Derivation of naïve bovine embryonic stem cells

Abstract

The present disclosure relates to methods, products, and compositions useful for the derivation of naive bovine embryonic stem cells. Described is an ECM-coated substrate that includes a positively charged biocompatible polymer that allows embryo attachment and outgrowth formation. Also provided is an outgrowth medium suitable for culturing bovine embryos and derivation of naive embryonic stem cells. Naive bovine embryonic stem cells can be used in in vitro breeding programs, including expanding preimplantation embryos with desired genetic characteristics; derivation of primordial germ cells and / or gametes for in vitro breeding programs, and developing and providing veterinary biologics and therapeutics.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 278,751, filed November 12, 2021, the contents of which are incorporated by reference herein in their entirety.

[0002] The present disclosure relates to bovine stem cells, and more particularly to naive bovine embryonic stem cells, related methods and compositions. [Background technology]

[0003] introduction Naïve embryonic stem cells can differentiate into any type of cell in the body, including extraembryonic cells such as trophoblast stem cells and extraembryonic endoderm lineage cells. Mouse blastocyst-like structures (also called induced blastoids) have been generated by differentiating expanded pluripotent stem (EPS) cells (Li et al., 2019), and more recently, human induced blastoid structures have been generated using either embryonic stem cell-derived naïve pluripotent stem cells (Yu et al., 2021) or induced pluripotent stem cells (iPS, Liu et al., 2021). However, unlike mice or humans, the derivation of bovine iPS cells has not yet been reported.

[0004] There are very few reports on bovine primed embryonic stem cells derived from preimplantation embryos, more specifically the embryonic inner cell mass (ICM) structure (Bogliotti et al. 2018; Soto et al., 2021). However, primed ESCs do not differentiate into extraembryonic cells and therefore cannot be directly used for the generation of induced blastoid structures. Furthermore, primed ESCs cannot be efficiently used for germ cell differentiation for use in in vitro breeding (Hou et al., 2018). Naïve embryonic stem cells, which represent the ground state of pluripotency (preimplantation ICM), can differentiate into germ cells more efficiently than primed ES cells (De Los Angeles, 2019).

[0005] In contrast to the use of nuclear transfer (cloning) approaches, which are labor intensive and generally limited by low embryo production rates (approximately 20-30% of blastocyst formation), the derivation of bovine embryonic stem cells may facilitate efficient propagation of embryos with desired characteristics. Desirable genetic characteristics in embryos may arise naturally, may result from processes such as meiosis, mutation, and introgression, which can be generated using assisted reproductive techniques such as Smith et al., WO 2020 / 168422, or genetic modification. Additionally, animal and veterinary sciences can realize broad benefits from naïve stem cell technology by providing an efficient platform for the large-scale production of genetically modified animals, providing a key component technology for in vitro breeding programs, and enabling the development and delivery of advanced veterinary biologics and therapeutics. There remains a need for methods for naïve bovine embryonic stem cells, and associated induced blastoid generation. Summary of the Invention

[0006] Described herein are materials and methods useful for achieving attachment and outgrowth formation using bovine embryos, such as morula and blastocyst stage embryos, to establish bovine embryonic stem cells.As demonstrated in the examples, the zona pellucida (ZP)-removed bovine embryos plated on layered ECM-coated substrates provide higher attachment rate and outgrowth formation compared to embryos plated on conventional ECM-coated substrates.

[0007] Further described are outgrowth medium compositions and associated methods that support embryo attachment and outgrowth formation, and the proliferation of inner cell mass (ICM) cells from such outgrowths, and allow for the derivation of naive embryonic stem cells from the ICM. Thus, embodiments described herein are useful for derivation of naive bovine embryonic stem cells, and optionally for use in breeding programs, such as, for example, to create induced blastoid structures, to grow preimplantation embryos with desirable genetic characteristics, to derive primordial germ cells and / or gametes for in vitro breeding programs, and / or to develop and provide veterinary biologics and therapeutics.

[0008] Thus, in one aspect, a method for deriving naive bovine embryonic stem cells is provided. In one embodiment, the method comprises: providing a zona pellucida (ZP)-removed bovine embryo containing naive bovine embryonic stem cells; contacting the ZP-removed bovine embryos with an extracellular matrix (ECM) coated substrate, the ECM coated substrate comprising a substrate comprising a negatively charged substrate surface adjacent to a positively charged biocompatible polymer layer and a negatively charged ECM layer adjacent to the positively charged biocompatible polymer layer; Culturing the ZP-removed bovine embryos in the presence of an elongation medium to induce attachment of the ZP-removed bovine embryos to the ECM-coated substrate and elongation of an inner cell mass (ICM) comprising induced naive bovine embryonic stem cells.

[0009] In one embodiment, the bovine embryo is genetically modified.

[0010] In another embodiment, the ZP removed bovine embryos are obtained from reconstituted diploid embryos.

[0011] In one embodiment, the biocompatible polymer is negatively charged at physiological pH, hi one embodiment, the biocompatible polymer is type A gelatin.

[0012] In one embodiment, the ECM comprises an EHS-ECM.

[0013] In one embodiment, the substrate comprises polystyrene.

[0014] In one embodiment, the outgrowth medium comprises a basal medium and one or more components described herein useful for inducing attachment of ZP-removed embryos to ECM-coated substrates and outgrowth of ICM in a feeder-free culture system. For example, in one embodiment, the outgrowth medium comprises one or more of: a 1:1 mixture of DMEM / F12 and Neurobasal medium; an N2B27 component; a Wnt activator component; a Wnt inhibitor component; a MEK / ERK inhibitor component; a ROCK inhibitor component; a LIF component; a PKC inhibitor component; and an insulin component. Optionally, the outgrowth medium further comprises an activin A component.

[0015] In one embodiment, the outgrowth medium comprises an N2B27 component; a Wnt activator component; a Wnt inhibitor component; a MEK / ERK inhibitor component; a ROCK inhibitor component; a LIF component; an activin A component; a PKC inhibitor; and an insulin component.

[0016] In one embodiment, the N2B27 component comprises B27 supplement and N2 supplement, optionally comprising about 1% B27 supplement and about 0.5% N2 supplement; the Wnt activator component comprises CHIR99021, BIO, CHIR-98014, LY2090314, and / or IM-12; the Wnt inhibitor component comprises XAV939, IWR-1, and / or IWP-2; the MEK / ERK inhibitor component comprises PD0325901, ravoxertinib, GSK1120212, MEK162, PD18435 2, trametinib, LY3214996, and / or ulixertinib; the ROCK inhibitor component comprises Y27632, thiazovivin, and / or blebbistatin; the LIF component comprises human LIF; the activin A comprises human activin A; the PKC inhibitor comprises Go6983, Go6976, LY317615, LY333531, PKC412, GSK690693, sotrastaurin, staurosporine, and / or bisindolylmaleimide; and / or the insulin component comprises insulin.

[0017] In one embodiment, the ZP removed bovine embryo is a morula (stage 4); a blastocyst (stage 5); an expanding blastocyst (stage 6); an expanding blastocyst (stage 7); a hatching blastocyst (stage 8); or a hatched blastocyst (stage 9).

[0018] In one embodiment, the ZP removed bovine embryos are obtained by enzyme-assisted ZP removal.

[0019] In one embodiment, the method comprises obtaining ZP-removed bovine embryos by enzyme-assisted ZP removal.

[0020] Also provided herein is a method for enzyme-assisted ZP removal, comprising the steps of: a) providing an embryo; b) contacting the embryo with a protease solution; c) incubating the embryo in the protease solution to partially digest the ZP and obtain a ZP-thinned embryo; d) contacting the ZP-thinned embryo with a protease inactivation medium to inactivate the protease; e) disrupting the ZP; and f) manipulating the embryo to separate the ZP from the embryo.

[0021] In one embodiment, the concentration of the protease in step c) is about 0.1% to about 0.5%, about 0.2% to 0.3%, or about 0.25%.

[0022] In one embodiment, the embryos and protease solution are incubated in step c) for about 30-60 seconds, optionally for about 45 seconds.

[0023] In one embodiment, the ZP is disrupted in step e) using a microblade.

[0024] In one embodiment, the manipulation of the embryo in step f) comprises pipetting.

[0025] In one embodiment, the method further comprises performing genetic testing to determine one or more genotypes of the ZP-removed bovine embryos for one or more biomarkers.

[0026] In one embodiment, the method further comprises selecting ZP removed bovine embryos based on genetic testing of one or more biomarkers.

[0027] In one embodiment, the method further comprises performing a genetic test to determine one or more genotypes of the induced naive bovine embryonic stem cells.

[0028] In one embodiment, the ZP removed bovine embryos are obtained from fresh embryos, optionally fresh biopsy embryos.

[0029] In one embodiment, the embryo in step a) is a genetically modified embryo.

[0030] In one embodiment, the ZP removed bovine embryos are obtained from frozen embryos, optionally biopsy frozen embryos.

[0031] In one embodiment, the method comprises thawing a frozen embryo and contacting the embryo with a recovery medium.

[0032] In one embodiment, the ZP-removed bovine embryo is obtained by a method comprising thawing a frozen embryo; contacting the frozen embryo with a recovery medium; manipulating the embryo to separate ZP from the embryo in the recovery medium; and incubating the ZP-removed bovine embryo in the recovery medium.

[0033] In one embodiment, the recovery medium comprises a glycogen synthase kinase 3 (GSK-3) inhibitor, a MEK / ERK kinase inhibitor, and a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor.

[0034] In one embodiment, the recovery medium comprises CHIR99021, PD0325901, and Y27632.

[0035] In one embodiment, the method further comprises incubating the ZP removed embryos with an adaptation medium, wherein the adaptation medium comprises a combination of recovery medium and elongation medium.

[0036] In one embodiment, the adaptation medium comprises a combination of recovery medium and elongation medium in a ratio between about 0.5:1 and 1.5:1, optionally in a ratio of about 1:1.

[0037] In another aspect, naive bovine stem cells derived using the methods described herein are provided. Further aspects include the use of naive bovine stem cells derived using the methods described herein in breeding schemes or genetic improvement programs, and for expanding preimplantation embryos with desirable genetic characteristics, deriving primordial germ cells and / or gametes for in vitro breeding programs, and / or for developing and providing veterinary biologics and therapeutics.

[0038] In another aspect, a method of preparing an extracellular matrix (ECM) coated substrate is provided, the method comprising: providing a substrate comprising a negatively charged surface; contacting the negatively charged surface with a first solution comprising a biocompatible polymer, the biocompatible polymer being positively charged; incubating the substrate in contact with the first solution such that a layer of the positively charged biocompatible polymer is deposited on the negatively charged surface of the substrate; removing the first solution and optionally washing the substrate; contacting the substrate with a second solution comprising an extracellular matrix (ECM), the ECM being negatively charged; and incubating the substrate in contact with the second solution such that a layer of the negatively charged ECM is deposited on the layer of the positively charged biocompatible polymer.

[0039] In one embodiment, the biocompatible polymer comprises type A gelatin.

[0040] In one embodiment, the ECM comprises an EHS-ECM.

[0041] In one embodiment, the substrate comprises polystyrene.

[0042] One embodiment includes an ECM coated substrate made according to the methods described herein.

[0043] Further embodiments include ECM coated substrates comprising: a positively charged surface; a layer of a positively charged biocompatible polymer in contact with a negatively charged surface of the substrate; and a layer of a negatively charged ECM in contact with the layer of positively charged biocompatible polymer.

[0044] In one embodiment, the biocompatible polymer comprises type A gelatin.

[0045] In one embodiment, the ECM comprises an EHS-ECM.

[0046] In one embodiment, the substrate comprises polystyrene.

[0047] One aspect of the present disclosure includes the use of an ECM coated substrate as described herein for culturing embryos, and optionally for inducing ICM outgrowth and / or inducing naive embryonic stem cells.

[0048] In one embodiment, the embryo is a bovine embryo, optionally between day 5 and day 7 of a bovine embryo.

[0049] In another embodiment, the bovine embryo is a reconstituted diploid embryo.

[0050] In one embodiment, the embryo is a genetically modified embryo.

[0051] In another aspect, a medium composition is provided that includes a basal medium and one or more components for culturing an embryo, and optionally for inducing ICM outgrowth and / or inducing naive embryonic stem cells. In one embodiment, the medium composition includes one or more of: an N2B27 component; a Wnt activator component; a Wnt inhibitor component; a MEK / ERK inhibitor component; a ROCK inhibitor component; a LIF component; a PKC inhibitor; and an insulin component. Optionally, the medium composition further includes an activin A component.

[0052] In one embodiment, the N2B27 component comprises about 1% B27 supplement and about 0.5% N2 supplement; the Wnt activator component comprises CHIR99021, BIO, CHIR-98014, LY2090314, or IM-12; the Wnt inhibitor component comprises XAV939, IWR-1, or IWP-2; and the MEK / ERK inhibitor component comprises PD0325901, ravoxertinib, GSK1120212, MEK162, PD184352, trametinib, LY321. 4996, or ulixertinib; the ROCK inhibitor component comprises Y27632, thiazovivin, or blebbistatin; the LIF component comprises human LIF; the activin A component comprises human activin A; the PKC inhibitor comprises Go6983, Go6976, LY317615, LY333531, PKC412, GSK690693, sotrastaurin, staurosporine, or bisindolylmaleimide; and / or the insulin component comprises insulin.

[0053] Further aspects include using the media compositions described herein to culture an embryo to induce ICM outgrowth formation, optionally wherein the embryo is a bovine embryo, a reconstituted diploid bovine embryo, and / or wherein the embryo is a genetically modified embryo.

[0054] Further embodiments include recovery medium comprising: a glycogen synthase kinase 3 (GSK-3) inhibitor, optionally CHIR99021, a MEK / ERK kinase inhibitor, optionally PD0325901, and a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor, optionally Y27632.

[0055] The preceding sections are provided by way of example only and are not intended to limit the scope of the disclosure and the appended claims. Additional objects and advantages associated with the compositions and methods of the present disclosure will be understood by those skilled in the art in view of the claims, description, and examples. For example, the various aspects and embodiments of the present disclosure can be utilized in numerous combinations, all of which are expressly contemplated by this specification. These additional advantages, objects, and embodiments are expressly included within the scope of the present disclosure. Publications and other materials used herein to clarify the background of the present disclosure and, in certain cases, to provide additional details regarding the practice are incorporated by reference and are listed in the accompanying reference section for convenience. [Brief description of the drawings]

[0056] Further objects, features and advantages of the present disclosure will become apparent from the following detailed description taken in conjunction with the accompanying drawings illustrating exemplary embodiments of the present disclosure, in which: [Figure 1] Shown are the results of an experiment to determine the attachment and outgrowth rates of embryos plated on different coating materials: (top left) Nunc 35 mm culture dish with coating solution, (top right) attachment and outgrowth efficiency as a function of coating material, (bottom left) unattached embryos, (bottom right) attached embryos. [Diagram 2] An image of the processed dish surface with Geltrex and a 30 um pore cell strainer is shown (image magnification; x100). [Diagram 3] A schematic diagram of the chemical structure of a plasma-treated polystyrene dish surface is shown. [Figure 4] A diagram of Layer-By-Layer (LbL) coating is shown. [Diagram 5] Improved induction efficiency using layer-by-layer ECM-coated substrates. LbL ECM-coated substrates showed higher and more stable attachment rates, TE (trophectoderm cells) and ICM (inner cell mass) growth compared to the control protocol using EHS-ECM alone. [Figure 6](Left) Schematic of the layout of the protease treatment dish and (right) images of protease-treated low-quality embryos in which the ZP was completely removed by enzymatic treatment. [Figure 7] Fresh embryos are shown on day 6 after ZP removal. All embryos appear healthy after ZP removal. [Figure 8] A schematic of the layout of the post-thaw recovery dish (top left) and an image showing the effect of 2iY medium on embryo recovery after thawing (top right). (Bottom) 2iY-treated embryos showed faster recovery and higher induction efficiency than control embryo culture medium, as observed by faster re-expansion of the embryos. 2iY-treated embryos showed a larger size and a more distinct ICM than the control group (dashed circle). [Figure 9] FIG. 1 is a graph showing the effect of 2iY on post-thaw embryo quality, with 2iY-treated embryos exhibiting a more advanced developmental stage after post-thaw recovery. [Figure 10] A schematic of the layout of adaptation medium dish #1 and dish #2 is shown. At the bottom is a graph showing the effect of adaptation time on induction of elongation. [Figure 11] Graph showing induction results with different types of serum sources. FBS=fetal bovine serum; KOSR=knock-out serum replacement; SR=serum replacement; N2B27=N2 supplement + B27 supplement (see examples). [Figure 12] FIG. 1 is a schematic diagram showing various Wnt signaling pathways and targets. [Figure 13] FIG. 1 is a graph showing the effect of forskolin and dual kinase Wnt pathway modulation on the induction of naive stem cells. [Figure 14] The figure shows the effect of IL-6 and SRC inhibitors on outgrowth induction, with the bottom part being a schematic showing various pathways downstream of SRC. [Figure 15] 1 shows the effect of MEK inhibitors on the formation of naive outgrowths. [Figure 16]FIG. 13 is a graph showing the induction efficiency between naive stem cell medium (left) and images showing outgrowth colonies induced by t2iL GoY medium. [Figure 17] (Left) Endodermal differentiation of outgrowths. ICM colonies (arrows) are surrounded by undifferentiated (arrowheads) and differentiated (stars) endodermal cells. (Right) ICM cell colonies (clusters of cells with light borders) after the first passage following insulin addition. [Figure 18] Based on the derivation of naive bovine embryonic stem cells, the steps involved in the derivation of cell lines to subsequently generate primordial germ cell lines and gametes and generate multiple genetically identical embryos (also called induced blastoids) are outlined. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] The following is a detailed description provided to assist those skilled in the art in practicing the present disclosure.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure belongs.The technical terms used in the description of this specification are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure.All publications, patent applications, patents, drawings and other references mentioned in this specification are expressly incorporated by reference in their entirety.

[0058] Moreover, it is intended that the definitions and embodiments described in a particular section are applicable to other embodiments described herein where they are suitable, as will be understood by those skilled in the art. For example, in the following passages, various aspects of the present disclosure are defined in more detail. Each aspect so defined can be combined with any other aspect, unless expressly indicated to the contrary. In particular, any feature described herein can be combined with any other feature described herein.

[0059] I. General Definitions As used herein, the following terms may have the meanings ascribed to them unless otherwise specified. However, it is understood that other meanings known or understood by those skilled in the art are possible and fall within the scope of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0060] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, as well as any other stated or intervening value in that stated range, is encompassed herein unless the context clearly dictates otherwise. Ranges from any lower limit to any upper limit are contemplated. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed herein, subject to any specific excluded limit in the stated range. Where a stated range includes one or both limits, ranges excluding either (or) both of those included limits are also included in the invention.

[0061] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0062] All numerical values ​​within the detailed description and claims herein are modified by "about" or "approximately" the indicated value to account for experimental error and variations that would be expected by one of ordinary skill in the art.

[0063] The term "and / or" as used in the specification and claims should be understood to mean "one or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether or not associated with the specifically identified elements.

[0064] "Or" as used herein in the specification and claims should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one of the elements or elements of the list, and optionally additional items not listed, but also including one or more. Only terms clearly indicating the contrary, such as "only one of" or "exactly one of," or "consisting of," when used in the claims, mean to include exactly one element of the elements or elements of the list. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by terms implying exclusivity such as "either," "one of," "only one of," or "exactly one of."

[0065] In the claims as well as in the above specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are used in an open-ended manner, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are closed or semi-closed transitional phrases, respectively.

[0066] As used herein and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from one or more elements in the list of elements, and not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for elements to be optionally present other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether or not related to those specifically identified elements.

[0067] As used herein, the term "about" means plus or minus 10% to 15%, 5 to 10%, or optionally about 5% of the referenced numerical value.

[0068] For certain methods described herein that include two or more steps or actions, it should be understood that the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described, unless otherwise indicated by context.

[0069] It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.

[0070] II. Method Described herein are methods for the outgrowth formation of naive embryonic stem cells and the derivation of naive embryonic stem cells. As described in the Examples, the inventors have demonstrated naive bovine stem cell outgrowth formation in a feeder-free culture system comprising a layered ECM-coated substrate and a specifically formulated outgrowth medium. The layered ECM-coated substrate and outgrowth medium support the ex vivo or in vitro attachment and outgrowth of inner cell mass (ICM) cells derived from embryos such as morula or blastocyst stage (e.g., bovine day 6 or day 7) embryos. Thus, the materials and methods described herein are useful for the derivation and maintenance of naive bovine embryonic stem cells, and optionally for use in breeding programs, such as, for example, for expanding preimplantation embryos with desired genetic characteristics and / or generating induced blastoid structures, for inducing primordial germ cells and / or gametes for in vitro breeding programs, and for developing and providing veterinary biologics and therapeutics. The desired genetic characteristics may arise by natural processes or by genetic modification.

[0071] Thus, in one aspect, a method for deriving naive bovine embryonic stem cells is provided. In one embodiment, the method comprises: providing a zona pellucida (ZP)-removed bovine embryo containing naive bovine embryonic stem cells; contacting the ZP-removed bovine embryos with an extracellular matrix (ECM) coated substrate, the ECM coated substrate comprising a substrate comprising a negatively charged substrate surface adjacent to a positively charged biocompatible polymer layer and a negatively charged ECM layer adjacent to the positively charged biocompatible polymer layer; Culturing the ZP-removed bovine embryos in the presence of an elongation medium to induce attachment of the ZP-removed bovine embryos to the ECM-coated substrate and elongation of an inner cell mass (ICM) comprising induced naive bovine embryonic stem cells.

[0072] As used herein, the term "naive embryonic stem cells" is used to refer to embryonic stem cells that substantially retain the molecular characteristics of cells of a morula stage embryo, e.g., a day 5 or 6 bovine embryo, where the cells are still in an undifferentiated state. Naive embryonic stem cells may be found within the inner cell mass (ICM) of a blastocyst (such as a day 7 embryo). Naive embryonic stem cells may be capable of developing into a complete organism and / or may retain the ability to give rise to a full complement of adult tissues and / or cell types. In contrast to induced pluripotent stem cells (iPSCs), naive embryonic stem cells can be derived and maintained in a self-renewing undifferentiated state without the need for expression of exogenous pluripotency factors. Similarly, the term "naive bovine embryonic stem cells" refers to naive embryonic stem cells from bovine.

[0073] Naive embryonic stem cells, for example naive bovine embryonic stem cells, can be derived from fully undifferentiated tissues, such as embryonic cells, for example, morula (stage 4); blastocyst (stage 5); expanding blastocyst (stage 6), expanding blastocyst (stage 7), hatching blastocyst (stage 8), or hatched blastocyst (stage 9). In one embodiment, naive bovine embryonic stem cells can be derived from bovine embryos at days 3-8, optionally at days 3, 4, 5, 6, 7, or 8, or at days 5-7. In one embodiment, the bovine embryo is a morula (stage 4); blastocyst (stage 5); expanding blastocyst (stage 6); expanding blastocyst (stage 7); hatching blastocyst (stage 8); or hatched blastocyst (stage 9). In one embodiment, the bovine embryo is a 3-7 day embryo, optionally a 5-7 day embryo, or a 6 or 7 day embryo. In one embodiment, the embryo is a pre-implantation embryo. In one embodiment, the embryo is a previously frozen and / or biopsied embryo. In one embodiment, the embryo is genetically modified. In one embodiment, the embryo is selected based on genetic testing of one or more biomarkers.

[0074] A "genetically modified embryo" refers to an embryo in which the genomic DNA of an embryonic cell has been manipulated to express one or more exogenous genes and / or introduce a mutation(s) into an endogenous gene or intergenic region that affects the expression or functional activity of one or more endogenous genes or gene products. Successful examples of genetic modification in bovine embryos include the introduction of transgenes by microinjection (US 7,067,713) and lentiviral infection (Park, 2007), and more recently, genome editing using transfection and genome editing tools, such as zinc finger nucleases, transcription activator-like effector nucleases (TALENs) (Bishop and Van Eenennaam, 2020), as well as the clustered regularly interspaced short palindromic repeats / CRISPR-associated gene (CRISPR / Cas) system. Common bovine targets for genetic modification are milk protein genes, e.g., β-lactoglobulin, β-casein, myostatin, horned / polled, prolactin receptors conferring a slick coat to improve heat tolerance, and various genes involved in disease susceptibility or resilience (Wang et al., 2022).

[0075] Similarly, a "genetically modified cell" refers to a cell in which the genomic DNA of the cell has been manipulated to express one or more exogenous genes and / or to introduce a mutation(s) into an endogenous gene or intergenic region that affects the expression or functional activity of one or more endogenous genes or gene products.

[0076] The zona pellucida (ZP) prevents attachment of embryonic cells to the culture substrate. Thus, in one embodiment, the ZP of the embryo is removed prior to contact with the layered ECM-coated substrate and / or the outgrowth medium. The ZP-removed embryo can be provided or obtained using any suitable method. For example, the ZP can be thinned and / or broken using enzymatic, chemical, and / or mechanical means and then separated from the embryo by mechanical manipulation to obtain the ZP-removed embryo. Suitable enzymatic or chemical means for thinning and / or breaking the ZP include, for example, the use of proteases such as pronase or acidic Tyrode's solution. Suitable mechanical methods for breaking the ZP include, for example, the use of a microblade, a micropipette, a microneedle, or a laser. The broken ZP can be separated from the embryo by agitation, such as, for example, pipetting, vortexing, or direct manipulation using a micropipette or microneedle.

[0077] In one embodiment, ZP-depleted embryos are obtained by enzyme-assisted ZP removal. As shown in the Examples, protease treatment of morula (day 6) bovine embryos, followed by mechanical disruption and separation of the ZP, results in ZP-depleted embryos suitable for deriving naïve embryonic stem cells as described herein.

[0078] In conventional embryo biopsy techniques, the ZP is disrupted, for example, using a microblade. Thus, in one embodiment, a ZP-removed embryo is obtained from a biopsy embryo.

[0079] Desirable genetic characteristics in an embryo may arise as a result of processes such as meiosis, mutation, and introgression, which may occur naturally or by genetic modification. Thus, in one embodiment, the ZP ablated embryo is obtained from a genetically modified embryo.

[0080] The embryos from which the ZP-removed bovine embryos are derived may be fresh or previously frozen, and may optionally be obtained from biopsy-frozen embryos, hi one embodiment, the embryos are genetically tested embryos.

[0081] In one embodiment, the bovine embryo is a reconstituted diploid embryo. Reconstituted diploid embryos are described, for example, in Smith et al., WO2020 / 168422, the entire contents of which are incorporated herein by reference.

[0082] Diploid embryos with a given genome can be generated in vitro by reconstituting biparental embryos using screened and selected androgenetic and parthenogenetic embryonic haploid cells (Smith et al., WO2020 / 168422). The genome of the reconstituted diploid embryo can be characterized by alleles, haplotypes, or other genetic information, such as, for example, production traits (e.g., milk, fat, protein, fat%, protein%, milk protein variant composition, e.g., A2A2 milk), meat quality traits, growth traits, health traits (e.g., somatic cell score, mastitis resistance, immune response, viability, disease resistance), reproduction traits (e.g., pregnancy rate, conception rate), calving traits (e.g., calving ease, calving to first insemination, stillbirth), conformation traits (e.g., hornless trait, udder and nipple traits, foot and leg traits, body size traits, size traits), efficiency traits, etc. Genetic or genomic traits that are uniquely characterized can be created to contain unique combinations of traits that meet stringent genetic criteria to significantly complement genetic or genomic features such as (e.g., feed utilization traits, workability, life span, productive life span), novel traits (e.g., robotic milking traits, heat tolerance, activity and behavior traits), and composite index traits (e.g., LPI (Lifetime Productivity Index), TPI (Total Productivity Index), and the absence of various deleterious alleles and haplotypes (e.g., dwarfism, monoclonal, hypotrichosis, brachyspondylosis, citrullinemia, bovine leukocyte adhesion deficiency).

[0083] Most cells in culture (except for stromal-derived cells) require a support layer to attach and grow in vitro, such as a tissue culture dish. This support layer can be created from stromal cells (that attach directly to the dish), commonly known as a feeder cell line. Mouse embryonic fibroblast (MEF) feeder systems are often used to culture embryonic stem cells. However, one drawback of using a feeder system is that cross-species contamination can occur when cells, such as embryonic stem cells, from a species different from the mouse are cultured on MEFs. Another option is to use a protein matrix as the support layer, also known as a feeder-free system.

[0084] Boggliotti et al. (2018) described the use of a feeder system for culturing bovine primed embryonic stem cells, where needles were used to press the embryos to the bottom of the culture dish to compensate for the low attachment rate. However, the use of such exogenous mechanical forces can damage the cells. In contrast, the embodiments described herein achieve attachment and outgrowth formation of less differentiated naive embryonic stem cells without mechanically pressing the cells onto the culture dish. In one embodiment, the methods and products described herein provide a feeder-free system for deriving naive embryonic stem cells.

[0085] For example, as shown in FIG. 5, ECM-coated substrates generated using a layer-by-layer (LbL) protocol with a positively charged biocompatible polymer binding layer showed higher and more stable attachment rates and growth of TE (trophectoderm cells) and ICM (inner cell mass) compared to control substrates using ECM extracted from Engelbreth-Holm-Swarm mouse sarcoma cells, e.g., Matrigel (Corning) or Geltrex (Invitrogen).

[0086] Thus, in one embodiment, a substrate is provided that includes: a positively charged surface; a layer of a positively charged biocompatible polymer in contact with the negatively charged surface of the substrate; and a layer of a negatively charged ECM in contact with the layer of positively charged biocompatible polymer.

[0087] Also provided is a method for preparing an ECM coated substrate suitable for embryo attachment and outgrowth as described herein. In one embodiment, the method comprises: a) providing a substrate comprising a negatively charged surface; b) contacting the negatively charged surface with a first solution comprising a biocompatible polymer, the biocompatible polymer being positively charged; c) contacting and incubating the substrate with the first solution such that a layer of a positively charged biocompatible polymer is deposited on the negatively charged surface of the substrate; d) removing the first solution and optionally washing the substrate; e) contacting the substrate with a second solution comprising an extracellular matrix (ECM), the ECM being negatively charged; and f) contacting and incubating the substrate with a second solution such that a layer of negatively charged ECM is deposited on the layer of positively charged biocompatible polymer.

[0088] As used herein, the term "substrate" generally refers to a physical surface onto which a layer or layers of material are deposited or adhered. The substrate may be rigid or flexible and may be made of any suitable material, for example a plastic such as polystyrene. The substrate may be treated to make it hydrophilic and / or to impart a charge, such as a negative charge, to the surface. Optionally, the substrate is plasma treated. In one embodiment, the substrate is plasma treated polystyrene (also known as tissue culture plastic).

[0089] As used herein, the term "biocompatible polymer" generally refers to a polymer that is compatible with living tissue or cells, e.g., a polymer that is non-toxic and does not cause undesirable effects on, e.g., survival, growth, proliferation, and / or other biological activities of cells. A biocompatible polymer may be inert with respect to such activities and / or may support a desired activity. A biocompatible polymer may be a naturally occurring polymer, may be prepared from a naturally occurring polymer, or may be a synthetic polymer with desired properties. A suitable biocompatible polymer has properties that result in deposition and / or adhesion of the polymer onto the surface of the substrate under the conditions used to coat the substrate with the polymer. The deposition or adhesion of the polymer may occur, for example, through electrostatic interactions, hydrogen bonding, or any other suitable type(s) of interaction. Thus, suitable properties of a biocompatible polymer may include, for example, a charge, such as a positive charge at the pH of the solution used for coating. The interaction between the polymer and the substrate needs to be maintained under the conditions (e.g., pH) used for the subsequent washing and ECM coating steps, and under the conditions (e.g., physiological pH) used for cell culture. Suitable polymers include gelatin type A (such as that derived from acid-hardened tissue). Thus, in one embodiment, the biocompatible polymer is type A gelatin, optionally type A porcine gelatin.

[0090] As understood in the art, the term "incubate" or "incubating" means maintaining, for example, a substance, material, composition, etc., at a particular temperature or within a temperature range for a period of time.

[0091] As used herein, the term "physiological pH" means a pH of about 7.1 to about 7.6, optionally about 7.15 to about 7.45, about 7.2 to about 7.4, about 7.25 to about 7.35, or about 7.3.

[0092] As used herein, the term "extracellular matrix" or "ECM" generally refers to a biocompatible matrix comprising one or more macromolecular components, such as, for example, proteins, glycosaminoglycans (GAGs), and / or proteoglycans, which provide attachment and support for the growth and proliferation of cells, such as cells grown ex vivo or in vitro. Common ECM components can include, but are not limited to, one or more of laminin, collagen (e.g., collagens I-XIV), fibronectin, vitronectin, entactin / nidogen, heparan sulfate proteoglycans, and / or one or more functional variants thereof. As known in the art, ECM generally includes basement membrane extracts, such as those isolated from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, hereinafter referred to as "EHS-ECM" (e.g., sold under the trade names Matrigel™ (Corning) and Geltrex™ (ThermoFisher)). In one embodiment, the EHS-ECM may include, for example, laminin, collagen IV, entactin / nidogen, and heparan sulfate proteoglycan. Alternatively, if desired, other sources of ECM with different compositions and / or purified components (e.g., FN) and / or synthetic ECM-like substrates (e.g., including RGD peptides) can also be used in the coating method. As will be appreciated by those skilled in the art, the selection of suitable ECM component(s) depends on multiple factors, including but not limited to cell type, stage of differentiation, and other experimental parameters. For example, EHS-ECM has been demonstrated herein to be suitable for culturing (e.g., attachment and outgrowth) bovine embryos. Thus, in one embodiment, the ECM is EHS-ECM, optionally Matrigel or Geltrex.

[0093] As shown herein, attachment and outgrowth formation from bovine embryos is influenced by the composition of the outgrowth medium in which the embryos are cultured. The outgrowth medium may, for example, comprise a basal medium and one or more small molecules, growth factors, and / or nutrients. Suitable basal media can be readily determined by one of skill in the art and include, but are not limited to, DMEM / F12, modified DMEM / F12, and Neurobasal medium. Suitable supplements can be readily determined by one of skill in the art and include, but are not limited to, MEM non-essential amino acids, L-glutamine, Glutamax, ascorbic acid, insulin, BSA (fraction V), beta-mercaptoethanol, and penicillin / streptomycin. In one aspect of the disclosure, outgrowth medium components useful for the derivation and maintenance of naive embryonic stem cells, including attachment and outgrowth of ICM, are provided. In one embodiment, the outgrowth medium comprises a basal medium and / or supplements, and one or more outgrowth medium components. In one embodiment, the outgrowth medium comprises one or more of a B27 component, optionally comprising about 1% B27 supplement and about 0.5% N2 supplement; a Wnt activator component, optionally CHIR99021, BIO, CHIR-98014, LY2090314, or IM-12; a Wnt inhibitor component, optionally XAV939, IWR-1, or IWP-2; a MEK / ERK inhibitor component, optionally PD0325901, ravoxertinib, GSK1120 212, MEK162, PD184352, trametinib, LY3214996, or ulixertinib; a ROCK inhibitor component, optionally Y27632, thiazovivin, or blebbistatin; a LIF component, optionally human LIF; a PKC inhibitor, optionally Go6983, Go6976, LY317615, LY333531, PKC412, GSK690693, sotrastaurin, staurosporine, or bisindolylmaleimide; and an insulin component, optionally insulin.Optionally, the outgrowth medium may include one or more of N2B27, CHIR99021 (Wnt activator), XAV939 (Wnt inhibitor), PD0325901 (MEK / ERK inhibitor), lavoxertinib (ERK specific inhibitor), Go6983 (PKC inhibitor), Y27362 (ROCK inhibitor), and / or LIF. In one embodiment, the outgrowth medium further comprises an activin A component, optionally human activin A.

[0094] II. Products and Compositions In one aspect of the disclosure, products and compositions useful for the derivation and culture of naive bovine embryonic stem cells are provided.

[0095] For example, in one embodiment, an ECM coated substrate is provided that includes a substrate comprising a negatively charged substrate surface adjacent to a positively charged biocompatible polymer layer and a negatively charged ECM layer adjacent to the positively charged biocompatible polymer layer. In one embodiment, the biocompatible polymer is type A gelatin. In one embodiment, the ECM includes EHS-ECM. In one embodiment, the substrate is a plastic substrate suitable for cell culture, such as polystyrene.

[0096] Also provided are medium formulations suitable for promoting bovine embryo attachment, outgrowth formation, and / or derivation of naive bovine embryonic stem cells as described herein.

[0097] In one embodiment, the elongation medium comprises a basal medium and one or more components identified herein. For example, in one embodiment, the elongation medium comprises one or more of an N2B27 component, a Wnt activator component, a Wnt inhibitor component, a MEK / ERK inhibitor component, a ROCK inhibitor component, a LIF component, a PKC inhibitor, and an insulin component. In one embodiment, the elongation medium comprises an N2B27 component, a Wnt activator component, a Wnt inhibitor component, a MEK / ERK inhibitor component, a ROCK inhibitor component, a LIF component, a PKC inhibitor, and an insulin component.

[0098] In one embodiment, the N2B27 component comprises B27 supplement and N2 supplement, optionally about 1% B27 supplement and about 0.5% N2 supplement.

[0099] In one embodiment, the Wnt activator component comprises CHIR99021, BIO, CHIR-98014, LY2090314, or IM- 12. Optionally, the Wnt activator component comprises CHIR99021, optionally at a concentration of about 0.1 uM to about 5 uM, optionally about 1 uM, to about 3 uM, optionally about 1 uM, about 2 uM, or about 3 uM.

[0100] In one embodiment, the Wnt inhibitor component comprises XAV939, IWR-1, or IWP-2. Optionally, the Wnt inhibitor component comprises XAV939, optionally at a concentration of about 0.2 uM to about 10 uM, optionally at a concentration of about 1 uM to about 5 uM, optionally at about 2 uM. Optionally, the Wnt inhibitor component comprises IWR-1, optionally at a concentration of about 0.25 uM to about 10 uM, optionally at a concentration of about 1 uM to about 5 uM, optionally at about 2.5 uM.

[0101] In one embodiment, the MEK / ERK inhibitor component comprises PD0325901, ravoxertinib, GSK1120212, MEK162, PD184352, trametinib, LY3214996, or ulixertinib. Optionally, the MEK / ERK inhibitor component comprises PD0325901 or ravoxertinib. Optionally, the MEK / ERK inhibitor component comprises PD0325901 at a concentration of about 0.05 uM to about 5 uM, optionally about 0.1 uM to about 2 uM, optionally about 1 uM. Optionally, the MEK / ERK inhibitor component comprises ravoxertinib at a concentration of about 0.25 uM to about 10 uM, optionally about 1 uM to about 5 uM, optionally about 2.5 uM.

[0102] In one embodiment, the ROCK inhibitor component comprises Y27632, thiazovivin, or blebbistatin. Optionally, the ROCK inhibitor component comprises Y27632, optionally at a concentration of about 0.5 uM to about 20 uM, optionally about 5 uM to about 10 uM, optionally about 5 uM or about 10 uM.

[0103] In one embodiment, the LIF component comprises human LIF, optionally at a concentration of about 1 ng / ml to about 1000 ng / ml, optionally about 5 ng / ml to about 100 ng / ml, optionally about 5 ng / ml, about 10 ng / ml, about 20 ng / ml, or about 100 ng / ml.

[0104] In one embodiment, activin A comprises human activin A, optionally at a concentration of about 1 ng / ml to about 50 ng / ml, optionally about 5 ng / ml to about 50 ng / ml, optionally about 10 ng / ml or about 20 ng / ml.

[0105] In one embodiment, the PKC inhibitor comprises Go6983, Go6976, LY317615, LY333531, PKC412, GSK690693, sotrastaurin, staurosporine, or bisindolylmaleimide. Optionally, the PKC inhibitor comprises Go6983, optionally at a concentration of about 0.2 uM to about 25 uM, optionally at a concentration of about 2 uM to about 2.5 uM, optionally at about 2 uM or about 2.5 uM.

[0106] In one embodiment, the insulin component optionally comprises an insulin peptide at a concentration of about 2 ug / ml to about 200 ug / ml, optionally about 20 ug / ml.

[0107] Also provided is a formulation suitable for post-thaw recovery of embryos. As shown in Figures 8 and 9, embryos treated with recovery medium formulated as described herein showed faster recovery and higher induction efficiency, as observed by faster re-expansion of embryos compared to controls. Embryos treated with recovery medium also appeared larger and had more defined ICM compared to controls.

[0108] In one embodiment, the recovery medium comprises a glycogen synthase kinase 3 (GSK-3) inhibitor, a MEK / ERK kinase inhibitor, and a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor. Suitable GSK-3 inhibitors include, for example, CHIR99021. Suitable MEK / ERK inhibitors include, for example, PD0325901. Suitable ROCK inhibitors include, for example, Y27632. In one embodiment, the recovery medium comprises CHIR99021, optionally at about 0.1 uM to about 5 uM, optionally at about 1 uM to about 3 uM, optionally at about 1 uM, about 2 uM, or about 3 uM; PD0325901, optionally at about 0.05 uM to about 5 uM, optionally at about 0.1 uM to about 2 uM, or about 1 uM; and Y27632, optionally at about 0.5 uM to about 20 uM, optionally at about 5 uM to about 10 uM, or about 10 uM.

[0109] Also provided are kits comprising one or more of the ECM coated substrates, outgrowth medium, and / or recovery medium described herein, hi one embodiment, the ECM coated substrates, outgrowth medium, and / or recovery medium are packaged in separate containers.

[0110] One aspect includes naive bovine stem cells derived using the methods described herein, hi one embodiment, the naive bovine stem cells are genetically modified stem cells.

[0111] III.Use Also provided is the use of a product, composition or kit described herein to aid in embryo attachment, embryonic cell outgrowth, and / or derivation of naive embryonic stem cells in culture.

[0112] For example, in one embodiment, there is provided a use of an ECM coated substrate as described herein for culturing embryos and / or cells derived from embryos, hi one embodiment, the ECM coated substrate is useful for promoting attachment and / or outgrowth of embryos and derivation of naive embryonic stem cells, optionally naive bovine embryonic stem cells.

[0113] In another embodiment, there is provided a use of the outgrowth medium described herein for culturing embryos and / or cells derived from embryos. In one embodiment, the outgrowth medium is useful for promoting attachment and / or outgrowth of embryos and derivation of naive embryonic stem cells, optionally naive bovine embryonic stem cells. In one embodiment, the outgrowth medium is for use in combination with an ECM-coated substrate described herein.

[0114] In another embodiment, there is provided a use of the recovery or adaptation medium described herein for treating embryos, optionally bovine embryos, and optionally reconstituted diploid bovine embryos. In one embodiment, the recovery medium is useful for treating fresh embryos, or embryos that have previously been biopsied and / or frozen. The recovery medium is also useful for processes in which the embryo can benefit from the recovery medium, such as ZP removal, thawing, biopsy, gene editing, or cell induction.

[0115] Further aspects include the use of naive bovine stem cells derived using the methods described herein in breeding schemes or genetic improvement programs, or to expand preimplantation embryos with desirable genetic characteristics, to derive primordial germ cells and / or gametes for in vitro breeding programs, and / or to develop and provide veterinary biologics and therapeutics.

[0116] The following non-limiting examples illustrate the present application. EXAMPLES

[0117] Example 1. Optimization of coating materials In this example, three different extracellular matrix coating materials were tested as coating materials: gelatin type A (Sigma, G1890), Geltrex™ (Thermo Fisher, A1413301) and Matrigel™ (Corning, 354277). Unless otherwise indicated, EHS-ECM (extracellular matrix) as used in the examples refers to Geltrex and / or Matrigel.

[0118] The deposition and elongation rates were determined for each type of coating material and at various concentrations of the material.

[0119] The coating protocol was as follows: 1. Apply 4 drops of 50uL of coating solution: 0.1% gelatin or various concentrations of EHS-ECM provided as a 100x concentrated solution onto the Nunc surface 35mm 2 Cell culture dishes were plated with the cells (see FIG. 1). 2. Incubate the coated substrates in a humidified 5% CO 2 The mixture was incubated in an incubator at 38.5°C for 1 hour. 3. After incubation, excess coating solution was removed and 50ul of cell culture medium was applied to the coated area, covered with 4mL of mineral oil, and 50ul of additional medium was added to each drop. 4. Pre-treat the medium in humidified 5% O 2 , 5% CO 2 The mixture was equilibrated in an incubator at 38.5°C for 2 hours. 5. After removal of the ZP and adaptation of the embryos, one embryo was placed into one microculture drop and elongation induction was initiated. 6. Initial attachment rates were determined after 48 h of outgrowth culture, and the first medium change was performed immediately after the initial attachment determination. 7. Attachment and outgrowth rates were assessed daily, and the medium was changed immediately after each daily assessment.

[0120] result As shown in Figure 1, a higher attachment rate was observed with the EHS-ECM coating (Geltrex) compared to the gelatin coating, but no difference in the rate of outgrowth formation was observed.

[0121] No differences in outgrowth efficiency were observed when different concentrations of EHS-ECM were used. The overall attachment rate was low, which resulted in reduced outgrowth efficiency.

[0122] Example 2. Grooved surface coating with mesh It was hypothesized that the smooth surface created by the thin layer of coating solution did not provide a suitable "topography" for the embryos to attach. To test whether attachment rates could be improved, a grooved surface was created by coating the mesh membrane with gelatin or the EHS-ECM coating material.

[0123] The protocol for processing the dishes was as follows: 1. Place a 1 cm cell strainer membrane (pore size 30 um) 2 was cut to size. 2. Cut the membrane into 35 mm pieces. 2 Placed on a plate. 3. 50ul of coating material (0.5% gelatin or 1x EHS-ECM) was applied to the cut membrane. 1x EHS-ECM showed the best adhesion rate in Example 1, so 1x EHS-ECM was chosen for this experiment. 4. The coating material was polymerized in a humidified incubator at 38.5° C. for 1 hour and then dried on the bench (under a laminar flow hood) at room temperature for 30 minutes. 5. The coated surface was sterilized under UV for 20 minutes. 6. The textured dish was placed on ice and the membrane was carefully removed.

[0124] result Using this protocol, a grooved dish surface could be easily created, as shown in Figure 2. However, upon addition of culture medium, the grooves rapidly collapsed.

[0125] Example 3. ECM-coated substrates produced using a layer-by-layer (LbL) protocol Although moderate attachment rates were obtained with EHS-ECM when fresh embryos were used, outgrowth was difficult, especially from frozen-thawed embryos. Using the coating protocol described in Example 1, some protein debris was observed at the bottom of the culture drop. We hypothesized that the protein matrix had detached from the surface of the culture dish and further hypothesized that the protein matrix did not adhere strongly enough to the culture dish. To address this issue, we modified the coating protocol to employ a layer-by-layer approach.

[0126] The Nunc™ Delta (plasma-treated) polystyrene dishes used in Examples 1 and 2 have a negatively charged surface (see, e.g., Figures 3 and 4). Common ECM components, such as basement membrane extracts, are also negatively charged under the conditions used for coating and / or at physiological pH. For example, Geltrex (A14133, Thermo Fisher) is negatively charged (isoelectric point of EHS-ECM: 4-5) at pH 7.2, which is the pH of the dilution buffer used for coating. Protein matrix molecules can adhere to the plasma-treated polystyrene surface by hydrogen bonding with surface -OH residues (Lerman et al., 2018).

[0127] However, because both components have similar electrostatic charges, the interaction between the two sides is relatively weak. Therefore, we investigated a "layer-by-layer" approach in which a positively charged material is placed as an "electrostatic adhesive" between both negatively charged components (see, for example, Figure 4). The isoelectric point of gelatin type A (usually extracted from pigs) is 7-9, whereas that of gelatin type B (usually extracted from cows) is 4.8-5.1. Thus, gelatin type A is positively charged at pH 7.2, making it a suitable candidate to act as an "electrostatic adhesive" in the layer-by-layer protocol. Furthermore, gelatin can not only provide electrostatic stability but also provide an abundance of protein motifs similar to EHS-ECM, which can enhance the biological interaction between the gelatin and EHS-ECM layers.

[0128] The protocol for generating LbL ECM coated substrates was as follows: 1. Add 40 ul of 0.1% gelatin type A (diluted in DMEM / F12 basal medium) to a Nunc 35mm 2 It was applied to the surface of a cell culture dish to create small drops. 2. Place the dish in a humidified 5% CO 2 The mixture was incubated in an incubator at 38.5°C for 1 hour. 3. After incubation, remove the gelatin solution and add Ca 2+ and Mg 2+ Excess gelatin was washed from the drop by adding 50ul of DPBS containing 0.01% ethanol, followed by removal using aspiration. The washing step was repeated two more times. 4. 50ul of 1xEHS-ECM was applied to the gelatin coated area and incubated for 1 hour at 38.5°C in a humidified 5% CO2 incubator. 5. After incubation, the EHS-ECM solution was removed and 50 ul of medium was applied; the drops were covered with 4 ml of mineral oil and 50 ul of additional medium was added to each drop. 6. Culture medium in 5% CO 2 The mixture was allowed to equilibrate in the incubator for 2 hours.

[0129] result As shown in Figure 5, embryo attachment was improved and more stable using LbL EHS-ECM coated substrates compared to control conditions (EHS-ECM only). The ability to generate outgrowths from embryos was also significantly improved with the use of LbL EHS-ECM coated substrates. Outgrowths showed better ICM growth that ultimately gave rise to embryonic stem cells compared to regular EHS-ECM coated substrates. Improved TE growth was also observed compared to the control.

[0130] Example 4. Preparation of embryos for elongation culture The embryo, also called the blastocyst, is surrounded by a thick membrane of glycoproteins that forms the zona pellucida (ZP). During development, the blastocyst begins to expand and eventually "hatches", allowing the blastocyst to leave its "shell". Generally, it is possible to obtain hatching and post-hatching blastocysts around day 8 of in vitro culture. However, bovine in vitro embryo culture medium can efficiently support development until day 7, after which the embryo must be transferred to a recipient or frozen. Preliminary experiments have shown that ZP blocks embryo attachment, and therefore ZP must be removed before elongation culture. Therefore, an investigation was carried out to improve ZP removal from fresh day 6 / 7 embryos and / or frozen / thawed day 7 embryos.

[0131] Enzyme-Based Zona Pellucida (ZP) Digestion Protocol for Fresh Embryos Enzyme-based approaches are a frequently used technique for ZP removal. This protocol typically uses a protease (from Streptomyces griseus, Pronase, Sigma P8811) at a concentration between 0.05-0.5%. However, proteases are not glycoprotein-specific enzymes and may cause damage to the embryo if the ZP is completely digested.

[0132] The ZP digestion protocol was as follows: 1. A protease treatment dish was prepared (see Figure 6). 2. Embryos were transferred from embryo culture medium to drop 1 of embryo handling medium (Hepes buffered medium). 3. Embryos were washed by transferring successively from drops 1 to 3. 4. Embryos were transferred to protease drops (test concentrations: 0.05%, 0.1%, 0.25% or 0.5%) and incubated until digestion of the ZP was observed (approximately 2 min). 5. Protease-treated embryos were transferred to drops 4, 5, and 6 of inactivation medium made with 10% FBS in embryo handling medium.

[0133] result As shown in Figure 6, prolonged exposure to proteases can cause dissociation of the embryo itself and damage the cells (indicated by dark, opaque cells).

[0134] Embryos at the morula stage (e.g., bovine day 6) have a very thick ZP (compared to the expanded blastocyst stage (e.g., bovine day 7), where the ZP begins to thin due to blastocyst expansion) and complete digestion was not possible at the lowest concentrations (<0.1%).

[0135] At higher concentrations (>0.25%), embryos were adversely affected by high concentrations of proteases because the enzyme was not completely inactivated after exposure to FBS, resulting in complete dissociation of embryos into single blastomeres.

[0136] The timing required for digesting the ZP also varies widely from embryo to embryo, making it very difficult to obtain a standardized protocol.

[0137] Enzyme-assisted ZP removal protocol when fresh or frozen / thawed Existing embryo biopsy protocols for day 7 embryos involve using a microblade to cut the ZP and perform the embryo biopsy. Once an opening is created in the ZP by the biopsy, it facilitates the release of the embryo from the ZP. However, this approach cannot be easily applied to embryos at the morula stage (e.g., day 6) because the ZP is much thicker / harder. Therefore, we combined two different protocols to develop an "enzyme-assisted ZP removal protocol" for morula stage embryos. Briefly, we treat the embryo with enough protease to thin and soften the ZP without causing embryo dissociation or damage to the cells. The morula with its thinned ZP can then be easily used in regular biopsy techniques using a microblade.

[0138] The ZP digestion protocol was as follows: 1. Protease dishes were prepared with 0.25% protease (e.g., as shown in Figure 6). Protease drops were prepared up to 2 hours prior to use. 2. One or more embryos were transferred from the embryo culture medium into drop 1 made of embryo handling medium. 3. Embryos were transferred from drop 1 to drop 3 and washed. 4. Embryos were transferred to 0.25% protease drops and incubated for 45 seconds. 5. Transfer treated embryos into inactivation medium drops 4, 5, and 6 made with 10% FBS in embryo handling medium. 6. Transfer the ZP thinned embryo to a biopsy dish.

[0139] result An enzyme-assisted ZP removal protocol can be used to generate ZP-removed morulae as shown in FIG. Example 5. Composition for improving the post-thaw quality of biopsy frozen embryos

[0140] For breeding applications, biopsy-frozen embryos are typically genetically identified (e.g., screened for biomarkers), allowing physicians to select embryos with desired genetic characteristics. Although it is possible to biopsy and freeze day 7 embryos, these embryos are exposed to more stress compared to fresh embryos. Biopsy-frozen embryos have an open ZP (due to the biopsy procedure), which allows the ZP to be easily removed by gentle pipetting. However, the quality of biopsy-frozen-thawed embryos is generally inferior to fresh embryos. Described herein is a post-thaw recovery medium (referred to herein as "2iY" medium or "recovery medium") that includes three inhibitors: CHIR99021, PD0325901, and Y27632. CHIR99021 and PD0325901 are well-characterized inhibitors, also known as "2i" in the stem cell field. These two inhibitors regulate two key pathways involved in transcription factor activity in naive embryonic stem cells (CHIR99021: Wnt pathway activator by inhibition of GSK3, PD0325901: MEK pathway inhibitor). Y27632 is a ROCK inhibitor and an actin filament stabilizer. We tested the ability of these three inhibitors to protect cells from post-thaw stress and promote more rapid cell recovery by regulating key stemness pathways. The experiments presented here demonstrate that this protocol significantly improves the quality of post-thaw embryos and allows for more efficient induction of elongation.

[0141] Protocol 1. 3 uM CHIR99021 (2520691, PeproTech), 1 uM PD0325901 (3911091, PeproTech), and 10 uM Y27632 (1293823, PeproTech) were added to embryo handling medium (2iY medium). 2. Thaw dishes were prepared using 2iY medium (see FIG. 8) or control embryo culture medium. 3. One or more embryos were thawed according to the protocol. 4. Embryos were placed in 2iY medium or control embryo handling medium. 5. Immediately after thawing, the ZP was removed by gentle pipetting. 6. Incubate the embryos in 6.8% CO 2 , 5% O 2 The plates were incubated at 38.5°C for 4 hours in a humidified incubator. 7. Once the embryos had fully recovered (re-expanded), they were transferred to elongation medium. Recovery was confirmed by the visible presence of a blastocyst cavity.

[0142] result As shown in Figure 8, the 2iY-treated group exhibited higher attachment and outgrowth formation rates than the control group when plated on LbL-ECM-coated dishes for 2 and 4-5 days, respectively. 2iY-treated embryos exhibit higher induction efficiency, as observed by faster recovery and faster re-expansion of embryos than in control embryo culture medium. Compared to the control group after 4 h of recovery, 2iY-treated embryos exhibit larger and more distinct ICMs (dashed circle in Figure 8).

[0143] The 2iY treatment group exhibits more advanced stage and improved quality embryos 4 hours after thawing from cryopreservation (see FIG. 9).

[0144] Example 6. Adaptation of embryos to a new culture environment Cells in culture, especially embryonic stem cells, are very sensitive to medium components and nutrient levels. However, there are two factors that are often overlooked in cell culture, especially when using complete / commercial media: medium osmolarity and pH. It was observed that transferring embryos directly from embryo handling medium to DMEM (embryonic stem cell basal medium) stressed the cells (data not presented), and measurements of osmolarity and pH confirmed the large differences of both media (see Table 1). It was therefore hypothesized that adaptation media may promote a gradual adaptation of cells to their new microenvironment.

[0145] Protocol 1. Thawing medium and elongation medium were mixed in a 1:1 ratio. 2. The adaptation dish was prepared as shown in Figure 10. 3. After post-thaw recovery, embryos were washed twice with adaptation medium (1:1 ratio of thawing medium (e.g., embryo handling medium to elongation medium (e.g., DMEM))) and then placed into the adaptation drop in dish #1 (Figure 10). 4. After 1 hour of adaptation, the embryos were washed 4 times by transferring them into the wash drops (outgrowth medium) of dish #2. 5. The embryos were then transferred to elongation medium (DMEM) and plated. 6. The adapted embryos were then plated onto LbL-coated microdrops.

[0146] result TIFF2024544563000001.tif48165

[0147] The induction efficiency as a function of adaptation treatment (attachment, TE outgrowth, and ICM outgrowth measured 4-6 days after embryo plating) is shown in Figure 10. After 1 hour of adaptation, the induction efficiency improves compared to 0.5 hours of adaptation or to the no adaptation control. Adaptation for more than 1 hour (2 and 4 hours of incubation) did not show any difference compared to 1 hour of adaptation.

[0148] Example 7. Testing media for ability to support outgrowth formation Cell culture media contain various components to support the general maintenance of cells, such as metabolism, survival and proliferation. Additional growth factors or inhibitors may be added to promote differentiation, self-renewal, or simply promote cell growth. In order to induce naive embryonic stem cells and maintain them in an undifferentiated state, a combination of growth factors and inhibitors is required in the culture media. An inappropriate combination of additives or differences in the concentrations of these molecules may induce irreversible differentiation of stem cells.

[0149] Serum / serum substitute type Five different serum source / concentration conditions were tested, as shown in Figure 11. The studies used FBS (10091, Gibco™), knock-out serum replacement (10828-10, Gibco™), serum replacement (S0638, Sigma), B27 supplement (17504-044, Gibco™), and N2 supplement (17502-048, Gibco™). Conventional mouse 2iL medium (DMEM / F12 + 1% MEM non-essential amino acids + 2mM Glutamax + 100μM beta-mercaptoethanol + 100IU penicillin / streptomycin + growth factors / inhibitors including 3μM CHIR99021, 1μM PD0325901 and 100ng / ml LIF) was used as the base medium for serum testing in the 1x-, 2x-SR groups, with 10% FBS and 20% KOSR. t2iLGoY medium (1:1 mixture of DMEM / F12 and Neurobasal medium + 1% MEM non-essential amino acids + 2mM Glutamax + 50μg / ml BSA, fraction V + 100μM beta-mercaptoethanol + 100IU penicillin / streptomycin + growth factors / inhibitors such as 1μM CHIR99021, 1μM PD0325901, 10μM Y27632, 2.5μM Go6983 and 10ng / ml LIF) was used to test the N2B27 group. The reason for this different combination is that the formulation of 2iL medium is much simpler (compared to t2iLGoY medium) and is more suitable for basic serum sources such as FBS, KOSR, or SR.

[0150] Knock-Out Serum Replacement (KOSR), a widely used serum source in other species, showed very low efficiency for the outgrowth of bovine embryos.

[0151] Serum replacement (SR) containing bovine-derived components showed much better efficiency compared to KOSR.

[0152] N2B27 medium containing 1% B27 supplement and 0.5% N2 supplement showed the best results for elongation induction (TE and ICM growth) of bovine embryos compared to other ingredients tested.

[0153] Additional basal media and other components / additives tested Conventional mouse 2iL medium: Medium composition: DMEM / F12 + 1x serum replacement + 100ng / ml leukemia inhibitory factor (LIF) + 3uM CHIR99021 + 1uM PD0325901. TIFF2024544563000002.tif38165

[0154] Although positive attachment rates were obtained, the rate of outgrowth formation was low, with ICM expansion at only 19% and TE expansion at only 50%.

[0155] Naïve human stem cell medium - NHSM (Gafni et al., 2013): Medium composition: DMEM / F12: Neurobasal medium (1:1 mixture) + N2B27 serum + 8ng / ml FGF + 1ng / ml TGF-b + 20ng / ml LIF + 3uM CHIR99021 + 1uM PD0325901 + 10uM SP600125 + 10uM + SB203580 TIFF2024544563000003.tif65165

[0156] This combination showed very low efficiency with 0% ICM expansion.

[0157] Forskolin medium Forskolin is an adenylyl cyclase stimulator, which increases intracellular cAMP levels, a second messenger involved in many signaling pathways. The combination of forskolin and 2iL medium showed good efficiency in inducing human naïve stem cells (Hanna et al., 2010) and reprogramming bovine pluripotent stem cells (Kawaguchi et al., 2015).

[0158] 2iLFk formulation: Conventional mouse 2iL medium + 10uM forskolin

[0159] This combination improved induction efficiency compared to conventional 2iL medium and NHSM medium (Figure 13). However, ICM-induced naive stem cell colonies did not show further expansion in 2iLFk medium after passaging.

[0160] Dual kinase regulation of Wnt Wnt is known to be a key player in naive stem cell signaling pathways. The combination of Wnt activator CHIR99021 and Wnt inhibitors such as IWR-1 or XAV939 (both inhibitors of the same protein complex, but each targeting a different unit) leads to cytoplasmic accumulation of beta-catenin, which may promote self-renewal of mouse pluripotent stem cells through stabilization of E-cadherin, a key component of adherens junctions (Kim et al. 2013). We tested the ability of dual Wnt regulation to overcome the poor expansion observed in 2iLFk medium.

[0161] Dual Wnt media formulation: 1.5uM CHIR99021+2.5uM IWR-1+1uM PD0325901+100ng / ml LIF+10uM Forskolin.

[0162] As shown in FIG. 13, this combination had a negative effect on bovine embryonic stem cells.

[0163] Bovine IL-6 (LIF superfamily) and SRCi: Bovine IL-6 (LIF superfamily) and an SRC inhibitor were tested as alternatives to hLIF and PD0325901, respectively, used in t2iL GoY medium for the induction of human naive stem cells (titrated 2i / LIF / Go6983 / Y27632, Guo et al., 2016).

[0164] Human LIF was initially used in the formulation of t2iLGoY, but the sequences of human and bovine LIF differ, therefore bovine IL-6, a member of the LIF superfamily, was tested as an alternative to human LIF.

[0165] SRC inhibitors, which are RTK inhibitors, are involved in most of the signaling pathways induced by growth factors (Theunissen et al., 2014). Since the endpoint of SRC inhibition was found to ultimately target the ERK / MEK pathway, we tested SRC inhibitors as a novel strategy to replace MEK pathway inhibition (by MEKi PD0325901).

[0166] t2iLGoY medium formulation: DMEM / F12:Neurobasal medium (1:1 mixture) + N2B27 serum + 1uM CHIR99021 + 1uM PD0325901 + 10ng / ml human LIF + 2.5uM Go6983 + 10uM Y27632.

[0167] SRCi media formulation: t2iLGoY media with PD0325901 replaced with 2uM CGP77675 (SRC inhibitor) and human LIF replaced with 10ng / ml bovine IL-6.

[0168] As shown in FIG. 14, substitution of either bovine IL-6 for human LIF or the SRC inhibitor CGP77675 for the MEK inhibitor PD0325901 had a negative effect on induction efficiency, either through a lower attachment rate or a lower rate of outgrowth formation, compared to t2iLGoY medium.

[0169] MEK inhibitors (MEKi) Zhao et al. (2021) published results suggesting that MEK inhibitors must be removed from bovine ES cell culture media, which contrasts with previously published results for human cells. We performed studies to test that approach by removing MEK inhibitors from the culture media. TIFF2024544563000004.tif69165

[0170] Removal of the MEK inhibitor, PD0325901, from the medium reduces the induction efficiency as indicated by germinal cavity formation, a clear sign of ICM cell differentiation (see FIG. 15).

[0171] PKC inhibitor, Go6983 Due to recent improvements in culture medium composition, the best culture conditions tested so far, i.e., 2iLFk and t2iLGoY media, were tested in parallel with 2i and NHSM media. Notably, the addition of the PKC inhibitor Go6983 (Guo et al., 2016) to t2iLGoY improved the induction efficiency, as shown in Figure 16.

[0172] t2iLGoY medium showed similar ICM growth rates but much higher rates of attachment and outgrowth formation, especially TE growth.

[0173] t2iLGoY medium formulation: DMEM / F12:Neurobasal medium (1:1 mixture) + N2B27 serum + 1uM CHIR99021 + 1uM PD0325901 + 10ng / ml LIF + 2.5uM Go6983 + 10uM Y27632.

[0174] Insulin The morphology of ICM cells was not maintained after the first passage using the various medium compositions described above. ICM cells rapidly differentiated into extraembryonic endoderm cells (hypoblast) during outgrowth culture and after passaging.

[0175] It was hypothesized that adjusting the insulin concentration in the medium may be able to overcome that type of differentiation ( Anderson et al., 2017 ).

[0176] As shown in FIG. 17, when 20 ug / ml of insulin was added to the medium, hypoblast formation was not observed during elongation culture, and ICM cells could be obtained after subculture.

[0177] ERK pathway inhibition Khan et al. (2021) used high-throughput chemical screening to identify the best culture conditions for human naïve stem cells and identified inhibition of the ERK pathway as a key factor for maintaining stable human naïve stem cell cultures (Khan et al., 2021).

[0178] Inhibition of the Wnt pathway has also been highlighted as a key factor in the human naive stem cell field (Bredenkamp et al., 2019). Combining the approaches of Khan et al. (2021) and Bredenkamp et al. (2019), we investigated and developed PXGRY / LA medium.

[0179] PXGRY / LA formulation: DMEM / F12:Neurobasal medium (1:1 mix) + N2B27 serum + 1uM PD0325901 + 2uM XAV939 + 2uM Go6983 + 2.5uM lavoxertinib + 10uM Y27632 + 10ng / ml LIF + 20ng / ml activin A.

[0180] As shown in Table 5, activation of the Wnt pathway (with t2iLGoY medium) or its inhibition (by using PXGRY / LA medium) did not show any significant difference in the efficiency of inducing naive outgrowth using a feeder-free system. However, naive stem cells are generally induced and maintained on feeder cells, and naive stem cell medium is technically designed for such a culture system. According to Cosin-Roger et al., 2019 and Talbot et al., 2012, feeder cells secrete several important growth factors and, more importantly, Wnt ligands. In PXGRY / LA medium with a feeder-free system, not only is the Wnt pathway inhibited, but the Wnt ligands normally secreted by feeder cells are also completely depleted. Therefore, the effect of Wnt inhibition may not be the same when using feeder-free conditions compared to cells maintained on feeder cells, especially after several passages. Therefore, t2iLGoY may be more suitable for inducing bovine eagle cells using feeder-free conditions. TIFF2024544563000005.tif93165

[0181] Example 8: Creation of induced blastoid structures Stable bovine ebony stem cells generated using the highly efficient protocol described in the preceding examples are used to reconstitute induced blastoid structures using approaches similar to those recently published by Liu et al. (2021), Yu et al. (2021), and Yanagida et al. (2021).

[0182] Protocol 1. Naïve stem cell colonies are dissociated into single cells using an enzymatic dissociation method. 2. Count the cells and adjust the cell density to 500 cells / ml with Induced Blastoid Medium 1. Induced Blastoid Medium 1 differentiates naïve stem cells into trophoblast stem cells, which form the outer layer (TE) of the blastocyst. 3. Using a multichannel pipette, plate 100 cells (200 ul) into each well of a non-adherent round bottom 96 well plate. 4. After 48 hours, remove Induced Blastoid Medium 1 from the culture plate and add 200 ul of Induced Blastoid Medium 2. 5. After 48 hours, well-established induced blastoids are transferred to induced blastoid manipulation medium (Hepes-buffered DMEM / F12+Neurobasal medium) and the quality of the induced blastoids is assessed based on a regular embryo grading system. 6. Freeze the induced blastoids according to the Boviteq embryo freezing protocol.

[0183] The formulation for Induced Blastoid Medium 1 is Modified-DMEM / F12 + 0.5% Serum Replacement + 1% MEM NEAA + 1% Glutamax™ + 0.1 mM beta-mercaptoethanol + gentamicin + 2 μM CHIR99021 + 5 μM Y27632 + 0.5 mM valproic acid + 1 μM A83-01 + 50 ng / ml EGF.

[0184] The formulation of induced blastoid medium 2 is a 1:1 mixture of DMEM / F12 and neurobasal medium + 1% MEM NEAA + 1% Glutamax™ + 0.1 mM beta-mercaptoethanol + 100 IU / ml Pen / Strep + 100 ng / ml Activin A + 3 μM CHIR99021 + 10 ng / ml LIF.

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Cosin-Roger J, Ortiz-Masia MD and Barrachina MD (2019) Macrophages as an emerging source of wnt ligands: Relevance in mucosal integrity. Frontiers in Immunology 10 2297. De Los Angeles A (2019) Frontiers of Pluripotency. Methods in Molecular Biology 2005 3-27. Gafni O, Weinberger L, Mansour AA, Manor YS, Chomsky E, Ben-Yosef D, Kalma Y, Viukov S, Maza I, Zviran A et al. (2013) Derivation of novel human ground state naive pluripotent stem cells. Nature 504. Guo G, Von Meyenn F, Santos F, Chen Y, Reik W, Bertone P, Smith A and Nichols J (2016) Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass. Stem Cell Reports 6. Hanna J, Cheng AW, Saha K, Kim J, Lengner CJ, Soldner F, Cassady JP, Muffat J, Carey BW and Jaenisch R (2010) Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Proceedings of the National Academy of Sciences of the United States of America 107 9222-9227. Hou Z, An L, Han J, Yuan Y, Chen D and Tian J (2018) Revolutionize livestock breeding in the future: an animal embryo-stem cell breeding system in a dish. Journal of Animal Science and Biotechnology 2018 9:1 9 1-11. Kawaguchi T, Tsukiyama T, Kimura K, Matsuyama S, Minami N, Yamada M and Imai H (2015) Generation of Naive Bovine Induced Pluripotent Stem Cells Using PiggyBac Transposition of Doxycycline-Inducible Transcription Factors. PLOS ONE 10 e0135403. Khan SA, Park K mi, Fischer LA, Dong C, Lungjangwa T, Jimenez M, Casalena D, Chew B, Dietmann S, Auld DS et al. (2021) Probing the signaling requirements for naive human pluripotency by high-throughput chemical screening. Cell Reports 35. Kim H, Wu J, Ye S, Tai CI, Zhou X, Yan H, Li P, Pera M and Ying QL (2013) Modulation of β-catenin function maintains mouse epiblast stem cell and human embryonic stem cell self-renewal. Nature Communications 4. Lerman MJ, Lembong J, Muramoto S, Gillen G and Fisher JP (2018) The Evolution of Polystyrene as a Cell Culture Material. Tissue Engineering - Part B: Reviews 24. Li R, Zhong C, Yu Y, Liu H, Sakurai M, Yu L, Min Z, Shi L, Wei Y, Takahashi Y et al. (2019) Generation of Blastocyst-like Structures from Mouse Embryonic and Adult Cell Cultures. Cell 179 687-702.e18. Liu X, Tan JP, Schroder J, Aberkane A, Ouyang JF, Mohenska M, Lim SM, Sun YBY, Chen J, Sun G et al. (2021) Modelling human blastocysts by reprogramming fibroblasts into iBlastoids. Nature 591 627-632. Park F. (2007) Lentiviral vectors: are they the future of animal transgenesis? Physiol Genomics. 31: 159-173. Smith, LC, Labrecque, R, Paredes LMA, Therrien J, and Vigneault, C. 2020. Use of haploid embryonic cells to generate offspring with predetermined genomes. WO 2020 / 168422. Solter D and Knowles BB (1975) Immunosurgery of mouse blastocyst. 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Claims

1. A method for deriving naive bovine embryonic stem cells, comprising: a) providing a zona pellucida (ZP)-removed bovine embryo comprising naive bovine embryonic stem cells; b) contacting the ZP-removed bovine embryo with an extracellular matrix (ECM)-coated substrate, the ECM-coated substrate comprising a substrate comprising a negatively charged substrate surface adjacent to a positively charged biocompatible polymer layer and a negatively charged ECM layer adjacent to the positively charged biocompatible polymer layer; and c) culturing the ZP-removed bovine embryos in the presence of an elongation medium to induce attachment of the ZP-removed bovine embryos to the ECM-coated substrate and elongation of an inner cell mass (ICM) comprising induced naive bovine embryonic stem cells; A method comprising:

2. The method of claim 1 , wherein the positively charged biocompatible polymer layer comprises type A gelatin.

3. The method of claim 1 , wherein the negatively charged ECM layer comprises an EHS-ECM.

4. The method of claim 1 , wherein the substrate comprises polystyrene.

5. The elongation medium comprises: N2B27 component; Wnt activator component; Wnt inhibitor component; MEK / ERK inhibitor component; ROCK inhibitor component; LIF components; Activin A component PKC inhibitors; and Insulin component; The method of claim 1 , comprising one or more of:

6. The elongation medium comprises: the N2B27 component; the Wnt activator component; the Wnt inhibitor component; the MEK / ERK inhibitor component; the ROCK inhibitor component; the LIF component; the PKC inhibitor; and the insulin component; The method of claim 5 , comprising:

7. 7. The method of claim 6, wherein the elongation medium further comprises an activin A component, optionally human activin A.

8. the N2B27 component comprises a B27 supplement and an N2 supplement, optionally about 1% B27 supplement and about 0.5% N2 supplement; the Wnt activator component comprises CHIR99021, BIO, CHIR-98014, LY2090314, and / or IM-12; the Wnt inhibitor component comprises XAV939, IWR-1, and / or IWP-2; the MEK / ERK inhibitor component comprises PD0325901, lavoxertinib, GSK1120212, MEK162, PD184352, trametinib, LY3214996, and / or ulixertinib; the ROCK inhibitor component comprises Y27632, thiazovivin, and / or blebbistatin; the LIF component comprises human LIF; the PKC inhibitor comprises Go6983, Go6976, LY317615, LY333531, PKC412, GSK690693, sotrastaurin, staurosporine, and / or bisindolylmaleimide; and / or the insulin component comprises insulin; The method of claim 5.

9. 2. The method of claim 1, wherein the ZP-removed bovine embryo is a day 3 to 8 embryo, optionally a day 6 or 7 embryo, or wherein the ZP-removed embryo is a morula (stage 4); a blastocyst (stage 5); an expanding blastocyst (stage 6); an expanding blastocyst (stage 7); a hatching blastocyst (stage 8); or a hatched blastocyst (stage 9).

10. 2. The method of claim 1, wherein the ZP-removed bovine embryos are obtained by enzyme-assisted ZP removal.

11. 11. The method of claim 10, comprising the steps of: a) providing an embryo; b) contacting the embryo with a protease solution; c) incubating the embryos in the protease solution to partially digest the ZP and obtain ZP-thinned embryos; d) contacting the ZP-thinned embryo with a protease-inactivating medium to inactivate the protease; e) breaking the ZP; and f) manipulating the embryo to separate the ZP from the embryo; obtaining said ZP-removed bovine embryos by enzyme-assisted ZP removal comprising:

12. 12. The method of claim 11, wherein the concentration of the protease in step c) is between 0.1% and 0.5%, optionally about 0.25%.

13. 12. The method of claim 11, wherein the embryos and the protease solution are incubated in step c) for about 30-60 seconds, optionally for about 45 seconds.

14. 12. The method of claim 11, wherein step e) uses a microblade to break the ZP.

15. 12. The method of claim 11, wherein the manipulation of the embryo in step f) comprises pipetting.

16. 10. The method of claim 1, wherein the ZP-removed embryo is genetically modified.

17. 10. The method of claim 1, wherein the ZP-depleted embryos are genetically tested to determine one or more genotypes of the ZP-depleted embryos for one or more biomarkers.

18. 10. The method of claim 1, further comprising performing genetic testing to determine one or more genotypes of the ZP-removed bovine embryos for one or more biomarkers.

19. 10. The method of claim 1, further comprising selecting the ZP-removed bovine embryos based on genetic testing of one or more biomarkers.

20. The method of claim 1, further comprising performing a genetic test to determine one or more genotypes of the induced naive bovine embryonic stem cells.

21. The method of claim 1 , wherein the ZP-removed bovine embryo is obtained from a reconstituted diploid embryo.

22. 10. The method of claim 1, wherein said ZP-removed bovine embryo is obtained from a fresh embryo, optionally a fresh biopsied embryo.

23. 10. The method of claim 1, wherein said ZP-removed bovine embryo is obtained from a frozen embryo, optionally a biopsy-frozen embryo.

24. 24. The method of claim 23, comprising thawing the frozen embryo and contacting the embryo with a recovery medium.

25. a) thawing the frozen embryo; b) contacting the frozen embryo with the recovery medium; c) manipulating the embryo to separate the ZP from the embryo in the recovery medium; and d) incubating said ZP-removed bovine embryos in said recovery medium; 25. The method of claim 24, comprising obtaining the ZP-removed bovine embryos by a method comprising:

26. 25. The method of claim 24, wherein the recovery medium comprises a glycogen synthase kinase 3 (GSK-3) inhibitor, a MEK / ERK kinase inhibitor, and a Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor.

27. 25. The method of claim 24, wherein the recovery medium comprises CHIR99021, PD0325901 and Y27632.

28. 25. The method of claim 24, further comprising incubating the ZP-removed embryos with an adaptation medium, wherein the adaptation medium comprises a combination of recovery medium and elongation medium.

29. 29. The method of claim 28, wherein the adaptation medium comprises a combination of recovery medium and elongation medium in a ratio of between about 0.3:1 and 1.7:1, between about 0.5:1 and 1.5:1, or optionally about 1:

1.

30. A naive bovine embryonic stem cell produced using the method according to any one of claims 1 to 29.

31. 31. Use of the naive bovine embryonic stem cells of claim 30 in a breeding scheme or genetic improvement program.

32. Use of naive bovine embryonic stem cells as described in claim 30 for growing preimplantation embryos, optionally with desirable genetic characteristics; deriving primordial germ cells and / or gametes, optionally for in vitro breeding programs, and / or developing and providing veterinary biological or therapeutic products.

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