passaged, and validated. Immunostaining for the TSC markers Cdx2 and Gata3 confirmed the TSC identity of this representative TSC line (Figure 5). Furthermore, the mTSC#3 line was maintained on MEFs and had classic TSC colony morphology.

[0627] Mouse XEN line #7 (XEN-ICR clone #7): This was established according to a previously published classical protocol (doi:10.1071 / rd06125). Briefly, modified XEN conditions were used to establish XEN cells from E3.5 blastocysts collected by flushing the uterus of 5-week-old female ICR mice. Mouse blastocysts were cultured on feeder cells until outgrowths formed on day 4. The outgrowths were then disaggregated by incubation in 0.1% trypsin-EDTA in an incubator for 5 minutes. Fresh mouse ES (FBS-LIF-based) was added to stop the trypsin reaction, and the medium was changed every other day until XEN cell colonies became visible. XEN cell colonies were subsequently picked and maintained as stocks in standard XEN medium: modified RPMI 1640 (11875-093, Thermo Fisher Scientific) or RPMI 1640 (Gibco, 11875085) supplemented with 15% (v / v) FBS (Gibco or Biological Industries), 0.1 mM β-mercaptoethanol (21985-023, Thermo Fisher Scientific or Sigma), 1× GlutaMAX (Invitrogen), 1% penicillin-streptomycin (15140122, Thermo Fisher Scientific or Biological Industries), 1× non-essential amino acids (Biological Industries), and 1 mM sodium pyruvate (BI, 03-042-1B). As previously reported (doi:10.1071 / rd06125), at least 15 days and three passages were required to establish individual mouse XEN early-passage lines. Immunostaining of the mouse XEN early-passage line, clone 7, at passage 5 confirmed the expected loss of pluripotency markers Oct4 and Nanog, and expression of XEN markers Foxa2 and Gata4 (Fig. 6A).In addition, real-time PCR analysis demonstrated upregulated expression of the XEN markers Gata6, PDGFRA, Sox17, and Gata4 (Figure 6B), further confirming their identity (10.1371 / journal.pone.0009794). As a reference control, mouse naive V6.5 naive ESCs grown in 2i-LIF conditions were used (value set to 1).

[0628] The mouse XEN line, XEN-Prep1, was established as described (in parallel) for XEN line #7. Real-time PCR analysis demonstrated upregulation of the expression of the XEN markers Gata6, PDGFRA, Sox17, and Gata4 (Figure 6B), confirming their identity (10.1371 / journal.pone.0009794). Mouse naive V6.5 ESCs grown under 2i-LIF conditions served as a reference control (value set to 1).

[0629] Mouse naive pluripotent stem cell (naive PSC) line: GFP-tagged pluripotent V6.5 mouse ES line (PMID: Cells (19427283) were maintained and expanded in N2B27-based serum-free, chemically defined medium (240 ml Neurobasal (ThermoFisher, 21103049) and 240 ml DMEM-F12 with HEPES (Sigma, D6421), 5 ml N2 supplement (Invitrogen, 17502048), 5–10 ml B27 supplement (Invitrogen, 17504044), 2 mM glutamine or 1 mM Glutamax (Invitrogen), 1% non-essential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma), and 1% penicillin-streptomycin (Invitrogen). Optionally, the medium also contained 5 mg / ml BSA (Sigma). 2i / LIF naive conditions for mouse PSCs included 20 ng / ml recombinant human LIF (in-house production). Where indicated, 2i was supplemented with the small molecule inhibitors CHIR99021 (CH, 3 μM—Axon Medchem) and PD0325901 (PD, 1 μM—Axon Medchem, 1408).

[0630] KH2-Gata4 Dox-inducible cells (KH2-GATA4): The KH2-wild type cell line is constructed in such a way that the Rosa26 locus is targeted to express M2Rtta, and the Colal locus contains the TetOn promoter and an invertible insert within the collagen locus, in this case Gata4 (SEQ ID NO: 1): The v6.5 mouse ES cell line was engineered to accept a plasmid containing Gata4 (PMID: 16400644). Target colonies were subjected to hygromycin selection for 10 days, and individual ES colonies were selected for further analysis to generate KH2-Gata4, which overexpresses Gata4 upon DOX addition (Figure 8). Real-time PCR analysis and immunostaining confirmed that the clones exhibited DOX-induced overexpression of the Gata4 insert (Figures 9-10).

[0631] Aggregation medium (AM): 240 ml of Neurobasal (ThermoFisher, 21103049), 240 ml of DMEM-F12 (Thermo, 21331-020 or SIGMA, D6421), 5 ml of N2 supplement (Invitrogen, 17502048), 10 ml of B27 supplement (Invitrogen, 17504044), 2 mM glutamine or 1x GlutaMAX (Invitrogen), 1x non-essential amino acids (NEAA) (Invitrogen), 0.1 mM β-mercaptoethanol (GIBCO, 31350-010), 1x penicillin-streptomycin (Invitrogen), 0.45% BSA (Sigma, A7979), 1% sodium pyruvate (BI, 03-042-1B). Where indicated, serum was added to the aggregation medium at the indicated percentages and origins.

[0632] IVC1 medium: Modified DMEM / F12 (Gibco, 12634010) supplemented with 1x Glutamax, 1x penicillin / streptomycin, 1x ITS-X (Gibco, 51500056), 8nM beta-estradiol, 20ng / ml progesterone, 25µM N-acetyl-L-cysteine, and FBS or HAS as indicated.

[0633] EUCM medium (also called EUCM1): 25% DMEM (GIBCO 11880) or modified DMEM / F12 (Thermo 12634010), 25% HAS (human adult serum or human umbilical cord serum), 50% rat serum (RAS), 1x Glutamax (GIBCO 35050038), 11 mM HEPES (GIBCO 15630056), and 1x penicillin / streptomycin. Where indicated, the medium was further supplemented with 4–12 mg / ml D-glucose, 1 mM sodium pyruvate, and / or 1x non-essential amino acids (NEAA). Aliquots of DMEM were stored at 4°C and used within 2 months. Rat serum was stored at -80°C, heat-inactivated at 56°C for 30 minutes, and filtered through a 0.22 μm PVDF filter (Millipore, SLGV033RS) before use. HAS should be freshly thawed immediately before the experiment. Rat serum and HAS can be thawed / frozen once.

[0634] Preparation of aggregation plates: To prepare 24-well AggreWell 400 plates (STEMCELL Technologies, 34415) or AggreWell 800 plates (STEMCELL Technologies), 500 ml of anti-adhesion rinse solution (STEMCELL Technologies, 07010) was added to each well. The plates were then centrifuged at 2,000 x g for 5 minutes and incubated at room temperature for 30 minutes. The anti-adhesion rinse solution (StemCell Technologies - #07010) was then aspirated from the wells, and 1 ml of PBS - / - was added to wash each well. This PBS wash was repeated twice. After aspirating the PBS, 500 ml of aggregation medium (AM) was added to each well.

[0635] Co-aggregation and culture (FIG. 7A): Co-aggregation was defined as time 0 of the protocol and was generated from three starting stem cell types. 1) V6.5-GFP (to give rise to the embryo proper); 2) mouse XEN line #7 cells (predicted to give rise to extraembryonic endoderm lineages), and 3) Mouse TSC line #1 cells (expected to give rise to the entire trophectoderm lineage).

[0636] Cells were trypsinized with 0.05% trypsin-EDTA for 3-5 minutes, followed by feeder depletion for 20 minutes at 37°C on gelatin-treated plates (if MEFs were used). Cells were then resuspended in aggregation medium (AM) supplemented with doxycycline (final concentration 2 μg / ml). After cell counting, a total of 18,000 TSCs, 6,000 XEN cells, and 6,000 v6.5-GFP wild-type cells were added per well of an AggreWell 400-microwell 24-well plate. A total of 4,500 TSCs, 1,500 XEN cells, and 1,500 v6.5 GFP cells were added per well of an AggreWell 800-microwell plate. The cell mixture was added at 25 cells / well (1 ml of the final volume in each well of the 24-well plate is 1.5 ml). Day 0: The plates were centrifuged at 100 x g for 3 minutes, which was designated as day 0, and then placed in a static incubator at 37°C, 5% CO2, and 20% O2. On day 1, 1 ml of medium was gently aspirated from each well and replaced with 1 ml of fresh pre-warmed AM + 20% PBS + DOX, as shown in Figure 7A. On day 2, 1 ml of medium was gently aspirated from each well and replaced with 1 ml of fresh prewarmed AM+20% PBS, as shown in Figure 7A. On day 3, 1 ml of medium was gently aspirated from each well and replaced with 1 ml of prewarmed IVC1 medium containing 20% ​​FBS, as shown in Figure 7A. On day 4, embryos were transferred to a 15 ml Falcon tube with a 3 ml plastic Pasteur pipette, allowed to sit for 5 minutes, and then most of the medium was gently removed, leaving the embryos in approximately 1 ml of medium. Embryos were then gently transferred with a pipette to a Petri dish (35 mm, Corning 430588 plate) or a 6-well plate (Costar 3736) or iBidi plate containing 4 ml of IVC1 medium containing 30% FBS and incubated on a rotary shaker at 60 rpm in a static incubator (37°C, 20% O2, and 5% CO2). On day 5, 2–3 ml of medium was gently aspirated with a manual pipette and replaced with fresh IVC1 medium containing 30% HAS (total volume returned to 4 ml), and incubated on a shaker (60 rpm) in a static incubator (37°C, 20% O2, and 5% CO2). Days 6 and 7: Each day, 2–3 ml of medium was gently aspirated with a manual pipette and replaced with fresh EUCM medium supplemented with 4 mg / ml D-glucose and 1 mM sodium pyruvate (total volume returned to 4 ml), as shown in Figure 7A. From day 8 onwards, high-quality embryos were selected and transferred to a previously described extrauterine roller culture system (Alejandro-Castrjon et al. Nature 2021) containing fresh EUCM medium supplemented with 4 mg / ml D-glucose and 1 mM sodium pyruvate and incubated at 20% O2, 5% CO2 and 6.5 Psi.

[0637] Coaggregation and culture (Fig. 7D): Instead of XEN cells, KH2-GATA4 Dox-inducible cells grown in 2i / LIF were used. For this purpose, KH2-GATA4 cells were subjected to the addition of DOX in 2i / LIF 12 hours before aggregation. Coaggregation was defined as time 0 of the protocol and was generated from three starting stem cells. 1) V6.5-GFP wild-type naive ESCs (to give rise to embryonic bodies); 2) DOX-pretreated KH2 tetO-GATA4 naive ESCs (to give rise to the extraembryonic endoderm lineage), and 3) TSCs (those predicted to give rise to the entire trophectoderm lineage).

[0638] Cells were trypsinized with 0.05% trypsin-EDTA for 3–5 minutes and then feeder-depleted (if using MEFs) for 20 minutes at 37°C on gelatin-treated plates. They were then resuspended in aggregation medium (AM) supplemented with doxycycline (final concentration 2 μg / ml). After cell counting, a total of 18,000 TSCs, 6,000 KH2-GATA4 cells, and 6,000 V6.5-GFP naive ES cells were added per well of an AggreWell 400-microwell 24-well plate. A total of 4,500 TSCs, 1,500 KH2-GATA4 cells, and 1,500 V6.5-GFP wild-type cells were added per well of an AggreWell 800-microwell 24-well plate. The cell mixture was added at 1 ml per well (final volume of each well of 24 wells is 1.5 ml), 25 cells / well.

[0639] Day 0: The plates were centrifuged at 100 x g for 3 minutes, which was designated as day 0, and then placed in a static incubator at 37°C, 5% CO2, and 20% O2. On day 1, 1 ml of medium was gently aspirated from each well and replaced with 1 ml of fresh pre-warmed AM+20% FBS+DOX, as shown in Figure 7D. On day 2, 1 ml of medium was gently aspirated from each well and replaced with 1 ml of fresh prewarmed AM+20% FBS, as shown in Figure 7D. On day 3, 1 ml of medium was gently aspirated from each well and replaced with 1 ml of prewarmed IVC1 medium containing 20% ​​FBS, as shown in Figure 7D. On day 4, embryos were transferred to a 15-ml Falcon tube using a 3-ml plastic Pasteur pipette, allowed to stand for 5 minutes, and then most of the medium was gently removed, leaving the embryos in approximately 1 ml of medium. The embryos were then gently transferred using a pipette to a Petri dish (35 mm, Corning 430588 plate) or a 6-well plate (Costar 3736) or iBidi plate containing 4 ml of IVC1 medium containing 30% FBS (as shown in Figure 7D), and incubated on a shaker (60 rpm) in a static incubator (37°C, 20% O2, and 5% CO2). On day 5, 2–3 ml of medium was gently aspirated with a manual pipette and replaced with fresh IVC1 medium containing 30% KSR (Knockout Serum Replacement - Invitrogen, as shown in Figure 7D; the total volume was returned to 4 ml) and maintained on a rotating shaker at 60 rpm in a static incubator (37°C, 20% O2, and 5% CO2). Days 6 and 7: Each day, 2–3 ml of medium was gently aspirated with a manual pipette and replaced with fresh EUCM medium supplemented with 4 mg / ml D-glucose and 1 mM sodium pyruvate (total volume returned to 4 ml), as shown in Figure 7D. From day 8 onwards, high-quality embryos were selected and transferred to a previously described extrauterine roller culture system (Alejandro-Castrjon et al. Nature 2021) containing fresh EUCM medium supplemented with 4 mg / ml D-glucose and 1 mM sodium pyruvate and incubated at 20% O2, 5% CO2 and 6.5 Psi.

[0640] Embryo whole-mount immunostaining: Embryos grown outside the uterus were dissected (with or without the removal of the yolk sac and amnion for E8.5 embryos), washed once with 1x PBS, transferred to iBidi glass-bottom 8-well slides (iBidi), and fixed overnight at 4°C with 4% EM-grade PFA (Electron Microscopy Sciences, Inc., 15710) in PBS. Embryos were then washed three times for 5 minutes each, permeabilized for 30 minutes with 0.5% Triton X-100 / 0.1 M glycine in PBS, blocked for 1 hour at room temperature (RT) with 10% normal donkey serum / 0.1% Triton X-100 in PBS, and incubated overnight at 4°C with primary antibodies diluted in blocking solution. Embryos were then rinsed three times for 5 minutes with PBS / 0.2% Triton X-100, incubated with secondary antibodies diluted 1:200 in blocking solution (all secondary antibodies were from Jackson ImmunoResearch) for 2 hours at room temperature, counterstained with DAPI (1 μg / ml in PBS) for 10 minutes, and washed three times for 5 minutes with PBS. If necessary, yolk sacs isolated from embryos were fixed and stained using this whole-mount immunostaining protocol. The following primary antibodies were used:1:100 rabbit monoclonal anti-Brachyury (Cell Signaling, 81694), 1:100 rabbit polyclonal anti-Cdx2 (Cell Signaling, 3977), 1:100 mouse monoclonal anti-Cdx2 (Biogenex, AU392A-UC), 1:100 goat polyclonal anti-Gata4 (Santa Cruz, SC-1237), 1:100 rabbit polyclonal anti-Gata4 (Abcam, Ab84593), 1:100 rabbit polyclonal anti-Foxa2 (Abcam, Ab40874), 1:100 mouse monoclonal anti-myosin heavy chain II (MF-20) (R&D, MAB4470), 1:200 goat polyclonal anti-Otx2 (R&D, AF1979), 1:100 rabbit polyclonal anti-Pax6 (Covance, PBR-278P), 1:2 Goat polyclonal anti-Sox2 (R&D, AF2018) at 1:00, rabbit polyclonal anti-Sox9 (Millipore, AB5535) at 1:100, goat polyclonal anti-Sox17 (R&D, AF1924) at 1:100, mouse monoclonal anti-Tuj1 (C-10) (Covance, MMS-435P) at 1:200, chicken polyclonal anti-GFP (Abcam, Ab13970) at 1:250, and mouse monoclonal anti-Oct4 (Santa Cruz, SC-5279), 1:100 goat polyclonal anti-Lefty1 (R&D, AF746), and 1:200 goat polyclonal anti-mCherry / Tomato (SiCGEN, AB0040-200).

[0641] E9.5–E11.5 embryos were cleared as follows: embryos were washed once with 1x PBS and fixed overnight at 4°C with 4% EM-grade PFA diluted in PBS. Samples were washed three times for 5 minutes each with PBS, followed by pretreatment without methanol. Specifically, samples were washed twice for 1 hour with PBS / 0.2% Triton X-100, then incubated overnight at 37°C in PBS / 0.2% Triton X-100 / 20% DMSO, and then incubated overnight at 37°C in PBS / 0.1% Tween-20 / 0.1% Triton X-100 / 0.1% deoxycholate / 0.1% NP40 / 20% DMSO, followed by two 1-hour washes with PBS / 0.2% Triton X-100. For immunostaining, embryos were permeabilized overnight at 37°C with PBS / 0.2% TritonX-100 / 20% DMSO / 0.3 M glycine and then blocked overnight at 37°C with PBS / 0.2% TritonX-100 / 10% DMSO / 6% donkey serum. After blocking, embryos were incubated at 37°C with primary antibodies diluted in PBS / 10 μg / ml heparin with 0.2% Tween-20 (PTwH) / 5% DMSO / 3% donkey serum [E9.5 / E10.5 = 24 h, E11.5 = 48 h (72 h for Sox17 and Foxa2 antibodies)]. Samples were then washed in PTwH for 24 h (15 min, 30 min, 1 h, 2 h, and overnight washes) and incubated with the appropriate secondary antibody diluted (1:200) in PTwH / 3% donkey serum for 48 h at 37°C.

[0642] For human cell-specific NUMA staining (if used), donkey anti-rabbit biotin and streptavidin-Cy3 (each incubated overnight) were used for signal enhancement. Embryos were then incubated with DAPI (1 μg / ml) diluted in PTwH for 30 minutes, washed daily in PTwH (5 min, 15 min, 30 min, 1 h, 2 h, and overnight washes), dehydrated in a methanol / HO series (20%, 40%, 60%, 80%, and 100%) for 1 hour each at room temperature, and then incubated overnight in 100% methanol. Embryos were incubated in 66.6% DCM / 33.3% methanol at room temperature for 3 hours on a shaker, followed by a 5-minute incubation in 100% DCM (Sigma, 270997), and finally cleared and stored in benzyl ether (Sigma, 108014). The following primary antibodies were used:1:100 rabbit monoclonal anti-Brachyury (Cell Signaling, 81694), 1:100 rabbit polyclonal anti-Cdx2 (Cell Signaling, 3977), 1:100 mouse monoclonal anti-Cdx2 (Biogenex, AU392A-UC), 1:100 goat polyclonal anti-Gata4 (Santa Cruz, SC-1237), 1:100 rabbit polyclonal anti-Gata4 (Abcam, Ab84593), 1:50 rabbit polyclonal anti-Foxa2 (Abcam, Ab40874), 1:100 mouse monoclonal anti-myosin heavy chain II (MF-20) (R&D, MAB4470), 1:200 goat polyclonal anti-Otx2 (R&D, AF1979), 1:100 rabbit polyclonal anti-Pax6 (Covance, PBR-278P), 1:200 goat polyclonal anti-Sox2 (R&D, AF2018), 1:100 rabbit polyclonal anti-Sox9 ( Millipore, AB5535), 1:50 goat polyclonal anti-Sox17 (R&D, AF1924), 1:200 mouse monoclonal anti-Tuj1 (C-10) (Covance, MMS-435P), 1:250 chicken polyclonal anti-GFP (Abcam, Ab13970), 1:200 goat polyclonal anti-mCherry / Tomato (SiCGEN, AB0040-200), 1:100 rabbit polyclonal anti-human TEMEM119 (Invitrogen, PA562505), and 1:100 rabbit anti-hNUMA (Abcam, ab84680).

[0643] Confocal Microscopy: Images were acquired on a Zeiss LSM700 inverted confocal microscope (Carl Zeiss) equipped with 405 nm, 488 nm, 555 nm, and 635 nm solid-state lasers. Images were acquired using a 20x Plan-Apochromat air objective (numerical aperture 0.8) for E5.5 / E6.5 embryos and a 10x EC Plan Neofluar air objective (numerical aperture 0.3) for E7.5–E11.5 embryos. Images were acquired at a resolution of 1024 × 1024. All images were acquired within the following parameters: laser power: 405 nm: 10–20%, 488 nm: 5–20%, 555 nm: 10–40%, 635 nm: 30–80%. Gain ranged from 350–600. The pixel size was 1.25 μm with a z-step of 15 μm using a 10× objective, or 0.5 μm with a z-step of 5 μm using a 20× objective. For confocal imaging, iDISCO-cleared embryos were mounted on 35 mm glass-bottom dishes (In Vitro Scientific, D35201.5N) and imaging solution was ethyl cinnamate (Sigma, 112372). For chimeric embryos, all parameters during image acquisition were compared with stained, uninjected control embryos imaged with equivalent parameters. Images and maximum intensity projections were processed using Zen 2 blue edition software 2011 (Carl Zeiss) and Adobe Photoshop CS4.

[0644] Immunostaining of ES, TSC, and XEN cell lines: Cells were grown on glass coverslips (13mm 1.5H, Marienfeld, 0117530) and fixed with 4% paraformaldehyde / phosphate buffer for 10 minutes at room temperature, washed three times with PBS, and permeabilized with PBS / 0.1% Triton for 10 minutes. Cells were blocked with blocking solution (2% normal goat serum, 0.1% BSA in PBS / 0.05% Tween) for 1 hour at room temperature and then incubated overnight at 4°C with primary antibodies diluted in blocking solution (all antibodies used in this study have been validated in the literature and by the inventors). Cells were then washed three times with PBS / 0.05% Tween, incubated with secondary antibodies for 1 hour at room temperature, washed with PBS / 0.05% Tween, counterstained with DAPI (1 μg / ml), washed three times again with PBS / 0.05% Tween, mounted with Shandon Immu-Mount (Thermo Scientific, 9990412), and imaged. Comparative experiments were performed simultaneously. All secondary antibodies were from Jackson ImmunoResearch.

[0645] Real-time PCR analysis: For RT-PCR, total RNA was isolated using Trizol (Ambion Life Technologies), and 1 μg of total RNA was reverse transcribed using the High-Capacity Reverse Transcription Kit (Applied Biosystems). Quantitativ...

Claims

1. 1. A method for producing a synthetic embryo, comprising: (a) inducing expression of a factor that induces differentiation into trophectoderm cells in a subpopulation of naive pluripotent stem cells (PSCs) to obtain trophectoderm primed cells; and (b) mixing the trophectoderm primed cells with naive PSCs under conditions that allow for the formation of aggregate cells; When the factor that induces differentiation into trophectoderm cells is an exogenous factor, the induction of the expression includes induction of transient expression that begins within a period between 14 days and 0 hours before the mixing and ends within 120 hours after the mixing, the factor that induces differentiation into trophectoderm cells is an endogenous factor, and the induction of expression comprises culturing the subpopulation of naive PSCs under conditions that permit expression of the factor, beginning within a period of 14 days to 6 hours prior to the mixing; Thereby producing said synthetic embryo.

2. 2. The method of claim 1, further comprising inducing in (a) expression of a factor that induces differentiation into extraembryonic primitive endoderm cells in a second subpopulation of naive PSCs to obtain extraembryonic primitive endoderm primed cells, and in (b) mixing the extraembryonic primitive endoderm primed cells with the cells.

3. When the factor that induces differentiation into extraembryonic primitive endoderm cells is an exogenous factor, the induction of the expression includes induction of transient expression that begins within a period between 14 days and 0 hours before the mixing and ends within 120 hours after the mixing, 3. The method of claim 2, wherein the factor that induces differentiation into extraembryonic primitive endoderm cells is an endogenous factor, and said inducing expression comprises culturing a subpopulation of said naive PSCs under conditions that permit expression of said factor, beginning 14 days to 6 hours prior to said mixing.

4. 1. A method for producing a synthetic embryo, comprising: (a) inducing transient expression of an exogenous factor that induces differentiation into trophectoderm cells in a subpopulation of naive pluripotent stem cells (PSCs) to obtain trophectoderm-primed cells; and (b) mixing the trophectoderm primed cells with naive PSCs under conditions that allow for the formation of aggregate cells; said induction of said transient expression is initiated within a period between 14 days and 0 hours before said mixing and is terminated within 120 hours after said mixing; Thereby producing said synthetic embryo.

5. The method of claim 1, further comprising inducing, in (a), transient expression of an exogenous factor that induces differentiation into extraembryonic primitive endoderm cells in a second subpopulation of naive PSCs to obtain cells primed towards extraembryonic primitive endoderm, and in (b), mixing the cells primed towards extraembryonic primitive endoderm with the cells.

6. The method of any one of claims 2 to 3 and 5, wherein the mixing of the naive PSCs, the trophectoderm primed cells, and the extraembryonic primitive endoderm primed cells is performed simultaneously.

7. The method of any one of claims 2 to 3 and 5, wherein the mixing of the naive PSCs with cells primed towards the extraembryonic primitive endoderm is performed prior to the mixing with cells primed towards the trophectoderm.

8. The method according to any one of claims 1 to 7, wherein the trophectoderm cells and / or the cells primed towards extraembryonic primitive endoderm express pluripotency markers upon said mixing.

9. The method according to any one of claims 1 to 8, wherein the cells primed towards trophectoderm express trophectoderm markers upon said mixing.

10. The method of any one of claims 2 to 3 and 5 to 9, wherein the cells primed towards extraembryonic endoderm express extraembryonic primitive endoderm markers upon said mixing.

11. A mixture or aggregate obtainable by the method according to any one of claims 1 to 10.

12. (i) naive pluripotent stem cells (PSCs); (ii) cells primed towards trophectoderm, including stem cells expressing endogenous trophectoderm markers and endogenous pluripotency markers; and (iii) Cells primed to extraembryonic primitive endoderm, including stem cells expressing endogenous extraembryonic primitive endoderm markers and endogenous pluripotency markers. A mixture or aggregate comprising: Not structurally organized like a natural embryo at any stage of development, and The mixture or aggregate is capable of forming a synthetic embryo that is structurally organized at least like a natural embryo at the post-implantation egg cylinder or bilayered scutellum developmental stage.

13. (i) trophectoderm-primed cells, including stem cells expressing exogenous factors that induce differentiation into trophectoderm cells, endogenous trophectoderm markers, and endogenous pluripotency markers; and (ii) Naïve PSCs The mixture or aggregate comprising:

14. (iii) Cells primed to extraembryonic primitive endoderm, including stem cells expressing exogenous factors that induce differentiation into extraembryonic primitive endoderm cells, endogenous extraembryonic primitive endoderm markers, and endogenous pluripotency markers.

14. The mixture or aggregate of claim 13, further comprising:

15. The method of any one of claims 2 to 3 and 5 to 10, or the mixture or aggregate of any one of claims 11 and 14, wherein the factors that induce differentiation into extraembryonic primitive endoderm cells include Gata4, Gata6, and / or SOX17.

16. 16. The method of any one of claims 2-3, 5-10, and 15, wherein inducing expression of a factor that induces differentiation into extraembryonic primitive endoderm cells comprises culturing a second subpopulation of naive PSCs with a factor that induces differentiation into extraembryonic primitive endoderm cells beginning within a period of 14 days to 6 hours prior to said mixing.

17. 17. The method of claim 16, wherein the factor that induces differentiation into extraembryonic primitive endoderm cells is selected from the group consisting of a GSK-3 inhibitor, a WNT ligand, heparin, FGF2, FGF4, PDGF, leukemia inhibitory factor (LIF), and insulin.

18. The method or mixture or aggregate according to any one of claims 1 to 11 and 13 to 17, wherein the factor that induces differentiation into trophectoderm cells is selected from the group consisting of Cdx2, Gata3, and Gata2.

19. 19. The method of any one of claims 1-10 and 15-18, wherein inducing expression of a factor that induces differentiation into trophectoderm cells comprises culturing a subpopulation of the naive PSCs with a factor that induces differentiation into trophectoderm cells beginning within a period of 14 days to 6 hours prior to the mixing.

20. The method of claim 19, wherein the factor that induces differentiation into trophectoderm cells is selected from the group consisting of a TGFR inhibitor, an FGFR inhibitor, a MEK / ERK inhibitor, BMP4, a JAK inhibitor, a ROCK pathway inhibitor, FGF4, FGF2, heparin, a SUMOylation inhibitor, a histone deacetylase inhibitor, a HIPPO signaling pathway inhibitor, and a factor that induces YAP nuclear translocation.

21. 21. The method of any one of claims 1 to 10 and 15 to 20, wherein the induction of the transient expression is initiated within a period of time between 120 hours and 0 hours before the mixing.

22. The method of any one of claims 1 to 10 and 15 to 20, wherein the induction of the transient expression is initiated within a period of 48 hours to 0 hours before the mixing.

23. The method according to any one of claims 1 to 10 and 15 to 22, wherein the induction of the transient expression is terminated within 48 hours after the mixing.

24. The method of any one of claims 1 to 10 and 15 to 23, wherein the induction of the transient expression is carried out for no more than 96 hours.

25. 25. The method of any one of claims 1-3, 6-10, and 15-24, wherein the induction of the expression of the endogenous factor is initiated within a period of 10 days to 1 day prior to the mixing.

26. 25. The method or mixture or aggregate of any one of claims 1 to 24, wherein the ratio of naive PSCs to trophectoderm primed cells is 1:1 to 1:

5.

27. 27. The method or mixture or aggregate of any one of claims 2-3, 5-12 and 14-26, wherein the ratio of naive PSCs to cells primed towards primitive endoderm is 1:1 to 2:

1.

28. The method according to any one of claims 1 to 10 and 15 to 27, further comprising, after (b), ex utero culture of the aggregated cells.

29. A method for ex utero culture of a synthetic embryo, comprising culturing ex utero a mixture or aggregate according to any one of claims 11 to 15, 18 and 26 to 27.

30. The method according to any one of claims 28 to 29, wherein the culturing is carried out by static culturing followed by dynamic culturing.

31. The method according to claim 30, wherein the static culture is carried out until the embryo reaches at least the early post-implantation egg cylinder stage or the bilayered blastodisc stage.

32. The method according to any one of claims 30 to 31, wherein the dynamic culture is initiated at the latest when the embryo reaches the early somite stage.

33. A method for culturing a synthetic embryo ex utero, comprising culturing a synthetic embryo at an aggregate stage ex utero by static culture followed by dynamic culture, said static culture being carried out until said embryo reaches at least the early egg cylinder stage or the bilayered blastodisc stage after implantation, and said dynamic culture being started at the latest when said embryo reaches the early somite stage.

34. The method according to any one of claims 30 to 33, wherein the dynamic culture comprises shaking culture, roller culture, or a sequential combination thereof.

35. 35. The method of any one of claims 30 to 34, wherein the dynamic cultivation is carried out under high pressure greater than 0.1 pounds per square inch (psi) and less than 10.2 psi.

36. The method according to any one of claims 28 to 35, wherein the culturing is carried out in an atmosphere containing 10 to 40% oxygen.

37. 37. The method of any one of claims 1-10 and 15-36, further comprising implanting said synthetic embryo into a uterus.

38. 38. The method or mixture or aggregate of any one of claims 1 to 37, wherein said cells are mammalian cells.

39. 39. The method or mixture or aggregate of claim 38, wherein said mammalian cells are mouse cells.

40. 39. The method or mixture or aggregate of claim 38, wherein said mammalian cells are human cells.

41. 39. The method or mixture or aggregate of claim 38, wherein said mammalian cells are non-human primate cells.

42. A synthetic embryo obtainable by the method according to any one of claims 1 to 10, 15 to 36 and 38 to 41.

43. A method for obtaining cells primed to extraembryonic primitive endoderm, comprising: ＋ GATA6 ＋ OCT4 ＋ Nanog － The method comprises culturing the naive PSCs in a basal medium lacking activin A under conditions that permit differentiation into cells, thereby obtaining cells primed towards the extraembryonic primitive endoderm.

44. 44. The method of claim 43, wherein the culturing is carried out for at least one day.

45. The method according to any one of claims 43 to 44, wherein the culturing is carried out for up to 10 days.

46. The cells are treated with ＋ GATA6 ＋ OCT4 ＋ Nanog － 45. The method of any one of claims 43 to 44, capable of being propagated in culture for at least five passages while maintaining the phenotype.

47. 47. The method of any one of claims 43 to 46, wherein the basal medium is supplemented with at least one of a GSK-3 inhibitor, leukemia inhibitory factor (LIF), and insulin.

48. 47. The method of any one of claims 43 to 46, wherein the basal medium is supplemented with at least one of a GSK-3 inhibitor, heparin, FGF4, PDGF, and BSA.

49. The method of any one of claims 43 to 45 and 46 to 48, wherein the cell is a human cell.

50. The method of any one of claims 43 to 48, wherein the cell is a mouse cell.

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