Non-cloning method for bovine embryo replication

Abstract

The present disclosure relates to methods, products, and compositions useful for deriving bovine induced blastoids. Methods are described that allow for juvenile stem cell aggregation and induced blastocyst formation. The invention further provides an aggregation culture medium and an induced blastocyst-like culture medium which are suitable for promoting aggregation of the young stem cells and preparing the induced blastocyst-like. The bovine induced blastoids can be used in selection and breeding procedures, including the replication of pre-implant embryos with desired characteristics and in vitro breeding including a cross-generation breeding cycle from parent germ cells and / or embryos to progeny embryos.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 456,624, filed April 3, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure relates to the derivation of bovine embryos, and more specifically to methods for producing multiple bovine embryos with desired characteristics from preparations of bovine juvenile stem cells, as well as related methods, selection and / or breeding procedures and compositions. Background Technology

[0004] The widespread use of artificial insemination (AI) has facilitated the precise genetic evaluation of male livestock for use in genetic improvement programs. However, for most genetic traits, AI lacks the ability to produce offspring with known traits due to recombination and crossing over during meiosis. Cloning techniques have been proposed as a solution to provide offspring with limited genetic diversity and greater homogeneity. Since the 1990s, numerous cloned animals have been produced using nuclear transfer methods. In recent years, researchers have used somatic cell nuclear transfer (SCNT) in the production of genetically modified livestock, which is considered by many to be the preferred technique because it produces genotypic homogeneity and lacks the likelihood of chimerism (Springer et al., 2021). However, SCNT generally results in low biological and economic efficiency due to the time-consuming nature of procedures such as oocyte enucleation and nuclear transfer, low embryo productivity (approximately 20-30% blastocyst formation), and the need for highly skilled specialists to perform the procedure. High rates of pregnancy loss, stillbirth, calf malformations (such as giant offspring syndrome), and suboptimal animal health and welfare are frequently associated with SCNT, particularly in cattle and other ruminants (Hill, 2014; Mueller and Van Eenennaam, 2022; Springer et al., 2021). In summary, this severely limits the use of nuclear transfer / SCNT as a tool for producing large numbers of identical embryos for large-scale / commercial purposes.

[0005] In 2021, two independent groups published in Nature their methods for generating induced blastoids (iblastoids) from human pluripotent stem cells (Yu et al., 2021 and Liu et al., 2021). Both successfully reassembled human blastocyst-like structures using either immature embryonic stem cells (Yu et al., 2021) or induced pluripotent stem cells (iPSCs, Liu et al., 2021), both exhibiting characteristics of preimplantation embryos. Furthermore, two additional papers have recently been published demonstrating improved quality of the produced human induced blastoids through protocol optimization (Yanagida et al., 2021 and Kagawa et al., 2022). Human induced blastoids expressed similar RNA transcriptome characteristics to in vitro-generated human blastoids. Moreover, induced blastoids could be induced into embryonic (immature and primed pluripotent stem cells) and extraembryonic (trophoblast stem cells and extraembryonic endoderm cells) cell lineages. Furthermore, when inoculated onto endometrial cells, induced blastocysts responded in a similar manner to conventional embryos in terms of their in vitro attachment ability. Based on these studies, researchers have concluded that induced blastocysts can serve as a substitute for real embryos in early human embryonic development research. However, due to ethical and regulatory concerns limiting the use of human stem cells and human embryos, it remains unknown whether human induced blastocysts can develop further after being transferred to the uterus.

[0006] In contrast, for farm animals, Pérez-Gómez et al. (2021) concluded that "primary bovine trophic ectodermal cell cultures can be established using relatively simple media… while the conditions required for true pluripotent ectodermal cell cultures in farm animals remain to be elucidated." Therefore, these researchers depicted significant differences in gene regulatory networks and roles in cell differentiation events from early embryonic development to implantation in humans, mice, and ungulates. Also noteworthy are the differences in placental structure in developing fetuses of ruminants, horses, pigs, and humans and mice. These findings on farm animals highlight the need for improved embryo and pluripotent cell culture and the development of species-specific assisted reproductive technologies, such as the generation of non-human induced blastocysts.

[0007] Furthermore, animal science can derive broad benefits from induced blastocyst technology by enhancing genetic improvement, expanding the reproduction and distribution of desired genetic resources, improving the consistency and predictability of genetic products, reducing genetic lag, and providing a platform for research and development in embryonic development, assisted reproductive technologies, and veterinary medical biopharmaceuticals and therapeutics. Key drivers of genetic change in genetic improvement systems include the accuracy and intensity of selection, genetic variation, the average age of parents at birth, and genetic lag. The genetic changes resulting from animal genetic improvement programs are significantly enhanced when genomic selection is combined with assisted reproductive technologies such as multiovulatory embryo transfer, oocyte retrieval, in vitro embryo production, and / or early-stage in vitro fertilization and embryo transfer (Mueller and Van Eenennam, 2022). When these technologies are combined, they enable accurate prediction of genetic value early in life, including for preimplantation embryos (Saadi et al., 2014b), resulting in shorter generation intervals and increased parental selection intensity. In recent years, genetic modification technology has provided new tools for targeted genetic alteration in animal populations (Mueller and Van Eenennam, 2022; Park, 2007; US 7,067,713).

[0008] New and / or improved methods are needed to produce large quantities of identical embryos for use in conventional and in vitro breeding systems, large-scale commercial applications, and the production and widespread distribution of healthy, superior, and / or genetically modified livestock. The preparation of bovine induced blastocysts can facilitate the efficient replication of embryos with desired characteristics. There remains a need for bovine induced blastocysts and related methods for their production. Summary of the Invention

[0009] One of the main advantages of the induced blastocysts described herein is that they are produced without the need for existing cloning methods. The disclosed induced blastocyst production technology represents a highly efficient and cost-effective method for producing large quantities of identical embryos, for generating and replicating superior genetic material (for small-scale use), for selection and breeding systems as described herein, and for large-scale applications in commercial settings, such as replicating genetically modified germplasm, reconstructed diploid embryos, and / or embryos with desired characteristics.

[0010] To enable this technology to be used in livestock, including cattle, as described in this article, techniques for using bovine juvenile stem cells have been developed and evaluated. 3D culture system for stem cells, culture conditions for producing bovine induced blastocysts, and selection and breeding systems for their use.

[0011] This document describes materials and methods that can be used to achieve the aggregation of bovine juvenile stem cells, the reassembly of blastocyst-like induced blastocysts, and the use of induced blastocysts in selection and breeding systems. As demonstrated in the examples, bovine induced blastocysts can be generated from bovine juvenile stem cells cultured under aggregation conditions, such as using hanging drop culture, low-attachment culture dishes, or microwell devices. As shown herein, juvenile stem cells seeded in microwell devices provide greater aggregation and consistency in aggregate size compared to cells aggregated using hanging drop or low-attachment culture dish methods. Furthermore, induced blastocyst culture medium compositions and related methods supporting the formation of induced blastocysts are described, thereby allowing the preparation of bovine induced blastocysts. Therefore, the embodiments described herein can be used to prepare bovine induced blastocysts and optionally for selection and breeding programs, such as for replicating preimplantation embryos with desired genetic and epigenetic characteristics. Thus, in one aspect, a method for preparing bovine induced blastocysts is provided, the method comprising:

[0012] a) Provide bovine juvenile stem cell populations;

[0013] b) The bovine juvenile stem cells were cultured in an aggregation medium under aggregation conditions to produce aggregated bovine juvenile stem cells;

[0014] c) Introduce a first induced blastocyst medium, optionally wherein the aggregated medium and non-aggregated cells are removed prior to the introduction of the first induced blastocyst medium;

[0015] d) The aggregated bovine juvenile stem cells were cultured in the first induced blastocyst medium under hypoxic conditions;

[0016] e) Introducing a second induced blastocyst medium, optionally wherein the first induced blastocyst medium is removed before the introduction of the second induced blastocyst medium; and

[0017] f) The aggregated bovine juvenile stem cells are cultured in the second induced blastocyst medium under hypoxic conditions to obtain the bovine induced blastocysts.

[0018] In one implementation, the bovine juvenile stem cell population in step a) is dissociated bovine juvenile stem cells, and / or the bovine juvenile stem cells are bovine embryo-derived juvenile stem cells.

[0019] In one embodiment, step b) includes introducing the cells into one or more wells of the microporous device and culturing the cells for about 24 hours. In one embodiment, the diameter of the wells in the microporous device is about 400 μm to about 600 μm. In one embodiment, the bovine juvenile stem cell population is added to the microporous device at a concentration of about 20-60 cells / well, optionally at a concentration of about 20-30 cells / well.

[0020] In one implementation, the cells are cultured for approximately 48 hours in step d) and / or step f).

[0021] In one embodiment, the aggregation medium comprises: a serum substitute component; and a Rho-associated coiled-coil protein kinase (ROCK) inhibitor component. In one embodiment, the serum substitute component comprises a) an N2B27 component, optionally about 1% B27 component and about 0.5% N2 component; b) an insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) a serum substitute (SR); or d) a knockout serum substitute (KOSR); and / or the ROCK inhibitor component comprises Y27632, fasudil, thiazovivin, or brebbistatin, optionally 5-10 μM Y27632.

[0022] In one embodiment, the first induced blastocyst culture medium comprises: a serum substitute component; a MEK / ERK inhibitor component; a TGFβ-1 inhibitor component; a HIPPO pathway inhibitor component; and a ROCK inhibitor component. In one embodiment, the serum substitute component comprises a) an N2B27 component, optionally about 1% B27 supplement and about 0.5% N2 supplement; b) an insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) a serum substitute (SR); or d) a knockout serum substitute (KOSR). In one embodiment, the MEK / ERK inhibitor component comprises PD0325901, ravoxertinib, GSK1120212, MEK162, PD184352, trametinib, LY3214996, or ulixertinib, optionally about 1 μM PD0325901. In one embodiment, the TGFβ-1 inhibitor component comprises A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736, optionally about 1 μM A83-01. In one embodiment, the HIPPO pathway inhibitor component comprises lysophosphatidylcholine (LPA), optionally about 1 μM LPA. In one embodiment, the ROCK inhibitor component includes Y27632, fasudil, thiazovirine, or brestatin, optionally 5-10 μM Y27632.

[0023] In one embodiment, the first induced blastocyst culture medium further comprises a second serum substitute component, optionally ITS-X, optionally 0.1-1% ITS-X; a LIF component, optionally human LIF, bovine LIF, goat LIF, porcine LIF, buffalo LIF, or recombinant LIF, optionally at a concentration of about 10 ng / ml; an epidermal growth factor (EGF) component, optionally about 50 ng / ml EGF; a Wnt agonist, optionally Wnt3a, Wnt agonist 1, or SKL2001, optionally 10-100 ng / mL Wnt3a; a GSK3β inhibitor, optionally CHIR99021, BIO, CHIR-98014, LY2090314, or IM-12, optionally 1-2 μM CHIR99021; and a histone deacetylase inhibitor, optionally valproic acid (VPA), sodium butyrate, or triamcinolone A, optionally 0.2-0.8 mM. VPA; and / or a second TGFβ-1 inhibitor, optionally 0.1-1 μM SB431542. In embodiments where the first inducible blastocyst medium contains an EGF component, the first inducible blastocyst medium may further contain a fibroblast growth factor (FGF) component, optionally basic fibroblast growth factor (bFGF) or FGF4, optionally at a concentration of 10-30 ng / ml.

[0024] In one embodiment, the second induced blastocyst culture medium comprises: a serum substitute component; a TGFβ-1 inhibitor component; a HIPPO pathway inhibitor component; and a ROCK inhibitor component. In one embodiment, the serum substitute component comprises a) an N2B27 component, optionally about 1% B27 supplement and about 0.5% N2 supplement; b) an insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) a serum substitute (SR); or d) a knockout serum substitute (KOSR). In one embodiment, the TGFβ-1 inhibitor component comprises A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736, optionally about 1 μM A83-01. In one embodiment, the HIPPO pathway inhibitor component comprises lysophosphatidylcholine (LPA), optionally about 1 μM LPA. In one embodiment, the ROCK inhibitor component comprises Y27632, fasudil, thiazovirine, or brestatin, optionally 5-10 μM Y27632.

[0025] In one embodiment, the second induced blastocyst medium further comprises one or more of the following: a second serum substitute component, optionally ITS-X, optionally 0.1-1% ITS-X; a fibroblast growth factor (FGF) component, optionally basic fibroblast growth factor (bFGF) or FGF4, optionally at a concentration of 10-30 ng / ml; a activin A component, optionally human activin A, bovine activin A or mouse activin A, optionally at a concentration of 5-50 ng / ml; and / or a GSK3β inhibitor, optionally CHIR99021, BIO, CHIR-98014, LY2090314 or IM-12, optionally 1-3 μM CHIR99021. In embodiments where the second induced blastocyst medium contains an FGF component, the second induced blastocyst medium may further contain an epidermal growth factor (EGF) component, optionally about 50 ng / ml EGF.

[0026] In one embodiment, the anoxic conditions comprise about 5%-10% CO2, optionally about 6.8% CO2, and about 1%-5% O2, optionally about 5% O2.

[0027] In one embodiment, the first induced blastocyst medium and / or the second induced blastocyst medium are replaced with fresh medium after approximately 24 hours.

[0028] In one embodiment, the bovine juvenile stem cell population is derived from primordial pluripotent stem cells or induced pluripotent stem cells.

[0029] In one embodiment, the bovine juvenile stem cell population is embryo-derived juvenile stem cells, optionally derived from preimplantation embryos, or embryo-derived expanded pluripotent stem cells.

[0030] In one implementation, the bovine juvenile stem cell population is derived by a method comprising the following steps:

[0031] Provide bovine embryos containing bovine juvenile stem cell-like cells, optionally the bovine embryos do not contain the zona pellucida (ZP);

[0032] The bovine embryo is brought into contact with an extracellular matrix (ECM)-coated substrate, wherein the ECM-coated substrate comprises a substrate including 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

[0033] The bovine embryos were cultured in the presence of an outgrowth medium to induce attachment of the bovine embryos to the ECM-coated matrix and the growth of the inner cell mass (ICM) containing derived bovine juvenile stem cells.

[0034] In one implementation, the bovine juvenile stem cells are genetically modified and / or genome-edited cells.

[0035] In one embodiment, the method further includes testing one or more biomarkers to determine one or more characteristics of the bovine juvenile stem cell population in response to one or more biomarkers, and optionally determining a score based on the determined characteristics.

[0036] In one embodiment, the method further includes selecting the bovine juvenile stem cell population based on one or more characteristics determined by testing one or more biomarkers and / or the score.

[0037] In one embodiment, the method further includes testing one or more biomarkers to determine one or more characteristics of the induced blastocyst, and optionally determining a score based on the determined characteristics.

[0038] In one embodiment, the method further includes selecting one or more of the induced blastocysts based on the one or more characteristics determined by testing one or more biomarkers and / or the scores.

[0039] One aspect includes multiple induced blastocysts prepared using the methods described herein.

[0040] Another aspect includes the use of the induced blastocyst technology described herein for selection and breeding systems. Therefore, this paper provides a bovine breeding method comprising:

[0041] a) Select at least one bovine parent from among multiple animals;

[0042] b) Obtain multiple gametes from the at least one bovine parent;

[0043] c) Generate at least one embryo from the plurality of gametes;

[0044] d) Derive a population of juvenile stem cells from at least one of the embryos;

[0045] e) Prepare one or more induced blastocysts according to the methods described herein; and

[0046] f) Select one or more of the induced blastocysts for breeding, optionally for in vitro breeding, and / or for transplantation into a female surrogate recipient to produce offspring.

[0047] In one embodiment, one or more biomarkers of the at least one embryo are tested to determine one or more characteristics of the at least one embryo, and optionally a score is determined based on the determined characteristics.

[0048] This document also provides an aggregation culture medium composition comprising: a serum substitute component; and a Rho-associated coiled-coil protein kinase (ROCK) inhibitor component. In one embodiment, the serum substitute component comprises a) an N2B27 component, optionally about 1% B27 component and about 0.5% N2 component; b) an insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) a serum substitute (SR); or d) a knockout serum substitute (KOSR); and / or the ROCK inhibitor component comprises Y27632, fasudil, thiazovivin, or brebbistatin, optionally 5-10 μM Y27632.

[0049] This document also provides an induced blastocyst culture medium composition 1 comprising: a serum substitute component; a MEK / ERK inhibitor component; a TGFβ-1 inhibitor component; a HIPPO pathway inhibitor component; and a ROCK inhibitor component. In one embodiment, the serum substitute component comprises a) an N2B27 component, optionally about 1% B27 component and about 0.5% N2 component; b) an insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) a serum substitute (SR); or d) a knockout serum substitute (KOSR). In one embodiment, the MEK / ERK inhibitor component comprises PD0325901, lavotinib, GSK1120212, MEK162, PD184352, trametinib, LY3214996, or uritinib, optionally about 1 μM PD0325901. In one embodiment, the TGFβ-1 inhibitor component comprises A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736, optionally about 1 μM A83-01. In one embodiment, the HIPPO pathway inhibitor component comprises lysophosphatidylcholine (LPA), optionally about 1 μM LPA. In one embodiment, the ROCK inhibitor component comprises Y27632, fasudil, thiazovirine, or brestatin, optionally 5-10 μM Y27632. In one embodiment, the induced blastocyst culture medium 1 composition further comprises one or more of the following: a second serum substitute component, optionally 0.1-1% ITS-X; a LIF component, optionally human LIF, bovine LIF, goat LIF, porcine LIF, buffalo LIF, or recombinant LIF, optionally at a concentration of about 10 ng / ml; an epidermal growth factor (EGF) component, optionally about 50 ng / ml EGF; a GSK3β inhibitor, optionally CHIR99021, BIO, CHIR-98014, LY2090314, or IM-12, optionally 1-2 μM CHIR99021; a Wnt activator, optionally Wnt3a, Wnt agonist 1, or SKL2001, optionally 10-100 ng / mL Wnt3a; and a histone deacetylase inhibitor, optionally valproic acid (VPA), sodium butyrate, or triamcinolone A, optionally 0.2-0.8 mM. VPA; and / or a second TGFβ-1 inhibitor component, optionally 0.1-1 μM SB431542.In an embodiment where the first induced blastocyst medium contains an EGF component, the first induced blastocyst medium may further contain a fibroblast growth factor (FGF) component, optionally basic fibroblast growth factor (bFGF) or FGF4, optionally at a concentration of 10-30 ng / ml.

[0050] This document also provides an induced blastocyst culture medium 2 composition comprising: a serum substitute component; a TGFβ-1 inhibitor component; a HIPPO pathway inhibitor component; and a ROCK inhibitor component. In one embodiment, the serum substitute component comprises a) an N2B27 component, optionally about 1% B27 component and about 0.5% N2 component; b) an insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) a serum substitute (SR); or d) a knockout serum substitute (KOSR). In one embodiment, the TGFβ-1 inhibitor component comprises A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736, optionally about 1 μM A83-01. In one embodiment, the HIPPO pathway inhibitor component comprises lysophosphatidylcholine (LPA), optionally about 1 μM LPA. In one embodiment, the ROCK inhibitor component comprises Y27632, fasudil, thiazolidin, or brestatin. In one embodiment, the induced blastocyst culture medium 2 composition further comprises one or more of the following: a second serum substitute component, optionally ITS-X, optionally 0.1-1% ITS-X; a fibroblast growth factor (FGF) component, optionally basic fibroblast growth factor (bFGF) or FGF4, optionally at a concentration of 10-30 ng / ml; an activator A component, optionally human activator A, bovine activator A or mouse activator A, optionally at a concentration of 5-50 ng / ml; a Wnt activator, optionally Wnt3a, Wnt agonist 1 or SKL2001; and / or a GSK3β inhibitor, optionally CHIR99021, BIO, CHIR-98014, LY2090314 or IM-12, optionally 1-3 μM CHIR99021. In an embodiment where the induced blastocyst culture medium 2 contains an FGF component, the induced blastocyst culture medium 2 may also contain an epidermal growth factor (EGF) component, optionally about 50 ng / ml EGF.

[0051] Another aspect includes the use of the culture medium composition described herein for the preparation of bovine induced blastocysts.

[0052] The foregoing portions are provided by way of example only and are not intended to limit the scope of this disclosure and the appended claims. Those skilled in the art will appreciate additional objects and advantages associated with the compositions and methods of this disclosure based on the claims, specification, and embodiments of the invention. For example, various aspects and embodiments of this disclosure can be utilized in many combinations, all of which are expressly covered by this specification. These additional advantages, objects, and embodiments are expressly included within the scope of this disclosure. Publications and other materials used to elucidate the background of this disclosure and, in particular, to provide additional details regarding practice, are incorporated by reference and, for convenience, are listed in the appended references section. Attached Figures

[0053] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description, taken in conjunction with the accompanying drawings illustrating illustrative embodiments of the present disclosure, in which:

[0054] Figure 1 This image shows organoids generated using the hanging drop method to produce trophoblast stem (TS) cells. A: Hanging drop culture dish (left) and stereomicroscopic image showing the cell suspension (right). B: Producing perfect droplets can be challenging (arrows indicate broken droplets). C: Cell aggregation after 48 hours. The organoids resemble the shape of a conventional embryo with a blastocoel (arrows) or have the appearance of a collapsed embryo (star-shaped). D: Bright field (left) and DNA staining (right) of TS organoids. E: DNA staining of a day 7 blastocyst (dashed circle indicates ICM).

[0055] Figure 2 Images show blastocyst-like structures derived from trophoblast stem (TS) cells produced in low-attachment culture dishes. Small cell clusters from day 0 had grown into blastocyst-like structures after 48 hours.

[0056] Figure 3 The image shows a microporous device (Stemfit3D, catalog number H853400) inserted into a 35 mm culture dish (left) and an image of the microporous device under a stereomicroscope (right).

[0057] Figure 4 Images of blastocyst-like structures produced using a microwell system are shown. A: Feeder stem (TS) cell aggregates in microwells after 24 hours. B: Images of several TS cell-derived blastocyst-like structures (after transfer to standard culture dishes) after 48 hours of culture in microwells. C: Aggregates of bovine juvenile stem cells (BSCs) derived from microwells after 24 hours. D: Aggregates of bovine juvenile stem cells (BSCs) derived from microwells after 48 hours. E: An image showing a blastocyst-like structure (red arrow) derived from bovine juvenile stem cells in a blastocoel after 96 hours of culture in microwells.

[0058] Figure 5This image shows SOX2 / CDX2 / SOX17 (S / C / S) staining for markers of the inner cell mass (ICM), trophectoderm (TE), and hypoblastomere in blastocysts (day 7 and day 11 embryos) and induced blastocysts (day 5). SOX2 (ICM), CDX2 (TE), and SOX17 (hypoblastomere) positive cells are well-organized in embryonic structures according to developmental stage. B: SOX2 (ICM) and CDX2 (TE) positive cells are well-organized in induced blastocyst structures. + / CDX2 - Structure of the area indicator cavity (arrow).

[0059] Figure 6 This diagram illustrates the steps involved in generating multiple genetically identical embryos (also known as induced blastocysts) from bovine juvenile stem cells.

[0060] Figure 7 The steps for characterizing candidate germplasm for a target market are shown. Candidate germplasm that meets specified requirements can be used for selection and breeding systems, such as... Figure 8 and Figure 9 Those shown.

[0061] Figure 8 A schematic diagram of a selection and breeding system is shown, which uses induced blastocysts to produce hybrid animals for target commercial markets with specific market requirements.

[0062] Figure 9 A schematic diagram of a selection and breeding system is shown, which uses induced blastocysts to produce purebred animals for target commercial markets with specific market requirements. Detailed Implementation

[0063] The following detailed description is provided to assist those skilled in the art in practicing this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. The terminology used in this description is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. All publications, patent applications, patents, drawings, and other references mentioned herein are expressly incorporated herein by reference in their entirety.

[0064] Furthermore, as those skilled in the art will understand, the definitions and embodiments described in certain sections are intended to apply to other embodiments to which they are suitable. For example, different aspects of this disclosure are defined in more detail in the following paragraphs. Unless expressly indicated to the contrary, each aspect so defined may be combined with any other one or more aspects. In particular, any feature described herein may be combined with any other one or more features described herein.

[0065] I. General Definition

[0066] As used herein, unless otherwise stated, the following terms may have the meanings assigned to them below. However, it should be understood that other meanings known or understood by one of ordinary skill in the art are also possible and are within the scope of this disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of any conflict, this specification (including definitions) shall prevail. Furthermore, the materials, methods and examples are illustrative only and are not intended to be limiting.

[0067] When a range of values ​​is provided, it should be understood that this specification covers every midpoint between the upper and lower limits of the range up to 1 / 10 of the lower limit unit (unless the context explicitly states otherwise), as well as any other stated value or midpoint within the range. A range covering any lower limit to any upper limit is included. The upper and lower limits of these smaller ranges, which may be independently included within smaller ranges, are also included in this specification, depending on any explicitly excluded limits within the range. Where the range includes one or both limits, ranges excluding any one or both of the included limits are also included in this specification.

[0068] It should be noted that, unless the context clearly specifies otherwise, the singular forms “a (a, an)” and “the” as used herein and in the appended claims include plural indications.

[0069] All numerical values ​​in the detailed description and claims herein are modified by the terms “about” or “approximately” and take into account experimental errors and variations that would be expected by one of ordinary skill in the art.

[0070] As used herein in the specification and claims, the phrase “and / or” should be understood to mean “any one or two” of the elements so combined, that is, elements that exist together in some cases and separately in others. Multiple elements listed with “and / or” should be interpreted in the same way, that is, “one or more” of the elements so combined. In addition to the elements specifically identified by the “and / or” clause, other elements may optionally exist, whether related to or unrelated to those specifically identified.

[0071] As used herein in the specification and claims, “or” 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 multiple elements or at least one of the elements in the list, but also including more than one, and optionally, additional unlisted items. Only terms explicitly stated to the contrary, such as “only one of them” or “exact one of them,” or when used in the claims, “consisting of…,” refer to including multiple elements or exactly one of the elements in the list. In general, the term “or” as used herein should be interpreted as indicating an exclusive alternative (i.e., “one or the other, but not both”) only when preceded by an exclusive term (such as “any one,” “one of them,” “only one of them,” or “exact one of them”).

[0072] In the claims, and in the foregoing description, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “constituting,” etc., shall be understood as open-ended, meaning including but not limited to. Only the transitional phrases “constituting of” and “constituting substantially of” shall be closed or semi-closed transitional phrases, respectively.

[0073] As used herein in the specification and claims, the phrase "at least one" in relation to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements referred to by the phrase "at least one," whether related to or unrelated to those specifically identified elements.

[0074] As used herein, the term “about” means ±10%–15%, 5–10%, or optionally about 5% of the referenced value.

[0075] It should be understood that in some methods described herein that include more than one step or action, unless the context otherwise indicates, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described.

[0076] It should also be understood that any methods and materials similar to or equivalent to those described and materials herein may also be used to practice or test this disclosure.

[0077] II. Methods

[0078] This document describes a method for preparing bovine induced blastocysts. As illustrated in the examples, the inventors have demonstrated the formation of bovine induced blastocysts with blastocyst-like structures comprising a blastocoel and containing cells expressing markers of ICM (expressing SOX2) and TE (expressing CDX2). Upon further culture, the induced blastocysts contain hypoblastocyst cells (expressing SOX17). Therefore, the materials and methods described herein can be used to derive and maintain bovine induced blastocysts, and optionally for breeding programs such as for replicating preimplantation embryos with desired characteristics.

[0079] Therefore, in one aspect, a method for preparing bovine induced blastocysts is provided. In one embodiment, the method includes:

[0080] a) Provide bovine juvenile stem cell populations;

[0081] b) The bovine juvenile stem cells were cultured in an aggregation medium under aggregation conditions to produce aggregated bovine juvenile stem cells;

[0082] c) Introduce a first induced blastocyst medium, optionally wherein the aggregated medium and non-aggregated cells are removed prior to the introduction of the first induced blastocyst medium;

[0083] d) The aggregated bovine juvenile stem cells were cultured under hypoxic conditions in the first induced blastocyst medium for approximately 48 hours;

[0084] e) Introducing a second induced blastocyst medium, optionally wherein the first induced blastocyst medium is removed before the introduction of the second induced blastocyst medium; and

[0085] f) The aggregated bovine juvenile stem cells are cultured under hypoxic conditions in the second induced blastocyst medium for approximately 48 hours to obtain the bovine induced blastocysts.

[0086] As used herein, the term "induced blastocyst" refers to a spherical 3D structure with a diameter of 100–200 μm (closely matching the size of a day 7 blastocyst), containing two morphologically distinct cell lineages (i.e., inner cell mass (ICM) and trophectoderm (TE) cells) and a cavity. Induced blastocysts are labeled with appropriate S / C / S staining when properly stained, for example, approximately 5–7 days after the onset of aggregation. This appropriate S / C / S staining consists of prominent SOX2 staining of ICM cells and CDX2 staining of TE cells, but without SOX17 signaling (similar to day 7 embryos). However, using extended in vitro embryo culture and staining, SOX17 signaling can become visible as early as approximately 7–9 days after the onset of aggregation (similar to day 9 embryos). When subjected to specific conditions for each cell type, induced blastocysts can be used to derive juvenile stem cells, primordial stem cells, and trophectoderm stem cells, or can be transplanted into recipients or surrogates for further growth and development.

[0087] As used herein, the term "immature stem cell" refers to a stem cell capable of being derived and maintained in a self-renewing, undifferentiated state without the need for exogenous expression of pluripotency factors, and capable of developing into a complete organism and / or retaining the ability to generate a full range of extraembryonic tissues, adult tissues, and / or cell types. Immature stem cells exhibit molecular characteristics substantially similar to those of early blastocyst morula and ICM cells, and female immature stem cells possess an X chromosome signature similar to that of preimplantation embryos. Bovine immature stem cells can be identified by, for example, round, dome-shaped cell colony morphology and the co-expression of one or more pluripotency markers (such as SOX2, OCT4, or NANOG) and one or more immature factors (SUSD2 and / or TFCP2L1 and / or KLF4 and / or other selected pluripotency and immature-specific factors, such as those described by Messimer et al. (2019)). It should be understood that immature stem cells are unlikely to exist in vivo, but can be derived from sufficiently undifferentiated ICM cells (referred to herein as "immature stem cell-like cells") under certain cell culture conditions.

[0088] Immature stem cells, such as bovine juvenile stem cells, can be derived directly from sufficiently undifferentiated tissues, such as, for example, blastomeres (2-4 cell embryos), or ICM cells of preimplantation embryos, such as morula (stage 4); blastocysts (stage 5); expanding blastocysts (stage 6); expanding blastocysts (stage 7); hatching blastocysts (stage 8); or hatched blastocysts (stage 9). In one embodiment, bovine juvenile stem cells can be derived from bovine embryos of 2 to 8 days, optionally 2, 3, 4, 5, 6, 7, or 8 days, or 5 to 7 days. In one embodiment, the bovine embryo is a blastomeres (2-4 cell embryos); morula (stage 4); blastocysts (stage 5); expanding blastocysts (stage 6); expanding blastocysts (stage 7); hatching blastocysts (stage 8); or hatched blastocysts (stage 9). In one embodiment, the bovine embryo is a 3 to 7 day embryo, optionally a 5 to 7 day embryo, or a 6 or 7 day embryo. In one embodiment, the embryo-derived cells are ICM-derived cells. In one embodiment, the bovine embryo is a bovine embryo without the zona pellucida. In one embodiment, the embryo is a preimplantation embryo. In one embodiment, the embryo is an embryo that has been previously frozen and / or biopsied.

[0089] The method described, for example, in Patent Cooperation Treaty application PCT / CA2022 / 051664 (filed November 11, 2022), or the method for expanding pluripotent stem cells described by Zhao et al. (2021), can be used to derive juvenile stem cells, such as bovine juvenile stem cells. In one embodiment, the bovine juvenile stem cells are embryo-derived juvenile stem cells. In another embodiment, the bovine juvenile stem cells are embryo-derived expanded pluripotent stem cells. In one embodiment, the embryo has been selected based on testing for one or more biomarkers.

[0090] Cellular potential resetting is considered an alternative method for deriving juvenile stem cells. For example, juvenile stem cells, such as bovine juvenile stem cells, can be derived from primordial embryonic stem cells as described by Guo et al. (2017), or from induced pluripotent stem cells (iPSCs) derived from fully differentiated cells such as fibroblasts, as disclosed in Liu et al. (2021) or WO2022 / 109667 A1 (Methods and Cell Structures). Thus, in one embodiment, bovine juvenile stem cells are iPSC-derived juvenile pluripotent stem cells. In another embodiment, bovine juvenile stem cells are derived from primordial pluripotent stem cells. Sources of pluripotent stem cells (e.g., primordial, expanded, or induced pluripotent stem cells) may include those derived using methods described, for example, those described in Soto et al. (2021), Bigliotti et al. (2018), Zhao et al. (2021), or Han et al. (2011).

[0091] Embryo-derived juvenile stem cells can also be generated in vitro using methods known in the art. For example, oocytes and sperm can be collected from mating pairs (female and male animals) and used to produce embryos via in vitro fertilization. In one embodiment, one or more biomarkers of the female and / or male animals are tested, and optionally, scores and / or selections for in vitro fertilization are based on the results of said tests.

[0092] Alternatively, juvenile stem cells may be derived from diploid reconstructed parental embryos. Diploid embryos with a predetermined genome can be generated in vitro by reconstructing parental embryos using selected androgynous and parthenogenetic embryonic haploid cells, as described, for example, in WO 2020 / 168422 (Use of Haploid Embryonic Cells to Generate Offspring with Predetermined Genomes), the contents of which are incorporated herein by reference in their entirety. Thus, in one embodiment, the bovine embryo is a reconstructed diploid embryo. A genome can be generated for the reconstructed diploid embryo to contain a unique combination of alleles, haplotypes, epigenetic markers, or traits that meet stringent genetic and / or predictive phenotypic criteria.

[0093] In the various embodiments described herein, juvenile stem cells are dissociated before being cultured under aggregated conditions. Various methods can be used to dissociate juvenile stem cells, such as those described in Example 1. Other methods may also be used.

[0094] In the various embodiments described herein, one or more biomarkers of naive stem cells have been tested, and optionally scored and / or selected based on the testing of one or more biomarkers.

[0095] As used herein, “testing for one or more biomarkers” includes testing for genetic, genomic, and / or epigenetic characteristics, including but not limited to the presence or absence of one or more specific alleles, single nucleotide polymorphisms (SNPs), genomic insertions and / or deletions, histone modifications, protamine modifications, DNA methylation, gene or mRNA expression levels, protein expression or modifications, and metabolite analysis. Testing for one or more biomarkers may be performed at any step of the methods described herein. For example, embryos, outgrowths, stem cells, induced blastocysts, sperm, oocytes, and / or animals (single female and male animals, or optionally mated pairs) may be tested.

[0096] As used herein, “trait” or “characteristic” refers to a specific feature of an animal, embryo, growth, stem cell, or induced blastocyst that may be influenced or determined by one or more genetic factors (e.g., allele variants, epigenetic markers, and combinations thereof) and / or environmental factors.

[0097] In some implementations, naive stem cells have been genetically modified.

[0098] "Genetically modified cells" refer to cells in which the genomic DNA of the cell has been manipulated to express one or more exogenous genes and / or to introduce mutations in endogenous genes or intergenic regions, said mutations affecting the expression or functional activity of one or more endogenous genes or gene products. Examples of successful gene modification in livestock include the introduction of transgenes via microinjection (US7,067,713) and lentiviral infection (Park, 2007), as well as more recent genome editing using transfection and genome editors such as zinc finger nucleases, transcription activator-like effector nucleases (TALEN), and clustered regularly spaced short palindromic repeats / CRISPR-associated genes (CRISPR / Cas) systems (reviewed by Bishop and Van Eenennaam, 2020, for livestock; and by Mueller and Van Eenennaam, 2022, for cattle). Common bovine targets for genetic modification include, for example, milk protein genes such as β-lactoglobulin, β-casein, myostatin, horned / polled, prolactin receptors that impart smooth fur to improve heat resistance, and various genes involved in disease susceptibility or resilience (Bishop and Van Eenennam, 2020 and Mueller and Van Eenennam, 2022).

[0099] Similarly, a “genetically modified embryo” refers to an embryo in which the genomic DNA of the cells in the embryo has been manipulated to express one or more exogenous genes and / or to introduce mutations in endogenous genes or intergenetic regions, said mutations affecting the expression or functional activity of one or more endogenous genes or gene products.

[0100] A dissociated bovine juvenile stem cell population can be obtained, for example, by using the methods described herein or other methods known in the art, through dissociation of the bovine juvenile stem cell population.

[0101] The term "aggregation conditions" refers to conditions that promote the formation of cell aggregates. For example, as shown in the embodiments herein, a hanging drop method can be used to aggregate cells, wherein small droplets of dissociated cells are suspended from a surface such as the underside of a tissue culture dish lid, and the cells come into contact with each other and adhere after settling to the bottom of the droplets. Thus, in one embodiment, aggregation conditions include hanging drops. In another embodiment shown herein, low-adhesion culture conditions can be used to aggregate cells, wherein cells are introduced into an environment (such as a low-adhesion culture dish) where cell adhesion to surfaces in the environment is inhibited, and the cells instead adhere to each other. Thus, in one embodiment, aggregation conditions include a low-adhesion environment, optionally an untreated, low-adhesion, or non-adhesive culture dish or plate. In yet another embodiment shown herein, culturing cells in a non-adhesive or ultra-low-adhesion microporous device results in the formation of relatively uniform blastocyst-like structures.

[0102] As used herein, the term "microwell device" refers to a container, tube, cell culture dish, or cell culture plate containing one or more culture spaces (e.g., wells) with diameters in the micrometer range. For example, a microwell device may include one or more wells with diameters ranging from about 100 μm to about 1000 μm, optionally from about 400 μm to about 600 μm. A microwell device may include any number of culture spaces (or wells) from one well to about 1000 or more wells. Standard configurations may include 6, 12, 24, 48, or 96-well plates or inserts. Microwell devices may contain non-adhesive, uncoated, or ultra-low adhesion materials, such as untreated or ultra-low adhesion treated plastics or non-plastic materials, such as silicon. Microwell devices may have circular or conical bottoms to induce efficient cell aggregation. Commercially available microporous devices that can be used in the methods described herein include, but are not limited to, Stemfit 3D (Microfit, catalog numbers: H389600L, H389600H, H853400, or H1613200); Aggrewell (Stemcell technology, catalog numbers 34411, 34421, 34811, or 34821); and non-adhesive 96-well plates (Thermo Scientific). TM (Catalogue numbers 174925, 174927; Corning, catalogue number CLS7007). Customized non-adhesive microporous devices optionally having a pore diameter range of 400 μm to about 600 μm can also be used. In one embodiment, the microporous device may include a plastic / silicone-based biocompatible polymer insert.

[0103] As understood in the art, the terms "incubate" or "incubating" refer to maintaining, for example, a substance, material, composition, etc., at a specific temperature or temperature range for a period of time.

[0104] 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.

[0105] As understood in the art, pH is affected by the concentration of carbon dioxide (CO2) in the environment. Typical concentrations used for tissue culture range from about 5% CO2 to about 10% CO2, optionally about 5% CO2 or about 6.8% CO2.

[0106] As used herein, the term "hypoxic condition" refers to a condition where the oxygen (O2) concentration is lower than the atmospheric oxygen concentration, i.e., lower than about 20.95% oxygen. For example, when cells are incubated under hypoxic conditions, they are incubated in a reduced oxygen environment (e.g., about 1% to about 15% oxygen, optionally about 1% to 10% oxygen, or about 1% to about 5% oxygen).

[0107] As illustrated herein, bovine juvenile stem cells aggregate and form induced blastocyst structures by sequentially culturing in various culture medium compositions, including aggregation medium, induced blastocyst medium 1, and induced blastocyst medium 2. The various culture media may contain, for example, a basal medium and one or more small molecules, growth factors, and / or nutrients. Suitable basal media can be readily determined by those skilled in the art and include, but are not limited to, DMEM / F12 (Dulbecco's Modified Eagle's Medium F12), advanced DMEM / F12, and Neurobasal medium. Suitable supplements can be readily determined by those skilled in the art and include, but are not limited to, minimum essential medium (MEM), non-essential amino acids (NEAA), L-glutamine (e.g., 1-2 mM), Glutamax, ascorbic acid, insulin, BSA (fraction V), β-mercaptoethanol, penicillin / streptomycin, and / or gentamicin. In one embodiment, the basal medium comprises a 1:1 mixture of DMEM / F12 and Neurobasal medium, 1x Glutamax, 1x NEAA, 0.1mM β-mercaptoethanol, and 50 IU / ml penicillin / streptomycin.

[0108] In one aspect of this disclosure, aggregation medium components are provided for use in the derivation and maintenance of induced blastocysts. In one embodiment, the aggregation medium comprises a basal medium and / or supplements, and one or more aggregation medium components. In one embodiment, the aggregation medium comprises one or more of the following: a serum substitute component; and a ROCK inhibitor component. In one embodiment, the serum substitute component comprises one or more of the following: a) an N2B27 component comprising a B27 supplement and an N2 supplement; b) an insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) a serum substitute (SR; Sigma); or d) a knockout serum substitute (KOSR; Gibco). In one embodiment, the N2B27 component comprises about 0.5% to about 2% of a B27 supplement, optionally about 1% of a B27 supplement, and about 0.5% to about 1% of an N2 supplement, optionally about 0.5% of an N2 supplement. In one embodiment, the ROCK inhibitor component includes Y27632, fasudil, thiazovirine, or brestatin. In one embodiment, the aggregation medium may contain N2B27 and Y27632.

[0109] In one aspect of this disclosure, an induced blastocyst culture medium component is provided for use in deriving and / or maintaining induced blastocysts.

[0110] In one embodiment, the first induced blastocyst medium comprises a basal medium and / or supplements, and one or more first induced blastocyst medium components. In one embodiment, the first induced blastocyst medium (also referred to herein as induced blastocyst medium 1) comprises a serum substitute component, optionally an N2B27 component, said N2B27 component comprising a B27 supplement and an N2 supplement, optionally comprising about 0.5% to about 2% of a B27 supplement, optionally about 1% of a B27 supplement, and about 0.5% to about 1% of an N2 supplement, optionally about 0.5% of an N2 supplement; a MEK / ERK inhibitor component, optionally PD0325901, lavotinib, GSK1120212, MEK16. 2. PD184352, trametinib, LY3214996 or unitinib; TGFβ-1 inhibitors, optionally A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3 or TP0427736; HIPPO pathway inhibitors, optionally lysophosphatidic acid (LPA); and ROCK inhibitor components, optionally Y27632, fasudil, thiazovirine or brestatin. In some embodiments, the induced blastocyst culture medium 1 may alternatively or additionally contain a serum substitute component (e.g., in addition to or in place of N2B27), said serum substitute component being selected from one or more of the following: insulin-transferrin-selenium component (optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G)); serum substitute (SR; Sigma); or knockout serum substitute (KOSR; Gibco). In some embodiments, the induced blastocyst culture medium 1 may further contain one or more of the following: a LIF component, optionally human LIF, bovine LIF, goat LIF, porcine LIF, buffalo LIF, or recombinant LIF, optionally at a concentration of about 10 ng / ml; an epidermal growth factor (EGF) component, optionally EGF; a GSK3β inhibitor, optionally CHIR99021, BIO, CHIR-98014, LY2090314, or IM-12; a Wnt activator, optionally Wnt3a, Wnt agonist 1, or SKL2001; and a histone deacetylase inhibitor (HDAC inhibitor), optionally valproic acid (VPA), sodium butyrate, or triamcinolone A, optionally at 0.2-0.8 mM. VPA; and / or a second TGFβ-1 inhibitor, optionally SB431542, A83-01, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3 or TP0427736.Optionally, the induced blastocyst medium 1 may contain one or more of N2B27, PD0325901, A83-01, LPA, human LIF, and / or Y27632. In embodiments where the first induced blastocyst medium contains an EGF component, the first induced blastocyst medium may further contain a fibroblast growth factor (FGF) component, optionally basic fibroblast growth factor (bFGF) or FGF4, optionally at a concentration of 10-30 ng / ml.

[0111] In another embodiment, the second induced blastocyst medium comprises a basal medium and / or a supplement, as well as one or more second induced blastocyst medium components. In one embodiment, the second induced blastocyst medium (also referred to herein as induced blastocyst medium 2) comprises one or more serum substitute components, optionally an N2B27 component, said N2B27 component comprising a B27 supplement and an N2 supplement, optionally comprising about 0.5% to about 2% of a B27 supplement, optionally about 1% of a B27 supplement, and about 0.5% to about 1% of an N2 supplement, optionally about 0.5% of an N2 supplement; a TGFβ-1 inhibitor, optionally A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736; a HIPPO pathway inhibitor, optionally LPA; and a ROCK inhibitor component, optionally Y27632, fasudil, thiazovirine, or brestatin. In some embodiments, the induced blastocyst medium 2 may alternatively or additionally contain a serum substitute component (e.g., in addition to or in place of N2B27), said serum substitute component being selected from one or more of the following: insulin-transferrin-selenium component (optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G)); serum substitute (SR; Sigma); or knockout serum substitute (KOSR; Gibco). In some embodiments, the induced blastocyst medium 2 may further comprise one or more of the following: a fibroblast growth factor (FGF) component, optionally basic fibroblast growth factor (bFGF) or FGF4; an activator A component, optionally human activator A, bovine activator A or mouse activator A; a Wnt activator, optionally Wnt3a, Wnt agonist 1 or SKL2001; and / or a GSK3β inhibitor, optionally CHIR99021, BIO, CHIR-98014, LY2090314 or IM-12. Optionally, the induced blastocyst medium 2 may comprise one or more of N2B27, A83-01, LPA and / or Y27632. In embodiments where the second induced blastocyst medium contains an FGF component, the second induced blastocyst medium may further comprise an epidermal growth factor (EGF) component, optionally about 50 ng / ml EGF.

[0112] III. Uses of Inducible Blastocysts in Bovine Breeding Programs

[0113] The method described in this article for preparing bovine induced blastocysts can be used to generate multiple genetically identical bovine embryos without cloning, which can then be used in bovine breeding programs.

[0114] To assess market suitability and its role in selection and breeding systems, germplasm can be characterized. Biopsies can be performed on embryos, fetuses, growths, stem cell lines, or induced blastocysts at any stage to remove a small number of cells (approximately 5 to 10), and the remaining cells can be used fresh or cryopreserved for future use in establishing cell lines and / or induced blastocysts. Alternatively, cell lines can be established from whole embryos or induced blastocysts, and a set of cells can then be used for genomic and / or epigenetic evaluation. Cell lines can be maintained in viable cultures while awaiting genomic results, or can be later frozen and thawed when genetic and / or predicted phenotypic information is available.

[0115] For genomic or epigenetic analysis, DNA is extracted from biopsied embryos, fetuses, sperm, animal or induced blastocyst cells, or established cell lines, and optionally amplified to obtain sufficient DNA for genomic analysis. When the amount of DNA collected is insufficient, DNA amplification protocols suitable for samples with small amounts of DNA can be used to amplify the DNA, such as those described in Saadi et al. (2014a) and Saadi et al. (2014b).

[0116] In one embodiment of this disclosure, target cells (e.g., embryos, growths, stem cells, induced blastocysts, animals, sperm, oocytes) and / or matings are characterized by testing one or more biomarkers, and optionally by scoring and / or selecting desired biomarkers (e.g., genetic, genomic, epigenetic, and / or efficaciousness characteristics).

[0117] A “score” generally refers to testing one or more biomarkers against multiple traits or characteristics, and then generating a score consisting of a series of traits weighted equally or differently according to their relative values. Scoring can be performed on embryos, growths, stem cells, induced blastocysts, animals, sperm, oocytes, and / or matings based on the results of testing one or more biomarkers. A “score” can also refer to a value consisting of hundreds or thousands of genomic markers and / or epigenetic markers that span the entire genome and individually or collectively constitute a genomic prediction of an individual’s breeding value, transmissibility, or phenotypic ability. In some embodiments, the score is a genomic score, optionally including a weighted combination of one or more single nucleotide polymorphisms and / or one or more epigenetic markers. Examples of such scores include, but are not limited to, genomic breeding value, genomic transmissibility, or genomic predicted phenotypic ability (gPPA), wherein optionally the gPPA may also include effects that may affect phenotypic ability but are not transmissible to offspring (Varona et al., 2018). Optionally, specific market, environmental and / or economic conditions, as well as related interactions, including interactions with genotypes (e.g., genotype-environment interactions), may be considered to construct a comprehensive index for scoring.

[0118] When obtaining genomic breeding values ​​from each cell and / or cross, the homozygosity of the cell or cross and its genomic imputation can be initially assessed (Sargolzaei et al., 2014).

[0119] Optionally, other genetic and / or non-genetic effects may be incorporated into genomic selection (Varona et al., 2018; David et al., 2019).

[0120] Alternatively, genomic breeding values ​​can be obtained from embryos, induced blastocysts, fetuses, sperm, oocytes, animals, stem cells, or mating without genomic filler. In other words, given a sufficient density of biomarker genotypes, it is not necessary to infer any biomarkers from neighboring biomarkers or implicit lineages based on genomic relationships.

[0121] Testing, scoring, and / or selection may also take into account the genetic makeup of target cells (e.g., embryos, growths, stem cells, induced blastocysts, animals, sperm, oocytes) and / or mating pairs (see [link to relevant documentation]). Figure 7For example, Khansefid et al. (2020) reported, “In summary, to improve hybrid GP [genome prediction], we recommend including hybrid animals and using a balanced breed reference set of SNPs [single nucleotide polymorphisms] that are close to QTLs [quantitative trait loci] and enriched with causal mutations.” Therefore, in one implementation, testing, scoring, and / or selection is performed considering one or more designated markets and / or one or more genetic (e.g., breed, family) compositions.

[0122] For example, candidate male-female pairs can be characterized as described above, and the gPPA score of future offspring can be determined. Optionally, the gPPA score may also include one or more of non-additive genetic effects, homozygous effects, and effects of epigenetic markers.

[0123] Hayes et al. (2009) describe genomic selection as “selection decisions based on genomic [estimated] breeding value (GEBV). GEBV is calculated as the sum of the effects of dense genetic markers or haplotypes of these markers throughout the genome, potentially capturing all quantitative trait loci (QTLs) leading to phenotypic variation.” Therefore, “selection” refers to identifying subpopulations of embryos, stem cells, induced blastocysts, animals, and / or matings with the most desirable scores in a population, and selecting one or more individuals from the identified subpopulations for the methods and / or breeding programs described therein. Animal subpopulations or matings may be selected based on one or more scores (e.g., as described above). In one embodiment, selection is performed considering one or more designated markets and / or one or more genetic (e.g., breed, family) compositions. Figure 7 Exemplary steps for characterizing (testing, screening, scoring, and selecting) candidate germplasm to determine its suitability for a target market are shown.

[0124] In one implementation, cells are scored, wherein genomic and / or epigenetic markers characterizing cells include genomic scoring of cells.

[0125] In one implementation, the mating pairs are scored, wherein genomic and / or epigenetic markers characterizing the mating pairs include genomic scoring of the mating pairs.

[0126] In another form of the method, cell selection is performed after assessing the homozygosity of the mating pairs or cells and / or their genomic incompleteness and / or estimating the genomic breeding value and / or genomic predictive performance of each embryo, fetus, induced blastocyst, sperm, oocyte, animal, stem cell line or mating pair.

[0127] In one form of the method, testing, scoring, and / or selection are performed to:

[0128] a) Producing subpopulations of induced blastocysts, juvenile stem cells, haploid embryos, animals, etc., enriched with specific traits, or

[0129] b) Generate subpopulations of induced blastocysts, juvenile stem cells, haploid embryos, animals, etc., that do not possess specific traits, or

[0130] c) To generate subpopulations of induced blastocysts, juvenile stem cells, haploid embryos, animals, etc., with a range of advantageous traits and / or without a range of disadvantageous traits, or

[0131] d) To generate a subpopulation of induced blastocysts, juvenile stem cells, haploid embryos, animals, etc., that do not possess one or more harmful haplotypes, or

[0132] e) Producing breeding animals or breeding lines (induced blastocysts, immature stem cells, haploid embryos, etc.) containing unique combinations of alleles, haplotypes, or traits, wherein the alleles, haplotypes, or traits typically occur at a "low" frequency, usually less than 50% of the population, but present at least 0.1% or at least 0.01%.

[0133] f) Generate subpopulations of induced blastocysts, immature stem cells, haploid embryos, animals, etc., that are particularly well-suited to specific environments or markets.

[0134] In another embodiment of this disclosure, genetic, genomic, or epigenetic traits include one or more 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, survival rate, disease resistance), reproductive traits (e.g., pregnancy rate, conception rate), calving traits (e.g., ease of calving, calving to first insemination, stillbirth), body type traits (e.g., hornless trait, mammary and teat traits, hoof traits, body traits, size traits), efficiency traits (e.g., feed efficiency-related, methane efficiency, processability, lifespan, productive lifespan), novel traits (e.g., robotic milking traits, heat tolerance, smooth coat, activity traits, and behavioral traits), and composite index traits (e.g., Life Production Index (LPI), Total Productive Index (TPI), LifetimeNet Merit). Dollars, NM$), heterozygosity, and / or the absence of various harmful alleles and haplotypes (e.g., haplotypes affecting fertility, dwarfism, mulefoot, sparse hair, brachyspina, citrullinemia, bovine leukocyte adhesion defect).

[0135] Once one or more mating pairs are selected based on one or more scores or traits (e.g., those mentioned above), they can be used to produce one or more embryos. For example, oocytes and sperm are collected from the females and males of the highest / most desirable 50% of offspring gPPA scores, respectively, using standard methods known in the art.

[0136] Embryos are produced via in vitro fertilization using germ cells collected from selected mating pairs with the most desired offspring gPPA scores. Optionally, the embryos may be characterized to determine the gPPA score (and / or other desired characteristics) of individual embryos.

[0137] Embryos (or juvenile stem cells derived from them) can be selected based on one or more desired traits, scores, etc., and then used to generate multiple genetically identical induced blastocysts using the methods described herein. These blastocysts can then be transferred to a female surrogate recipient and conceived to produce genetically identical offspring. The performance of the offspring can then be determined for one or more traits (e.g., lactation traits, meatiness traits, etc., optionally based on fitness for a given environment or market, as needed). Alternatively, or additionally, phenotypic data from the initial cohort will be incorporated into the analysis to calculate updated scores. The offspring can then be included in future breeding programs, if desired. Alternatively, or additionally, induced blastocysts can be selected and used in in vitro breeding programs.

[0138] If desired, for example, if performance and / or updated scores of genetically identical offspring are particularly desired, additional genetically identical induced blastocysts may also be generated from a bank of naive stem cells and / or induced blastocysts.

[0139] A representative selection and breeding system with the potential to produce market-specific genetic products (including embryos and offspring) using the disclosed induced blastocyst technology is outlined in Figure 8 (for hybrids) and Figure 9 (For purebreds) In addition to utilizing molecular and advanced reproductive techniques such as genomic selection, genomically predicted performance capabilities, oocyte retrieval, in vitro embryo production, and reassembly of blastocyst-like induced blastocysts, the system also includes optional gene modification steps. Breeding systems described by others (e.g., Hou et al., 2018; Bishop and Van Eenennam, 2020; Mueller and Van Eenennam, 2022, to name a few) can also benefit from the use of the disclosed induced blastocyst technology when the production of multiple genetically identical embryos is desired.

[0140] IV. Products and compositions of matter and their uses

[0141] In one aspect of this disclosure, products and compositions of substances that can be used for the aggregation, formation, and / or maintenance of bovine induced blastocysts are provided.

[0142] Formulations suitable for promoting the aggregation of bovine juvenile stem cells, the formation of bovine induced blastocysts, and / or for preparing culture media as described herein are also provided.

[0143] In one embodiment, the aggregation medium comprises a basal medium and one or more components identified herein. For example, in one embodiment, the aggregation medium comprises one or more of a serum substitute component (such as an N2B27 component) and a Rho-associated coil-and-coil protein kinase (ROCK) inhibitor component.

[0144] In one embodiment, the serum substitute component comprises one or more of the following: a) an N2B27 component; b) an insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) a serum substitute (SR; Sigma); or d) a knockout serum substitute (KOSR; Gibco). In one embodiment, the N2B27 component comprises a B27 supplement and an N2 supplement, optionally about 0.5% to about 2% of the B27 supplement, optionally about 1% of the B27 supplement, and about 0.5% to about 1% of the N2 supplement, optionally about 0.5% of the N2 supplement.

[0145] In one embodiment, the ROCK inhibitor component includes Y27632, fasudil, thiazovirine, or brestatin. Optionally, the ROCK inhibitor component includes Y27632, optionally at a concentration of about 0.5 μM to about 20 μM, optionally about 5 μM to about 10 μM, optionally about 5 μM, or about 10 μM.

[0146] In one embodiment, the induced blastocyst medium 1 comprises a basal medium and one or more components identified herein. For example, in one embodiment, the induced blastocyst medium 1 comprises a serum substitute component; a MEK inhibitor; a TGFβ-1 inhibitor; a HIPPO pathway inhibitor; and a ROCK inhibitor.

[0147] In one embodiment, the serum substitute component comprises a) an N2B27 component; b) an insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X), optionally 0.1-1% ITS-X, or insulin-transferrin-selenium (ITS-G); c) a serum substitute (SR; Sigma); or d) a knockout serum substitute (KOSR; Gibco). In one embodiment, the N2B27 component comprises a B27 supplement and an N2 supplement, optionally about 0.5% to about 2% of the B27 supplement, optionally about 1% of the B27 supplement, and about 0.5% to about 1% of the N2 supplement, optionally about 0.5% of the N2 supplement.

[0148] In one embodiment, the MEK / ERK inhibitor component includes PD0325901, lavotinib, GSK1120212, MEK162, PD184352, trametinib, LY3214996, or unitinib. Optionally, the MEK / ERK inhibitor component includes PD0325901 or lavotinib. Optionally, the MEK / ERK inhibitor component includes PD0325901 at concentrations of about 0.05 μM to about 5 μM, optionally about 0.1 μM to about 2 μM, or optionally about 1 μM. Optionally, the MEK / ERK inhibitor component includes lavotinib at concentrations of about 0.25 μM to about 10 μM, optionally about 1 μM to about 5 μM, or optionally about 2.5 μM.

[0149] In one embodiment, the TGFβ-1 inhibitor includes A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736. Optionally, the TGFβ-1 inhibitor component includes A83-01 at concentrations of about 0.1 μM to about 5 μM, optionally about 0.5 μM to about 2 μM, and optionally about 1 μM.

[0150] In one embodiment, the HIPPO pathway inhibitor includes lysophosphatidylcholine (LPA) at concentrations of optionally about 0.1 to about 5 μM, optionally about 0.5 μM to 2 μM, and optionally about 1 μM.

[0151] In one embodiment, the ROCK inhibitor component includes Y27632, fasudil, thiazovirine, or brestatin. Optionally, the ROCK inhibitor component includes Y27632, optionally at a concentration of about 0.5 μM to about 20 μM, optionally about 5 μM to about 10 μM, optionally about 5 μM, or about 10 μM.

[0152] In one embodiment, the induced blastocyst culture medium 1 further comprises one or more of the following: a second serum substitute component, optionally ITS-X, optionally 0.1-1% ITS-X; a LIF component, optionally human LIF, bovine LIF, goat LIF, porcine LIF, buffalo LIF, or recombinant LIF, optionally at a concentration of about 10 ng / ml; an epidermal growth factor (EGF) component, optionally about 50 ng / ml EGF; a Wnt activator, optionally 10-100 ng / mL Wnt3a; a GSK3β inhibitor, optionally 1-2 μM CHIR99021; a histone deacetylase inhibitor (HDAC inhibitor), optionally valproic acid (VPA), sodium butyrate, or triamcinolone A, optionally 0.2-0.8 mM VPA; and / or a second TGFβ-1 inhibitor, optionally 0.1-1 μM. SB431542, optionally A83-01, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736. In embodiments where the first inducible blastocyst medium contains an EGF component, the first inducible blastocyst medium may also contain a fibroblast growth factor (FGF) component, optionally basic fibroblast growth factor (bFGF) or FGF4, optionally at a concentration of 10-30 ng / ml.

[0153] In one embodiment, the LIF component includes human LIF, bovine LIF, porcine LIF, goat LIF, buffalo LIF, or recombinant 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.

[0154] In one embodiment, the induced blastocyst medium 2 comprises a basal medium and one or more components identified herein. For example, in one embodiment, the induced blastocyst medium 2 comprises one or more of the following: a serum substitute component, such as an N2B27 component; a TGFβ-1 inhibitor component; a HIPPO pathway inhibitor; and a ROCK inhibitor.

[0155] In one embodiment, the serum substitute component comprises one or more of the following: a) an N2B27 component; b) an insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) a serum substitute (SR; Sigma); or d) a knockout serum substitute (KOSR; Gibco). In one embodiment, the N2B27 component comprises a B27 supplement and an N2 supplement, optionally about 0.5% to about 2% of the B27 supplement, optionally about 1% of the B27 supplement, and about 0.5% to about 1% of the N2 supplement, optionally about 0.5% of the N2 supplement.

[0156] In one embodiment, the TGFβ-1 inhibitor includes A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736. In one embodiment, the TGFβ-1 inhibitor includes A83-01, optionally at a concentration of about 0.5 μM to about 2 μM, optionally about 1 μM.

[0157] In one embodiment, the HIPPO pathway inhibitor includes lysophosphatidylcholine (LPA), optionally at a concentration of about 0.5 μM to about 2 μM, optionally about 1 μM.

[0158] In one embodiment, the ROCK inhibitor component includes Y27632, fasudil, thiazovirine, or brestatin. Optionally, the ROCK inhibitor component includes Y27632, optionally at a concentration of about 0.5 μM to about 20 μM, optionally about 5 μM to about 10 μM, optionally about 5 μM, or about 10 μM.

[0159] In one embodiment, the induced blastocyst medium 2 further comprises one or more of the following: a second serum substitute component, optionally ITS-X, optionally 0.1-1% ITS-X; a fibroblast growth factor (FGF) component, optionally basic fibroblast growth factor (bFGF) or FGF4, optionally at a concentration of 10-30 ng / ml; an activator A component, optionally 5-50 ng / ml activator A; a Wnt activator, optionally Wnt3a, Wnt agonist 1 or SKL2001; and / or a GSK3β inhibitor, optionally 1-3 μM CHIR99021. In embodiments where the induced blastocyst medium 2 contains an FGF component, the induced blastocyst medium 2 may also contain an epidermal growth factor (EGF) component, optionally about 50 ng / ml EGF.

[0160] In one embodiment, the activator A component includes human activator A, bovine activator A, or mouse activator 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.

[0161] In one embodiment, the GSK3β inhibitor component includes CHIR99021, BIO, CHIR-98014, LY2090314, or IM-12. Optionally, the GSK3β inhibitor component includes CHIR99021, optionally at a concentration of about 0.1 μM to about 5 μM, optionally about 1 μM to about 3 μM, optionally about 1 μM, about 2 μM, or about 3 μM.

[0162] In one embodiment, the Wnt activator component includes Wnt3a, Wnt agonist 1, or SKL2001. In one embodiment, the Wnt activator component includes Wnt3a, optionally at a concentration of about 10 ng / mL to about 100 ng / mL.

[0163] Kits are also provided that contain one or more of the aggregation medium, induced blastocyst medium 1, and / or induced blastocyst medium 2 as described herein. In one embodiment, the aggregation medium, induced blastocyst medium 1, and / or induced blastocyst medium 2 are packaged in separate containers. In one embodiment, the kit further includes a microporous device for preparing induced blastocysts.

[0164] The products, compositions or kits described herein are also provided for use in promoting the aggregation of naive stem cells in culture and / or for forming induced blastocyst structures, including bovine induced blastocysts.

[0165] For example, in one embodiment, the use of an aggregation medium as described herein for promoting the aggregation of dissociated juvenile stem cells (optionally, bursal juvenile stem cells) is provided.

[0166] In another embodiment, the use of induced blastocyst media (e.g., induced blastocyst media 1 and induced blastocyst media 2) as described herein is provided for culturing aggregated juvenile stem cells and promoting the formation of induced blastocysts. In one embodiment, the induced blastocyst media can be used to prepare induced blastocysts, optionally keratocysts.

[0167] The following non-limiting embodiments exemplify this application:

[0168] Example

[0169] Example 1. Preparation of a 3D culture system

[0170] When attached to feeder cells or a protein matrix, juvenile stem cells proliferate into flattened / dome-shaped colonies. Generating 3D structures from attached colonies is not efficient because cells tend to proliferate through a given supporting structure (scaffold). To efficiently generate blastocyst-like structures from attached cells, various 3D culture systems can be used, such as hanging drop cultures, low-attachment cell culture dishes, or microporous devices made of synthetic polymers. The first objective is to develop a protocol for generating bovine induced blastocysts from established bovine juvenile stem cells and established genetically modified bovine juvenile stem cells. The next step is to adapt this protocol for large-scale production of bovine induced blastocysts for commercial purposes.

[0171] In the examples below, three different 3D culture systems were tested using bovine trophoblast stem cells (TSCs). One reason for using TSCs in these tests is the ease of culturing them. Trophoblast stem cells form blastocyst cavities under appropriate conditions, thus TSCs can represent an ideal model for testing techniques of induced blastocyst production. TSCs are also available from embryos of different stages and qualities, providing a robust testing system.

[0172] To avoid any confusion, the protocols described below are based on tests performed using TSC. Then, in Example 5, a protocol for induced blastocyst production is described.

[0173] Cell dissociation protocol (for providing dissociated cells for Examples 2-5)

[0174] 1. Aspirate the culture medium from the petri dish and use a calcium-free solution. 2+ / Mg 2+ Wash the cells three times with DPBS.

[0175] Note: Other calcium-free alternatives can be used here. 2+ and Mg 2+ Buffers / culture media, such as those without Ca 2+ / Mg 2+ Hank's balanced salt solution (HBSS) or Earl's balanced salt solution (EBSS).

[0176] 2. Add TrypLE to the washed cells and incubate the cells in a humidified incubator containing 5% CO2 at 38.5°C for 5 minutes.

[0177] Note: Other types of dissociation buffers, such as Accutase, collagenase IV, and 0.05% trypsin, can be used here.

[0178] 3. Gently pipette to dissociate the cells into small clusters. Transfer them to a conical tube. Add 10 ml of solution containing Ca. 2+ / Mg 2+ The DPBS was centrifuged at 300xg for 5 minutes.

[0179] Note: Other substances containing Ca can be used here. 2+ and Mg 2+ Buffers / culture media such as Hank Balanced Salt Solution (HBSS), Earl Balanced Salt Solution (EBSS), DMEM, DMEM / F12, Advanced DMEM, Advanced DMEM / F12, Neurobasal medium, RPMI 1640, α-MEM, Opti-MEM and other cell culture media.

[0180] 4. Discard the supernatant and resuspend the cell pellet in 1 ml of fresh N2B27 culture medium.

[0181] Note 1: N2B27 medium is a 1:1 mixture of basal medium DMEM / F12 and Neurobasal medium, supplemented with 1% B27 supplement, 0.5% N2 supplement, 1x Glutamax, 1x NEAA, 0.1mM β-mercaptoethanol and 50 IU / ml penicillin / streptomycin.

[0182] Note 2: The N2B27 basal medium can be replaced by DMEM / F12 or advanced DMEM / F12, and other supplements can be used.

[0183] 5. Centrifuge at 300x g for 5 minutes.

[0184] 6. Discard the supernatant and resuspend the cell pellet in 1 ml of fresh N2B27 culture medium.

[0185] Example 2. Hanging Drop System

[0186] Hanging drop systems are commonly used to generate 3D structures from many different cell types, such as mouse embryonic stem cells (Wang and Yang, 2008), kidney cells (Wang et al., 2017), cancer cells (Foty 2011), and hepatocytes (Shri et al., 2017). To use this method, dissociated cell droplets (20-30 μl) containing 100-500 cells are prepared on the lid of a culture dish. The lid is then very carefully inverted back onto the culture dish to create the hanging droplet. This confined environment generates microgravity within the droplet, causing the contained cells to settle to the bottom of each droplet. After several hours to several days, depending on the cell type (24-48 h), cell aggregation begins to form through confluence contact conditions induced by microgravity.

[0187] This article describes protocols for generating blastocyst-like structures using a hanging drop system. Cell lines used in all protocols may include those that are genetically selected or genetically selected with modifications.

[0188] preparation of hanging drops

[0189] For the hanging drop method, with 1x10 4 A concentration of cells / ml was used with dissociated cells.

[0190] 1. Place 20 μl of dissociated cell suspension (200 cells / drop) on the lid of the culture dish.

[0191] 2. Add sterile water to cover the bottom of the petri dish.

[0192] Note: Any sterile liquid can be used to create a saturated humidity environment, such as physiological saline, DPBS, HBSS, EBSS, or Milli-Q (ultrapure) water.

[0193] 3. Turn the cap in very carefully (to avoid breaking any adhering droplets, see below). Figure 1 B) and place it on top of a petri dish containing sterile water.

[0194] 4. In a humidified incubator containing 5% CO2, the cells in the hanging drop were cultured at 38.5°C for up to 48 hours until the blastocyst-like structure became visible.

[0195] 5.48 hours later, the blastocyst-like structure was transferred to a conventional embryo culture dish.

[0196] result

[0197] Aggregation efficiency. Each 20 μL hanging drop contains approximately 10-15 aggregates.

[0198] The diameter of the structures produced using this method ranges from 50 to 150 μm.

[0199] The target diameter for blastocyst-like structures is approximately 100-150 μm (similar to the size of a day 7 blastocyst). Using this method, only one or two structures per 20 μL droplet are within the target size range.

[0200] Another important factor in the formation of blastocyst-like structures is the presence of a blastocoel. Using the pendant drop method, approximately 50% of the structures exhibit a cavity.

[0201] In summary, the pendant drop method has a relatively low aggregation efficiency (0.03 structures / μL), almost no control over the size of the generated structures, and only a small fraction of the structures exhibit blastocyst cavities.

[0202] Summary: Number of blastocyst-like structures produced: 4 culture dishes (10 drops / dish) per person / hour. 2 good structures / drop. Total 80 aggregates / hour (estimated maximum capacity).

[0203] Figure 1 D and Figure 1 Image E shows a side-by-side comparison of Hoechst-stained vesicles produced using this method with a day 7 embryo.

[0204] like Figure 1 As shown in D and 1E, the structures produced after DNA staining exhibit a cell organization pattern that closely matches the cell organization pattern observed from the blastocyst on day 7. Figure 1 D: TS organoid-blastocyst-like structure, ICM not expected here; 1E: Day 7 blastocyst with ICM, which can be easily observed through the high abundance of cells indicated by the dashed circle.

[0205] Other observations

[0206] It is worth noting that when the lid of the culture dish is flipped back to create a "hanging drop," the droplets can easily be disturbed or broken, rendering them unusable. Extensive training is required to obtain reliable results from the hanging drop method. Furthermore, the hanging drop method is labor-intensive, which may make it unsuitable for large-scale production of induced blastocysts.

[0207] Finally, in the hanging drop method, the cells were cultured for 48 hours with 20 μl of culture medium. Unbound by theory, juvenile stem cells are known to have a very active metabolism, and the culture conditions, particularly the limited availability of nutrients in the culture medium, may explain the low yield of blastocyst-like structures.

[0208] Example 3. Low-adhesion petri dish method

[0209] Low-attachment culture dishes can be used as an alternative to hanging drop systems. It has been successfully applied to many different cell types, such as embryonic stem cell differentiation (Valamehr et al., 2008), liver organoids (Thompson and Takebe, 2020), and brain organoids (Giandomenico et al., 2021). When coated with a hydrogel layer, the surface of the low-attachment culture dish completely inhibits cell adhesion. Because cells cannot attach to the coated culture dish, they will grow into 3D structures in the suspension culture system. Compared to the hanging drop method, the low-attachment culture dish method has many potential advantages, especially because it eliminates the need to prepare individual droplets, thus significantly increasing processing speed.

[0210] Scheme for low-adhesion culture dish method

[0211] For the low-adhesion culture dish method, use 1x106 A concentration of cells / ml was used with dissociated cells.

[0212] 1. Add 500 μl of fresh N2B27 medium to each well of a non-adherent dish (such as Corning, catalog 3473; NUNC, catalog 174930; Sciencell, catalog 0383).

[0213] 2. Add 100 μl of dissociated cells (1 x 10⁻⁶) 5 (One cell / well) is placed in each well.

[0214] 3. In a humidified incubator containing 5% CO2, culture the cells at 38.5°C for 48 hours until blastocyst-like structures become visible.

[0215] 4. After 48 hours of culture, the blastocyst-like structure is transferred to an embryo culture dish.

[0216] result

[0217] Aggregation efficiency: Each well contains approximately 300 good structures / 500 μL (the volume of culture medium contained in one well).

[0218] The structures produced using this method range in diameter from 50 to 150 μm, with 80% of the structures having a diameter of approximately 100 to 150 μm.

[0219] When observing the proportion of structures with blastocoel, 80% of the structures show a cavity.

[0220] In summary, the low-attachment culture dish method showed higher aggregation efficiency (0.38 structures / μL) and higher cavity formation efficiency (80% vs. 50%) compared to the pendant drop method (0.03 structures / μL). However, a large difference in structure size was observed.

[0221] To produce structures that are more uniform in size, and more importantly, closely match the size of day 7 embryos, different techniques may be required.

[0222] Example 4. Microporous System

[0223] While the methods of Examples 2 and 3 are frequently used to generate relatively large organoids (millimeter-scale), researchers have developed other methods to generate much smaller structures (micrometer-scale). Micropore systems have been used to generate cell aggregates, particularly for embryonal formation (Antonchuk 2013) or adult stem cell spheroids (Futrega et al., 2017). Recently, a group studying human embryonic stem cells has used micropore systems to generate human induced blastocysts (Yu et al., 2021; Liu et al., 2021). It is assumed that pores of a defined size will provide consistent culture conditions for the cells, thus generating aggregates of uniform size. Therefore, the ability of micropore systems to improve the size uniformity of the resulting bovine blastocyst-like structures was tested.

[0224] plan

[0225] 1. Add 1 ml of 0.1% BSA from N2B27 medium to StemFIT 3D. TM The microporous device (Microfit, catalog number H853400) was then incubated overnight at 38.5°C in a humidified incubator containing 5% CO2 to prepare the microporous device. Figure 3 )

[0226] 2. Cells were prepared using the cell dissociation protocol described in Example 1.

[0227] 3. Dilute the cell pellet to obtain 1x10 5 Concentration per ml.

[0228] 4. Add the dissociated cells to the microporous device to obtain approximately 20-60 cells / well.

[0229] 5. Cell aggregates can be observed after culturing at 38.5°C for 24 hours in a humidified incubator containing 5% CO2, and structures with blastocyst cavities can be observed after 48 hours of culture.

[0230] result

[0231] Aggregation efficiency ( Figure 4 )

[0232] Each device contains approximately 600 good structures per 1 mL (device volume).

[0233] The structures produced using this method have diameters ranging from 50 to 150 μm, with 70% of the structures having diameters of approximately 100 to 150 μm.

[0234] Blastocyst cavity was observed in approximately 75% of the generated structures.

[0235] In summary, compared with the low-attachment culture method (0.38 structures / μl), the microporous system exhibited similar aggregation efficiency (0.32 structures / μl) and produced structures with smaller size differences.

[0236] We did not want to be limited by theory, because the initial cell number and culture space size of all wells were similar, so the resulting blastocyst-like structures showed a greater degree of size uniformity than when using low-attachment culture dishes.

[0237] When compared to low-attachment culture dish methods, the microwell system requires similar production time and produces a greater number of uniformly sized blastocyst-like structures. Another advantage may relate to the abundance of nutrients available for cells when the microwell volume is approximately 5 μL and all wells “share” a total of 1 mL of culture medium (compared to 20 μL for the hanging drop method or 500 μL for the low-attachment method).

[0238] Example 5. Generation of induced blastocysts

[0239] Immature embryonic stem cells can originate from preimplantation ICM cells or early morula blastomeres, and can then differentiate into TE cells (Dong et al., 2020) and extraembryonic endoderm cells (XEN, Linneberg-Agerholm et al., 2019). In 2021, two studies reported the generation of human induced blastocysts by applying TE and XEN differentiation techniques to immature pluripotent stem cells (Liu et al., 2021 and Yu et al., 2021). In short, not wanting to be limited by theory, immature stem cells can be induced into the TE lineage by inhibiting the MEK / ERK and TGFβ-1 pathways. Inhibition of the HIPPO pathway induces TE cell polarization and coelom formation into induced blastocysts by activating the TEAD4 transcription factor. Then, activation of the MEK / ERK pathway by removing MEK inhibitors induces the initiation of immature stem cell differentiation, followed by endoderm differentiation.

[0240] The plan is as follows:

[0241] 1. (Day 0) Bovine juvenile stem cells were generated as described in Patent Cooperation Treaty Application No. PCT / CA2022 / 051664. The cells were dissociated as described in Example 1 and resuspended in N2B27 medium containing 5-10 μM Y27632.

[0242] Note: Immature stem cells derived from iPSCs, primordial embryonic stem cells, embryonic-derived expanded juvenile stem cells, or other suitable derived juvenile stem cells may also be used, and / or the cells may be genetically modified / genome-edited.

[0243] 2. Add 20-30 single juvenile stem cells to StemFIT 3D medium containing N2B27 + 5-10 μM MY27632 (ROCK inhibitor). TM The sample was placed in each well of the microfit device (Catalogue No. H853400) and incubated at 38.5°C for 24 hours in a humidified incubator containing 6.8% CO2 and 5% O2.

[0244] Note: ROCK inhibitors can reduce cell death caused by mechanical damage (Chen 2010).

[0245] 3. After 24 hours, the culture medium and non-aggregated cells were gently removed from the microporous device by pipetting.

[0246] 4. Add induced blastocyst-like medium 1 (N2B27 basal medium + 1μM PD0325901 (MEK inhibitor) + 1μM A83-01 (TGFβ-1 inhibitor) + 1μM LPA (lysophosphatidylcholine, HIPPO pathway inhibitor) + 10ng / ml LIF (leukemia inhibitory factor) + 5-10μM Y27632 (ROCK inhibitor)). Optional additives can improve efficiency: 0.1-1% ITS-X (insulin-transferrin-selenoethanolamine), 50ng / ml EGF (epidermal growth factor), 1-2μM CHIR99021 (GSK3β inhibitor), 0.2-0.8mM VPA (histone deacetylase inhibitor valproic acid) or 0.1-1μM SB431542 (TGFβ-1 inhibitor).

[0247] 5. Incubate the microporous device at 38.5°C for up to 24 hours in a humidified incubator containing 6.8% CO2 and 5% O2.

[0248] 6. On the second day, the medium was changed by repeating the induction blastocyst medium 1.

[0249] 7. On day 3 (approximately 48 hours after starting in induced blastocyst medium 1), replace the medium with induced blastocyst medium 2 (N2B27 basal medium + 1 μM A83-01 + 1 μM M PA + 5-10 μM Y27632). Optional additives may improve efficiency: second serum substitute components, such as 0.1-1% ITS-X; 10-30 ng / ml bFGF (basic fibroblast growth factor), 5-50 ng / ml activator A, or 1-3 μM CHIR99021. Incubate the samples in a humidified incubator containing 6.8% CO2 and 5% O2 at 38.5°C for up to 24 hours.

[0250] 8. On day 4, the culture medium was changed again using induced blastocyst medium 2.

[0251] Evaluation of induced blastocysts (S / C / S staining).

[0252] Depending on the developmental stage, embryos are composed of different cell types. Day 7 embryos primarily contain two cell types (ICM and TE), while day 9 through 15 embryos contain three cell types (ICM, TE, and hypoblastomere). To detect the presence of each cell type in induced blastocysts, day 5 induced blastocysts were morphologically evaluated and compared with day 7 and day 11 embryos stained according to a standard protocol.

[0253] The S / C / S staining protocol for induced blastocysts is as follows:

[0254] 1. Using Ca 2+ / Mg 2+ The induced blastocysts were washed three times with DPBS.

[0255] 2. Fix the induced blastocysts with cold 2-4% paraformaldehyde (PFA) at room temperature for 15 minutes.

[0256] 3. Use calcium-free 2+ / Mg 2+ Wash the sample with DPBS for 5 minutes, 3 times.

[0257] 4. Use 0.5% Triton X in a Ca-free environment. 2+ / Mg 2+ Permeabilize the sample in DPBS for 30 minutes to 1 hour.

[0258] 5. Place the sample in a blocking solution (in a Ca-free environment). 2+ / Mg 2+ Incubate in 1-3% BSA in DPBS for 1 hour.

[0259] 6. Incubate the samples with primary antibodies (SOX2 for ICM [Invitrogen, 14-9811-82]; CDX2 for TE [Biogenex, AM382]; and SOX17 for hypoblastoderm [R&D system, AF1924]) in blocking solution overnight at 4°C.

[0260] 7. Use DPBST (in Ca-free) 2+ / Mg 2+ Wash the sample with 0.05% Twin20 in DPBS for 5 minutes, 3 times.

[0261] 8. Incubate the sample with the secondary antibody in DPBST at room temperature for 1 hour.

[0262] 9. Use DPBST (in Ca-free) 2+ / Mg 2+ Wash the sample with 0.05% Twin20 in DPBS for 5 minutes, 3 times.

[0263] 10. Deposit the sample onto a glass slide and cover it with a sealing medium (Vectashield + DAPI).

[0264] result

[0265] S / C / S staining ( Figure 5 A) Each type of cell (ICM, TE, and hypoblastomere) was detected in the embryo on day 11.

[0266] SOX2 (ICM marker) and CDX2 (TE marker) were detected in embryos on days 7 and 11. However, SOX17 (hypoblastic marker) (not shown) was detected from day 9 onwards, and a very clear signal was obtained from day 11 embryos as hypoblastic cells began to appear after blastocyst expansion.

[0267] In summary, S / C / S staining provides good visualization of the embryo and is highly specific for each desired cell type. This tool has the potential to be used to evaluate induced blastocysts.

[0268] The structure of bovine induced blastocysts can be further identified by SOX2 and CDX2 staining. Figure 5 B). Five days after induction, bovine juvenile stem cell aggregates expressed SOX2 and CDX2. Each aggregate exhibited a cavity-like structure lacking SOX2 expression and containing fewer cells. Figure 5 (Arrow in B). The size of the induced blastocyst is similar to that of a day 7 embryo. Figure 5 (Scale bar: 50μm)

[0269] Example 6. Generation of multiple genetically identical induced blastocysts from a single mating

[0270] When used in conjunction with genomic selection, other assisted reproductive technologies and / or in vitro breeding (such as Hou et al., 2018), the induced blastocyst technique reported in this paper offers additional opportunities to improve phylogenetic genetic gains by increasing selection intensity, shortening generation intervals, and reducing genetic lag. Therefore, as Figures 6-9As shown, induced blastocyst technology has the potential to replace somatic cell nuclear cloning (SCNT) or AI, and provides significant value to breeding systems and the commercial environment by offering an efficient platform for the large-scale production of genetically modified animals (when a specific genome is required), for hybridization breeding (when a specific breed composition is desired (e.g., F1, ternary hybrids, 5 / 8:3 / 8, etc.), for replicating important components and products within conventional and in vitro breeding systems, and for improving the delivery of advanced veterinary medical biopharmaceuticals and therapeutics, particularly for animal species with relatively low fertility and medium to long generation intervals.

[0271] like Figure 8 As shown, using the above-described method for generating induced blastocysts, multiple genetically identical induced blastocysts are generated from a single mating as follows:

[0272] 1. For example, optionally characterizing or evaluating the desired traits of one or more candidate male / female pairs by testing one or more biomarkers (e.g., SNP analysis), and optionally further scoring and / or selecting based on one or more selection criteria (e.g., estimated breeding value, genomic predictive performance ability, etc.).

[0273] 2. Collect oocytes and sperm from selected females and males using techniques known in the art.

[0274] 3. Producing one or more embryos via in vitro fertilization using techniques known in the art.

[0275] 4. For example, optional characterization or assessment of the desired traits of individual embryos by testing one or more biomarkers, and optional further scoring and / or selection based on one or more selection criteria (e.g., estimated breeding value, genomic predictive performance ability, etc.).

[0276] 5. Using methods such as those taught in Patent Cooperation Treaty application number PCT / CA2022 / 051664, or methods for expanding pluripotent stem cells as described by Zhao et al. (2021), to derive and propagate juvenile stem cells.

[0277] 6. For example, optionally characterizing or assessing the desired traits of naive stem cells by testing for one or more biomarkers, and optionally further scoring and / or selecting based on one or more selection criteria (e.g., estimated breeding value, genomic predictive performance ability, etc.), and / or optionally cryopreserving a subgroup of naive stem cells for storage.

[0278] 7. Using the methods described herein, such as those described in Example 5, to generate multiple genetically identical induced blastocysts from juvenile stem cells.

[0279] 8. For example, optionally characterizing or evaluating the desired traits of one or more induced blastocysts by testing one or more biomarkers, and optionally further scoring and / or selecting based on one or more selection criteria (e.g., estimated breeding value, genomic predictive performance ability, etc.).

[0280] Genetically identical induced blastocysts are then cryopreserved for storage and / or transferred to female surrogate recipients for pregnancy to produce genetically identical offspring. Optionally, the performance and / or updated scores of the offspring are then determined (e.g., for a given environment or market), and additional genetically identical induced blastocysts are produced from a selected bank of naive stem cells and / or induced blastocysts.

[0281] Example 7. Generation of multiple genetically identical embryos and their use in selection and breeding systems.

[0282] Given the scope and scale of global livestock farming, many species have limited asexual or bisexual reproduction rates. This means that traditional selection and breeding systems have limited capacity to assess and subsequently allocate germplasm suitable for many specific markets. Induced blastocyst technology, by enabling the production of large numbers of identical induced blastocysts in vitro, will greatly enhance the ability to deliver offspring with desired phenotypic abilities when specific genomes (e.g., offspring from a given cross, offspring with one or more desired traits such as resistance to a given disease and / or high methane efficiency, genome-edited offspring, etc.) and / or specific breed compositions (e.g., F1, ternary hybrids, 5 / 8:3 / 8, etc.) are required.

[0283] The selected induced blastocysts were generated as in Example 6.

[0284] High-quality, genetically identical induced blastocysts can be frozen for future transfer, or fresh induced blastocysts can be transferred into the reproductive tract of one or more recipient females (surrogate mothers) in a designated market to achieve pregnancy and viable offspring using conventional embryo transfer protocols.

[0285] Optionally, one or more biomarkers may be tested on offspring (e.g., to confirm initial test results for one or more biomarkers). Alternatively or additionally, performance data of offspring in one or more designated markets (e.g., milk production, etc.) may be recorded and optionally used to update scores.

[0286] result

[0287] Table 1 illustrates the improved accuracy of the new induced blastocysts in predicting performance on three different traits as an increasing number of genetically identical induced blastocyst groups are generated and tested.

[0288] Table 1. Accuracy of new induced blastocysts in predicting performance for three different traits by testing different numbers of genetically identical induced blastocyst groups.

[0289]

[0290]

[0291] The calculation formula comes from Van Vleck, 1999.

[0292] Table 2 illustrates the accuracy of the predicted average performance of induced blastocyst groups on three different traits when generating and testing an increasing number of genetically identical induced blastocyst groups.

[0293] Table 2. Accuracy of predicted average performance for three different traits by testing different numbers of genetically identical induced blastocyst groups.

[0294]

[0295] The calculation formula comes from VanVleck, 1999.

[0296] References:

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Claims

1. A method for preparing bovine induced blastocysts, the method comprising: a) Provide bovine juvenile stem cell populations; b) The bovine juvenile stem cells were cultured in an aggregation medium under aggregation conditions to produce aggregated bovine juvenile stem cells; c) Introduce a first induced blastocyst medium, optionally wherein the aggregated medium and non-aggregated cells are removed prior to the introduction of the first induced blastocyst medium; d) The aggregated bovine juvenile stem cells were cultured in the first induced blastocyst medium under hypoxic conditions; e) Introducing a second induced blastocyst medium, optionally wherein the first induced blastocyst medium is removed before the introduction of the second induced blastocyst medium; and f) The aggregated bovine juvenile stem cells are cultured in the second induced blastocyst medium under hypoxic conditions to obtain the bovine induced blastocysts.

2. The method according to claim 1, wherein the bovine juvenile stem cell population in step a) is dissociated bovine juvenile stem cells, and / or the bovine juvenile stem cells are bovine embryo-derived juvenile stem cells.

3. The method according to claim 1 or claim 2, wherein step b) comprises introducing the cells into one or more wells of the micropore device and culturing the cells for about 24 hours.

4. The method of claim 3, wherein the diameter of the pores in the microporous device is about 400 μm to about 600 μm.

5. The method according to claim 3 or claim 4, wherein the bovine juvenile stem cell population is added to the microporous device at a concentration of about 20-60 cells / well, optionally at a concentration of about 20-30 cells / well.

6. The method according to any one of claims 1 to 5, wherein the cells are cultured for about 48 hours in step d) and / or step f).

7. The method according to any one of claims 1 to 6, wherein the aggregation culture medium comprises: Serum substitute components; and Rho-related components of coiled-coil protein kinase (ROCK) inhibitors.

8. The method of claim 7, wherein the serum substitute component comprises a) an N2B27 component, optionally about 1% B27 component and about 0.5% N2 component; b) an insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) a serum substitute (SR); or d) a knockout serum substitute (KOSR); and / or the ROCK inhibitor component comprises Y27632, fasudil, thiazovirine or brestatin, optionally 5-10 μM Y27632.

9. The method according to any one of claims 1 to 8, wherein the first induced blastocyst culture medium comprises: Serum substitute components; MEK / ERK inhibitor components; TGFβ-1 inhibitor components; HIPPO pathway inhibitor components; and ROCK inhibitor components.

10. The method of claim 9, wherein The serum substitute components include a) an N2B27 component, optionally about 1% B27 supplement and about 0.5% N2 supplement; b) an insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) a serum substitute (SR); or d) a knockout serum substitute (KOSR); The MEK / ERK inhibitor component includes PD0325901, lavotetinib, GSK1120212, MEK162, PD184352, trametinib, LY3214996 or unitinib, optionally about 1 μM PD0325901; The TGFβ-1 inhibitor component includes A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3 or TP0427736, optionally about 1 μM of A83-01; The HIPPO pathway inhibitor component includes lysophosphatidic acid (LPA), optionally about 1 μM LPA; and / or The ROCK inhibitor component includes Y27632, fasudil, thiazovirine, or brestatin, optionally 5-10 μM Y27632.

11. The method according to claim 9 or claim 10, wherein the first induced blastocyst culture medium further comprises a second serum substitute component, optionally ITS-X, optionally 0.1-1% ITS-X; a LIF component, optionally human LIF, bovine LIF, goat LIF, porcine LIF, buffalo LIF or recombinant LIF, optionally at a concentration of about 10 ng / ml; an epidermal growth factor (EGF) component, optionally about 50 ng / ml EGF; a Wnt agonist, optionally Wnt3a, Wnt agonist 1 or SKL2001, optionally 10-100 ng / mL Wnt3a; and a GSK3β inhibitor, optionally CHIR99021, BIO, CHIR-98014, LY2090314 or IM-12, optionally 1-2 μM. CHIR99021; a histone deacetylase inhibitor, optionally valproic acid (VPA), sodium butyrate or trichostatin A, optionally 0.2-0.8 mM VPA; and / or a second TGFβ-1 inhibitor, optionally 0.1-1 μM SB431542.

12. The method of claim 11, wherein the first induced blastocyst culture medium comprises an EGF component and further comprises a fibroblast growth factor (FGF) component, optionally basic fibroblast growth factor (bFGF) or FGF4, optionally at a concentration of 10-30 ng / ml.

13. The method according to any one of claims 1 to 12, wherein the second induced blastocyst culture medium comprises: Serum substitute components; TGFβ-1 inhibitor components; HIPPO pathway inhibitor components; and ROCK inhibitor components.

14. The method of claim 13, wherein The serum substitute components include a) an N2B27 component, optionally about 1% B27 supplement and about 0.5% N2 supplement; b) an insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) a serum substitute (SR); or d) a knockout serum substitute (KOSR); The TGFβ-1 inhibitor component includes A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3 or TP0427736, optionally about 1 μM of A83-01; The HIPPO pathway inhibitor component includes lysophosphatidic acid (LPA), optionally about 1 μL PA; and / or The ROCK inhibitor component includes Y27632, fasudil, thiazovirine, or brestatin, optionally 5-10 μM Y27632.

15. The method according to claim 13 or claim 14, wherein the second induced blastocyst culture medium further comprises one or more of the following: a second serum substitute component, optionally ITS-X, optionally 0.1-1% ITS-X; a fibroblast growth factor (FGF) component, optionally basic fibroblast growth factor (bFGF) or FGF4, optionally at a concentration of 10-30 ng / ml; an activin A component, optionally human activin A, bovine activin A or mouse activin A, optionally at a concentration of 5-50 ng / ml; and / or a GSK3β inhibitor, optionally CHIR99021, BIO, CHIR-98014, LY2090314 or IM-12, optionally 1-3 μM CHIR99021.

16. The method of claim 15, wherein the second induced blastocyst culture medium comprises an FGF component and further comprises an epidermal growth factor component, optionally about 50 ng / ml EGF.

17. The method according to any one of claims 1 to 16, wherein the hypoxic conditions comprise about 5%-10% CO2, optionally about 6.8% CO2, and about 1%-5% O2, optionally about 5% O2.

18. The method according to any one of claims 1 to 17, wherein the first induced blastocyst medium and / or the second induced blastocyst medium are replaced with fresh medium after about 24 hours.

19. The method according to any one of claims 1 to 18, wherein the bovine juvenile stem cell population is derived from primordial pluripotent stem cells or induced pluripotent stem cells, or wherein the bovine juvenile stem cell population is embryo-derived juvenile stem cells, optionally derived from preimplantation embryos or embryo-derived expanded pluripotent stem cells, optionally wherein the bovine juvenile stem cell population is derived by a method comprising the following steps: a) Provides a bovine embryo containing bovine juvenile stem cell-like cells, optionally wherein the bovine embryo does not contain the zona pellucida (ZP); b) Contacting the bovine embryo with an extracellular matrix (ECM)-coated substrate, wherein the ECM-coated substrate comprises a substrate including 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) The bovine embryos are cultured in the presence of a growth medium to induce attachment of the bovine embryos to the ECM-coated substrate and the growth of the inner cell mass (ICM) containing derived bovine juvenile stem cells, thereby generating a bovine juvenile stem cell population.

20. The method according to any one of claims 1 to 19, wherein the bovine juvenile stem cells are genetically modified and / or genome-edited cells.

21. The method according to any one of claims 1 to 20, further comprising testing one or more biomarkers to determine one or more characteristics of the bovine juvenile stem cell population in response to one or more biomarkers, and optionally determining a score based on the determined characteristics.

22. The method of claim 2v, further comprising selecting the bovine juvenile stem cell population based on the one or more characteristics determined by testing one or more biomarkers and / or the score.

23. The method according to any one of claims 1 to 20, further comprising testing one or more biomarkers to determine one or more characteristics of the induced blastocyst, and optionally determining a score based on the determined characteristics.

24. The method of claim 23, further comprising selecting one or more of the induced blastocysts based on the one or more characteristics determined by testing one or more biomarkers and / or the score.

25. A plurality of induced blastocysts prepared using the method according to any one of claims 1 to 24.

26. A method for breeding cattle, the method comprising: a) Select at least one bovine parent from among multiple animals; b) Obtain multiple gametes from the at least one bovine parent; c) Generate at least one embryo from the plurality of gametes; d) Derive a population of juvenile stem cells from at least one of the embryos; e) Prepare one or more induced blastocysts according to the method of any one of claims 1 to 24; and f) Select one or more of the induced blastocysts for breeding, optionally for in vitro breeding, and / or for transfer to a female surrogate recipient to produce offspring.

27. The method of claim 26, wherein one or more biomarkers of the at least one embryo are tested to determine one or more characteristics of the at least one embryo, and optionally a score is determined based on the determined characteristics.

28. An aggregation culture medium composition comprising: Serum substitute components; and Rho-related components of coiled-coil protein kinase (ROCK) inhibitors.

29. The culture medium composition according to claim 28, wherein... The serum substitute components include a) an N2B27 component, optionally about 1% B27 component and about 0.5% N2 component; b) an insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) a serum substitute (SR); or d) a knockout serum substitute (KOSR); and / or The ROCK inhibitor component includes Y27632, fasudil, thiazovirine, or brestatin, optionally 5-10 μM Y27632.

30. A composition for inducing blastocyst culture medium 1, comprising: Serum substitute components; MEK / ERK inhibitor components; TGFβ-1 inhibitor components; HIPPO pathway inhibitor components; and ROCK inhibitor components.

31. The induced blastocyst culture medium 1 composition according to claim 30, wherein... The serum substitute components include a) an N2B27 component, optionally about 1% B27 component and about 0.5% N2 component; b) an insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) a serum substitute (SR); or d) a knockout serum substitute (KOSR); The MEK / ERK inhibitor component includes PD0325901, lavotetinib, GSK1120212, MEK162, PD184352, trametinib, LY3214996 or unitinib, optionally about 1 μMP PD0325901; The TGFβ-1 inhibitor component includes A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3 or TP0427736, optionally about 1 μM of A83-01; The HIPPO pathway inhibitor component includes lysophosphatidic acid (LPA), optionally about 1 μL PA; and / or The ROCK inhibitor component includes Y27632, fasudil, thiazovirine, or brestatin, optionally 5-10 μM Y27632.

32. The induced blastocyst culture medium 1 composition according to claim 30 or claim 31, further comprising one or more of the following: The second serum substitute component, optionally 0.1-1% ITS-X; LIF component, optionally human LIF, bovine LIF, goat LIF, porcine LIF, buffalo LIF or recombinant LIF, optionally at a concentration of about 10 ng / ml; Epidermal growth factor (EGF) component, optionally 50 ng / ml EGF; GSK3β inhibitors, optionally CHIR99021, BIO, CHIR-98014, LY2090314 or IM-12, optionally 1-2 μM CHIR99021; Wnt activator, optionally Wnt3a, Wnt agonist 1 or SKL2001, optionally 10-100 ng / mL Wnt3a; Histone deacetylase inhibitors, optionally valproic acid (VPA), sodium butyrate, or triamcinolone A, optionally 0.2-0.8 mM VPA; and / or The second TGFβ-1 inhibitor component, optionally 0.1-1 μM SB431542.

33. The induced blastocyst culture medium 1 composition according to claim 32, wherein the induced blastocyst culture medium 1 comprises an EGF component and further comprises a fibroblast growth factor (FGF) component, optionally basic fibroblast growth factor (bFGF) or FGF4, optionally at a concentration of 10-30 ng / ml.

34. A composition for an inducible blastocyst culture medium 2, comprising: Serum substitute components; TGFβ-1 inhibitor components; HIPPO pathway inhibitor components; and ROCK inhibitor components.

35. The induced blastocyst culture medium 2 composition according to claim 34, wherein... The serum substitute components include a) an N2B27 component, optionally about 1% B27 component and about 0.5% N2 component; b) an insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) a serum substitute (SR); or d) a knockout serum substitute (KOSR); The TGFβ-1 inhibitor component includes A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3 or TP0427736, optionally about 1 μM of A83-01; The HIPPO pathway inhibitor component includes lysophosphatidic acid (LPA), optionally about 1 μL PA; and / or The ROCK inhibitor components include Y27632, fasudil, thiazovirine, or brestatin.

36. The induced blastocyst culture medium 2 composition according to claim 34 or claim 35, further comprising one or more of the following: Second serum substitute component, optionally ITS-X, optionally 0.1-1% ITS-X; Fibroblast growth factor (FGF) component, optionally basic fibroblast growth factor (bFGF) or FGF4, optionally at a concentration of 10-30 ng / ml; The activator A component may be human activator A, bovine activator A, or mouse activator A, with a concentration of 5-50 ng / ml. Wnt activator, optionally Wnt3a, Wnt agonist 1 or SKL2001; and / or GSK3β inhibitors, optionally CHIR99021, BIO, CHIR-98014, LY2090314 or IM-12, optionally 1-3 μM CHIR99021.

37. The induced blastocyst culture medium 2 composition according to claim 36, wherein the induced blastocyst culture medium 2 comprises an FGF component and further comprises an epidermal growth factor component, optionally about 50 ng / ml EGF.

38. Use of the culture medium composition according to any one of claims 28 to 37 for the preparation of bovine induced blastocysts.

Images