Bovine trophoblast stem cells and application thereof

Abstract

The invention relates to bovine trophoblast stem cells and application thereof.

Description

[0001] This application is an international application claiming priority to U.S. Provisional Patent Application No. 63 / 370,192, filed on August 2, 2022, and U.S. Provisional Patent Application No. 63 / 413,789, filed on October 6, 2022. The contents of these U.S. Provisional Patent Applications are hereby incorporated by reference in their entirety.

[0002] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications are hereby incorporated by reference in their entirety into this application to more fully describe the state of the art known to those of ordinary skill in the art of the present invention as of the date described and claimed herein.

[0003] This patent disclosure contains copyrighted material. The copyright owner does not object to the facsimile reproduction by anyone of the patent document or patent disclosure in the patent files or records of the United States Patent and Trademark Office, but reserves any and all copyright rights in other respects.

[0004] Government Interests

[0005] This invention was made with government support under award number 2019-67016-29863 from the U.S. Department of Agriculture (USDA), National Institute of Food and Agriculture, and award number R01HD102533 from the National Institutes of Health (NIH), Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD). The government has certain rights in this invention. Technical Field

[0006] The present invention relates to bovine trophoblast stem cells and their uses. Summary of the Invention

[0007] Aspects of the present invention relate to bovine trophoblast stem cells and their uses.

[0008] In embodiments, aspects of the present invention relate to methods of culturing, expanding, or growing a population of cells derived from a mammalian blastocyst. For example, the method includes culturing these cells derived from a mammalian blastocyst in a culture medium for a period of time, wherein the culture medium contains human leukemia inhibitory factor (hLIF), an inhibitor of glycogen synthase kinase-3 (GSK-3), an antagonist of muscarinic M2 and histamine H1 receptors, an inhibitor of matrix metalloproteinases (MMP), or any combination thereof.

[0009] In an embodiment, the cultured cells remain undifferentiated.

[0010] In an embodiment, the cultured cells are capable of differentiating.

[0011] In an embodiment, the method further comprises placing a mammalian blastocyst in a container seeded with fibroblasts and adding the culture medium, thereby providing a cell population derived from the mammalian blastocyst. For example, the fibroblasts include mouse embryonic fibroblasts. In an embodiment, trophoblast stem cells are cultured in the absence of fibroblast feeder cells.

[0012] In an embodiment, the cell population comprises trophoblast stem cells, trophoblast stem cell-like cells, or derivatives thereof.

[0013] In an embodiment, the mammal is a cow.

[0014] In an embodiment, the GSK-3 inhibitor comprises CHIR99021. For example, the amount of glycogen synthase kinase-3 inhibitor is about 3 μM.

[0015] In an embodiment, the antagonists of muscarinic M2 and histamine H1 receptors comprise dimethindene maleate (DiM). For example, the amount of the antagonists of muscarinic M2 and histamine H1 receptors is about 2 μM.

[0016] In an embodiment, the inhibitor of matrix metalloproteinase (MMP) comprises minocycline hydrochloride (MiH). For example, the amount of the matrix metalloproteinase inhibitor is about 2 μM.

[0017] For example, the amount of human leukemia inhibitory factor (hLIF) is about 10 ng / ml.

[0018] In an embodiment, the container comprises a culture dish, flask, well, tube, or plate.

[0019] In an embodiment, the container comprises a solid surface or a porous surface.

[0020] In an embodiment, the cells are cultured on a surface coated with an extracellular matrix or a component of the extracellular matrix. For example, the extracellular matrix is Matrigel TM or Matrigel TM -like substance. For example, the surface is not Matrigel TM .

[0021] Aspects of the invention also relate to an in vitro cell culture. For example, the in vitro cell culture comprises a cell population derived from a mammalian blastocyst produced by the method as described herein.

[0022] In an embodiment, the cells of the in vitro cell culture comprise trophoblast stem cells, trophoblast stem cell-like cells, or derivatives thereof.

[0023] In an embodiment, the cells of the in vitro cell culture comprise undifferentiated cells.

[0024] In an embodiment, the cells of the in vitro cell culture are capable of self-renewal.

[0025] Aspects of the invention also relate to isolated cells. In an embodiment, the isolated cells are derived from a mammalian blastocyst.

[0026] In an embodiment, the isolated cells express at least one pluripotency marker. For example, the at least one marker comprises GATA3, CDX2, ELF3, TFAP2A, KLF5, KRT8, SFN, DNMT1, DNMT3A, PAG2, PAG11, PAG12, CYP17A1, HSD3B1, HAND1, or any combination thereof. For example, the at least one marker comprises a marker of the Wnt signaling pathway, the LIF signaling pathway, the HIF-1 signaling pathway, the AMPK signaling pathway, or any combination thereof.

[0027] In an embodiment, the isolated cells comprise trophoblast stem cells, trophoblast stem cell-like cells, or derivatives thereof.

[0028] In an embodiment, the isolated cells are undifferentiated.

[0029] In an embodiment, the isolated cells are capable of self-renewal.

[0030] In an embodiment, the isolated cells are capable of differentiating into cells of the trophoblast lineage in vitro and in vivo.

[0031] In an embodiment, the isolated cells are bovine cells.

[0032] Aspects of the invention further relate to a method of evaluating a candidate compound.

[0033] In an embodiment, the method comprises contacting an in vitro cell culture or isolated cells as described herein with a quantity of a candidate compound and evaluating a characteristic of the in vitro cell culture or isolated cells. For example, the characteristic is cell growth, cell development, differentiation, apoptosis, trophoblast development, trophoblast activity, or any combination thereof.

[0034] Still further, aspects of the invention relate to a cell culture comprising a population of bovine embryonic stem cells in a culture medium.

[0035] In an embodiment, the culture medium comprises one or more factors selected from the group consisting of: human leukemia inhibitory factor (hLIF), an inhibitor of glycogen synthase kinase-3 (GSK-3), an antagonist of muscarinic M2 and histamine H1 receptors, and an inhibitor of matrix metalloproteinase (MMP) or any combination thereof.

[0036] For example, the amount of human leukemia inhibitory factor (hLIF) is about 10 ng / ml.

[0037] For example, the amount of the glycogen synthase kinase-3 inhibitor is about 3 μM.

[0038] For example, the amount of the muscarinic M2 and histamine H1 receptor antagonist is about 2 μM.

[0039] For example, the amount of the matrix metalloproteinase inhibitor is about 2 μM.

[0040] In an embodiment, the cell culture is in a microplate.

[0041] In an embodiment, the cell culture further comprises a trophoblast stem cell population. For example, the trophoblast stem cell population comprises a bovine trophoblast stem cell population.

[0042] Other objects and advantages of the present invention will become apparent from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0044] Figure 1 Illustrations showing the derivation and characterization of in vitro bovine trophoblast stem cells (bTSCs) are provided. Panel A provides a schematic diagram representative of the derivation of bovine TSCs from blastocysts. Panel B provides bright-field images of the typical morphology of blastocyst growth and bovine TSCs on feeder cells or without feeder cells. D5: growth after 5 days of culture; P15: passage 15; P60: passage 60. (Scale bar: 100 μm) Panel C provides an illustration showing immunofluorescent staining of GATA3, KRT8, CDX2, and SOX2 in bovine day 7 IVF blastocysts and bTSCs. (Scale bar: 50 μm). Panel D provides RT-PCR analysis of the expression of CDX2, SFN, ELF5, GATA3, ASCL2, GATA2, ETS2, and GAPDH in bovine TSCs. BEF: bovine embryonic fibroblasts; bESC: bovine embryonic stem cells. Panel E provides an illustration showing flow cytometry analysis of GATA3 in bTSCs.

[0045] Figure 2 provides illustrations showing the in vitro developmental potential of bovine TSCs. Panel A provides an illustration of an immunofluorescence (IF) image showing binucleation of differentiated bTSCs (P27). (Scale bar: 100 μm) Panel B provides an illustration of an IF staining image showing differentiated bTSCs (P27) stained for PTGS2 and PL-1. (Scale bar: 75 μm) Panel C provides an illustration showing the IFNT activity secreted by bTSCs and trophoblast lineages (from day 2 to day 6 of in vitro differentiation of bTSCs), n = 6. IFNT: interferon tau. Panel D provides an illustration showing the expression levels of IFNT during neutralization and in vitro differentiation of bTSCs. Panel E provides an illustration showing the expression levels of the binucleated trophoblast markers BEVR-k1env, ERVE-A, and PAG1. Panel F provides an illustration showing the expression levels of the trophoblast markers PAG2, PAG11, and PAG12 according to RNAseq data. Panel G provides an illustration showing the gene expression kinetics of the functional trophoblast cell markers CYP11A1, CYP17A1, FURIN, HAND1, PTGS2, and HSD3B1 during differentiation according to RNAseq data. Panel H provides an illustration showing the top 10 enriched gene ontology (GO) terms in Diff_D4 trophoblasts compared to bTSCs. Panel I provides an illustration showing gene set enrichment analysis (GSEA) of bTSCs and Diff_D4 trophoblast cells. The green line shows the enrichment curve. The vertical black bars show the positions of the genes in the given gene set. Data are represented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

[0046] Figure 3 Provides an illustration showing the implantation of bTSCs into NOD-SCID mice. Panel A provides an illustration of a NOD-SCID mouse with a tumor formed after injection of bTSCs. (Left) The tumor was removed from the mouse 9 days after injection. (Right) Panel B provides an illustration showing hematoxylin and eosin (H&E) staining in TS-derived lesions. Asterisk: necrotic area. (Scale bar: 200 μm) (Panel C) Blood-filled lacunae (arrows). (Panel D) Binucleated cells. (Panel E) IF images stained for MMP2, PL-1, and PTGS2. (Scale bar: 75 μm).

[0047] Figure 4 provides illustrations showing the transcriptomic features of bovine TSCs. Panel A provides an illustration of the principal component analysis (PCA) of global gene expression (RNA-seq) showing bTSCs, trophectoderm of day 7 IVF blastocysts (D7_TE), day 7 IVF blastocysts (BL), trophectoderm from day 14 elongated embryos (D14_TE), bESCs, and bEPSCs. Panel B provides an illustration of the PCA plot of RNA-seq data from mouse ESCs / TSCs, human ESCs / TSCs, and bovine ESCs / TSCs. Panel C provides an illustration of the expression patterns of trophoblast and pluripotency marker genes in bTSCs, D7_TE, BL, D14_TE, bESCs, and bEPSCs. Panel D provides an illustration of the heatmap of highly expressed genes in bTSCs. Panel E provides an illustration of the KEGG pathways enriched in the highly expressed genes in bTSCs. Panel F provides an illustration of the top 5 enriched and depleted GO terms in bTSCs compared to D7_TE or D14_TE. Panel G provides an illustration of the GSEA comparison between bTSCs and bESCs and bEPSCs ES Genes in the Hippo signaling pathway, lysosome, and tight junction were upregulated in bTSCs.

[0048] Figure 5 provides illustrations showing the ATAC-seq-based chromatin accessibility of bTSCs. Panel A provides an illustration of the sample Pearson correlation analysis of bTSCs, D7_TE, D14_TE, and Diff_D4 trophoblast cells. Panel B provides an illustration of the enrichment of ATAC-seq peaks at the transcription start sites (TSSs) in bTSCs, D7_TE, and D14_TE. Panel C provides an illustration of the enriched motifs of bTSCs. Panel D provides an illustration of the genomic view of genes selected based on the ATAC-seq data in bTSCs, D7_TE, and D14_TE. Panels E and F show the enriched and depleted KEGG in bTSCs compared to D7_TE (Panel E) or D14_TE (Panel F) according to chromatin accessibility.

[0049] Figure 6 provides a diagram showing the DNA methylome profiling of bTSCs. Panel A provides a diagram showing the Pearson correlation coefficients for comparing the DNA methylation levels between bTSCs, D7_TEs, D14_TEs, and bEPSCs. Panel B provides a diagram showing the DNA methylation levels in bTSCs, D7_TEs, D14_TEs, and bEPSCs. (bTSCs, D7_TEs, and bEPSCs: 2 replicates; D14_TE: 3 replicates). Panel C provides a diagram showing the genomic features in bTSCs, D7_TEs, D14_TEs, and bEPSCs: methylation levels at promoters, exons, introns, and intergenic regions. Panel D provides a diagram showing the DNA methylation gene expression in bTSCs, D7_TEs, D14_TEs, and bEPSCs. Panel E provides a diagram showing the number of DMRs and corresponding genes between different groups. Panels F and G provide diagrams of the KEGG enrichment of hypermethylated DMRs in D7_TE (Panel F) and D14_TE (Panel G).

[0050] Figure 7 A diagram showing that LCDM supports bESCs is provided. Panel A provides a schematic diagram showing the transition of primed bESCs to LCDM-ESCs. (Scale bar: 100 μm). Panel B provides a diagram showing the IF staining images of SOX2, NANOG, CDX2, and GATA3 in LCDM-ESCs, primed ESCs, and bTSCs. (Scale bar: 75 μm). Panel C provides a diagram showing the PCA analysis of bovine inner cell mass (ICM), ESCs, EPSCES, EPSCiPS, and TSCs. Panel D provides a diagram showing the following: left: a Venn diagram of the upregulated genes in three groups; right: the top 10 GO terms of the upregulated genes in LCDM-ESCs. Panel E provides a diagram showing the following: left: a heat map of specific genes in bESCs, bEPSCs ES , LCDM-ESCs, and bTSCs respectively. Right: the enriched GO terms of the LCDM-ESC specific genes.

[0051] Figure 8 . Panel A provides a diagram showing the screening of basal media, growth factors, and inhibitors required for in vitro bovine TSCs and the growth rate of the media. C1: Combination 1; DM: dimenhydrinate maleate; MH: minocycline hydrochloride. Panel B provides a diagram showing the growth of blastocysts (top row) and passage 3 (P3) cells (bottom row) cultured in C9, C10, and C11 media for 7 days. (Scale bar: 100 μm).

[0052] Figure 9Panel A provides a diagram showing IF analysis of GATA3, KRT8, CDX2, and SOX2 in bTSCs at passage 10 (P10) and passage 55 (P55). (Scale bar: 50 μm). Panel B provides a diagram showing the karyotypes of bTSCs at passage 15 and passage 45, respectively. Panel C provides a diagram showing the bright-field image of differentiated TSCs. (Scale bar: 50 μm). Panel D provides a diagram showing the IF image of binucleate formation in differentiated bTSCs (P55). (Scale bar: 25 μm) Panels E and F provide diagrams showing the IF staining images of differentiated bTSCs (P55) for PL-1 and PTGS2. (Scale bar: 25 μm)

[0053] Figure 10 Panel A provides a diagram showing the PCA of global gene expression (RNA-seq) in bTSCs and differentiated TSCs. Panel B provides a diagram showing the Pearson correlation analysis of bTSCs and differentiated TSCs. Panel C provides a diagram showing the Pearson correlation analysis of bTSCs, day 7 blastocysts (BL), D7_TE, and D14_TE. Panel D provides a diagram showing the differential expression profiles between bTSCs and Diff_D4. Panel E provides a diagram showing the GO analysis of the top 50 upregulated genes in Diff_D4.

[0054] Figure 11 Panel A provides a diagram showing the expression levels of SOX2, OCT4, and NANOG in bESCs and LCDM-ESCs. Panel B provides a diagram showing the relative expression levels of tight junction-related claudin family genes in bESCs and LCDM-ESCs. Panels C and D provide diagrams showing the transcriptome analysis of selected naive pluripotency markers (Panel C) and (Panel D) primed pluripotency markers in bESCs (2 replicates) and LCDM-ESCs (3 replicates). Data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

[0055] Figure 12 provides a diagram showing the derivation and characterization of bovine TSCs. Panel A provides a display of the derivation of bTSCs from blastocysts. Panel B provides bright-field images of the typical morphology of blastocyst growth and bTSCs on feeder cells or without feeder cells. D7, growth after 7 days of culture; P15, passage 15; P60, passage 60. Scale bar: 100 μm. Panel C provides immunostaining of the epiblast marker SOX2 and trophoblast markers (GATA3, KRT8, and CDX2) in bovine day 7 IVF blastocysts and bTSCs. Scale bar: 50 μm. Panel D provides GATA3 in bTSCs +Flow cytometry quantification of cell populations. Panel E provides immunostaining of bovine mature trophoblast markers (PL-1 and PTGS2) in differentiated bTSC (P55). Arrow: binucleated cells. Scale bar: 25 μm. Panel F provides a graph showing the IFNτ activity secreted by bTSC and trophoblast cells differentiated from bTSC from day 2 to day 6 (n = 6). IFNτ, interferon t. Panel G provides a graph showing the expression levels of IFNτ, ERVE-A, BEVR-k1 env, and PAG1 during in vitro differentiation (n = 3). Panel H provides a graphical representation showing the expression kinetics of mature trophoblast cell marker genes (PAG1, PAG11, PAG12, CYP17A1, HAND1, PTGS2, CYP11A1, FURIN, and HSD3B1) during in vitro differentiation of bTSC (n = 2). Panel I provides H&E staining analysis of bovine TSC-derived lesions. Asterisk: necrotic area; arrow: blood-filled lacuna; arrow: binucleated cells. Scale bar: 200 μm. Panel J provides immunostaining of mature trophoblast markers (PL-1 and PTGS2) and trophoblast endometrial regulator (MMP2) in TSC-derived lesions. Scale bar: 75 μm. (Panels F and G) Data are represented as mean ± SD. *p < 0.001, **p < 0.0001, ***p < 0.00001, ****p < 0.000001.

[0056] Figure 13 provides a graphical representation showing the transcriptomic characteristics of bovine TSC. Panel A provides principal component analysis (PCA) of global gene expression (RNA-seq) of bTSC, trophectoderm of day 7 IVF blastocysts (D7_TE), day 7 IVF blastocysts (BL), trophoblast from day 14 elongated embryos (D14_TE), bESC, and bEPSC. Panel B provides a PCA plot showing RNA-seq data from mouse ESC / TSC, human ESC / TSC, and bovine ESC / TSC. Panel C provides the expression patterns of trophoblast and pluripotency marker genes in bTSC, D7_TE, BL, D14_TE, bESC, and bEPSC. Panel D provides gene set enrichment analysis (GSEA) of the transcriptomes between bTSC, bESC, and bEPSC. Genes in the Hippo signaling pathway and tight junction context are upregulated in bTSC. Panel E provides a heat map of highly expressed genes in bTSC (left). Enriched KEGG of highly expressed genes in bTSC (right). Panels F and G provide graphical representations showing the top 7 enriched and depleted KEGG in bTSC compared to bEPSC ES (Panel F) or bEPSC Xiang (Panel G). ES (Panel G).

[0057] Figure 14 provides illustrations showing the epigenomic features of bovine TSCs. Panel A provides a motif enrichment analysis of ATAC-seq peaks from bTSCs. Panel B provides an illustration showing the enriched pathways in genes with more accessible chromatin or closed chromatin in bTSCs compared to D7_TE. Panel C provides a graph showing the average genome-wide DNA methylation levels of bTSCs, D7_TE, D14_TE, and bEPSCs (bTSCs, D7_TE, and bEPSCs: n = 2; D14_TE: n = 3). Panel D provides a graph showing the expression levels of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) in bTSCs, D7_TE, D14_TE, and bEPSCs. Panel E provides an illustration showing the total number of identified differentially methylated regions (DMRs) and their annotated genes between bTSCs and D7_TE or D14_TE. Panels F and G provide illustrations showing the enriched pathways associated with genes annotated to hypomethylated DMRs in bTSCs compared to D7_TE (Panel F) or D14_TE (Panel G).

[0058] Figure 15 Illustrations showing the characterization of bovine TSCs are provided. Panel A provides representative images of the growth of blastocysts (upper row) cultured for 7 days in C9, C10, and C11 media and cells after 3 passages (P3) (lower row). Scale bar: 100 μm. Panel B provides karyotype analyses of bTSCs at passage 15 and passage 45, respectively. Panel C provides RT-PCR analyses of bovine trophoblast marker genes (CDX2, SFN, ELF5, GATA3, ASCL2, GATA2, and ETS2) in bovine TSCs. GAPDH was used as a control. BEF: bovine embryonic fibroblasts; bESC: bovine embryonic stem cells. Panel D provides immunostaining of the epiblast marker SOX2 and trophoblast markers (GATA3, KRT8, CDX2) in bTSCs at passage 10 (P10) and passage 55 (P55) (Scale bar: 50 μm). Panel E provides a bright-field image of differentiated TSCs. Scale bar: 50 μm. Panel F provides a representative immunostaining image showing binucleation in differentiated bTSCs (P27). Scale bar: 100 μm. Panel G provides representative immunostaining of mature trophoblast markers (PTGS2 and PL-1) in differentiated bTSCs (P27). Scale bar: 75 μm.

[0059] Figure 16 provides a diagram showing the transcriptomic and epigenetic features of bovine TSCs. Panel A provides a PCA analysis of the transcriptomes of bTSCs, D7_TE, D14_TE, and differentiated TSCs on days 2, 3, 4, 5, and 6. Panel B provides a diagram showing the top 10 enriched gene ontology (GO) terms in Diff_D4 trophoblasts compared to bTSCs. Panel C provides a gene set enrichment analysis (GSEA) of bTSC and Diff_D4 trophoblast cells. The green line shows the enrichment curve. The vertical black bars show the positions of the genes in the given gene set. Panel D provides a diagram showing the enriched GO terms of the upregulated genes in Diff_D5 or Diff_D6. Panel E provides a diagram showing NOD-SCID mice with tumors formed after injection of bTSCs (top row). Tumors removed from the mice 9 days after injection (bottom row). Panel F provides a diagram showing the top 5 enriched and depleted GO terms in bTSCs compared to D7_TE or D14_TE. Panel G provides a diagram showing the pathways enriched in genes with more accessible chromatin or closed chromatin in bTSCs compared to D14_TE. Panels H and I provide diagrams showing the pathways enriched in genes annotated with hypermethylated DMRs in bTSCs compared to D7_TE (Panel H) or D14_TE (Panel I).

[0060] Figure 17 A schematic diagram is provided showing that bovine trophoblast stem cells (bTSCs) retain the developmental potency to differentiate into mature trophoblast cells and exhibit transcriptomic and epigenetic features characteristic of trophectoderm cells from early bovine embryos. Detailed Description

[0061] Abbreviations and Definitions

[0062] This document provides a detailed description of one or more preferred embodiments. However, it should be understood that the present invention may be embodied in various forms. Accordingly, the specific details disclosed herein are not to be construed as limiting, but rather as a basis for the claims and a representative basis for teaching those skilled in the art to use the present invention in any appropriate manner.

[0063] The singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise. In the claims and / or the specification, the use of the word "a / an" when combined with the term "comprising" may refer to "one", but is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0064] Wherever the phrases "such as", "e.g.", "including", or any of the like are used in this document, unless specifically stated otherwise, it should be understood that the phrase "and without limitation" follows. Similarly, "example", "exemplary", etc. should be understood as non-limiting.

[0065] The term "substantially" permits deviation from the descriptor, but without negatively affecting the intended purpose. Even if the word "substantially" is not explicitly mentioned, descriptive terms should be understood to be modified by the term "substantially".

[0066] The terms "comprising", "including", "having", and "involving" (and similarly "comprises", "includes", "has", and "involves") are used interchangeably and have the same meaning. In particular, the definition of each term is consistent with the common definition of "comprising" in U.S. patent law and is thus interpreted as an open-ended term meaning "at least the following" and is also considered not to exclude additional features, limitations, aspects, etc. Thus, for example, "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Wherever the term "a" or "an" is used, it should be understood to mean "one or more" unless such an interpretation is meaningless in the context.

[0067] The term "about" is used herein to mean approximately, roughly, around, or in the vicinity of. When the term "about" is used in connection with a numerical range, it modifies that range by extending the upper and lower boundaries of the stated numerical values. Generally, the term "about" is used herein to modify numerical values above and below the stated value by a variation of, for example, more than or less than 20% (higher or lower).

[0068] Aspects of the present invention relate to methods of culturing, expanding, or growing a cell population derived from a mammalian blastocyst. After fertilization, the fertilized egg moves down the fallopian tube and undergoes multiple mitotic divisions to form a cell population called a blastocyst. The blastocyst consists of an inner cell mass that develops into an embryo, while the outer layer develops into tissues that nourish and protect the embryo. The blastocyst attaches to the uterine wall and obtains nutrients through the mother's blood. The major systemic structures of a calf develop during the embryonic period in a process called differentiation. At this stage, the kidneys, brain, spinal cord, nerves, heart, and blood cells begin to develop, and the gastrointestinal tract begins to form.

[0069] The trophoblast is the cell layer that forms the outer layer of the blastocyst. The trophoblast provides nutrients for the embryo and develops into a large part of the placenta. The trophoblast forms during the first stage of pregnancy and is the first group of cells to differentiate from the fertilized egg into extra-embryonic structures and does not directly contribute to the embryo.

[0070] The embodiments described herein include culturing cells derived from mammalian blastocysts (e.g., bovine blastocysts). "Culturing" a cell or cell population can refer to propagating or growing a cell, cell aggregate, tissue, or organ by incubating it for a period of time in an environment and conditions that support cell viability or reproduction. For example, culturing cells can keep the cells under conditions where they can proliferate, differentiate, and avoid senescence. For example, the environment and conditions that support cell viability and / or reproduction can include culturing a cell or cell population in a culture medium that contains human leukemia inhibitory factor (hLIF), an inhibitor of glycogen synthase kinase-3 (GSK-3), an antagonist of muscarinic M2 and histamine H1 receptors, an inhibitor of matrix metalloproteinases (MMPs), or any combination thereof.

[0071] Culturing can include one or more of the following steps: amplifying and proliferating a cell or cell population, and / or collecting a cell, cell population, tissue, or organ.

[0072] "Amplifying" a cell population can refer to culturing a cell population for a period of time under conditions that not only allow cell growth and development but also allow cell proliferation, such that more cells are obtained at the end of the amplification than before the amplification. For example, one cell can be amplified into two cells by cell division. In an embodiment, when certain cells proliferate in a culture, the amplification of the cell population can occur spontaneously. In other embodiments, the amplification of the cell population can require certain growth conditions, including but not limited to a minimum cell density, cell confluence on the surface of the culture vessel, or the addition of chemical factors (e.g., growth factors, differentiation factors, or signaling factors).

[0073] As described herein, embodiments include culturing cells derived from mammalian blastocysts. As used herein, "mammal or mammalian" can refer to any mammal, non-limiting examples of which include humans, primates, mice, rats, dogs, cats, cows, cattle, horses, pigs, fish, or birds. For example, "bovine" can refer to an animal from the bovine species, non-limiting examples of which include cows, buffalo, and bison.

[0074] In an embodiment, the methods described herein include culturing mammalian cells in a culture medium. The terms "culture medium", "cell culture medium", "medium" can refer to a solution containing nutrients that nourish cells in growth. In certain embodiments, the culture medium can be used to grow mammalian cells. The culture medium can provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required for minimal cell growth and / or survival. The culture medium can also contain supplementary components (see discussion of "supplementary components" below) that enhance growth and / or survival above the minimal rate, including but not limited to hormones and / or other growth factors, specific ions (e.g., sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds present at very low final concentrations), amino acids, lipids, and / or glucose or other energy sources. In certain embodiments, the culture medium is advantageously formulated to have a pH and salt concentration most suitable for cell survival and proliferation.

[0075] In an embodiment, the culture medium can contain "supplementary components", which can refer to components that enhance growth and / or survival above the minimal rate. Non-limiting examples of supplementary components include hormones and / or other growth factors, ions (e.g., sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds present at very low final concentrations), amino acids, lipids, and / or glucose or other energy sources. In certain embodiments, the supplementary components are added to the initial cell culture. In certain embodiments, the supplementary components are added after the start of cell culture.

[0076] The term "defined component culture medium" can refer to a culture medium in which the composition of the medium is known and controlled. See, for example, the culture medium of Example 4.

[0077] In an embodiment, the cell culture can contain a "nutrient source", which can refer to a composition that nourishes mammalian cells in growth, including the source itself. Non-limiting examples of nutrient sources include DMEM, IDMEM, MEM, M199, RPMI 1640, Ham's F12, DMEM / F12, Ham's F10, McCoy's 5A, NCTC 109, and NCTC 135.

[0078] "Culture medium" can refer to a solution used for growing, storing, processing, and maintaining cells, cell populations, and / or cell lines. In an embodiment, the solution can include factors required or contributing to cell attachment, cell growth, cell proliferation, maintaining the undifferentiated state of cells, and / or maintaining the cell environment. Non-limiting examples of such factors include salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins such as cytokines, growth factors, and hormones.

[0079] In an embodiment, the culture medium comprises human leukemia inhibitory factor, an inhibitor of glycogen synthase kinase-3, an antagonist of muscarinic M2 and histamine H1 receptors, an inhibitor of matrix metalloproteinases, or any combination thereof.

[0080] In an embodiment, the culture medium can be a liquid solution that supports the growth and / or maintains stem cells (such as trophoblast stem cells) in an undifferentiated state. For example, a "mammalian cell culture" can refer to a liquid culture medium containing a variety of mammalian cells maintained or proliferated under a set of controlled physical conditions. In an embodiment, the culture medium can be a water-based culture medium.

[0081] A "cell culture" can refer to cells growing in suspension or adhering to various surfaces or matrices in a container (such as roller bottles, tissue culture flasks, culture dishes, multi-well plates, etc.). For example, a cell culture can refer to a cell population derived from a mammalian blastocyst and a culture medium, such as trophoblast stem cells, the culture medium comprising human leukemia inhibitory factor (hLIF), an inhibitor of glycogen synthase kinase-3 (GSK-3), an antagonist of muscarinic M2 and histamine H1 receptors, an inhibitor of matrix metalloproteinases (MMP), or any combination thereof.

[0082] In an embodiment, the cell culture can comprise a plurality of cells in an undifferentiated state. For example, cells in an "undifferentiated state" can refer to cells that do not have a specialized structure or function. Thus, undifferentiated cells can refer to cells that have the ability to differentiate and have not yet entered the process of differentiating into cells with specific functions of tissues or organs. In an embodiment, undifferentiated cells are capable of self-renewal.

[0083] In an embodiment, the cell culture can comprise a plurality of cells capable of differentiating. "Differentiation" or "differentiating" can refer to the process by which less specialized cells (such as stem cells, embryonic cells) undergo maturation to become more distinct in form and function (such as acquiring specialized structures and / or functional characteristics characteristic of mature cells). For example, less specialized cells (such as stem cells or cells maintaining stemness) can progress from a stage with the potential to differentiate into cells of different cell lineages to a stage of becoming specialized and terminally differentiated cells. During differentiation, the cell structure changes, and tissue-specific proteins appear.

[0084] Non-limiting examples of undifferentiated cells include pluripotent stem cells, embryonic stem cells, progenitor cells, induced pluripotent stem cells, germline stem cells, and the like.

[0085] The embodiments described herein provide methods for culturing a cell population derived from a mammalian blastocyst, wherein the cell population maintains cell viability. "Cell viability" can refer to the ability of cells in a culture to survive under a given set of culture conditions or experimental variations. The term can also refer to the portion of cells that are alive at a particular time relative to the total number of cells (alive and dead) in the culture at that time.

[0086] In an embodiment, the method as described herein can include placing a mammalian blastocyst in a container, thereby providing a container containing a cell population derived from a mammalian blastocyst. Any suitable container can be used in the embodiments described herein. For example, a "container" can refer to a type of culture vessel that provides a contamination barrier to protect the culture from the external environment while maintaining an appropriate internal environment. Non-limiting examples of containers include flasks, tubes, culture dishes, roller bottles, and / or multi-well plates.

[0087] For example, a cell population derived from a mammalian blastocyst (e.g., a trophoblast stem cell population) can be cultured in a multi-well plate. In some cases, the multi-well plate is a V-bottom multi-well plate. In some cases, the V-bottom multi-well plate is an AggreWell plate.

[0088] In an embodiment, culturing the cell population as described herein can include centrifuging a culture container containing the cell population and a culture medium. For example, after adding the cells and the medium to the plate, the culture container can be centrifuged at about 50 x g. For example, after adding the cells and the medium to the plate, the culture container can be centrifuged at about 100 x g. For example, after adding the cells and the medium to the plate, the culture container can be centrifuged at about 150 x g. For example, after adding the cells and the medium to the plate, the culture container can be centrifuged at about 200 x g. For example, after adding the cells and the medium to the plate, the culture container can be centrifuged at about 250 x g. For example, after adding the cells and the medium to the plate, the culture container can be centrifuged at about 300 x g. For example, after adding the cells and the medium to the plate, the culture container can be centrifuged at about 350 x g. For example, after adding the cells and the medium to the plate, the culture container can be centrifuged at about 400 x g. For example, after adding the cells and the medium to the plate, the culture container can be centrifuged at about 450 x g. In some cases, after adding the cells and the medium to the plate, the V-bottom plate is centrifuged at about 500 x g.

[0089] In an embodiment, the container can be seeded with fibroblasts, such as mouse embryonic fibroblasts. "Cell seeding" can refer to spreading cells within or onto the surface of a container. "Fibroblasts" can refer to cells that contribute to the formation of connective tissue and are not terminally differentiated. Fibroblasts are heterogeneous mesenchymal cells that play an important role in the production and maintenance of the extracellular matrix.

[0090] In an embodiment, trophoblast stem cells can be cultured with or without fibroblast feeder cells. A "fibroblast feeder cell" can refer to a type of cell that can be co-cultured with another type of cell to provide an environment in which the second type of cell can grow. For example, trophoblast stem cells can be co-cultured with MEFs such that the trophoblast stem cells grow. The feeder cells can be human feeder cells or can be non-human feeder cells. In one embodiment, the feeder cells can be mouse embryonic fibroblasts.

[0091] As used herein, the phrase "feeder cell support" refers to the ability of feeder cells (e.g., fibroblasts) to maintain pluripotent stem cells in a proliferative and undifferentiated state when the pluripotent stem cells are co-cultured on the feeder cells or when the pluripotent stem cells are cultured on a substrate (e.g., extracellular matrix, synthetic matrix) in the presence of conditioned medium generated by the feeder cells. The support by the feeder cells depends on the structure of the feeder cells when cultured (e.g., the three-dimensional matrix formed by culturing the feeder cells in a tissue culture plate), the function of the feeder cells (e.g., growth factors, nutrients, and hormones secreted by the feeder cells, the growth rate of the feeder cells, the ability of the feeder cells to expand before senescence), and / or the attachment of the pluripotent stem cells to the feeder cell layer.

[0092] As used herein, the phrase "absence of feeder cell support" refers to a culture medium and / or cell culture that lacks feeder cells and / or the conditioned medium generated therefrom.

[0093] In an embodiment, a container can contain a "surface" to which a cell or cell population can attach. For example, the surface can be a solid matrix, a porous matrix, or another non-solid matrix.

[0094] For example, a solid surface can be coated with an insoluble matrix, optionally, which matrix can in turn be coated with one or more additional surface coatings of the matrix, or with any other chemical or biological material that allows cells to proliferate or be stabilized in culture. Non-limiting examples of the matrix include any one or a combination of polyornithine, laminin, polylysine, purified collagen, gelatin, fibronectin, tenascin, vitronectin, nestin, heparan sulfate proteoglycan, polyglycolic acid (PGA), polylactic acid (PLA), and polylactic-co-glycolic acid (PLGA).

[0095] For example, a "porous surface" can refer to a surface that allows in vitro partitioning of the cell microenvironment while still allowing physical and biochemical crosstalk between cells.

[0096] In an embodiment, cells can be cultured on a surface coated with an extracellular matrix or a component of the extracellular matrix as described herein.

[0097] As described herein, aspects of the present invention relate to culturing a cell population derived from a mammalian blastocyst. The terms "cell" and "cell population" can refer to multiple cells (i.e., more than one cell). In embodiments, the population can be a pure population containing one cell type. In other embodiments, the population can include multiple cell types. Thus, there is no limitation on the cell types that a cell population can contain. In embodiments, the cell population can contain trophoblast stem cells or trophoblast stem cell-like cells.

[0098] Any suitable cell population can be used in methods for culturing, expanding, or growing a cell population (such as the trophoblast stem cells described herein) derived from a mammalian blastocyst. In embodiments, the cell population can contain germ cells, such as, for example, female germline stem cells and their progeny. Examples of germ cells include, but are not limited to, embryos, oocytes, fertilized eggs, blastomeres, morulas, and blastocysts.

[0099] In embodiments, the cell population can contain somatic cells, such as fibroblasts (e.g., embryonic fibroblasts or skin fibroblasts). Somatic cells can be obtained from different organs (e.g., skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra, and other urinary organs) by well-known methods. Examples of somatic cells include, but are not limited to, adult stem cells, Sertoli cells, endothelial cells, granulosa epithelium, neurons, islet cells, epidermal cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B lymphocytes and T lymphocytes), red blood cells, macrophages, monocytes, mononuclear cells, fibroblasts, cardiomyocytes, and other muscle cells.

[0100] A suitable cell population can be obtained from any suitable source. In embodiments, the cell population can be obtained from a subject, such as from the subject's tissue (e.g., embryonic tissue), bone (e.g., bone marrow), blood (e.g., peripheral blood or cord blood), body fluid (e.g., tears, urine, or saliva), serum, plasma, or protein by any method known in the art. Subjects include, but are not limited to, human or non-human mammals, such as rodents (e.g., mice or rats), ungulates (e.g., horses or pigs), or cattle (e.g., cows).

[0101] "Stem cells" can refer to undifferentiated cells that are capable of substantially unlimited proliferation in vivo or in vitro and are capable of differentiating into other cell types. This can be certain differentiated, committed, immature, progenitor, or mature cell types present in isolated tissues, or significantly differentiated cell types derived from common progenitor cells, or even cell types at any stage in tissues completely different from the tissue from which the stem cells are obtained. Stem cells can retain a constant differentiation potential even after undergoing cell division. Examples of stem cells include pluripotent embryonic stem cells (ES cells) derived from fertilized eggs or cloned embryos, somatic stem cells and pluripotent stem cells present in living tissues, hepatic stem cells, dermal stem cells, and germline stem cells that underlie various tissues, pluripotent stem cells derived from germline stem cells, pluripotent stem cells obtained by nuclear reprogramming of somatic cells, etc.

[0102] "Stem cell-like cells" can refer to cells that possess some of the characteristics of stem cells. For example, stem cell-like cells have some ability for self-renewal. Examples of stem cell-like cells include, but are not limited to, progenitor cells, multipotent stem cells, cells undergoing the process of induced pluripotency, cancer cells, cancer stem cells, hematopoietic stem cells, iPS, and some antibody-producing hybridoma cells.

[0103] "Trophoblast stem cells" can refer to precursors of differentiated cells of the placenta that mediate the interaction between the fetus and the mother.

[0104] "Pluripotent stem cells" can refer to stem cells that permit in vitro culture. Pluripotent stem cells can differentiate into the cells that make up the body. "Pluripotent stem cells" can be obtained from fertilized eggs, cloned embryos, germline stem cells, or stem cells in tissues. Also included are cells that have a differentiation pluripotency similar to that of embryonic stem cells and are artificially conferred by transferring several different genes into somatic cells.

[0105] "Embryonic stem cells" can refer to cells obtained from embryonic tissues (e.g., blastocysts) formed after pregnancy before implantation (i.e., pre-implantation blastocysts), expanded blastocyst cells (EBCs) obtained from blastocysts at the post-implantation / gastrulation-stage-previous stage, and embryonic germ (EG) cells obtained from fetal germ tissues at any time during pregnancy (preferably before 10 weeks of pregnancy). "Fetus" can refer to a mammalian organism at a developmental stage after the embryonic stage and before birth, whose organs are fully differentiated but not yet fully grown.

[0106] Examples of stem cells that can be used in the embodiments described herein include mammalian embryonic stem cells established by culturing early pre-implantation embryos, embryonic stem cells established by culturing early embryos (prepared by nuclear transfer of the nucleus of a somatic cell), trophoblast stem cells established from various species (including cows, mice, humans, and non-human primates), and induced pluripotent stem cells (iPS cells) established by transferring several different genes into somatic cells.

[0107] In an embodiment, cells are cultured on a surface coated with an extracellular matrix or a component of the extracellular matrix (ECM). For example, the ECM can be composed of a variety of polysaccharides, water, elastin, and glycoproteins. Non-limiting examples of glycoproteins can include collagen, nidogen (nidogen), fibronectin, and laminin. The ECM can be secreted by connective tissue cells. Different types of ECM are known, each of which contains a different composition, including different types of glycoproteins and / or different combinations of glycoproteins. The ECM can be provided by culturing cells that produce the ECM (such as fibroblasts) in a container, then removing these cells and adding isolated tissue fragments or isolated epithelial stem cells (such as mammalian blastocysts).

[0108] Non-limiting examples of cells that produce the extracellular matrix include chondrocytes that mainly produce collagen and proteoglycans; fibroblasts that mainly produce type IV collagen; laminin; interstitial procollagen; fibronectin; colonic myofibroblasts that mainly produce collagen (types I, III, and V); chondroitin sulfate proteoglycan; hyaluronic acid; fibronectin; and tenascin-C.

[0109] In an embodiment, the ECM can be commercially available. Non-limiting examples of commercially available extracellular matrices include extracellular matrix proteins (Invitrogen) and basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g., MATRIGEL TM (BD Biosciences)). Synthetic extracellular matrix materials such as ProNectin (Sigma Z378666) can be used. Mixtures of extracellular matrix materials can be used.

[0110] In an embodiment, culturing stem cells using the ECM can enhance the long-term survival of the stem cells and the persistence of undifferentiated stem cells.

[0111] Aspects of the present invention can further relate to an in vitro cell culture comprising a cell population derived from a mammalian blastocyst and a culture medium as described herein. In embodiments, the in vitro cell population is capable of self-renewal. "Self-renewal" can refer to the process by which stem cells self-perpetuate (e.g., replenish mature cells to maintain tissue homeostasis throughout the life cycle of an organism). Self-renewal is the division while maintaining an undifferentiated state. This may require cell cycle control and / or maintenance of pluripotency or multipotency, depending on the stem cell.

[0112] Aspects of the present invention further relate to isolated cells derived from a mammalian blastocyst. "Isolated cells" can refer to cells removed from their natural environment, e.g., as part of an organ, tissue, or organism.

[0113] Markers (i.e., biomarkers) can be used to identify and isolate different cell types. For example, the embodiments described herein can comprise isolated cells that express at least one pluripotency marker. "Pluripotency" can refer to the ability of a cell to develop into the lineages of the body or body (i.e., the embryonic body). For example, pluripotent cells can develop into the three primary germ cell layers of the early embryo and thus into the cells of the adult body. Pluripotent stem cells can self-renew and give rise to the cells of body tissues. Non-limiting examples of pluripotency markers include CDX2, GATA3, ELF3, TFAP2A, KLF5, KRT8, SFN, DNMT1, DNMT3A, PAG2, PAG11, PAG12, CYP17A1, HSD3B1, HAND1, or any combination thereof. Other exemplary markers that the isolated cells can express include markers of signaling pathways. Non-limiting examples of signaling pathways include the Wnt signaling pathway, the LIF signaling pathway, the HIF-1 signaling pathway, the AKT signaling pathway, the AMPK signaling pathway, or any combination thereof.

[0114] In other embodiments described herein, the cell population can comprise at least one marker of trophoblast stem cells. Referring to Example 1, for example, the resulting cell line has TSC characteristics, including but not limited to trophoblast marker gene expression, self-renewal, long-term stable morphology, karyotype, and transcriptomic and epigenomic characteristics. Additionally, the cell population can generate functional mononuclear and binuclear trophoblasts in vitro and in vivo.

[0115] Aspects of the present invention also relate to methods of evaluating a candidate compound. "Candidate compound" can refer to a compound or agent whose activity is to be tested for a purpose.

[0116] In an embodiment, the method includes contacting a cell culture or isolated cells as described herein with an amount of a candidate compound and evaluating a characteristic of the cell culture or isolated cells. Non-limiting examples of characteristics that can be evaluated include cell growth, cell development, differentiation, apoptosis, trophoblast development, trophoblast activity, or any combination thereof.

[0117] Aspects of the invention further provide cell populations that can be used to assemble artificial blastocysts for use in various assisted reproductive technology (ART) applications. "Assisted reproductive technology" can refer to technologies that assist in achieving pregnancy, including but not limited to in vitro fertilization (IVF), embryo transfer (e.g., embryo transfer at any stage, including blastocysts), gamete intrafallopian transfer (GIFT), tubal embryo transfer (TET), intracytoplasmic sperm injection (ICSI), and intrauterine insemination (IUI).

[0118] In an embodiment, trophoblast stem cells can be used to assemble artificial blastoid structures. A "blastoid" can refer to a stem cell-based blastocyst-like structure that is similar to a blastocyst in terms of morphology, size, cell number, and lineage composition and distribution. A "blastocyst" can refer to a thin-walled hollow structure in early embryonic development that contains a cluster of cells called the inner cell mass from which the embryo develops.

[0119] As described herein, the culture medium can include human leukemia inhibitory factor, an inhibitor of glycogen synthase kinase-3, an antagonist of muscarinic M2 and histamine H1 receptors, an inhibitor of matrix metalloproteinases, or any combination thereof. Non-limiting examples of GSK-3 inhibitors include CHIR99021, CHIR98014, CHIR98023, SB-216763, and SB-415286. Non-limiting examples of antagonists of muscarinic M2 and histamine H1 receptors include dimenhydrinate maleate (DiM). Non-limiting examples of matrix metalloproteinase (MMP) inhibitors include minocycline hydrochloride (MiH).

[0120] In an embodiment, the culture medium contains two or more factors selected from the group consisting of human leukemia inhibitory factor (hLIF), an inhibitor of glycogen synthase kinase-3 (GSK-3), an antagonist of muscarinic M2 and histamine H1 receptors, and an inhibitor of matrix metalloproteinases (MMPs).

[0121] In an embodiment, the culture medium contains three or more factors selected from the group consisting of human leukemia inhibitory factor (hLIF), an inhibitor of glycogen synthase kinase-3 (GSK-3), an antagonist of muscarinic M2 and histamine H1 receptors, and an inhibitor of matrix metalloproteinases (MMPs).

[0122] In an embodiment, the culture medium comprises human leukemia inhibitory factor (hLIF), an inhibitor of glycogen synthase kinase-3 (GSK-3), an antagonist of muscarinic M2 and histamine H1 receptors, and an inhibitor of matrix metalloproteinase (MMP). Referring to Example 1, for example, a modified chemical mixture (comprising bLCDM:hLIF, CHIR99021, dimenhydrinate maleate (DiM), minocycline hydrochloride (MiH)) allows the derivation and long-term culture of trophoblast stem cells (TSCs) from large animals, such as cattle. In certain embodiments, long-term culture means that the cells and / or cell aggregates can be maintained in a viable state for a longer duration than conventional methods of trophoblast stem cell culture, for example, for more than 1 week to 6 weeks or longer.

[0123] The methods described herein include culturing a cell population derived from a mammalian blastocyst in a culture medium for a period of time, the culture medium comprising at least one human leukemia inhibitory factor, at least one inhibitor of glycogen synthase kinase-3 (GSK-3), at least one antagonist of muscarinic M2 and histamine H1 receptors, and at least one inhibitor of matrix metalloproteinase (MMP). For example, the cell population can be cultured for a period of time sufficient to assemble an artificial blastocyst. For example, the period of time can be at least 18 hours, at least about 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, or longer. In a further aspect of the method of culturing trophoblast stem cells, the cell population is cultured for a suitable period of time sufficient to form a blastoid. In some cases, the culture is carried out for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or longer as needed. In some cases, the culture is carried out for about 1 - 5 days, about 2 - 6 days, about 3 - 7 days, about 4 - 8 days, about 5 - 9 days, or about 6 - 10 days.

[0124] In an embodiment, human leukemia inhibitory factor, an inhibitor of glycogen synthase kinase-3 (GSK-3), an antagonist of muscarinic M2 and histamine H1 receptors, and / or an inhibitor of matrix metalloproteinase (MMP) can be provided in an effective amount in the culture medium. As used herein, "effective amount", "effective dose", or "effective... amount" can refer to the amount of an agent that effectively provides at least one characteristic of trophoblast stem cells (e.g., cell growth, cell development, differentiation, apoptosis, trophoblast development, trophoblast activity, or any combination thereof). Such characteristics can be monitored by conventional methods or can be monitored according to the methods described herein. The effective amount can vary depending on, for example, the human leukemia inhibitory factor, the inhibitor of glycogen synthase kinase-3 (GSK-3), the antagonist of muscarinic M2 and histamine H1 receptors, and the inhibitor of matrix metalloproteinase (MMP) used.

[0125] For example, compared to the proportion of cells in the pluripotency formation stage when culturing a cell population in the absence of an inhibitor of human leukemia inhibitory factor, glycogen synthase kinase-3 (GSK-3), an antagonist of muscarinic M2 and histamine H1 receptors, and an inhibitor of matrix metalloproteinase (MMP), using an effective amount of an inhibitor of human leukemia inhibitory factor, glycogen synthase kinase-3 (GSK-3), an antagonist of muscarinic M2 and histamine H1 receptors, and an inhibitor of matrix metalloproteinase (MMP) as described herein can result in an increase in the proportion of cells in the pluripotency formation stage of at least 10% or more, including, for example, 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%, at least about 90% or more.

[0126] In embodiments, the effective amount of human leukemia inhibitory factor can be between about 0.1 and about 10,000 ng / ml. For example, the effective amount of human leukemia inhibitory factor can be between about 1 and about 10,000 ng / ml, between about 10 and about 10,000 ng / ml, between about 100 and about 10,000 ng / ml, between about 1,000 and about 10,000 ng / ml, between about 5,000 and about 10,000 ng / ml, between about 0.1 and about 5,000 ng / ml, between about 1 and about 5,000 ng / ml, between about 10 and about 5,000 ng / ml, between about 1,000 and about 5,000 ng / ml, or between about 2,500 and about 5,000 ng / ml. In some embodiments, the effective amount of human leukemia inhibitory factor for the methods described herein can be between 0.1 and 100 μM. In some embodiments, the effective amount of human leukemia inhibitory factor for the methods described herein can be between 0.1 and 90 μM, between 0.1 and 80 μM, between 0.1 and 70 μM, between 0.1 and 60 μM, between 0.1 and 50 μM, between 0.1 and 40 μM, between 0.1 and 30 μM, between 0.1 and 20 μM, between 0.1 and 10 μM, between 0.1 and 1 μM, and between 0.1 and 0.5 μM. In some embodiments, the effective amount of human leukemia inhibitory factor for the methods described herein can be between 0.5 and 100 μM, between 1 and 100 μM, between 10 and 100 μM, between 20 and 100 μM, between 30 and 100 μM, between 40 and 100 μM, between 50 and 100 μM, between 60 and 100 μM, between 70 and 100 μM, between 80 and 100 μM, and between 90 and 100 μM.

[0127] An effective amount of an inhibitor of glycogen synthase kinase-3 (GSK-3) for the methods described herein can be between 0.1 and 10,000 ng / ml. In some embodiments, an effective amount of an inhibitor of glycogen synthase kinase-3 (GSK-3) for the methods described herein can be between 1 and 10,000 ng / ml, between 10 and 10,000 ng / ml, between 100 and 10,000 ng / ml, between 1,000 and 10,000 ng / ml, between 5,000 and 10,000 ng / ml, between 0.1 and 5,000 ng / ml, between 1 and 5,000 ng / ml, between 10 and 5,000 ng / ml, between 1,000 and 5,000 ng / ml, or between 2,500 and 5,000 ng / ml. In some embodiments, an effective amount of an inhibitor of glycogen synthase kinase-3 (GSK-3) for the methods described herein can be between 0.1 and 100 μM. In some embodiments, an effective amount of an inhibitor of glycogen synthase kinase-3 (GSK-3) for the methods described herein can be between 0.1 and 90 μM, between 0.1 and 80 μM, between 0.1 and 70 μM, between 0.1 and 60 μM, between 0.1 and 50 μM, between 0.1 and 40 μM, between 0.1 and 30 μM, between 0.1 and 20 μM, between 0.1 and 10 μM, between 0.1 and 1 μM, and between 0.1 and 0.5 μM. In some embodiments, an effective amount of an inhibitor of glycogen synthase kinase-3 (GSK-3) for the methods described herein can be between 0.5 and 100 μM, between 1 and 100 μM, between 10 and 100 μM, between 20 and 100 μM, between 30 and 100 μM, between 40 and 100 μM, between 50 and 100 μM, between 60 and 100 μM, between 70 and 100 μM, between 80 and 100 μM, and between 90 and 100 μM.

[0128] The effective amount of the antagonist of muscarinic M2 and histamine H1 receptors for the methods described herein can be between 0.1 and 10,000 ng / ml. In some embodiments, the effective amount of the antagonist of muscarinic M2 and histamine H1 receptors for the methods described herein can be between 1 and 10,000 ng / ml, between 10 and 10,000 ng / ml, between 100 and 10,000 ng / ml, between 1,000 and 10,000 ng / ml, between 5,000 and 10,000 ng / ml, between 0.1 and 5,000 ng / ml, between 1 and 5,000 ng / ml, between 10 and 5,000 ng / ml, between 1,000 and 5,000 ng / ml, or between 2,500 and 5,000 ng / ml. In some embodiments, the effective amount of the antagonist of muscarinic M2 and histamine H1 receptors for the methods described herein can be between 0.1 and 100 μM. In some embodiments, the effective amount of the antagonist of muscarinic M2 and histamine H1 receptors for the methods described herein can be between 0.1 and 90 μM, between 0.1 and 80 μM, between 0.1 and 70 μM, between 0.1 and 60 μM, between 0.1 and 50 μM, between 0.1 and 40 μM, between 0.1 and 30 μM, between 0.1 and 20 μM, between 0.1 and 10 μM, between 0.1 and 1 μM, and between 0.1 and 0.5 μM. In some embodiments, the effective amount of the antagonist of muscarinic M2 and histamine H1 receptors for the methods described herein can be between 0.5 and 100 μM, between 1 and 100 μM, between 10 and 100 μM, between 20 and 100 μM, between 30 and 100 μM, between 40 and 100 μM, between 50 and 100 μM, between 60 and 100 μM, between 70 and 100 μM, between 80 and 100 μM, and between 90 and 100 μM.

[0129] An effective amount of an inhibitor of matrix metalloproteinase (MMP) for the methods described herein can be between 0.1 and 10,000 ng / ml. In some embodiments, an effective amount of an inhibitor of matrix metalloproteinase (MMP) for the methods described herein can be between 1 and 10,000 ng / ml, between 10 and 10,000 ng / ml, between 100 and 10,000 ng / ml, between 1,000 and 10,000 ng / ml, between 5,000 and 10,000 ng / ml, between 0.1 and 5,000 ng / ml, between 1 and 5,000 ng / ml, between 10 and 5,000 ng / ml, between 1,000 and 5,000 ng / ml, or between 2,500 and 5,000 ng / ml. In some embodiments, an effective amount of an inhibitor of matrix metalloproteinase (MMP) for the methods described herein can be between 0.1 and 100 μM. In some embodiments, an effective amount of an inhibitor of matrix metalloproteinase (MMP) for the methods described herein can be between 0.1 and 90 μM, between 0.1 and 80 μM, between 0.1 and 70 μM, between 0.1 and 60 μM, between 0.1 and 50 μM, between 0.1 and 40 μM, between 0.1 and 30 μM, between 0.1 and 20 μM, between 0.1 and 10 μM, between 0.1 and 1 μM, and between 0.1 and 0.5 μM. In some embodiments, an effective amount of an inhibitor of matrix metalloproteinase (MMP) for the methods described herein can be between 0.5 and 100 μM, between 1 and 100 μM, between 10 and 100 μM, between 20 and 100 μM, between 30 and 100 μM, between 40 and 100 μM, between 50 and 100 μM, between 60 and 100 μM, between 70 and 100 μM, between 80 and 100 μM, and between 90 and 100 μM.

[0130] In an embodiment, the culture medium can be changed after culturing a cell population for a period of time. For example, the content of the culture medium can be changed about 8 hours, about 12 hours, about 16 hours, about 24 hours, about 36 hours, or about 48 hours after culturing the cell population. In an embodiment, the culture medium is replaced with a culture medium without human leukemia inhibitory factor. In an embodiment, the culture medium is replaced with a culture medium without a GSK-3 inhibitor. In an embodiment, the culture medium is replaced with a culture medium without an antagonist of the muscarinic M2 and histamine H1 receptors. In an embodiment, the culture medium is replaced with a culture medium without an inhibitor of matrix metalloproteinase.

[0131] In embodiments, the trophoblast cells described herein can be used in methods for determining drug toxicity. For example, the method can include (a) obtaining or providing trophoblast cells produced by a method according to any of the methods described herein, (b) contacting the trophoblast cells as described herein with a drug; and (c) detecting signs of toxicity.

[0132] The methods described herein encompass genetic manipulation of any cell population described herein. Genetic manipulation includes modifying, inserting, or deleting at least one gene in a cell.

[0133] Genetic manipulation can include transduction with a vector (e.g., a non-integrating vector (e.g., an episomal vector) or an integrating vector (e.g., a lentiviral vector)). In some embodiments, the methods described herein involve genetic manipulation of a cell population using an episomal vector. Thus, in some embodiments, the cell population involved in the methods described herein is a genetically modified cell.

[0134] As used herein, a "vector" is any nucleic acid mediator (DNA or RNA) capable of facilitating the transfer of a nucleic acid molecule into a cell. Generally, vectors include, but are not limited to, episomal vectors, plasmids, phagemids, viral vectors, and other mediators derived from viral or bacterial sources that are engineered by insertion or incorporation of a target nucleotide sequence. Viral vectors include, but are not limited to, vectors comprising nucleotide sequences derived from the genomes of the following viruses: retroviruses; lentiviruses; adenoviruses; adeno-associated viruses; SV40-type viruses; polyomaviruses; Epstein-Barr viruses; papillomaviruses; herpesviruses; vaccinia viruses; polioviruses. Other vectors not named but known in the art can be readily employed.

[0135] The methods described herein encompass reprogramming a cell population (e.g., a somatic cell population) to a less differentiated state. As used herein, reprogramming refers to the process of altering or reversing the differentiation state of a cell (e.g., a somatic cell), which can be either partially differentiated or terminally differentiated. Reprogramming includes both complete and partial reversal of the cell differentiation state.

[0136] Aspects of the invention also provide a kit for culturing, expanding, or growing a cell population derived from a mammalian blastocyst. Non-limiting examples of the components of the kit include cells as described herein, media, containers and components, and instructions for use. The kit can be used to practice the methods described herein.

[0137] The cells can be packaged in the kit by any suitable method for transporting and storing cells. For example, the cells can be provided in a frozen form, such as cryopreserved; a dried form, such as lyophilized; or a liquid form, such as in a buffer. For example, cryopreserved cells can be viable after thawing.

[0138] The culture medium can be included in the kit. For example, the culture medium can contain essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required for minimal cell growth and / or survival. The culture medium can contain human leukemia inhibitory factor, an inhibitor of glycogen synthase kinase-3, an antagonist of muscarinic M2 and histamine H1 receptors, an inhibitor of matrix metalloproteinases, or any combination thereof. The culture medium can be packaged by any suitable means for transporting and storing the culture medium.

[0139] The container can be a type of culture vessel that provides a contamination barrier to protect the culture from the external environment while maintaining an appropriate internal environment. For example, the container can be a flask, tube, Petri dish, roller bottle, and / or multi-well plate.

[0140] The instruction manual can include one or more of the following: a description of the cells of the kit; methods for thawing or preparing the cells; a culture schedule; precautions; warnings; and / or references. The instruction manual can be printed directly on the container (if present), or as a label affixed to the container, or as a separate sheet, booklet, card, or folder provided inside or with the container. The kit described herein also includes packaging. In some embodiments, the kit includes a sterile container that contains a therapeutic or prophylactic composition; such a container can be a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container form known in the art. Such a container can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing cells or drugs.

[0141] Other Embodiments

[0142] Although the present invention has been described in connection with its specific embodiments, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0143] The present invention will be further illustrated in the following examples, which do not limit the scope of the present invention described in the claims.

[0144] Examples

[0145] Examples are provided below to assist in a more comprehensive understanding of the present invention. The following examples illustrate exemplary modes of making and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only, since alternative methods can be utilized to obtain similar results.

[0146] Example 1

[0147] Trophoblast stem cells (TSCs) are precursors of the differentiated cells of the placenta, which mediate the interaction between the fetus and the mother. Here, we demonstrate that a modified chemical mixture (bLCDM: hLIF, CHIR99021, dimenhydrinate (DiM), minocycline hydrochloride (MiH)) permits the derivation and long-term culture of trophoblast stem cells (TSCs) from the large animal bovine. The resulting cell line exhibits TSC characteristics (trophoblast marker gene expression, self-renewal, long-term stable morphology, karyotype, and transcriptomic and epigenomic features), and can give rise to functional mononuclear and binuclear trophoblasts in vitro and in vivo. The bovine TSCs established here will provide a powerful tool for studying placental trophoblast differentiation and function, as well as early pregnancy failure. Together with embryonic stem cells, the established TSCs and bLCDM conditions can be used to assemble artificial blastocysts for various assisted reproductive technology (ART) applications.

[0148] Uses of the bovine TSC cell line include, for example, 1) pregnancy drug screening and testing, 2) serving as an in vitro model for basic and translational research on placental development (deposited with ATCC for sale), 3) assembly of artificial bovine blastocysts for assisted reproductive technology (in vitro breeding).

[0149] To date, there have been no bovine TSCs and conditions to support bovine TSCs.

[0150] Currently available bovine trophoblast cells, including trophoblast cell lines (CT-1 and CT-5, BT-1), and more recently undifferentiated trophectoderm cells supported by irradiated mouse embryonic fibroblast feeder cells (MEFs) are all derived from bovine blastocysts. However, none of these cells meet the TSC criteria, namely i) the ability to maintain long-term self-renewal, and 2) the ability to develop into functional trophoblasts in vitro and in vivo. Therefore, the generation of self-renewing and stable bovine TSC lines remains unexplored.

[0151] Example 2

[0152] The placental trophoblast plays a crucial role in the communication between the fetus and the mother. In cattle, insufficient placental trophoblast development and subsequent dysfunction lead to a series of adverse consequences for the conceptus / offspring; for example, the abnormalities seen in in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) embryos. The biggest obstacle to progress in this field in cattle is the low feasibility of in vivo experimental systems or the lack of an operational in vitro cell culture model that recapitulates placental cell differentiation. To date, trophoblast stem cells (TSCs) have been established from multiple species, including mice, humans, and non-human primates, but the generation of self-renewing and stable bovine TSC lines has yet to be explored. Based on the signaling required for mouse and human TSC pluripotency, we screened 11 culture conditions and found that a culture condition containing a chemical mixture of human leukemia inhibitory factor (hLIF), CHIR99021 (an inhibitor of glycogen synthase kinase-3 (GSK-3)), DiM (an antagonist of muscarinic M2 and histamine H1 receptors), and MiH (an inhibitor of matrix metalloproteinases (MMPs)) allowed for the long-term culture (more than 55 passages) of bovine TSCs without altering their morphology and differentiation from bovine IVF embryos. Three stable cell lines were maintained and used for downstream characterization (n = 3). Real-time quantitative (qRT)-PCR and immunostaining assays showed that the resulting cells highly expressed bovine trophectoderm markers, including CDX2, GATA3, and KRT8. These cells had the ability to differentiate in vitro into multinucleated trophoblast cells that secrete IFN-τ and express high levels of the binucleate trophoblast cell marker genes PAG2, PAG11, PAG12, and the mononucleate trophoblast cell marker genes CYP17A1, HSD3B1, and HAND1. To validate the pluripotent state of bovine TSCs and map bovine trophoblast differentiation, we generated transcriptomes and accessible chromatin of in vitro trophoblast cell cultures (including TSCs, multinucleated cells from day 2 and day 6 in vitro differentiation, and trophoblast lineages (including the trophectoderm (TE) of day 7 IVF blastocysts and trophoblast cells of day 14 elongated embryos)) by RNA-seq and assay for transposase-accessible chromatin (ATAC)-seq, respectively. Sequencing analysis was performed at least twice for each developmental stage (n ≥ 2). The transcriptomes of bovine pluripotent stem cell models representing epiblast, naive bovine embryonic stem cells (ESCs), and expanded potential stem cells (EPSCs) were also compared to identify the bovine trophoblast stem cell niche. Our analysis revealed important transcription factors (such as GATA3, ELF3, TFAP2A, KLF5, KRT8, SFN, DNMT1, and DNMT3A; adjusted P < 0.05) and signaling pathways (Wnt, LIF, HIF-1, and AMPK signaling pathways; adjusted P < 0.05) required to capture the bovine TSC state and reconstructed the molecular trajectory of bovine placental trophoblast development. Without wishing to be bound by theory, the bovine TSCs established in this study provide a powerful model for bovine early placental establishment and early pregnancy failure.

[0153] Example 3

[0154] Placental trophoblast cells are specialized cells that mediate the interaction between the fetus and the mother in the placenta and are derived from the trophectoderm (TE) of the blastocyst. Here, we developed a platform that allows the derivation and long-term culture of trophoblast stem cells (TSCs) from the large animal cow. The resulting cell lines have TSC characteristics and can give rise to functional mononuclear and binuclear trophoblasts in vivo and in vitro. The bovine TSCs established here will provide a powerful tool for studying placental trophoblast differentiation and function as well as early pregnancy failure. TSCs can also be used together with embryonic stem cells to assemble artificial blastocysts for various assisted reproductive technology (ART) applications such as in vitro breeding.

[0155] Example 4

[0156] Protocols / Conditions for Bovine Trophoblast Stem Cells

[0157] Cell culture reagents:

[0158]

[0159]

[0160] LCDM Medium Volume Neurobasal Medium 48 mL DME / F-12 48 mL MEM NEAA 1 mL GlutaMAX 1 mL N2 Supplement 0.5 mL B27 Supplement 1 mL Bovine Albumin, Low Endotoxin 0.1g 2-Mercaptoethanol 181.8 μL 10 ng / mL LIF (stock solution: 200 μg / mL) 5 μL 3 μM CHIR99021 (stock solution: 3 mM) 100 μL 2 μM Dimethindene Maleate (stock solution: 10 mM) 20 μL 2 μM Minocycline Hydrochloride (stock solution: 10 mM) 20 μL

[0161]

[0162]

[0163] Bovine IVF Embryo Production

[0164] The IVM-IVF embryos used in this study were generated using cumulus-oocyte complexes (COCs) and maintained in BO-Bioscience medium. The blastocysts were collected, the zona pellucida was removed by pipetting, and the blastocysts were processed for TSC derivation.

[0165] Derivation and Culture of Bovine TSCs

[0166] Each blastocyst was placed in a separate well of a 12-well plate seeded with mitomycin C-treated mouse embryonic fibroblasts (MEFs) (ATCC, catalog number: SCRC-1040).

[0167] bTSC Thawing

[0168] 1. Prepare 5 mL of 10% FBS in DMEM in a 15 mL tube and set the water bath to 37 °C.

[0169] 2. Thaw the bTSC in the vial in a water bath, sterilize it with 70% ETOH, and then wipe it.

[0170] 3. Add all the medium in the vial to 5 mL of 10% FBS in DMEM.

[0171] 4. Centrifuge at 1000 rpm for 5 minutes.

[0172] 5. Resuspend the cells with LCDM and plate them in a 6-well plate with feeder cells.

[0173] Subculture of bTSC

[0174] 1. Prepare MEF feeder cells one day before subculture. Fresh feeder cells are better.

[0175] 2. When about 80%-90% confluent, aspirate the medium and wash once with 1 mL of PBS.

[0176] 3. Add 1 mL of Accutase to each well of the 6-well plate and place it back in the incubator for 4-5 minutes. Then, inactivate Accutase with the same volume of LCDM medium.

[0177] 4. Collect the cells and centrifuge at 1000 rpm for 5 minutes.

[0178] 5. Resuspend the cells in 1.5 mL of LCDM medium in each well of the 6-well plate. Usually subculture the cells at a 1:6 ratio every 6 days.

[0179] 6. Culture the cells in an incubator at 38.5 °C and 5% CO2.

[0180] Changing the medium

[0181] 1. Heat the medium at room temperature for at least 30 minutes.

[0182] 2. Aspirate the old medium.

[0183] 3. Add 2 mL of LCDM medium to the wells and change the medium every day.

[0184] bTSC culture without feeder cells

[0185] For feeder-free conditions, subculture the bTSC cultured on feeder cells into a plate coated with Matrigel (Corning, 354234), where MEF-conditioned bTSC medium (MEF-bTSC) is used.

[0186] 1. Culture the LCMD medium on MEF feeder cells for 24 hours, then collect and store it at -20 °C, and call it conditioned LCDM.

[0187] 2. Coat the plate with matrigel at room temperature for 1 hour.

[0188] 3. Dissociate the cells using Accutase according to the passage steps.

[0189] 4. Aspirate the Matrigel.

[0190] 5. Add 10 μM Y-27632 to the conditioned LCDM, resuspend the cells with this medium and seed the cells on the Matrigel-coated plate. After 24 hours, remove Y-27632 and replace with fresh conditioned LCDM every day.

[0191] Cryopreservation

[0192] 1. Dissociate the cells using 1 mL Accutase and neutralize the reaction using 1 mL LCDM medium.

[0193] 2. Centrifuge at 1000 rpm for 5 minutes.

[0194] 3. The cryopreservation medium is 1:1 (1.7 mL ProFreeze cryopreservation medium + 0.3 mL DMSO) and LCDM medium.

[0195] 4. Use 1 mL of the cryopreservation medium for each well of a 6-well plate. Add the cells to the cryovial.

[0196] 5. Place the tube at -80 °C for 12 - 24 h, then transfer the tube to the liquid nitrogen tank.

[0197] bTSC differentiation

[0198] 1. Coat the plate with 2.5 μg / mL Col IV at room temperature for 1 hour.

[0199] 2. Dissociate 80%-90% confluent bTSC with TrypLE in the incubator for 15 minutes.

[0200] 3. Inactivate TrypLE using 10% FBS in the same volume of DMEM.

[0201] 4. Centrifuge at 1000 rpm for 5 minutes.

[0202] 5. Aspirate Col IV and rinse once with PBS.

[0203] 6. Resuspend the cells with the differentiation medium and seed the cells at a density of 1–1.5×10 5 cells / well of a 6-well plate.

[0204] 7. Change the medium every two days.

[0205] Example 5

[0206] Abstract

[0207] The ability of human and mouse extended pluripotent stem (EPS) cells to differentiate into the trophoblast lineage has been controversial. Here, we demonstrate that a modified chemical cocktail (LCDM: hLIF, CHIR99021, DiM, and MiH) for human and mouse EPS cells permits derivation and long-term culture of trophoblast stem cells (TSCs) from the large animal cow. The resulting cell lines have trophoblast stem cell characteristics and are capable of generating functional mononuclear and binuclear trophoblasts in vivo and in vitro. Molecular analysis of the transcriptome and epigenome reconstructed the trajectory of bovine placental trophoblast development and revealed enrichment of bovine trophoblast stem cell-specific features. Notably, the LCDM conditions support long-term culture of bovine TSCs and formative ESCs. The bovine TSCs established in this study will provide a powerful model for studying early placental establishment and early pregnancy failure in cattle.

[0208] Introduction

[0209] Placental trophoblast cells are specialized cells that mediate the interaction between the fetus and the mother in the placenta and are derived from the trophectoderm (TE) of the blastocyst. In cattle, the hatched blastocyst enters a special stage, the elongation stage, during which trophoblast cells differentiate and rapidly proliferate to optimally attach to the maternal caruncle. During this process, undifferentiated trophectoderm cells or trophoblast stem cells differentiate into mononuclear trophoblast cells and subsequently into binuclear giant cells [1, 2], which drive embryo elongation and ultimately fuse with uterine epithelial cells to form a fetal-maternal hybrid cell to establish the fetal-maternal interface [3]. The development and function of the trophoblast are crucial for the success of pregnancy. Insufficient development and subsequent dysfunction of the placental trophoblast lead to a series of adverse consequences for the conceptus / offspring, such as abnormalities seen in bovine in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) embryos [4 - 6]. Our major gap in understanding bovine trophoblast differentiation and function is the low feasibility of in vivo experimental systems or the lack of an operational in vitro cell culture model that recapitulates placental cell differentiation.

[0210] To date, trophoblast stem cells (TSCs) have been established from multiple species, including mice [7], humans [8], and non-human primates [9]. Thus, in ruminants, numerous cell lines, including CT-1 and CT-5

[10] , trophoblast cell line BT-1

[11] , and more recently undifferentiated trophectoderm cells supported by irradiated mouse embryonic fibroblast feeder cells (MEFs)

[12] , have been derived from bovine blastocysts. However, none of these cells meet the TSC criteria, namely i) the ability to maintain long-term self-renewal and 2) the ability to develop into functional trophoblasts in vitro and in vivo. Thus, the generation of self-renewing and stable bovine TSC lines remains unexplored.

[0211] Results in Conflict with EPS Conditions

[0212] One group derived EPS with in vivo embryonic and extraembryonic potency under LCDM conditions

[13] .

[0213] Another group captured human trophoblast development with EPSCs

[14] .

[0214] Another group challenged the ability of human EPSCs to differentiate into human trophoblast cells in vitro

[15] .

[0215] Yet another group challenged the ability of mouse EPSCs to generate trophoblast cells in vitro and in vivo

[16] .

[0216] In this study, we applied N2B27 basal medium supplemented with LIF, CHIR99021, dimenhydrinate (DM), and minocycline hydrochloride (MH) to derive bovine TSCs. This medium robustly derived TSCs from bovine IVF blastocysts, and the TSCs maintained long-term stable morphological, karyotypic, transcriptomic, and epigenomic characteristics as well as in vitro and in vivo developmental potential. The bovine TSCs established in this study will provide a powerful model for studying early placental establishment and early pregnancy failure in cattle and, without wishing to be bound by theory, can be used in assisted reproductive technologies.

[0217] Results

[0218] Derivation of Bovine Trophoblast Stem Cells (bTSCs) from In Vitro-Generated Blastocysts

[0219] We first tested the culture conditions that allowed robust growth of cells derived from bovine TE of blastocysts ( Figure 1 , inset A). We examined fourteen small molecules (eleven culture conditions) based on important signaling pathways enriched in primary undifferentiated trophoblast cells

[12] and pathways involved in the derivation of mouse and human trophoblast stem cells and expanded potential stem cells [7, 8, 17, 18]. Figure 8, small panel A). Supported by MEF feeder cell layers, four conditions (8 (C8: N2B27, LIF, and Wnt inhibitor (CHIR99021), muscarinic M2 / histamine H1 inhibitor (DM), and MMP inhibitor (MH)); 9 (C9: N2B27, 10% KSR, LIF, bFGF, and Wnt inhibitor (CHIR99021), MEK1 / MEK2 inhibitor (PD0325901), TGF-β inhibitor (Activin A)); 10 (C10: N2B27, 10% KSR, LIF, bFGF, and Wnt inhibitor (CHIR99021) and TGF-β inhibitor (Activin A)); 11 (C11: N2B27, bFGF, and Wnt inhibitor (CHIR99021) and TGF-β inhibitor (Activin A)) can easily derive cells from bovine blastocysts and form colonies ( Figure 1 , small panel B and Figure 8 , small panel B). However, C9 cannot support TE-derived cells for more than five passages without differentiation. The other three conditions (C8, C10, and C11) allow blastocysts to attach to the feeder cell layer on day 2 and form colonies on day 7, with growth rates of 89.3% (25 / 28), 66.7% (8 / 12), and 53.3% (8 / 15) for C8, C10, and C11, respectively ( Figure 8 , small panel A). Notably, under these three conditions, blastocysts that did not attach on day 2 could be physically squeezed to attach to the feeder cell layer and maintain growth. Further maintenance of these TE-derived cells demonstrated that cells under C10 and C11 conditions could not survive cryopreservation, had low cell viability, and a low passage ratio (1:2). Overall, cells under C8 conditions had a high growth rate, formed vesicles in the center of colonies, self-renewed at a passage ratio of 1:6, maintained a flat morphology without change, and maintained a normal morphology after long-term culture and several rounds of cryopreservation ( Figure 1 , small panel B). Therefore, we named the cells derived and maintained in C8 (LCDM: LIF, CHIR99021, DM, MH) medium as bovine trophoblast stem cells (bTSC).

[0220] We also verified whether small molecules were required in C8. Our screening demonstrated that removing any one of the small molecules could not maintain the morphology and self-renewal of bTSC, indicating that LCDM is indispensable for the derivation and maintenance of bTSC. Additionally, bTSC could be maintained in long-term culture under feeder-free conditions by supplementing with MEF-conditioned bTSC medium ( Figure 1 , small panel B).

[0221] We successfully derived bTSCs and maintained five lines for further characterization. First, bTSCs exhibited a colony morphology of flattened trophoblasts and continuously proliferated for more than 60 passages ( Figure 1 , panel B). During long-term culture, they had a normal karyotype ( Figure 9 , panel B). Second, bTSCs expressed bovine TE markers, including CDX2, GATA3, and KRT8, but did not express the inner cell mass (ICM) marker SOX2 (showing the same expression pattern as the TE of blastocysts) ( Figure 1 , panel C and Figure 9 , panel A). Third, they expressed bovine trophectoderm-related transcription factors (CDX2, SFN, ELF3, GATA3, ASCL2, GATA2, and ETS2), ( Figure 1 , panel D and panel E), and showed homogeneity ( Figure 1 , panel E). Collectively, these results indicate that the LCDM medium supports the long-term culture of bTSCs.

[0222] Differentiation of bTSCs into Functional Trophoblast Cells

[0223] The bovine placenta consists of two cell populations (mononuclear and binuclear trophoblast cells). Binuclear trophoblast cells account for 20% of trophoblast cells throughout gestation [2]. Next, we verified whether bTSCs could differentiate into functional binuclear trophoblasts in vitro. Initial culture of bTSCs in N2B27 basal medium could not maintain cell differentiation. It has been reported that forskolin, a cAMP agonist, can reduce lipid content and induce cell fusion [19, 20]. In a culture system containing forskolin, Y27632, and 4% KSR, bTSCs underwent differentiation into binuclear cells (Figure 2, panel A; Figure 9 , panel C and Figure 9 , panel D). The differentiated cells expressed the bovine trophoblast markers PTGS2 and placental lactogen 1 (PL-1) (Figure 2, panel B; Figure 9 , panel E and Figure 9, small panel F). Compared with days 7 to 13, the abundance of PTGS2 was significantly increased on days 16 to 19, which had the same pattern as IFNT2, showing its role in maternal recognition

[21] . PL-1 expressed in bovine trophoblast cells plays a crucial role in placentation

[22] . Interferon tau (IFNT), produced by the mononuclear trophoblast cells of the ruminant conceptus during mid-gestation, is the signal for maternal recognition of pregnancy

[23] . By testing IFNT activity using a luciferase-based IFN-stimulated response element (ISRE) assay during differentiation

[24] , we found that the daily release of IFNT increased significantly during differentiation and peaked on day 5 (Figure 2, small panel C). The mRNA expression level of IFNT also increased significantly during differentiation (Figure 2, small panel D). qRT-PCR analysis further demonstrated that the expression of binucleate cell-specific genes (BEVR-k1 env and bEPVE-A

[25] ) and pregnancy-associated glycoprotein 1 (PAG1, a pregnancy-associated glycoprotein (PAG) produced only by the specialized trophoblast binucleate cells of the bovine placenta

[26] ) was significantly upregulated towards differentiation (Figure 2, small panel E). Collectively, these data indicate that bTSC has the ability to give rise to functional binucleate trophoblasts.

[0224] Next, we analyzed the overall transcriptome during a 6-day time course of bTSC differentiation. Hierarchical clustering analysis (PCA) and Pearson correlation confirmed that bTSC entered an intermediate differentiation stage on day 2 and then further differentiated into trophoblasts from day 3 to day 6 ( Figure 10 , small panel A and Figure 10 , small panel B). RNA-seq analysis of trophoblast cell differentiation demonstrated an enhanced expression pattern of PAG family genes (including PAG2, PAG11, and PAG12) during trophoblast differentiation (Figure 2, small panel F). Genes well-known to be upregulated and abundant in the bovine placenta during pregnancy [26 - 28] demonstrated increased expression during differentiation, including CYP11A1, CYP17A1, FURIN, HAND1, PTGS2, and HSD3B1 (Figure 2, small panel G). From PCA and Pearson correlation analysis, day 4 was the farthest from bTSC ( Figure 7 , small panel A and Figure 1 , small panel B). Annotation of the upregulated genes in trophoblast cells (day 4) revealed significant enrichment in morphogenesis, cell migration, and motility (Figure 2, small panel H and Figure 10 , small panel D). In addition, enrichment of the top 50 differentially expressed genes in trophoblast cells on day 4 highlighted spindle organization, tissue development and morphogenesis, and microtubule cytoskeleton organization ( Figure 10, small panel E). All of the above confirmed the existence of invasive trophoblast binucleate cell types. Additionally, compared with bTSCs, differentiated trophoblast cells had enriched genes involved in signaling pathways (including ECM-receptor interaction, TNF, IL-17, and MAPK signaling) (Figure 2, small panel I), which was consistent with the reported increase in ECM-receptor interaction and TNF-α and IL-17 during implantation and placental development in ruminants and humans, respectively [29 - 31].

[0225] To verify the developmental potential of bTSCs in vivo, we subcutaneously injected 5 × 106 bTSCs into non-obese diabetic (NOD)-severe combined immunodeficient (SCID) mice. The injected bTSCs formed a lesion of approximately 0.5 cm at day 9 ( Figure 3 , small panel A), and then gradually resorbed. Immunobiostaining analysis revealed that the central region of the lesion was necrotic ( Figure 3 , small panel B), and the lesion contained blood-filled lacunae that mimicked nutrient exchange between the fetus and the mother ( Figure 3 , small panel C). Similar structures were also found in lesions formed by mouse

[32] and human TSCs [8]. Binucleate cells, which represent typical mature trophoblast cells in cattle, were also found in some regions of the lesion ( Figure 3 , small panel D). Additionally, PL-1-positive cells and PTGS2-positive cells were observed in the peripheral region of the lesion ( Figure 3 , small panel E). At the edge of the lesion, we also found MMP2-positive cells ( Figure 3 , small panel E). MMP2 expression increased significantly during the peri-implantation stage, which is a key factor in trophoblast cell-endometrial epithelia talk and endometrial stromal remodeling

[33] . These data indicate that bTSCs injected into NOD-SCID mice mimic some key characteristics of trophoblast cells during pregnancy.

[0226] Transcriptional Profile of bTSCs

[0227] To elucidate the transcriptional features of bTSCs and whether or at which developmental stage bTSCs mimic in vivo cytotrophoblasts, we performed RNA-seq analysis on bTSCs, trophectoderm (TE) from bovine day 7 blastocysts (D7_TE) and day 14 elongated embryos (D14_TE), and compared our findings with publicly available RNA-seq datasets of bovine naive embryonic stem cells (bESCs) and blastocyst-derived pluripotent stem cells (bEPSCs) [34, 35]. Day 7 blastocysts (BL, where most cells are trophectoderm cells) were also included. Principal component analysis (PCA) and Pearson correlation analysis of the transcriptomic data indicated that measurements between biological replicates were consistent across developmental stages (Figure 4, panels A and Figure 10 , panel C). Transcriptomic data along the PC1 dimension showed that bTSCs clustered tightly together, separate from both ESCs / EPSCs and TE / BL (Figure 4, panel A). Transcriptomic data along the PC2 dimension seemed to position bTSCs between two distinct developmental trophoblast groupings, representing the trophectoderm stem cells of pre-implantation embryos (D7_TE) and the stage of elongated trophoblasts during the peri-implantation period (D14_TE) (Figure 4, panel A), demonstrating that bTSCs exist in different trophoblast stem cell transitions. Additionally, PCA analysis of bTSCs and TSCs / ESCs from humans and mice revealed that bTSCs clustered together with both human and mouse TSCs but were distinct from ESCs (Figure 4, panel B), further indicating the molecular identity of bTSCs as trophoblast stem cells.

[0228] Next, we analyzed well-known pluripotency markers and trophoblast transcription factors in bTSCs, TE, and ESCs. bTSCs expressed trophoblast markers (including KLF5, SFN, GATA2, GATA3, TBX3, KRT7, TEAD4, CDX2, and TFAP2A) but not pluripotency markers (POU5F1, SOX2, and NANOG), with a few exceptions (e.g., LIN28A and SALL4), while their expression showed an opposite trend in ESCs and EPSCs (Figure 4C). Notably, Lin28A has been reported to play a functional role in regulating ruminant trophoblast differentiation and function

[36] . Differences were also observed between bTSCs and TE, where the expression of trophoblast transcription factors (KRT7, TEAD3, ELF3, CDX2, and TFAP2A) was lower or absent in TE (Figure 4, panel C), indicating different stemness properties of bTSCs in vitro and that bTSCs are derived from mature TE.

[0229] In addition, we identified genes specifically expressed in bTSCs compared to TE and EPSC (Figure 4, panel D), and their unique transcriptional trends indicate the molecular characteristics of bTSCs and their specific regulatory functions in trophoblast stem cell development. KEGG pathway analysis of these transcripts revealed key signalings representing trophoblast cell fate, including focal adhesion, HIF-1, Hippo, VEGF, and Wnt signaling pathways, actin cytoskeleton, and tight junctions (Figure 4, panel E). For example, Hippo signaling stimulation initiates TE differentiation in human, bovine, and mouse

[37] . Actin is one of the important functional cytoskeletal proteins, maintaining kinetic state and vesicle transport during blastocoel formation, blastocyst hatching, and embryo implantation

[12] . Trophoblast cells are connected by tight junctions to prevent fluid exchange and allow fluid accumulation within the blastocyst. Focal adhesion is upregulated during placental development in goats and sheep

[29] , and dysregulated focal adhesion proteins may affect binucleate organization and trophoblast polarity

[38] .

[0230] Finally, we performed pairwise comparisons of the bTSC transcriptome with those of D7_TE, D14_TE, bESC, and bEPSC. GO analysis of genes upregulated in bTSCs compared to D7_TE and D14_TE revealed significant enrichment in intracellular transport and metabolic processes, while the GO-enriched terms for downregulated genes involved mitochondrial function (Figure 4, panel F). Compared to bESC and bEPSC, significantly upregulated pathways in bTSCs included Hippo signaling pathway, lysosome, and tight junctions (Figure 4, panel G). These specific enriched signaling and metabolic mechanisms represent another unique feature of bTSCs.

[0231] Collectively, these data indicate that bTSCs represent a unique stem cell state of trophoblast fate and are similar to mature TE in terms of transcriptomic characteristics.

[0232] Epigenomic Profile of bTSCs

[0233] To gain insights into the epigenetic regulation of the bovine trophoblast program, we performed ATAC-seq and WGBS analyses on bTSC, D7_TE, and D14_TE. ATAC-seq analysis revealed that bTSC displayed global chromatin accessibility features very similar to those of D7_TE and D14_TE (Figure 5, panels A and B), while different from those of differentiated trophoblasts (Figure 5, panel A). We found that the binding motifs of trophectoderm lineage markers (including GATA1, 2, 3, 4, 6, TEAD 1, 3, 4, and KLF1 and 3) were among the top ten most enriched in bTSC (Figure 5, panel C). By leveraging the chromatin accessibility of trophoblast marker genes, we found that bTSC showed more similarities to D14_TE than to D7_TE (Figure 5, panel D). Additionally, we analyzed the differential enrichment of ATAC-seq peaks among bTSC, D7_TE, and D14_TE. Genes with open chromatin accessibility in bTSC were involved in MAPK, HIF-1, TGF-β, focal adhesion, and signaling pathways regulating PSCs compared to D7_TE and D14_TE (Figure 5, panels E and F), which was consistent with the results of our transcriptome analysis. Genes with lower chromatin accessibility in bTSC compared to D7_TE represented pathways including cGMP-PKG, Hippo, and calcium signaling pathways, arginine and proline metabolism, and cellular senescence, while genes involved in Rap1, oxytocin, apelin, estrogen, Wnt, and GnRH signaling pathways had more accessible chromatin in D14_TE compared to bTSC (Figure 5, panels E and F). This analysis identified important candidate regulators and signaling networks guiding bovine trophoblast lineage specification.

[0234] Comparing the WGBS data of bTSC, D7_TE, and D14_TE with those of bEPSC (Figure 6, panel A), we found that the DNA methylome of bTSC was closer to those of D7_TE and D14_TE, but significantly different from those of bEPSC. The average methylation level of bTSC (56.75%) was significantly higher than that of D7_TE (29.90%) and D14_TE (28.03%), but lower than that of bEPSC (79.80%) (Figure 6, panel B). In addition, we found that the promoter and exon regions were consistently hypomethylated in bTSC, D7_TE, D14_TE, and bEPSC, and much lower in bEPSC, while the methylation levels of introns and intergenic regions were high (Figure 6, panel C). These results suggest that the overall methylation level mainly reflects those in the non-coding regions. By analyzing the gene expression of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) and DNA methylcytosine dioxygenases (TET1, TET2, and TET3) according to RNA-seq data, we found that the expression levels of DNMT1, DNMT3A, and DNMT3B were high in bTSC and bEPSC, consistent with the high DNA methylation levels in bTSC and bEPSC (Figure 6, panel D). We identified the differentially methylated regions (DMRs) between bTSC and D7_TE (2068), D14_TE (2630), and bEPSCs (864) respectively (Figure 6E). Compared with bTSC, the hypermethylated DMRs in D7_TE were enriched in Ras signaling, cGMP-PKG signaling, Hippo signaling, and mTOR signaling (Figure 6, panel F). While the hypermethylated DMRs in D14_TE were mainly concentrated in calcium signaling, Ras signaling, cGMP-PKG signaling, Notch signaling, and estrogen signaling (Figure 6, panel G). These results were consistent with those of transcriptome and chromatin accessibility. Collectively, these signaling pathways are important factors regulating trophoblast development.

[0235] LCDM Conditions Support Bovine ESCs

[0236] LCDM was previously used to generate mouse EPSC

[39] , and we next attempted to test whether LCDM could maintain bovine ESCs. Under LCDM conditions, bovine naive ESCs maintained stable growth kinetics and exhibited the colony morphology of dome-shaped naive ESCs during long-term self-renewal ( Figure 7 , panel A). Compared with naive bESCs, LCDM-ESCs had increased SOX2 expression but were negative for NANOG ( Figure 7, small panel B). Compared with the naive bESC, the mRNA expression level of SOX2 was significantly increased, while the expression levels of OCT4 and NANOG were significantly decreased ( Figure 11 , small panel A). It was confirmed that LCDM-ESC did not express the trophoblast markers CDX2 and GATA3, which was different from TSC cultured under the same conditions ( Figure 7 , small panel B). In humans, compared with naive ESC, primed ESC formed tight junctions and expressed high levels of tight junction-related genes

[40] . When compared with primed ESC, the mRNA expression levels of some tight junction-related genes (CLDN6, CLDN7, and CLDN10) in LCDM-ESC were significantly decreased ( Figure 11 , small panel B). Then, we performed RNA-seq analysis on LCDM-ESC and compared it with the transcriptomes of bESC, bEPSC, ICM, and bTSC. PCA analysis showed LCDM-ESC as a separate group and placed LCDM-ESC between ICM and bESC and closer to bEPSC ( Figure 7 C), indicating that LCDM-ESC could be a specific embryonic stem cell type between primed ESC and naive ESC. To verify the pluripotent state of LCDM-ESC, we analyzed the expression of some naive and primed pluripotency markers used in mice and humans [35, 40, 41]. Compared with primed ESC, LCDM-ESC expressed higher levels of naive pluripotency genes and lower levels of primed pluripotency genes ( Figure 11 , small panels C and D). We identified 2,504 genes that were upregulated in three paired groups, and these genes were enriched in GO terms (including glycosylation, cell projection organization, cell projection assembly, and cellular lipid catabolic process) ( Figure 7 , small panel D). When performing GO analysis on genes specifically upregulated only in LCDM-ESC, we found that these genes were mainly related to cell projection organization, nervous system development, cellular part morphogenesis, and histone modification ( Figure 7 , small panel D). These results indicated that LCDM-ESC was in a pluripotent state different from that of primed ESC and EPSC.

[0237] Discussion

[0238] In this study, we found that LCDM could efficiently establish bovine TSCs from in vitro blastocysts. Bovine TSCs in our system had the ability of unlimited self-renewal, maintained normal karyotypes in long-term culture, and had the potential to differentiate into functional mono- and binucleated trophoblasts. In addition to the above, transcriptome and epigenome analyses indicated that bovine TSCs had some characteristics specific to mature trophoblast cells. Moreover, TSCs injected into NOD-SCID mice mimicked some important processes of trophoblast cells during elongation and implantation.

[0239] The transcription factors of bovine TSCs were different from those of mouse and human TSCs. We found that the expression of CDX2 was high in bovine TSCs (>209 TPM), but much lower in trophoblast cells and even undetectable (0 TPM) in bESCs and bEPSCs, indicating that CDX2 was an important regulator in bovine TSCs. This was consistent with mouse TSCs, where Cdx2 was required for self-renewal

[42] . However, the expression of CDX2 was very low in human TSCs and cytotrophoblast cells, and it was not essential for the proliferation of human TSCs [8]. More strikingly, SOX2 was undetectable (0 TPM) in bovine TSCs, but much higher in bESCs (about 400 TPM) and bEPSCs (>220 TPM), indicating that SOX2 was not essential for the maintenance of bovine TSCs. This view was supported by the following: the expression of bovine SOX2 began at the 16-cell stage and then was restricted to the ICM of blastocysts

[43] . In mouse TSCs, Sox2 could support TSC self-renewal independent of FGF

[44] , and could also regulate TSC-specific genes in combination with Tfap2c

[42] .

[0240] We found that LIF, Wnt activator (CHIR99021), and PARP or MMP inhibitor (MiH) as well as muscarinic and histamine H1 receptor inhibitor (DiM) are important for the derivation and maintenance of bovine TSCs. Transient inhibition of Rho-associated protein kinase (ROCK) is beneficial for passage survival. For human TSCs, activation of Wnt and EGF and inhibition of TFG-β, histone deacetylase (HDAC), and ROCK are important [8], while activation of FGF and TGF-β and inhibition of Wnt and ROCK are required for the derivation of mouse TSCs

[45] . Only Wnt signaling is conserved between humans and cattle. There are obvious differences in placental development among mice, humans, and cattle. In mice, the polar TE generates the extraembryonic ectoderm (ExE) and ectoplacental cone (EPC), and there are four different trophoblast cell types, each with its role in establishing an exchange surface, covering the implantation site, and contacting the maternal decidua

[46] . In humans, the initial invasion is completed by the syncytiotrophoblast cells in the initial state, and the migration of mononuclear cytotrophoblast cells forms primary villi

[46] . However, for cattle, there are significant differences after the blastocyst stage. The embryo remains implanted and enters the elongation stage on day 13, and the embryo morphology finally transforms into a filamentous shape, with a significant increase in TE length and weight. After elongation, the conceptus length can reach over 20 cm

[47] . Therefore, it is reasonable that mice, humans, and cattle require different pathways to support TSC self-renewal.

[0241] PARP1 plays multiple roles, such as DNA damage, chromatin modification, transcription, etc.

[48] . Parp 1 is involved in the extraembryonic developmental potency of mouse EPSCs

[39] , and PARP activity can be detected in the bovine placenta

[49] , but its exact function still requires more research. MiH is also a non-selective inhibitor of MMP. MMP2 and MMP9 are expressed in the tissue cohorts of the bovine placenta and its appendages

[50] , and their activities play a crucial role during maternal implantation in cows. When MMP is inhibited, bovine TSCs will maintain self-renewal instead of differentiating into binucleated or multinucleated cells. Compared with differentiated cells, a downregulation of MAPK signaling was observed in bovine TSCs (Figure 2, inset I). MAPK signaling is one of the important downstream signaling pathways of muscarinic receptors and histamine receptors

[51] .

[0242] Differentiated TSCs lose their proliferative ability, but some differentiated cells have two nuclei. IFNT (pregnancy recognition signal) activity was detected in the culture medium, and the expression level increased significantly during differentiation. PTGS2 regulated by IFNT during early pregnancy was detected at both the transcriptional and protein levels. However, little is known about the molecular mechanism that controls the balance between mononuclear and binuclear trophoblast cells, and this balance determines the success of elongation. In addition, more precise markers at different pregnancy stages need to be identified to support pregnancy testing.

[0243] In addition, we found that LCDM could support the growth of bovine ESCs. Under LCDM, bovine naive ESCs changed into more compact and dome-like colonies, in which the expression of SOX2 was significantly increased. Moreover, compared with naive bESCs, LCDM-ESCs had higher expression levels of naive state markers and lower levels of primed state markers ( Figure 11 , panels C and D). Our results indicate that LCDM-ESCs have several distinct cellular and molecular characteristics from primed bESCs and bEPSCs. Recently, Smith proposed that there is a specific stage between naive and primed pluripotency stages, called the formative stage

[52] . There are some criteria to evaluate formative pluripotency. The important rule is that formative stage cells have the ability to form chimeras and germ cells.

[0244] In summary, we have established bovine TSCs from IVF blastocysts, which opens up new avenues for studying the molecular and functional mechanisms of bovine trophoblast cells. In addition, our bovine TSCs provide new possibilities for understanding the pathogenesis of embryonic developmental failure associated with trophoblast defects.

[0245] Materials and Methods

[0246] Generation of Bovine IVF Embryos

[0247] The IVM-IVF embryos used in this study were generated as previously described

[53] . Briefly, bovine cumulus-oocyte complexes (COCs) were aspirated from selected follicles of ovaries from a slaughterhouse. Oocytes were matured in vitro using BO-IVM medium (IVF Bioscience), and then IVF was performed using cryopreserved semen from Holstein bulls with proven fertility. The embryos were then washed and cultured in BO-IVC medium (IVF Bioscience) at 38.5 °C and 6% CO2. The blastocysts were collected, and the zona pellucida was removed by pipetting and processed for TSC derivation.

[0248] Mouse trophoblast stem cells (mTSCs) are derived from blastocysts in the presence of fibroblast growth factor 4 (FGF4) [7]. mTSCs are the best in vitro model for molecular and functional analysis of mouse trophoblast cells. Recently, human trophoblast stem cells (hTSCs) have been established from blastocysts and cytotrophoblast (CT) cells by activating WNT and EGF and inhibiting TGF-β, histone deacetylase (HDAC), and Rho-associated protein kinase (ROCK) [8]. hTSCs have the ability to generate CT, extravillous CT (EVT), and syncytiotrophoblast (ST) in vitro. For mice, Cdx2, Gata3, Eomes, and Elf5 are essential for maintaining the undifferentiated state of mTSCs

[42] , while for humans, TP63, GATA3, and TEAD4 have been reported as important players in the undifferentiated state [8].

[0249] Derivation and culture of bovine TSCs

[0250] Place each blastocyst in a separate well of a 12-well plate seeded with mitomycin C-treated mouse embryonic fibroblasts (MEFs). Culture the embryos in bovine TSC medium, which contains DMEM:F12 (Gibco), Neurobasal medium (Gibco) (1:1), 0.5x N2 supplement (Gibco), 0.5x B27 supplement (Gibco), 1x NEAA (Gibco), 1x GlutaMAX (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), 0.1% BSA (MP Biomedicals), 10 ng / mL LIF (Peprotech, 300-05), 3 μM CHIR99021 (Sigma, SML1046), 2 μM dimemindene maleate (DM) (Tocris, 1425), and 2 μM minocycline hydrochloride (MH) (Santa Cruz, sc-203339). Incubate the cells at 38.5 °C and 5% CO2. After 48 hours of plating, press the unattached embryos against the bottom of the plate with a needle under the microscope. Change the medium daily. On day 7 or 8, dissociate the outgrowths with dispase (STEMCELL Technologies) at 38.5 °C for 5 - 10 min and then wash twice with DMEM / F12. Mechanically passage bTSCs under the microscope. For optimal viability, add 10 μM Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Tocris, 1254) to the medium for 24 hours.

[0251] Once established, bTSCs were passaged every 6 days at a split ratio of 1:6 using Accutase (Gibco, A1110501). Each well of bTSCs was dissociated with 1 mL of Accutase at 38.5 °C for 5 min, and the reaction was neutralized by diluting Accutase with the same volume of bTSC medium. According to the manufacturer's instructions, bTSCs were cryopreserved using ProFreeze cryomedium (Lonza Group, 12 - 769E).

[0252] For feeder - free conditions, bTSCs cultured on feeder cells were passaged into plates coated with Matrigel (Corning, 354234), where MEF - conditioned bTSC medium (MEF - bTSC) was used.

[0253] Differentiation of bovine TSCs

[0254] Bovine TSCs were grown to 80% - 90% confluence in bTSC medium and dissociated with TrypLE (Gibco, 12605 - 010) at 38.5 °C for 15 min. Then, bTSCs were seeded at a density of 1 - 1.5×10⁵ cells / well into 6 - well plates coated with 2.5 μg / mL Col IV (Corning, 354233) and cultured in 2 mL of differentiation medium containing DMEM:F12 and Neurobasal medium (1:1), with 0.5x N2 supplement, 0.5x B27 supplement, 1x NEAA, 1x GlutaMAX, 0.1 mM 2 - mercaptoethanol, 0.1% BSA, 2.5 μM Y27632, 2 μM forskolin (Sigma, F3917), and 4% KSR (Invitrogen, 10828028). The medium was changed every two days.

[0255] Culture of bovine naive ESCs

[0256] Bovine naive ESCs (bESCs) were maintained on mitomycin C - treated MEFs in mTeSR1 (StemCell Technologies, 85851) supplemented with 2.5 μM IWR1 (Sigma, I0161) and 20 ng / mL FGF2 (Peprotech, 100 - 18B). bESCs were passaged every 4 days at a ratio of 1:3 using TrypLE (Gibco, 12605 - 010), and fresh medium was changed daily. The cells were incubated at 37 °C and 5% CO₂.

[0257] Culture of bESCs in LCDM

[0258] Twenty-four hours after the passage of bESCs, the culture medium was replaced with LCDM. On the 4th day, the culture medium was aspirated, and 1 mL of 0.5 mM EDTA (Invitrogen, 15575020) was added to each well, followed by incubation at 37 °C for 4 - 5 min. The EDTA was aspirated and the wells were washed twice with PBS. LCDM was added to collect the cells by pipetting. The cells were centrifuged at 1000 rpm for 5 min. The cell pellet was resuspended using LCDM plus 10 μM Y27632. The cells were plated on mitomycin C-treated MEFs. On the 7th day, dome-shaped colonies appeared and expanded. These colonies were picked for purification and expansion. The purified LCDM-ESCs were passaged every 3 days at a ratio of 1:4.

[0259] Immunofluorescence staining

[0260] The cells or blastocysts were fixed with 4% paraformaldehyde (PFA) at room temperature for 20 min and then rinsed three times in wash buffer (0.1% Triton X-100 and 0.1% polyvinylpyrrolidone in PBS). After fixation, the cells were permeabilized with 1% Triton X-100 in PBS for 30 min and then rinsed with the wash buffer. The cells were then transferred to blocking buffer (0.1% Triton X-100, 1% BSA, and 0.1 M glycine) at room temperature for 2 h. Subsequently, the cells were incubated with the primary antibodies overnight at 4 °C. The primary antibodies used in this experiment included anti-SOX2 (Bethyl Laboratories, an833), anti-CDX2 (Bethyl Laboratories, MU392A; 1:200), anti-GATA3 (Cell Signaling, D13C9; 1:200), and anti-KRT8 (Origene, BP5075; 1:300). For secondary antibody incubation, the cells were incubated with the Fluor 488 or 555 or 647-conjugated secondary antibodies at room temperature for 1 h. The cell nuclei were stained with ProLong Diamond Antifade Mountant (containing DAPI). Images were taken using a fluorescence confocal microscope (Leica).

[0261] The paraffin sections were deparaffinized and then boiled in sodium citrate buffer (pH 6.0) for 20 min for antigen retrieval. The sections were blocked with 5% goat serum in TBST for 1 h and incubated with the primary antibody overnight at 4 °C. Then, the sections were incubated with the fluorescence-conjugated secondary antibody at room temperature for 1 h. The cell nuclei were stained with DAPI (Invitrogen, D1306).

[0262] Quantitative real-time PCR

[0263] Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. First-strand cDNA was synthesized using the iScript cDNA Synthesis Kit (BIO-RAD). qRT-PCR was performed using SYBR Green PCR Master Mix (BIO-RAD) and specific primers (Table S1). Data were analyzed using the BIO-RAD software provided with the instrument. Relative gene expression values were calculated using the ΔΔCT method and normalized to the internal control GAPDH.

[0264] IFNT activity assay

[0265] IFNT activity was measured by an established IFN-stimulated response element - reporter gene assay

[24] . Briefly, 5 - 10×105 Madin-Darby bovine kidney cells (MDBK) stably transduced with the ISRE-Luc reporter gene were plated into the wells of a 96-well polystyrene plate (Corning) with an opaque wall and optically clear bottom and cultured in MDBK growth medium (high glucose DMEM, 10% FBS, and 1% Pen / Strep) at 37 °C for 4 h. After removing the MDBK growth medium, 50 μL of sample or standard (recombinant human IFN-α, IFNA: Millipore, IF007) was added. A standard curve was generated by 1:3 serial dilutions of IFNA. The cells were incubated at 37 °C for 16 h, then 50 μL of One-Glow luciferase reagent (Promega Corp; E6120) was added to each well for a final volume of 100 μL. After mixing on an orbital shaker platform for 10 min, measurements were taken on a plate reader.

[0266] TSC lesion assay

[0267] bTSC cells were grown to approximately 80% confluence in bTSC medium and dissociated with TrypLE. 5×106 bovine TS cells were resuspended in 200 μL of a 1:1 mixture of bTSC medium and Matrigel and injected subcutaneously into 6-month-old non-obese diabetic (NOD)-severe combined immunodeficient (SCID) mice. Lesions were collected on days 7 and 9 and fixed overnight in 4% PFA at 4 °C for analysis.

[0268] Karyotype analysis assay

[0269] The bTSCs were incubated with bTSC medium containing 1 mL of KaryoMAX colcemid solution (Gibco, 15212012) at 38.5 °C for 4 - 5 hours. Then the cells were dissociated with 1 mL of trypsin (Gibco, 25200 - 056) at 38.5 °C and centrifuged at 300×g for 5 min. The cells were resuspended in 1 mL of PBS solution and centrifuged at 400×g for 2 min. The supernatant was aspirated, and 500 μL of 0.56% KCl was added to resuspend the cells. The cells were incubated for 15 min and then centrifuged at 400×g for 2 min. 1 mL of cold fresh Carnoy’s fixative (3:1 methanol:acetic acid) was added to resuspend the cells, and then incubated on ice for 10 min. After centrifugation, 200 μL of Carnoy’s fixative was added to resuspend the cells. The cells were dropped onto clean glass slides and air-dried, and then soaked in the solution (1:25 Giemsa stain (Sigma, GS500): deionized water) for 9 min. The glass slides were rinsed with deionized water and air-dried. Images were taken at 1000× magnification using a Leica DM6B under oil immersion.

[0270] RNA-seq analysis

[0271] Total RNA of bovine TSCs and ESCs was extracted using the RNeasy Micro Kit (Qiagen). Day 7 pure trophectoderms were isolated by placing the embryos in a culture dish containing phosphate-buffered saline and performing microsurgery using a microsurgical blade under a microscope. RNA-seq libraries were generated using the Smart-seq2 v4 kit, with minor modifications according to the manufacturer's instructions. Briefly, mRNA was captured and amplified using the Smart-seq2 v4 kit (Clontech). After purification with AMPure XP beads, the amplified RNA was quality-checked using the Agilent High Sensitivity D5000 kit (Agilent Technologies). Library preparation was performed on the high-quality amplified RNA (Nextera XT DNA Library Preparation Kit; Illumina) and multiplexed with Nextera XT Indexes (Illumina). After purifying the library with AMPure XP beads (Beckman Coulter), the concentration of the sequencing library was determined using the Qubit dsDNA HS Assay Kit (Life Technologies). The size of the sequencing library was determined by high-sensitivity D5000 assay in the Tapestation 4200 system (Agilent). The pooled indexed library was then sequenced on the Illumina Novaseq platform with 150-bp paired-end reads.

[0272] The Salmon tool

[54] was applied to quantify gene expression profiles from the raw sequencing data using the Ensembl bovine genome annotation (ARS-UCD1.2). Transcripts per million reads (TPM) were used as the unit of gene expression. The edgeR tool

[55] was applied to identify differentially expressed genes. The TMM algorithm implemented in the edgeR package was used to normalize read counts and estimate the effective library size. Differential expression analysis was performed by the likelihood ratio test implemented in the edgeR package. Conventional statistical analysis was performed based on the R platform. The Spearman rank correlation test was performed using the "cor.test" function. Principal component analysis (PCA) of gene expression profiles was performed using the "dudi.pca" function in the "ade4" package. Heatmaps were drawn using the "heatmap.2" function in the "gplots" package. Gene ontology and pathway analysis were performed using the David tool

[56] .

[0273] Overall, we sequenced two biological replicates of bTSC, trophoblasts differentiated on days 2, 3, 4, 5, and 6, three biological replicates of whole blastocysts and day 7 trophectoderm cells selected from the same batch used for bTSC derivation, and three biological replicates of bovine ESCs cultured under LCDM conditions. RNA-seq datasets of bovine day 14 trophectoderm

[57] , ESCs

[35] , and EPSCs

[34] were downloaded from previous publications, respectively.

[0274] ATAC-seq analysis

[0275] ATAC-seq libraries of fresh cells were prepared as previously described

[53] . Briefly, cells or embryos were lysed on ice and then incubated with Tn5 transposase (TDE1, Illumina) and tagging buffer. Tagmented DNA was purified using the MinElute Reaction Cleanup kit (Qiagen). ATAC-seq libraries were amplified with Illumina TrueSeq primers and multiplexed with index primers. Finally, high-quality indexed libraries were pooled together and sequenced with 150-bp paired-end reads on the Illumina Novaseq platform.

[0276] ATACseq analysis followed our established analysis pipeline

[53] . Quality-assessed ATAC-seq reads were aligned to the bovine reference genome using Bowtie 2.3 with the following options: –very-sensitive -X 2000 –no-mixed –no-discordant. Alignments caused by PCR duplicates or positions in mitochondria were excluded. Only unique alignments within each sample were retained for subsequent analysis. MACS2 called ATAC-seq peaks for each sample separately with the following options: –keep-dup all –nolambda –nomodel. ATAC-seq bigwig files were generated using bamcoverage from deeptools. ATAC-seq signals were visualized in the Integrative Genome Viewer genome browser. Annotations of genomic features (including transcription start sites, transcription termination sites (TESs), promoters, CDSs, introns, 5′UTRs, 3′UTRs, and intergenic regions) were downloaded from the UCSC Genome Browser. The enrichment of transcription factor motifs in the peaks was evaluated using HOMER (http: / / homer.ucsd.edu / homer / motif / ). For downstream analysis, we normalized read counts by calculating counts scaled by the number of sequenced fragments multiplied by one million (CPM).

[0277] Whole-genome bisulfite sequencing (WGBS) analysis

[0278] The WGBS library was prepared using the TruSeq DNA Methylation Library Preparation Kit (Illumina). Briefly, genomic DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions. Then, approximately 500 ng of DNA was bisulfite-treated using the EZ DNA Methylation Kit (Zymo Research). The bisulfite-converted DNA was end-repaired, dA-tailed, and ligated with adapters according to the instructions of the TruSeq DNA Methylation Library Preparation Kit. Finally, high-quality indexed libraries were pooled and sequenced on the Illumina Novaseq platform with 150-bp paired-end reads.

[0279] WGBS data analysis followed our established analysis pipeline [58, 59]. Briefly, the first 12 bp at both 5' ends of the WGBS raw data were removed, and reads with adapters and low-quality bases were removed using TrimGalore-0.4.3. The trimmed sequences were mapped to the bovine genome (ARS-UCD1.2) using Bismark. Then, unique mapped reads, PCR duplicates, and unconverted reads were removed using deduplicate_bismark and filter_non_conversion. To avoid sequencing bias, only reads with 10-fold coverage were used for downstream analysis. Methylation at each CpG site was calculated, and methylation of each sample was calculated by averaging the methylated genomic windows of 300-bp contiguous blocks. Genomic features, including promoters (1000 bp upstream of the transcription start site), exons, introns, CpG islands, and intergenic regions, were downloaded from the University of California, Santa Cruz (UCSC) Genome Browser. Gene ontology and pathway analysis were performed using the David tool

[56] .

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[0308] 28. Brooks, K., G. Burns, and T. E. Spencer, Conceptus elongation in ruminants: roles of progesterone, prostaglandin, interferon tau and cortisol. J Anim Sci Biotechnol, 2014. 5(1): p. 53.

[0309] 29. Luo, N., et al., Screening Candidate Genes Regulating Placental Development from Trophoblast Transcriptome at Early Pregnancy in Dazu Black Goats (Capra hircus). Animals (Basel), 2021. 11(7).

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[0316] 36. Ali, A., et al., The Role of LIN28-let-7-ARID3B Pathway in Placental Development. Int J Mol Sci, 2020. 21(10).

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[0340] Example 6

[0341] Establishment of Bovine Trophoblast Stem Cells

[0342] Summary

[0343] Here, we report that a chemical mixture (LCDM: leukemia inhibitory factor [LIF], CHIR99021, dimenhydrinate maleate [DiM], minocycline hydrochloride) previously developed for mouse and human extended pluripotent stem cells (EPSCs) permits de novo derivation and long-term culture of bovine trophoblast stem cells (TSCs). Bovine TSCs retain the developmental potency to differentiate into mature trophoblast cells and exhibit transcriptomic and epigenetic (chromatin accessibility and DNA methylome) features characteristic of trophectoderm cells from early bovine embryos. The bovine TSCs established in this study will provide a model for studying bovine placental formation and early pregnancy failure.

[0344] Introduction

[0345] Trophoblast cells are specialized cells that mediate maternal-fetal crosstalk in the placenta and are derived from the trophectoderm (TE) of the blastocyst. Establishment of pregnancy in cattle requires TE elongation, a unique process in ruminants prior to apposition, attachment, and implantation. 1 During this process, undifferentiated TE cells or so-called trophoblast progenitors will differentiate into mononuclear trophoblast cells and subsequently into binucleate giant cells 2,3 , to drive embryo elongation and ultimately fuse with uterine epithelial cells to establish the fetal-maternal interface. 4

[0346] Embryo loss and early pregnancy failure are the major causes of cattle infertility 5,6 , and most embryo losses occur within the first few weeks of pregnancy. 7 9Proper trophoblast development and function are crucial for pregnancy success. However, due to the technical and logistical difficulties of in vivo experiments in cattle, and the lack of an operational cell culture model that can recapitulate placental cell differentiation in vitro, we still lack a complete understanding of early bovine placental development. To date, trophoblast stem cells (TSCs) have been established in several rodent and primate species, including mice 10 , humans 11 and non-human primates 12 . Despite several attempts 13–15 , true bovine TSCs that can withstand the rigors of long-term culture have not been successfully derived.

[0347] In this study, we found that the LCDM conditions previously reported for mouse and human extended pluripotent stem cells (EPSCs) (composed of leukemia inhibitory factor [LIF], CHIR99021, dimemidine maleate [DiM], and minocycline hydrochloride [MiH]) 16 supported the de novo derivation of TSCs from bovine blastocysts generated by in vitro fertilization (IVF). Bovine TSCs grown under LCDM conditions maintained stable morphological, karyotypic, transcriptomic, and epigenomic characteristics, as well as in vitro and in vivo developmental potential after long-term passage. The bovine TSCs generated and characterized in this study provide a valuable source of material for studying ruminant trophoblast development and function in vitro and enable the assembly of bovine blastocyst-like structures (blastoids). 17

[0348] Results

[0349] De Novo Derivation of Bovine TSCs from Blastocysts

[0350] The TE cells of bovine blastocysts retained the plasticity to generate inner cell mass (ICM) cells, and vice versa, 18,19 which prompted us to test the de novo derivation of bovine TSCs using different combinations of basal media, growth factors, and chemicals previously used to culture pluripotent stem cells (PSCs) (Table 1). We identified four conditions that could support the growth of bovine blastocysts on mouse embryonic fibroblast (MEF) feeder cells for several generations (Figure 12, insets A and B; Figure 15 , inset A).

[0351] Table 1. Screening of culture conditions for the derivation of bovine TSCs.

[0352]

[0353] Interestingly, the extended pluripotent stem cell (EPSC) culture condition LCDM (human LIF [hLIF], CHIR99021, DiM, and MiH) 16Most effective in supporting long-term (>70) passaging of bovine TSC-like cells (bTSC-LC) from blastocysts. Removal of each of hLIF, CHIR99021, DiM, and MiH failed to maintain the morphology and self-renewal of bTSCLC. In the presence of MEF-conditioned LCDM medium, bTSC-LC could also be maintained feeder-free on Matrigel (Figure 12, inset B).

[0354] Additional characterization revealed that (1) bTSC-LC maintained a stable colony morphology and normal chromosomal diploid number (60) after long-term in vitro culture (Figure 12, inset B; Figure 15 , inset B); (2) bTSC-LC highly expressed TE-related transcription factors (TFs) (CDX2, SFN, ELF5, GATA3, ASCL2, GATA2, and ETS2)( Figure 15 , inset C); (3) at the protein level, similar to TE cells in bovine blastocysts, bTSC-LC expressed CDX2, GATA3, and KRT8, but not SOX2 (Figure 12, inset C; Figure 15 , inset D); and (4) most bTSC-LC were found to be GATA3 + (Figure 12, inset D).

[0355] Overall, we found that EPSC culture conditions supported de novo derivation and long-term self-renewal of stable bTSC-LC in vitro. bTSC-LC expressed bona fide TE-related TFs at the mRNA and protein levels, and hereafter we refer to them as bTSCs.

[0356] Directed Differentiation of bTSCs into Functional Trophoblast Cells

[0357] Next, we evaluated the in vitro differentiation potential of bTSCs. By using conditions containing forskolin, Y27632, and 4% knockout serum replacement (KSR), we were able to differentiate bTSCs into binucleated cells that expressed the trophoblast markers PTGS2 and placental lactogen 1 (PL-1) (Figure 12, inset E; Figure 15 , inset E - inset G). In ruminants, interferon tau (IFNτ) produced by mature trophoblast cells is considered a signal for maternal recognition of pregnancy. 20 By using a luciferase-based IFN-stimulated response element (ISRE) assay, 21 we found that IFNτ production increased significantly during bTSC differentiation and peaked around day 5 (Figure 12, inset F). qRT-PCR analysis further demonstrated that after bTSC differentiation, IFNτ and mature trophoblast markers (BEVR-k1 env, bEPVE-A 22 and pregnancy-associated glycoprotein 1 [PAG1]23 ) was significantly upregulated (Figure 12, panel G).

[0358] We performed RNA sequencing (RNA-seq) at six time points during bTSC differentiation and found that bTSCs transitioned to an intermediate stage on day 2 and then further differentiated into more mature trophoblast cells between days 3 and 6 (Figure 16, panel A). RNA-seq analysis demonstrated that PAG family genes (PAG2, PAG11, and PAG12) and well-known bovine placental marker genes (CYP11A1, CYP17A1, FURIN, HAND1, PTGS2, and HSD3B1) 23–25 were upregulated during differentiation (Figure 12, panel H). Differentiated trophoblasts (day 4) had upregulation of genes enriched in gene ontology (GO) terms related to morphogenesis, cell migration, and motility (Figure 16, panel B), indicating the presence of invasive trophoblast cells. Additionally, differentiated trophoblast cells expressed many genes involved in extracellular matrix (ECM)-receptor interaction, tumor necrosis factor (TNF), interleukin-17 (IL-17), and MAPK signaling pathways (Figure 16, panel C), which is consistent with increased activity of these signaling pathways during implantation and placental development in ruminants and humans. 26–28 Notably, the top several GO terms enriched in upregulated genes on day 6 compared to day 5 during bTSC differentiation were related to apoptosis (Figure 16, panel D), indicating decreased cell viability. These were consistent with the decline in IFNτ activity (Figure 12, panel F) and the downregulation of mature trophoblast marker gene expression (Figure 12, panel G).

[0359] We also determined the differentiation potential of bTSCs by subcutaneously injecting bTSCs into NOD-SCID mice. By day 9, the injected bTSCs formed lesions approximately 0.5 cm in size (Figure 16, panel E). Immunohistological analysis revealed that the central region of the lesion was necrotic and that the lesion contained blood-filled lacuna-like structures (Figure 12, panel I), similar to the lesions formed by mouse 29 and human TSCs. 11 Binucleated cells were identified in the perilesional area and expressed PL-1 and PTGS2, indicating trophoblast maturation (Figure 12, panels I and J). We also identified cells positive for MMP2 (a key factor in trophoblast-endometrial epithelial cell crosstalk and endometrial stromal remodeling 30 ) staining in the periphery of the lesion (Figure 12, panel J).

[0360] Collectively, these results revealed the differentiation potential of bTSCs and demonstrated the ability of bTSCs to generate mature trophoblast cells in vitro and in vivo.

[0361] Transcriptional and Epigenomic Features of bTSCs

[0362] We compared the transcriptome of bTSCs with those derived from: (1) early placental cells at two different developmental stages: TE of pre-implantation blastocysts (D7_TE) and TE of day 14 conceptuses (D14_TE), 31 (2) day 7 IVF blastocysts, and (3) two types of pluripotent stem cells: bovine expanded pluripotent stem cells (bEPSCs: bEPSCs ES 、bEPSCs iPS,32 and bEPSCs 33 ) and naïve bovine ESCs (bESC) 34 (Figure 13, panel A). Principal component analysis revealed that bTSCs were separated from D7_TE, day 7 IVF blastocysts, D14_TE, bESC, and bEPSC (Figure 13, panel A). Additionally, bTSCs were different from bEPSCs cultured under LCDM conditions supplemented with KSR serum and higher concentrations of DiM (2 mM), MiH (2 mM), and CHIR99021 (1 mM) 33 (Figure 13, panels A, F, and G), while bEPSCs ES and bEPSCs Xiang derived from two different conditions both showed similar transcriptomic profiles (Figure 13, panel A).

[0363] Bovine 34 、Human 11,35 and murine 36,37 additional transcriptomic comparisons of TSCs and ESCs confirmed the lineage identity of bTSCs (Figure 13, panel B). bTSCs highly expressed two pluripotency-related genes, LIN28A and SALL4 (Figure 13, panel C), as well as trophoblast-related genes, KRT7, TEAD3, ELF3, CDX2, and TFAP2A, which was in contrast to TE cells of early embryos (Figure 13, panel C). Additionally, when compared with D7_TE and D14_TE, GO terms related to intracellular transport and metabolic processes were enriched in the bTSC transcriptome (Figure 16, panel F), and when compared with bESC and bEPSC ES , GO terms related to the Hippo signaling pathway and tight junctions were enriched in the bTSC transcriptome (Figure 13, panel D). Notably, signaling pathways including focal adhesions and HIF-1 were also uniquely enriched in bTSCs (Figure 13, panel E).

[0364] We also performed assays of transposase-accessible chromatin using sequencing (ATAC-seq) and whole-genome bisulfite sequencing (WGBS) analysis and investigated the epigenomic features of bTSCs. We confirmed that trophoblast TFs are one of the most enriched binding motifs in bTSCs (Figure 14, panel A). Differential enrichment analysis of ATAC-seq peaks between bTSCs and D7_TE / D14_TE further confirmed the overrepresentation of focal adhesion and HIF-1 signaling pathways in bTSCs (Figure 14, panel B; Figure 16, panel G). WGBS analysis showed that the overall methylation level of bTSCs (56.75%) was much higher than those of D7_TE (29.90%) and D14_TE (28.03%), but lower than that of bEPSCs (79.80%) (Figure 14, panel C). This was consistent with the higher levels of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) in bTSCs and bEPSCs (Figure 14, panel D). We were able to identify differentially methylated regions (DMRs) between bTSCs and D7_TE / D14_TE (Figure 14, panel E). Hypomethylated regions in bTSCs compared with D7_TE and D14_TE included genes involved in metabolism (including Ras, cGMP-PKG, calcium signaling, and purine metabolism) (Figure 14, panels F and G). In contrast, hypermethylated regions of bTSCs compared with D7_TE were enriched for adherens junctions, insulin resistance, sphingolipids, and IL-17 signaling, while hypermethylated regions of bTSCs compared with D14_TE were enriched for MARK, oxytocin, glycosaminoglycan biosynthesis, gap junctions, and chemokine signaling (Figure 16, panels H and I).

[0365] Our RNA-seq, ATAC-seq, and WGBS analyses together provided a comprehensive transcriptomic and epigenomic profile of bTSCs and revealed molecular features of the earliest steps of bovine placental development.

[0366] Discussion

[0367] Here, we demonstrated that the EPSC culture condition (LCDM) 16 can support the de novo derivation of stable bTSCs from blastocysts. LCDM-derived bTSCs showed the ability of long-term self-renewal in culture while retaining the potential to differentiate into mature trophoblast cells. Comprehensive transcriptomic and epigenomic analyses of bTSCs and TEs revealed molecular features during early bovine placental development and predicted regulators of bovine trophoblast differentiation. As a community resource, the data presented here fill in the gaps and add a reliable stem cell model for studying placental development in ungulate species.

[0368] The LCDM conditions were originally developed for the derivation and long-term culture of mouse and human EPSCs, which have intra- and inter-species developmental potencies towards embryonic and extra-embryonic tissues, respectively. 16 Recently, the LCDM conditions have been successfully used to generate porcine induced pluripotent stem cells (iPSCs) from pericytes and embryonic fibroblasts 38 , as well as porcine PSCs (pLCDM) from in vivo blastocysts. 39 Interestingly, unlike mouse and human EPSCs, pLCDM is prone to trophoblast differentiation, and TSC-LCs can be generated from pLCDM using human TSC conditions. 11 The LCDM conditions have also recently been tested in cattle. One study showed that bovine iPSCs can be cultured and adapted to EPSCs in LCDM medium, or can be reprogrammed directly from fetal fibroblasts that exhibit embryonic and extra-embryonic potencies in bovine-mouse chimeras. 33 Interestingly, however, the LCDM conditions cannot support the direct derivation of EPSCs from bovine blastocysts. 33 Consistent with this, under the LCDM conditions, we did not find EPSC-like colonies, but TSC-like cells could be easily observed from blastocyst outgrowths. Thus, similar to pigs, the trophoblast lineage seems to be favored in LCDM-cultured bovine blastocysts due to the action of DiM (an inhibitor of muscarinic and histamine H1) and / or MiH (inhibitors of PARP and MMP). 39 However, the ability of LCDM to support the generation and long-term culture of bovine iPSCs suggests that with further optimization in future studies, bovine EPSCs could potentially be derived from blastocysts. The ability to support both TSCs and PSCs demonstrates the "permissive" nature of the LCDM culture conditions, which has recently been observed in other cultures. 40–42 The ability to grow more than one embryonic and extra-embryonic stem cell under the same conditions will help to facilitate the study of lineage crosstalk during early development.

[0369] In this study, we found that LIF / STAT3, CHIR99021 (a GSK3 inhibitor that activates the canonical Wnt signaling pathway), DiM, and MiH are indispensable for the derivation and maintenance of bTSCs. Notably, in addition to EGF pathway activation and constitutive inhibition of TGF-β, HDAC, and Rho-associated protein kinase (ROCK) activities, the derivation and culture of human TSCs also require activation of the Wnt signaling. 11 In contrast, although transient ROCK inhibition during passage is beneficial, the long-term maintenance of bTSCs does not require continuous ROCK inhibition. MiH is known to inhibit PARP and its family member PARP1, which plays multiple roles such as DNA damage, chromatin modification, transcriptional regulation, and histone modification. 43PARP1 promotes the binding of SOX2 to intractable genomic loci, which drives the expression of key pluripotency genes. 44 Inhibition of PARP1 is necessary to maintain the extraembryonic developmental potency of mouse EPSCs but does not affect self-renewal. 16 MiH is also a non-selective inhibitor of MMP9 expressed in trophoblast cells. MMP9 can degrade components of the ECM, providing a suitable environment for tissue remodeling and migration of binucleate trophoblast cells. 45 Supplementation with DiM in porcine IVF embryo culture increased the proportion of trophoblast cells and the total cell number of blastocysts, 39 indicating that DiM promotes differentiation towards the trophoblast lineage. In addition, MAPK signaling has been reported to be one of the important downstream pathways of muscarinic and histamine receptor signaling (which can be inhibited by DiM). 46 Consistent with this, we also observed downregulation of MAPK signaling-related genes in bTSCs. The exact roles of MiH and DiM in stem cell biology remain elusive and warrant future investigation.

[0370] In summary, we have established stable bTSCs from IVF blastocysts, which can not only serve as a model for studying the unique placental formation process and early pregnancy failure in ruminants but also allow the generation of the first-generation blastocyst-like structures (blastoid) from large domestic animal species. 17

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[0430] Key resource table

[0431]

[0432]

[0433]

[0434] Experimental model and subject details

[0435] Animal Care and Use

[0436] In vivo embryos were collected from non-lactating 3-year-old crossbred (Bos taurus x Bos indicus) cows. Six-month-old NOD-SCID mice (male and female) were used for the TSC lesion assay. The mice were housed on a 12-hour light / 12-hour dark cycle.

[0437] Method details

[0438] Bovine IVF Embryo Production

[0439] The IVF embryos used in this study were generated as previously described. 55 Briefly, bovine cumulus-oocyte complexes (COCs) were aspirated from selected follicles of abattoir ovaries. BO-IVM medium (IVF Biosciences) was used for in vitro maturation of oocytes. IVF was performed using cryopreserved semen from Holstein bulls with proven fertility. The embryos were then washed and cultured in BO-IVC medium (IVF Biosciences) at 38.5 °C and 6% CO2. Day 7 blastocysts were collected, zona pellucida removed, and processed for TSC derivation.

[0440] Bovine Day 14 Elongated Embryo Production

[0441] As previously described, Day 14 elongated embryos were collected from crossbred cows 55 . Briefly, superovulation was achieved using five doses of FSH administered intramuscularly, starting five days after insertion of a controlled intravaginal drug release (CIDR) device. Two doses of prostaglandin F2α were given concurrently with the last two FSH treatments, and then the CIDR was removed. Standing estrus was observed approximately 48 h after prostaglandin injection (Day 0). GnRH was then administered at estrus. Each cow was inseminated within 12 h and 24 h after standing estrus. Day 14 (D14) elongated embryos were collected by conventional non-surgical uterine flushing.

[0442] Derivation and Culture of Bovine TSCs

[0443] Each blastocyst was placed in a separate well of a 12-well plate seeded with mitomycin C-treated mouse embryonic fibroblasts (MEFs). The embryos were cultured in bovine TSC medium, which contained DMEM:F12 (Gibco), Neurobasal medium (Gibco) (1:1), 0.5x N2 supplement (Gibco), 0.5x B27 supplement (Gibco), 1x NEAA (Gibco), 1x GlutaMAX (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), 0.1% BSA (MP Biomedicals), 10 ng / mL LIF (Peprotech, 300-05), 3 mM CHIR99021 (Sigma, SML1046), 2 mM dimethindene maleate (DiM) (Tocris, 1425), and 2 mM minocycline hydrochloride (MiH) (Santa Cruz, sc-203339). The cells were cultured at 38.5 °C and 5% CO2. Forty-eight hours after plating, unattached embryos were pressed against the bottom of the plate with a needle under a microscope. The medium was changed daily. On day 7 or 8, the outgrowths were dissociated with dispase (StemCell Technologies) at 38.5 °C for 5–10 min and then washed twice with DMEM / F12. bTSCs were mechanically passaged under a microscope. For optimal survival rate, 10 mM Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Tocris, 1254) was added to the medium for 24 h.

[0444] Once established, bTSCs were passaged every 6 days at a split ratio of 1:6 using Accutase (Gibco, A1110501). Each well of bTSCs was dissociated with 1 mL Accutase at 38.5 °C for 5 min, and the reaction was neutralized by diluting Accutase with the same volume of bTSC medium. According to the manufacturer's instructions, bTSCs were cryopreserved with ProFreeze cryopreservation medium (Lonza, 12-769E).

[0445] For feeder-free conditions, bTSCs cultured on feeders were passaged into plates coated with Matrigel (Corning, 354234), where MEF-conditioned bTSC medium (MEF-bTSC) was used.

[0446] Differentiation of Bovine TSCs

[0447] Bovine TSCs were grown to 80%–90% confluence in bTSC medium and dissociated with TrypLE (Gibco, 12605-010) at 38.5 °C for 15 min. Then, the bTSCs were seeded at 1–1.5×10 5Cells were seeded at a density of

[0448] Immunofluorescence Analysis

[0449] Cells or blastocysts were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature and then rinsed three times in wash buffer (0.1% Triton X-100 and 0.1% polyvinylpyrrolidone in PBS). After fixation, cells were permeabilized with 1% Triton X-100 in PBS for 30 min and then rinsed with wash buffer. Cells were then transferred to blocking buffer (0.1% Triton X-100, 1% BSA, and 0.1 M glycine) for 2 h at room temperature. Subsequently, cells were incubated with primary antibodies overnight at 4°C. Primary antibodies used in this experiment included anti-SOX2 (Bethyl Laboratories, an833), anti-CDX2 (Bethyl Laboratories, MU392A; 1:200), anti-GATA3 (Cell Signaling Technology, D13C9; 1:200), and anti-KRT8 (Origene, BP5075; 1:300). For secondary antibody incubation, cells were incubated with secondary antibodies conjugated to Fluor 488 or 555 or 633 for 1 h at room temperature. Nuclei were stained with ProLong Diamond Antifade Mountant (with DAPI). Images were taken using a fluorescence confocal microscope (Leica).

[0450] Paraffin sections were deparaffinized and then boiled in sodium citrate buffer (pH 6.0) for 20 min for antigen retrieval. Sections were blocked in 5% goat serum in TBST for 1 h and incubated with primary antibodies overnight at 4°C. Primary antibodies used in this experiment included anti-MMP2 (Cell Signaling Technology, 40994; 1:200), anti-PL-1 (Santa Cruz Biotechnology, sc-376436; 1:200), and anti-PTGS2 (Sigma-Aldrich, SAB2500267; 1:100 - 1:200). Then, sections were incubated with fluorescently conjugated secondary antibodies for 1 h at room temperature. Nuclei were stained with DAPI (Invitrogen, D1306).

[0451] Quantitative Real-Time PCR

[0452] Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. First-strand cDNA was synthesized using the iScript cDNA Synthesis Kit (BIO-RAD). qRT-PCR was performed using SYBR Green PCR Master Mix (BIO-RAD) and specific primers (Table 2). Data were analyzed using the BIO-RAD software provided with the instrument. Relative gene expression values were calculated using the ΔΔCT method and normalized to the internal control GAPDH.

[0453] Table 2. Primer list.

[0454]

[0455] IFNT Activity Analysis

[0456] IFNT activity was measured by an established IFN-stimulated response element-reporter gene assay. 21 Briefly, 5 - 10×10 5 Madin-Darby bovine kidney cells (MDBK) stably transduced with the ISRE-Luc reporter gene were plated into the wells of a 96-well polystyrene plate (Corning) with an opaque wall and an optically clear bottom and cultured in MDBK growth medium (high glucose DMEM, 10% FBS, and 1% Pen / Strep) at 37 °C for 4 hours. After removing the MDBK growth medium, 50 μL of sample or standard (recombinant human IFN-α, IFNA: Millipore, IF007) was added. A standard curve was generated by 1:3 serial dilutions of IFNA. The cells were incubated at 37 °C for 16 hours, and then 50 μL of One-Glow luciferase reagent (Promega Corp; E6120) was added to each well for a final volume of 100 μL. After mixing on an oscillator platform for 10 minutes, measurements were taken on a plate reader.

[0457] TSC Focus Assay

[0458] bTSC cells were grown to approximately 80% confluence in bTSC medium and dissociated with TrypLE. 5×10 6The bovine TS cells were resuspended in 200 μL of a 1:1 mixture of bTSC medium and Matrigel and subcutaneously injected into 6-month-old non-obese diabetic (NOD)-severe combined immunodeficient (SCID) mice. Lesions were collected on days 7 and 9 and fixed overnight in 4% PFA at 4 °C for analysis.

[0459] Karyotype Analysis Assay

[0460] The bTSCs were incubated in bTSC medium containing 1 mL of KaryoMAX colcemid solution (Gibco, 15212012) at 38.5 °C for 4 - 5 hours. The cells were then dissociated using 1 mL of trypsin (Gibco, 25200-056) at 38.5 °C and centrifuged at 300×g for 5 min. The cells were resuspended in 1 mL of PBS solution and centrifuged at 400×g for 2 min. The supernatant was aspirated and 500 μL of 0.56% KCl was added to resuspend the cells. The cells were incubated for 15 min and then centrifuged at 400×g for 2 min. 1 mL of cold fresh Carnoy's fixative (3:1 methanol:acetic acid) was added to resuspend the cells, and then the cells were incubated on ice for 10 min. After centrifugation, 200 μL of Carnoy's fixative was added to resuspend the cells. The cells were dropped onto clean slides and air-dried, and then soaked in a solution (1:25 Giemsa stain (Sigma, GS500): deionized water) for 9 min. The slides were rinsed with deionized water and air-dried. Images were taken at 1000× magnification using a Leica DM6B under oil immersion.

[0461] RNA Sequencing Analysis

[0462] Total RNA of bovine TSCs was extracted using the RNeasy Micro Kit (Qiagen). The trophectoderm from day 7 blastocysts was isolated as follows: embryos were placed in a Petri dish containing phosphate-buffered saline and microsurgery was performed using a microsurgical blade under a microscope. RNA-seq libraries were generated using the Smart-seq2 v4 kit with minor modifications according to the manufacturer's instructions. Briefly, mRNA was captured and amplified using the Smart-seq2 v4 kit (Clontech). After purification with AMPure XP beads, the amplified RNA was quality-checked using the Agilent High Sensitivity D5000 kit (Agilent Technologies). Library preparation was performed on the high-quality amplified RNA (Nextera XT DNA Library Preparation Kit; Illumina) and multiplexing was carried out using Nextera XT Indexes (Illumina). After purification of the library with AMPure XP beads (Beckman Coulter), the concentration of the sequencing library was determined using the Qubit dsDNA HS Assay Kit (Life Technologies). The size of the sequencing library was determined by high-sensitivity D5000 assay in the Tapestation 4200 system (Agilent). The pooled indexed library was then sequenced on the Illumina NovaSeq platform with 150-bp paired-end reads.

[0463] StringTie was applied to quantify gene expression profiles from the raw sequencing data by using the Ensembl bovine genome annotation (ARS-UCD1.2). Transcripts per million reads (TPM) was used as the unit of gene expression. DESeq2 48 was applied to identify differentially expressed genes. The TMM algorithm implemented in the DESeq2 package was used to normalize read counts and estimate the effective library size. Differential expression analysis was performed by the likelihood ratio test implemented in the DESeq2 package. Conventional statistical analysis was performed based on the R platform. The Spearman rank correlation test was performed using the "cor.test" function. Principal component analysis (PCA) of gene expression profiles was performed using the "dudi.pca" function in the "ade4" package. Heatmaps were drawn using the "heatmap.2" function in the "gplots" package. Gene ontology and pathway analysis were performed by the David tool. 49 56

[0464] Overall, we sequenced two replicates of bTSC, trophoblasts differentiated on days 2, 3, 4, 5, and 6, three replicates of whole blastocysts and day 7 trophectoderm cells selected from the same batch used for bTSC derivation. Bovine day 14 trophectoderm was downloaded from a previous publication 31 , ESC 34 and EPSC 32 RNA-seq datasets.

[0465] ATAC-seq Analysis

[0466] ATAC-seq libraries of fresh cells were prepared as previously described. 55 Briefly, cells or embryos were lysed on ice and then incubated with Tn5 transposase (TDE1, Illumina) and tagging buffer. Tagmented DNA was purified using the MinElute Reaction Cleanup kit (Qiagen). ATAC-seq libraries were amplified with Illumina TrueSeq primers and multiplexed with index primers. Finally, high-quality indexed libraries were pooled together and sequenced on the Illumina NovaSeq platform with 150-bp paired-end reads.

[0467] ATACseq analysis followed our established analysis pipeline. 55 Quality-assessed ATAC-seq reads were aligned to the bovine reference genome using Bowtie 2.3 with the following options: –very-sensitive -X 2000 –no-mixed –no-discordant. Alignments caused by PCR duplicates or positions in mitochondria were excluded. Only unique alignments within each sample were retained for subsequent analysis. MACS2 was used to call ATAC-seq peaks separately for each sample with the following options: –keep-dup all –nolambda –nomodel. ATAC-seq bigwig files were generated using bamcoverage of deeptools. ATAC-seq signals were normalized in the Integrative Genome Viewer genome browser. Annotations of genomic features (including transcription start sites, transcription termination sites (TES), promoters, CDS, introns, 5’UTR, 3’UTR, and intergenic regions) were downloaded from the UCSC genome browser. The enrichment of transcription factor motifs in the peaks was evaluated using HOMER (http: / / homer.ucsd.edu / homer / motif / ). For downstream analysis, we normalized read counts by calculating counts scaled by the number of sequenced fragments multiplied by one million (CPM).

[0468] Whole Genome Bisulfite Sequencing (WGBS) Analysis

[0469] The WGBS library was prepared using the TruSeq DNA Methylation Library Preparation Kit (Illumina). Briefly, genomic DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions. Then, approximately 500 ng of DNA was bisulfite-treated using the EZ DNA Methylation Kit (Zymo Research). The bisulfite-converted DNA was end-repaired, dA-tailed, and ligated with adapters according to the TruSeq DNA Methylation Library Preparation Kit protocol. Finally, high-quality indexed libraries were pooled and sequenced on the Illumina NovaSeq platform with 150-bp paired-end reads.

[0470] WGBS data analysis followed our established analysis pipeline. 57,58 Briefly, the WGBS raw data had the first 12-bp at both 5' ends removed and reads with adapters and low-quality bases were removed using TrimGalore-0.4.3. The trimmed sequences were mapped to the bovine genome (ARS-UCD1.2) using Bismark. Then, unique mapped reads, PCR duplicates, and unconverted reads were removed using deduplicate_bismark and filter_non_conversion. To avoid sequencing bias, only reads with 10x coverage were used for downstream analysis. Methylation at each CpG site was calculated, and methylation of each sample was calculated by averaging the methylated genomic windows of 300-bp contiguous blocks. Differentially methylated regions (DMRs) were defined as 300-bp blocks common between two comparison groups, with a methylation level ≥75% in one group and ≤25% in the other group, and with a significant difference by Fisher's exact test (P value ≤0.05, FDR ≤0.05). Hypermethylated and hypomethylated blocks refer to those with DNA methylation levels ≥75% and ≤25%, respectively. Gene ontology and pathway analysis were performed using the David tool. 56

[0471] Quantitative and statistical analysis

[0472] Statistical differences between datasets were analyzed by two-tailed unpaired t-tests. Values with p < 0.05 were considered statistically significant. Quantitative data are presented as mean ± SD. Replicate numbers are indicated as "n" in the legends.

[0473] *****

[0474] Equivalents

[0475] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

1. A method for culturing, expanding, or growing a cell population derived from a mammalian blastocyst, the method comprising culturing these cells derived from a mammalian blastocyst in a culture medium for a certain period of time, wherein the culture medium contains human leukemia inhibitory factor (hLIF), an inhibitor of glycogen synthase kinase-3 (GSK-3), an antagonist of muscarinic M2 and histamine H1 receptors, an inhibitor of matrix metalloproteinase (MMP), or any combination thereof.

2. The method according to claim 1, wherein these cultured cells remain undifferentiated.

3. The method according to claim 1, wherein these cultured cells are capable of differentiating.

4. The method according to claim 1, wherein the method further comprises placing a mammalian blastocyst in a container seeded with fibroblasts and adding the culture medium, thereby providing a cell population derived from a mammalian blastocyst.

5. The method according to claim 1, wherein the cell population comprises trophoblast stem cells, trophoblast stem cell-like cells, or derivatives thereof.

6. The method according to claim 1, wherein the mammalian is a cow.

7. The method according to claim 2, wherein these fibroblasts comprise mouse embryonic fibroblasts.

8. The method according to claim 1, wherein the GSK-3 inhibitor comprises CHIR99021.

9. The method according to claim 1, wherein the antagonist of muscarinic M2 and histamine H1 receptors comprises dimenhydrinate maleate (DiM).

10. The method according to claim 1, wherein the inhibitor of matrix metalloproteinase (MMP) comprises minocycline hydrochloride (MiH).

11. The method according to claim 1, wherein the amount of human leukemia inhibitory factor (hLIF) is about 10 ng / ml.

12. The method according to claim 1, wherein the amount of the glycogen synthase kinase-3 inhibitor is about 3 μM.

13. The method according to claim 1, wherein the amount of the antagonist of muscarinic M2 and histamine H1 receptors is about 2 μM.

14. The method according to claim 1, wherein the amount of the matrix metalloproteinase inhibitor is about 2 μM.

15. The method according to claim 2, wherein the container comprises a culture dish, a flask, a well, a tube, or a plate.

16. The method according to claim 2, wherein the container comprises a solid surface or a porous surface.

17. The method according to claim 1, wherein these cells are cultured on a surface coated with an extracellular matrix or a component of an extracellular matrix.

18. The method according to claim 15, wherein the extracellular matrix is Matrigel TM or a Matrigel TM -like substance.

19. The method according to claim 1, wherein the surface is not Matrigel TM .

20. The method according to claim 1, wherein these trophoblast stem cells are cultured in the absence of fibroblast feeder cells.

21. An in vitro cell culture comprising a cell population derived from a mammalian blastocyst produced by the method according to claim 1.

22. The in vitro cell culture according to claim 21, wherein the cells comprise trophoblast stem cells, trophoblast stem cell-like cells or derivatives.

23. The in vitro cell culture according to claim 21, wherein the cells comprise undifferentiated cells.

24. The in vitro cell culture according to claim 21, wherein the cells are capable of self-renewal.

25. An isolated cell derived from a mammalian blastocyst, wherein the isolated cell expresses at least one pluripotency marker.

26. The isolated cell according to claim 25, wherein the isolated cell comprises trophoblast stem cells, trophoblast stem cell-like cells or derivatives thereof.

27. The isolated cell according to claim 25, wherein the at least one marker comprises GATA3, CDX2, ELF3, TFAP2A, KLF5, KRT8, SFN, DNMT1, DNMT3A, PAG2, PAG11, PAG12, CYP17A1, HSD3B1, HAND1 or any combination thereof.

28. The isolated cell according to claim 25, wherein the at least one marker comprises a marker of the Wnt signaling pathway, the LIF signaling pathway, the HIF-1 signaling pathway, the AMPK signaling pathway or any combination thereof.

29. The isolated cell according to claim 25, wherein the cell is undifferentiated.

30. The isolated cell according to claim 25, wherein the cell is capable of self-renewal.

31. The isolated cell according to claim 25, wherein the cell is capable of differentiating into cells of the trophoblast lineage in vitro and in vivo.

32. The isolated cell according to claim 25, wherein the mammal is a cow.

33. A method for evaluating a candidate compound, the method comprising contacting an in vitro cell culture as described in claim 21 or an isolated cell as described in claim 25 with a quantity of the candidate compound, and evaluating a characteristic of the in vitro cell culture or the isolated cell.

34. The method according to claim 33, wherein the characteristic is cell growth, cell development, differentiation, apoptosis, trophoblast development, trophoblast activity, or any combination thereof.

35. A cell culture comprising a population of bovine embryonic stem cells in a culture medium, wherein the culture medium comprises one or more factors selected from the group consisting of: human leukemia inhibitory factor (hLIF), an inhibitor of glycogen synthase kinase-3 (GSK-3), an antagonist of muscarinic M2 and histamine H1 receptors, and an inhibitor of matrix metalloproteinase (MMP), or any combination thereof.

36. The cell culture according to claim 35, wherein the culture medium comprises human leukemia inhibitory factor (hLIF), an inhibitor of glycogen synthase kinase-3 (GSK-3), an antagonist of muscarinic M2 and histamine H1 receptors, and an inhibitor of matrix metalloproteinase (MMP).

37. The cell culture according to claim 35, wherein the amount of the human leukemia inhibitory factor (hLIF) is about 10 ng / ml.

38. The cell culture according to claim 35, wherein the amount of the glycogen synthase kinase-3 inhibitor is about 3 μM.

39. The cell culture according to claim 35, wherein the amount of the antagonist of muscarinic M2 and histamine H1 receptors is about 2 μM.

40. The cell culture according to claim 35, wherein the amount of the matrix metalloproteinase inhibitor is about 2 μM.

41. The cell culture according to claim 35, wherein the inhibitor of glycogen synthase kinase-3 (GSK-3) is CHIR99021, the antagonist of muscarinic M2 and histamine H1 receptors is dimenhydrinate maleate (DiM), and the inhibitor of matrix metalloproteinase (MMP) is minocycline hydrochloride (MiH).

42. The cell culture according to claim 35, wherein the culture is in a microplate.

43. The cell culture according to claim 35, wherein the cell culture further comprises a population of trophoblast stem cells.

44. The cell culture according to claim 43, wherein the population of trophoblast stem cells comprises a population of bovine trophoblast stem cells.

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