Non-cloning methods for bovine embryo replication

Abstract

This disclosure relates to methods, products, and compositions useful for inducing bovine induced blastoids. Methods enabling naive stem cell aggregation and induced blastoid formation are described. Aggregation and induced blastoid media suitable for promoting naive stem cell aggregation and preparing induced blastoids are also provided. Bovine induced blastoids may be used in selection and breeding programs, including the replication of pre-transplant embryos with desirable characteristics, as well as in vitro breeding, including cross-generation breeding cycles from parental germ cells and / or embryos to offspring embryos.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 456,624, filed on 3 April 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] This disclosure relates to the induction of bovine embryos, and more specifically, to a method for generating multiple bovine embryos having desirable properties from a preparation of bovine eve stem cells, as well as related methods, selection and / or breeding programs, and compositions.

[0003] preface The widespread use of artificial insemination (AI) has helped enable accurate genetic assessment of male parents for use in genetic modification programs. However, for most genetic traits, AI does not have the ability to produce offspring with known traits as a result of recombination and crossover during meiosis. Cloning techniques have been presented as a solution to produce offspring with limited genetic variation and high uniformity. Since the 1990s, numerous cloned animals have been produced using nuclear transfer approaches. More recently, when producing genetically modified livestock, researchers have used somatic cell nuclear transfer (SCNT), which is considered by many to be the preferred technique due to the resulting genotypic uniformity and lack of mosaicism (Springer et al. 2021). However, SCNT generally results in low biological and economic efficiency as a result of the time-intensive nature of procedures such as oocyte enucleation and nuclear transfer, low embryo production rates (approximately 20-30% blastocyst formation), and the need for skilled experts to complete the operation. High rates of pregnancy loss, high stillbirth rates, high incidence of calf abnormalities such as overweight syndrome, and undesirable animal health and welfare are often associated with SCNT, most notably in cattle and other ruminants (Hill, 2014; Mueller and Van Eenennaam, 2022; Springer et al., 2021). Overall, this significantly limits the use of nuclear transfer / SCNT as a tool for producing a large number of identical embryos for large-scale / commercial purposes.

[0004] In 2021, the generation of induced blastoid-like cells (iblastoids) from human pluripotent stem cells was published in Nature by two independent groups (Yu et al., 2021 and Liu et al., 2021). Both groups successfully reconstructed human blastocyst-like structures using either naive embryonic stem cells (Yu et al., 2021) or induced pluripotent stem cells (iPSCs, Liu et al., 2021), both of which exhibited pre-transplant embryo characteristics. Furthermore, two additional papers have recently been published (Yanagida et al., 2021 and Kagawa et al., 2022) concerning improvements in the quality of the produced human iblastoids through protocol optimization. Human iblastoids express similar RNA transcriptome characteristics to human blastocysts produced in vitro. Furthermore, induced blastoids can be induced into embryonic (naive and primed pluripotent stem cells) and extraembryonic (trophoblast stem cells and extraembryonic endoderm cells) cell lines. Additionally, induced blastoids respond similarly to normal embryos in terms of in vitro adhesion when seeded on endometrial cells. Based on these studies, researchers concluded that induced blastoids could be used as a substitute for actual embryos in early human embryonic development research. However, due to ethical and regulatory issues that limit what can be done with human stem cells and human embryos, it remains unclear whether human induced blastoids can further differentiate after being transferred to the uterus.

[0005] In contrast, regarding livestock, Perez-Gomez et al. (2021) concluded that "while primary bovine trophoectoderm cell cultures can be established using relatively simple media, the conditions necessary for true pluripotent epiblast cell culture in livestock remain unclear." As a result, these researchers clarified key differences in gene regulatory networks and their roles in cell differentiation events from early embryonic development to embryo transfer in humans, mice, and ungulates. Notably, the placental structure of developing fetuses differs among ruminants, horses, and pigs, as well as in humans and mice. These findings regarding livestock highlight the need to improve embryonic and pluripotent cell cultures, as well as to develop species-specific assisted reproductive technologies, such as the generation of non-human induced blastoids.

[0006] Furthermore, animal science can realize various benefits from inducible blastoid technology through enhanced genetic improvement, expansion and distribution of desired genetic resources, improved consistency and predictability of gene products, reduction of genetic lag, and provision of a platform for research and development in embryonic development, improvement of assisted reproductive technologies, and veterinary biologics and therapeutics. Key drivers of genetic change in genetic improvement systems include the accuracy and strength of selection, genetic variation, the mean age of parents at the birth of offspring, and genetic lag. When genomic selection is combined with assisted reproductive technologies such as multiple ovulation embryo transfer, oocyte retrieval, in vitro embryo production, and / or juvenile in vitro fertilization and embryo transfer (Mueller and Van Eenennam, 2022), the genetic change from animal genetic improvement programs becomes much greater. When combined, these technologies enable accurate prediction of genetic advantages in the immediate postnatal period, including pre-transplant embryos (Saadi et al., 2014b), shorten generation intervals, and increase the strength of parental selection. More recently, genetic modification technology has provided new tools for inducing targeted genetic changes in animal populations (Mueller and Van Eenennam, 2022; Park, 2007; US7, 067, 713).

[0007] Novel and / or improved methods are needed for conventional and / or in vitro breeding systems, large-scale and commercial applications, and for generating large numbers of identical embryos for the production and widespread distribution of healthy, selected, and / or genetically modified livestock. The preparation of bovine-induced blastoids can facilitate the efficient replication of embryos with desirable characteristics. Bovine-induced blastoids and related methods for their production remain in need. [Overview of the Initiative]

[0008] One of the main advantages of the induced blastoids described herein is that they are produced without the need to use existing cloning methods. The disclosed induced blastoid production techniques present a highly effective and cost-effective method for producing a large number of identical embryos, generating and replicating selected genetic traits (small-scale use), for the selection and breeding systems described herein, and for commercial large-scale applications such as genetically modified germplasm, reconstructed diploid embryos, and / or replication of embryos with desirable traits.

[0009] To enable this technology for livestock, including cattle, 3D culture systems for bovine stem cells, culture conditions for producing bovine-induced blastoids, and selection and breeding systems for their use have been developed and evaluated as described herein.

[0010] This specification describes materials and methods useful for achieving aggregation of bovine naive stem cells, reconstruction of blastocyst-like induced blastoids, and use of induced blastoids in selection and breeding systems. As shown in the examples, bovine induced blastoids can be generated from cultured bovine naive stem cells under aggregation conditions, for example, using hanging drop culture, low-adhesion dishes, or microwell devices. As shown herein, naive stem cells seeded in microwell devices result in higher aggregation and uniformity of aggregate size compared to cells aggregated using the hanging drop method or low-adhesion dish method. Furthermore, induced blastoid medium compositions and related methods that support induced blastoid formation and enable the preparation of bovine induced blastoids are described. Accordingly, the embodiments described herein are useful for preparing bovine induced blastoids and, optionally, for use in selection and breeding programs such as the replication of pre-transplant embryos with desired genetic and epigenetic characteristics. Accordingly, one embodiment provides a method for preparing bovine induced blastoids, comprising: a) To provide a population of bovine stem cells, b) Culturing bovine stem cells in an aggregation medium under aggregation conditions to produce aggregated bovine stem cells. c) Introducing a first induction blastoid medium, wherein optionally, the aggregated medium and non-aggregated cells are removed before introducing the first induction blastoid medium. d) Culture the aggregated bovine eve stem cells in the first induction blastoid medium under hypoxic conditions. e) Introducing a second induction blastoid medium, wherein optionally, the first induction blastoid medium is removed before introducing the second induction blastoid medium, and f) Aggregated bovine stem cells are cultured in a second induction blastoid medium under hypoxic conditions to obtain bovine induction blastoids.

[0011] In one embodiment, the bovine naive stem cell population in step a) is dissociated bovine naive stem cells, and / or the bovine naive stem cells are bovine embryo-derived naive stem cells.

[0012] In one embodiment, step b) includes introducing cells into one or more wells of a microwell device and culturing the cells for about 24 hours. In one embodiment, the wells of the microwell device have a diameter of about 400 μm to about 600 μm. In one embodiment, a population of bovine stem cells is added to the microwell device at a concentration of about 20 to 60 cells / well, optionally about 20 to 30 cells / well.

[0013] In one embodiment, the cells are cultured for approximately 48 hours in step d) and / or step f).

[0014] In one embodiment, the agglutination medium comprises a serum substitute component and a Rho-related coiled-coil-containing protein kinase (ROCK) inhibitor component. In one embodiment, the serum substitute component comprises a) an N2B27 component, optionally about 1% of the B27 component and about 0.5% of the 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, thiazovibin, or brevistatin, optionally 5-10 μM of Y27632.

[0015] In one embodiment, the first inducible blastoid 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 additive and about 0.5% N2 additive, 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 includes PD0325901, laboxerutinib, GSK1120212, MEK162, PD184352, trametinib, LY3214996, or urixerutinib, optionally comprising about 1 μM of PD0325901. In one embodiment, the TGFβ-1 inhibitor component includes A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736, optionally comprising about 1 μM of A83-01. In one embodiment, the HIPPO pathway inhibitor component includes lysophosphatidic acid (LPA), optionally comprising about 1 μM of LPA. In one embodiment, the ROCK inhibitor component includes Y27632, fasudil, thiazovibin, or brevistatin, optionally comprising 5-10 μM of Y27632.

[0016] In one embodiment, the first inducible blastoid 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 approximately 10 ng / ml; an epidermal growth factor (EGF) component, optionally at approximately 50 ng / ml EGF; and a Wnt agonist, optionally. The formulation includes, optionally, Wnt3a, Wnt agonist 1, or SKL2001; optionally, 10–100 ng / mL of Wnt3a; a GSK3β inhibitor; optionally, CHIR99021, BIO, CHIR-98014, LY2090314, or IM-12; optionally, 1–2 μM of CHIR99021; a histone deacetylase inhibitor; optionally, valproic acid (VPA), sodium butyrate, or trichostatin A; optionally, 0.2–0.8 mM of VPA; and / or a second TGFβ-1 inhibitor; optionally, 0.1–1 μM of SB431542. In embodiments in which the first inducible blastoid medium contains an EGF component, the first inducible blastoid medium may further contain a fibroblast growth factor (FGF) component, optionally basic fibroblast growth factor (bFGF) or FGF4, at an optionally selected concentration of 10-30 ng / ml.

[0017] In one embodiment, the second induced blastoid medium contains a serum replacement component, a TGFβ-1 inhibitor component, a HIPPO pathway inhibitor component, and a ROCK inhibitor component. In one embodiment, the serum replacement component includes a) N2B27 component, optionally about 1% B27 additive and about 0.5% N2 additive, b) insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G), c) serum replacement (SR), or d) knockout serum replacement (KOSR). In one embodiment, the TGFβ-1 inhibitor component includes A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736, optionally containing about 1 μM of A83-01. In one embodiment, the HIPPO pathway inhibitor component includes lysophosphatidic acid (LPA), optionally containing about 1 μM of LPA. In one embodiment, the ROCK inhibitor component includes Y27632, fasudil, thiazovivin, or brevystatin, optionally containing 5-10 μM of Y27632.

[0018] In one embodiment, the second induced blastoid medium further includes one or more of the following: a second serum replacement 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 of CHIR99021. In embodiments where the second induced blastoid medium contains an FGF component, the second induced blastoid medium may further include an epidermal growth factor (EGF) component, optionally containing about 50 ng / ml of EGF. [[ID=⑤]] [[ID=⑥]]

[0019] [[ID=⑦]] In one embodiment, the hypoxic conditions include about 5% - 10% CO2, optionally about 6.8% CO2, and about 1% - 5% O2, optionally about 5% O2.

[0020] In one embodiment, the first induced blastoid medium and / or the second induced blastoid medium are replaced with fresh medium after about 24 hours.

[0021] In one embodiment, the population of bovine naive stem cells is derived from prime pluripotent stem cells or induced pluripotent stem cells.

[0022] In one embodiment, the population of bovine naive stem cells is embryonic naive stem cells and optionally derived from pre-implantation embryos or expanded pluripotent stem cells derived from embryos.

[0023] In one embodiment, the population of bovine naive stem cells is induced by a method comprising: providing a bovine embryo comprising bovine naive stem cell-like cells, optionally wherein the bovine embryo does not comprise a zona pellucida (ZP); contacting the bovine embryo with a substrate coated with an extracellular matrix (ECM), wherein the substrate coated with ECM comprises 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 culturing the bovine embryo in the presence of a growth medium to induce attachment of the bovine embryo to the substrate coated with ECM and growth of an inner cell mass (ICM) comprising the resulting bovine naive stem cells.

[0024] In one embodiment, the bovine naive stem cells are genetically modified and / or genome-edited cells.

[0025] In one embodiment, the method further includes testing one or more biomarkers to determine one or more characteristics of a population of bovine stem cells for one or more biomarkers, and optionally determining a score based on the determined characteristics.

[0026] In one embodiment, the method further includes selecting a population of bovine eve stem cells based on one or more characteristics and / or scores determined by testing for one or more biomarkers.

[0027] In one embodiment, the method further includes testing one or more biomarkers to determine one or more characteristics of the induced blastoid, and optionally determining a score based on the determined characteristics.

[0028] In one embodiment, the method further includes selecting one or more induced blastoids based on one or more characteristics and / or scores determined by testing one or more biomarkers.

[0029] One embodiment includes a plurality of induction blastoids prepared using the method described herein.

[0030] Further embodiments include the use of the induction blastoid technology described herein for selection and breeding systems. Thus, a method for breeding cattle is provided herein, which includes: a) Select at least one parent cattle from multiple animals. b) Obtaining multiple gametes from at least one parent cattle, c) Creating at least one embryo from the multiple gametes, d) Inducing a population of naive stem cells from at least one embryo, e) Prepare one or more induction blastoids according to the method described herein, and f) Selecting one or more induction blastoids for reproduction, optional in vitro reproduction, and / or for transplantation into a female surrogate animal to produce offspring.

[0031] In one embodiment, at least one embryo is tested for one or more biomarkers to determine one or more characteristics of the at least one embryo, and optionally, a score is determined based on the determined characteristics.

[0032] This specification also provides agglutination medium composition comprising a serum substitute component and a Rho-related coiled-coil-containing protein kinase (ROCK) inhibitor component. In one embodiment, the serum substitute component comprises a) an N2B27 component, optionally about 1% of the B27 component and about 0.5% of the 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, thiazovibin, or brevistatin, optionally 5-10 μM of Y27632.

[0033] This specification also provides an induction blastoid medium 1 composition 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% of the B27 component and about 0.5% of the 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 includes PD0325901, laboxerutinib, GSK1120212, MEK162, PD184352, trametinib, LY3214996, or urixerutinib, optionally comprising about 1 μM of PD0325901. In one embodiment, the TGFβ-1 inhibitor component includes A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736, optionally comprising about 1 μM of A83-01. In one embodiment, the HIPPO pathway inhibitor component includes lysophosphatidic acid (LPA), optionally comprising about 1 μM of LPA. In one embodiment, the ROCK inhibitor component includes Y27632, fasudil, thiazovibin, or brevistatin, optionally comprising 5-10 μM of Y27632.In one embodiment, the induction blastoid medium composition further comprises: 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 at about 50 ng / ml EGF; a GSK3β inhibitor, optionally CHIR99021, BIO, CHIR-98014, LY2090314, or I The medium comprises one or more of the following: M-12, optionally 1-2 μM CHIR99021; Wnt activator, optionally Wnt3a, Wnt agonist 1, or SKL2001, optionally 10-100 ng / mL Wnt3a; 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 component, optionally 0.1-1 μM SB431542. In embodiments where the first inducible blastoid medium contains an EGF component, the first inducible blastoid 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.

[0034] This specification also provides two inducible blastoid medium compositions 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% of the B27 component and about 0.5% of the 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 includes A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736, optionally comprising about 1 μM of A83-01. In one embodiment, the HIPPO pathway inhibitor component includes lysophosphatidic acid (LPA), optionally comprising about 1 μM of LPA. In one embodiment, the ROCK inhibitor component includes Y27632, fasudil, thiazovibin, or brevistatin. In one embodiment, the inducible blastoid medium 2 composition further comprises 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; 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 at a concentration of 1-3 μM CHIR99021. In embodiments where the inducible blastoid medium 2 contains an FGF component, the inducible blastoid medium 2 may further contain an epidermal growth factor (EGF) component, optionally at approximately 50 ng / ml of EGF.

[0035] Further embodiments include the use of the culture medium compositions described herein for preparing bovine-induced blastoids.

[0036] The preceding sections are provided merely as examples and are not intended to limit the scope of this disclosure and the accompanying claims. Further purposes and advantages relating to the compositions and methods of this disclosure will be understood by those skilled in the art, taking into consideration the claims, description and examples. For example, various aspects and embodiments of this disclosure can be used in numerous combinations, all of which are expressly contemplated in this description. These additional advantages, purposes and embodiments are expressly included within the scope of this disclosure. Publications and other materials used herein to provide background to this disclosure and, in particular, to provide additional details relating to the practice are incorporated by reference and, for convenience, listed in the accompanying references section.

[0037] Further objectives, features, and advantages of this disclosure will become apparent from the following detailed description, together with the accompanying drawings illustrating exemplary embodiments of this disclosure, which are as follows: [Brief explanation of the drawing]

[0038] [Figure 1] This shows the generation of trophoblast stem (TS) cell organoids using the hanging drop method. A: Stereomicroscope image (right) showing the hanging drop dish (left) and cell suspension. B: It can be difficult to create complete droplets (arrows indicate broken droplets). C: Cell aggregation after 48 hours. Organoids have a shape similar to a normal embryo with a blastocoel (arrows) or have the appearance of a disintegrated embryo (stars). D: Bright-field (left) and DNA staining (right) of TS organoids. E: DNA staining of a blastocyst on day 7 (dotted circles indicate ICM). [Figure 2] Images of blastocyst-like structures derived from trophoblast stem (TS) cells generated by a low-adhesion dish are shown. Small cell clusters on day 0 grew into blastocyst-like structures after 48 hours. [Figure 3]The image on the left shows a microwell device (Stemfit3D, catalog number H853400) inserted into a 35mm dish, and the image on the right shows the microwell device under a stereomicroscope. [Figure 4] Images of blastocyst-like structures generated using a microwell system are shown. A: Trophoblast stem (TS) cells aggregate in a microwell after 24 hours. B: Images of several TS cell-derived blastocyst-like structures after 48 hours of culture in a microwell (after being transferred to a standard culture dish). C: Aggregates derived from bovine stem cells after 24 hours in a microwell. D: Aggregates derived from bovine stem cells after 48 hours in a microwell. E: Image of a blastocyst-like structure showing the blastocoel (red arrow) derived from bovine stem cells after 96 hours of culture in a microwell. [Figure 5] This image shows SOX2 / CDX2 / SOX17(S / C / S) staining for the inner cell mass (ICM), trophectoderm (TE), and hypoblast markers in blastocysts (embryos on day 7 and 11) and induced blastoids on day 5. SOX2(ICM), CDX2(TE), and SOX17(hypoblast)-positive cells are well-constructed throughout the embryonic structure according to the developmental stage. B: SOX2(ICM), CDX2(TE)-positive cells are well-constructed throughout the structure of the induced blastoid. The SOX2+ / CDX2- region shows a cavity structure (arrowhead). [Figure 6] This diagram outlines the steps involved in generating multiple genetically identical embryos (also known as induced blastoids) from bovine stem cells. [Figure 7] Steps are outlined for characterizing candidate germplasm for its suitability to the target market. Candidate germplasm that meets specific requirements can be used in selection and breeding systems, such as those shown in Figures 8 and 9. [Figure 8] This diagram illustrates a selection and breeding system using induced blastoids for the production of brood animals for targeted commercial markets with specific market requirements. [Figure 9]A schematic diagram of a selection and breeding system using induced blastoids for the production of purebred animals for targeted commercial markets with specific market requirements is shown. [Modes for carrying out the invention]

[0039] The following is a detailed description provided to assist those skilled in the art in implementing this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this disclosure belongs. The terms used herein are for the purpose of describing specific embodiments and are not intended to limit this disclosure. All publications, patent applications, patents, figures, and other references referenced herein are incorporated expressly by reference in their entirety.

[0040] Furthermore, the definitions and embodiments described in certain sections are intended to be applicable to other embodiments described herein in which they are preferred, as can be understood by those skilled in the art. For example, different embodiments of this disclosure are defined in more detail in the following sections. Each of the embodiments thus defined may be combined with any other embodiment unless expressly otherwise stated. In particular, any feature described herein may be combined with any other feature described herein.

[0041] I. General definition As used herein, the following terms may have the following meanings unless otherwise specified. However, other meanings known or understood by those skilled in the art are also possible and should be understood to be within the scope of this disclosure. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In case of any conflict, this specification shall prevail, including definitions. In addition, materials, methods, and examples are illustrative and not intended to be limiting.

[0042] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, it is understood that each intermediate value between the upper and lower limits of that range, up to one-tenth of the lower limit unit, as well as any other indicated or intermediate values ​​within that range, are included in this description. A range from any lower limit to any upper limit is contemplated. The upper and lower limits within these smaller ranges, which may be independently contained within smaller ranges, are also included in this description and subject to any specific exclusion limits within the range. Where a range includes one or both limit values, ranges excluding one or both of those limit values ​​are also included in this description.

[0043] When used in this specification and the appended claims, the singular forms "a," "an," and "the" refer to multiple objects, unless the context clearly indicates otherwise.

[0044] All numerical values ​​in the embodiments and claims for carrying out the inventions herein are modified by their indicated values ​​to be "approximately" or "about," taking into account experimental errors and variations that can be expected by those skilled in the art.

[0045] As used herein and in the claims, the phrase “and / or” should be understood to mean “either or both” of the elements thus combined, that is, elements that exist conjunctly in some cases and disjunctly in others. Multiple elements enumerated by “and / or” should be interpreted similarly, that is, “one or more” of the elements thus combined. In addition to the elements specifically identified by the “and / or” phrase, other elements may exist at will, whether related to or unrelated to the elements specifically identified.

[0046] Where used herein and in the 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 non-exclusive, meaning that it includes at least one of several elements or a list of elements, but also more than one of several elements or a list of elements, and that additional unlisted items are included at will. Only terms that explicitly indicate the opposite, such as “only one of” or “exactly one of,” or, where used in the claims, the term “consists of,” refer to including exactly one element of several elements or a list of elements. In general, where used herein, the term “or” should be interpreted as indicating an exclusive choice (i.e., “one or the other, but not both”) only when accompanied by an exclusive word such as “either,” “one of,” “only one of,” or “exactly one of.”

[0047] In the claims and the above specification, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and similar phrases are open-ended, meaning that “including” is not limited. Only the transitional phrases “consist of” and “essentially consist of” are closed or semi-closed transitional phrases, respectively.

[0048] As used herein and in the claims, the phrase “at least one” referring to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, and not necessarily having to include at least one of each element specifically enumerated in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows for the existence 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 the specifically identified elements, at the discretion of the element.

[0049] As used herein, the term “about” means plus or minus 10% to 15%, 5% to 10%, or optionally about 5% of the number being referenced.

[0050] In certain methods described herein that involve multiple steps or actions, please understand that the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions are listed, unless otherwise indicated by the context.

[0051] It should also be understood that any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this disclosure.

[0052] II. Method This specification describes a method for preparing bovine-induced blastoids. As shown in the examples, the inventors have demonstrated the formation of bovine-induced blastoids having a blastocyst-like structure, containing blastocoel, and containing cells expressing the markers ICM (expressing SOX2) and TE (expressing CDX2). After further culture, the induced blastoids contain hypoblast cells (expressing SOX17). Therefore, the materials and methods described herein are useful for inducing and maintaining bovine-induced blastoids and, optionally, for use in breeding programs such as the replication of pre-transplant embryos with desirable characteristics.

[0053] Therefore, in one embodiment, a method for preparing bovine-induced blastoids is provided. In one embodiment, the method includes: a) To provide a population of bovine stem cells, b) Culturing bovine stem cells in an aggregation medium under aggregation conditions to produce aggregated bovine stem cells. c) Introducing a first induction blastoid medium, wherein optionally, the aggregated medium and non-aggregated cells are removed before introducing the first induction blastoid medium. d) The aggregated bovine eve stem cells are cultured in the first induction blastoid medium under hypoxic conditions for approximately 48 hours, as optional. e) Introducing a second induction blastoid medium, wherein optionally, the first induction blastoid medium is removed before introducing the second induction blastoid medium, and f) Aggregated bovine eve stem cells are cultured in a second induction blastoid medium under hypoxic conditions for approximately 48 hours, optionally, to obtain bovine induction blastoids.

[0054] As used herein, the term “inducible blastoid” refers to a spherical 3D structure having a diameter of 100–200 μm (closely corresponding to the size of a 7-day blastocyst), as well as a blastocyst-like structure containing two morphologically distinct cell lineages, namely inner cell mass (ICM) cells and trophectoderm (TE) cells, and a cavity. For example, approximately 5–7 days after the onset of aggregation, with appropriate staining, the inducible blastoid is characterized by appropriate S / C / S staining, i.e., clear SOX2 staining of ICM cells and CDX2 staining of TE cells, without SOX17 signaling (similar to a 7-day embryo). However, with prolonged in vitro embryo culture and staining, the SOX17 signal appears as early as approximately 7–9 days after the onset of aggregation (similar to a 9-day embryo). When the inducible blastoid is subjected to specific conditions for each cell type, it can be used to induce naive stem cells, primed stem cells, and trophoblast stem cells, or transplanted into a recipient or surrogate animal for further proliferation and development.

[0055] As used herein, the term “naive stem cell” refers to a stem cell that can be induced and maintained in an undifferentiated state of self-renewal without requiring exogenously expressed pluripotency factors, can develop into a complete organism, and / or can retain the ability to produce a complete complement of extraembryonic tissue, adult tissue, and / or cell type. Naive stem cells exhibit molecular properties substantially similar to those of early blastocyst morula and ICM cells, and female naive stem cells have an X chromosome signature similar to that of pre-transplant embryos. Bovine naive stem cells can be identified, for example, by the morphology of a round, dome-shaped cell colony, as well as by the co-expression of one or more pluripotency markers such as SOX2, OCT4, or NANOG, and naive factors such as SUSD2 and / or TFCP2L1 and / or KLF4 and / or other selected pluripotency and naive-specific factors, such as those described in Messmer et al. (2019). Naive stem cells are unlikely to exist in vivo, but it is understood that under certain cell culture conditions, they can be induced from well-undifferentiated ICM cells, which are referred to herein as “naive stem cell-like cells.”

[0056] Naive stem cells, such as bovine eve stem cells, may be directly induced from well-undifferentiated tissues, such as blastomeres (2-4 cell embryos), or cells from the ICM of a pre-transplant embryo, such as a morula (stage 4), blastocyst (stage 5), expanding blastocyst (stage 6), expanding blastocyst (stage 7), hatching blastocyst (stage 8), or post-hatching blastocyst (stage 9). In one embodiment, bovine eve stem cells may be induced from a 2-8 day bovine embryo, optionally a 2, 3, 4, 5, 6, 7, or 8 day bovine embryo, or a 5-7 day bovine embryo. In one embodiment, the bovine embryo is a blastomere (2-4 cell embryo), morula (stage 4), blastocyst (stage 5), expanding blastocyst (stage 6), expanding blastocyst (stage 7), hatching blastocyst (stage 8), or post-hatching blastocyst (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 that does not contain the zona pellucida. In one embodiment, the embryo is a pre-transplant embryo. In one embodiment, the embryo is a previously frozen and / or biopsied embryo.

[0057] Naive stem cells, such as bovine stem cells, can be induced, for example, using the method described in Patent Cooperation Treaty Application No. PCT / CA2022 / 051664, filed November 11, 2022, or the method for proliferated pluripotent stem cells described in Zhao et al. (2021). In one embodiment, bovine stem cells are embryo-derived naive stem cells. In one embodiment, bovine stem cells are embryo-derived proliferated pluripotent stem cells. In one embodiment, embryos are selected based on testing for one or more biomarkers.

[0058] Resetting differentiation potential is recognized as an alternative method for inducing naive stem cells. For example, naive stem cells such as bovine naive stem cells may be induced from primed embryonic stem cells, as described in Guo et al. (2017), or from induced pluripotent stem cells (iPSCs) that can be induced from fully differentiated cells such as fibroblasts, as disclosed in Liu et al. (2021) or WO2022 / 109667A1 (method and cell structure). Thus, in one embodiment, bovine naive stem cells are iPSC-derived naive pluripotent stem cells. In another embodiment, bovine naive stem cells are derived from primed pluripotent stem cells. Sources of pluripotent stem cells (e.g., primed, proliferated, or induced pluripotent stem cells) may include those induced using methods described, for example, Soto et al. (2021), Bigliotti et al. (2018), Zhao et al. (2021), or Han et al. (2011).

[0059] Naive stem cells can also be derived from embryos produced in vitro using methods known in the art. For example, egg cells and sperm cells may be collected from mating pairs (female and male parents) and used to produce embryos by in vitro fertilization techniques. In one embodiment, the female and / or male parents are tested for one or more biomarkers and optionally scored and / or selected for in vitro fertilization based on the results of said testing.

[0060] Alternatively, naive stem cells can be derived from diploid reconstructed diploid embryos. Diploid embryos having a given genome can be generated in vitro by reconstructing diploid embryos using screened and selected androgenic and parthenogenic haploid cells, for example, as described in WO2020 / 168422 (Use of Haploid Embryonic Cells to Generate Offspring with Predetermined Genomes), the entire contents of which are incorporated herein by reference. Thus, in one embodiment, the bovine embryo is a reconstructed diploid embryo. The genome for the reconstructed diploid embryo can be produced to include unique combinations of alleles, haplotypes, epigenetic marks, or traits that meet strict genetic and / or predictive performance capability criteria.

[0061] In the various embodiments described herein, naive stem cells are dissociated before being cultured under aggregated conditions. Various methods can be used to dissociate the naive stem cells, for example, as described in Example 1. Other methods may also be used.

[0062] In various embodiments described herein, naive stem cells are tested for one or more biomarkers and, optionally, are scored and / or selected based on the testing of one or more biomarkers.

[0063] As used herein, “testing for one or more biomarkers” includes testing for genetic, genomic, and / or epigenetic features, 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 modification, and metabolite analysis. Testing for one or more biomarkers may be performed at any step of the methods described herein. For example, testing may be performed on embryos, growth products, stem cells, induced blastoids, sperm cells, oocytes, and / or animals (individual female and male parents, or optionally, mating pairs).

[0064] As used herein, “trait” or “feature” refers to a specific characteristic of an animal, embryo, growth, stem cell, or induced blastoid that is influenced by or determined by one or more genetic factors (e.g., allele variants, epigenetic marks, and / or combinations thereof) and / or environmental factors.

[0065] In some embodiments, naive stem cells are genetically modified.

[0066] "Genetically modified cells" refer to cells whose genomic DNA has been manipulated to express one or more exogenous genes, and / or to introduce one or more mutations that affect the expression or functional activity of an endogenous gene or gene product into an endogenous gene or intergenetic region. Successful examples of genetic modification in livestock include microinjection (US7,067,713) and lentivirus infection (Park, 2007), and more recently, the introduction of introduced genes through transfection and genome editing using genome editors, such as zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and clustered, regularly spaced short palindromic repeats / CRISPR-related gene (CRISPR / Cas) systems (verified by Bishop and Van Eenennaam, 2020 for livestock and Mueller and Van Eenennaam, 2022 for cattle). Common cattle targets for genetic modification include, for example, milk protein genes such as β-lactoglobulin, β-casein, myostatin, horn / pollen, prolactin receptor, conferring smooth coat to improve heat tolerance, and various genes involved in disease susceptibility or resilience (Bishop and Van Eenennam, 2020 and Mueller and Van Eenennam, 2022).

[0067] Similarly, a "genetically modified embryo" refers to an embryo in which the genomic DNA of cells within the embryo has been manipulated to express one or more exogenous genes, and / or to introduce one or more mutations into an endogenous gene or intergenetic region that affect the expression or functional activity of an endogenous gene or gene product.

[0068] A population of dissociated bovine stem cells can be obtained, for example, by dissociating a population of bovine stem cells using the method described herein or other methods known in the art.

[0069] The term "aggregation conditions" refers to conditions that promote the formation of cell aggregates. For example, as shown in the examples herein, cells can be aggregated using the hanging-drop method, in which 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, the aggregation conditions include hanging drops. In another embodiment shown herein, cells can be aggregated using low-adhesion culture conditions, under which cells are introduced into an environment (e.g., a low-adhesion dish) where cell adhesion to surfaces in the environment is inhibited, and instead the cells adhere to each other. Thus, in one embodiment, the aggregation conditions include a low-adhesion environment, optionally, an untreated, low-adhesion, or non-adhesion dish or plate. In further embodiments shown herein, relatively homogeneous blastocyst-like structures are formed by culturing cells in a non-adhesion or ultra-low-adhesion microwell device.

[0070] 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) having a diameter in the range of micrometers. For example, a microwell device may contain one or more wells with a diameter ranging from about 100 μm to about 1000 μm, and optionally from about 400 μm to about 600 μm. A microwell device may have any number of culture spaces (or wells) from 1 well to about 1000 or more wells. A typical configuration may include a 6-well, 12-well, 24-well, 48-well, or 96-well plate or insert. A microwell device may contain non-adhesive, uncoated, or very low-adhesive materials, such as untreated or very low-adhesive treated plastics or non-plastic materials, such as silicon. A microwell device may have a rounded or conical base to induce efficient cell aggregation. Commercially available microwell devices that may be used in the methods described herein include, but are not limited to, Stemfit3D (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®, catalog numbers 174925, 174927, Corning, catalog number CLS7007). Customized non-adhesive microwell devices having well diameters in the range of 400 μm to about 600 μm may also be used, at an optional rate. In one embodiment, the microwell device may include a plastic / silicone-based biocompatible polymer insert.

[0071] As understood in the art, the terms “incubate” or “to incubate” mean, for example, maintaining a substance, material, composition, etc., at a specific temperature or within a temperature range for a certain period of time.

[0072] As used herein, the term “physiological pH” means a pH of approximately 7.1 to approximately 7.6, optionally approximately 7.15 to approximately 7.45, approximately 7.2 to approximately 7.4, approximately 7.25 to approximately 7.35, or approximately 7.3.

[0073] As is understood in this art, pH is affected by the concentration of carbon dioxide (CO2) in the environment. Typical concentrations used in tissue culture range from about 5% CO2 to about 10% CO2, and optionally about 5% CO2 or about 6.8% CO2.

[0074] As used herein, the term “hypoxic conditions” means conditions with an oxygen (O2) concentration lower than the oxygen concentration of the atmosphere, i.e., less than approximately 20.95% oxygen. For example, when incubating cells under hypoxic conditions, the cells are incubated in an environment with reduced oxygen, e.g., approximately 1% to approximately 15% oxygen, optionally approximately 1% to 10% oxygen, or approximately 1% to approximately 5% oxygen.

[0075] As described herein, bovine stem cells are aggregated by continuous culture in various culture medium compositions, including aggregation medium, inducible blastoid medium 1, and inducible blastoid medium 2, to form inducible blastoid structures. The various media may include, for example, basal media and one or more low molecular weight, growth factors, and / or nutrients. Suitable basal media may be readily determined by those skilled in the art and are not limited to, but include DMEM / F12 (Dulbecco's modified Eagle medium F12), improved DMEM / F12, and Neurobasal medium. Suitable additives may be readily determined by those skilled in the art and are not limited to, but include minimal essential medium (MEM), non-essential amino acids (NEAA), L-glutamine (e.g., 1-2 mM), Glutamax, ascorbic acid, insulin, BSA (fraction V), beta-mercaptoethanol, penicillin / streptomycin, and / or gentamicin. In one embodiment, the basal medium comprises 1:1 DMEM / F12 and Neurobasal medium, 1x Glutamax, 1x NEAA, 0.1 mM β-mercaptoethanol, and 50 IU / ml Pen / Strep.

[0076] In one aspect of the present disclosure, agglutination medium components useful for inducing and maintaining induced blastoids are provided. In one embodiment, the agglutination medium comprises a basal medium and / or additives, as well as one or more agglutination medium components. In one embodiment, the agglutination medium comprises one or more serum substitute components and a ROCK inhibitor component. In one embodiment, the serum substitute component comprises one or more of the following: a) an N2B27 component including B27 and N2 additives, 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 B27 additive, optionally about 1% of B27 additive, and about 0.5% to about 1% of N2 additive, optionally about 0.5% of N2 additive. In one embodiment, the ROCK inhibitor component includes Y27632, fasudil, thiazovibin, or brevistatin. In one embodiment, the agglutination medium may include N2B27 and Y27632.

[0077] One aspect of the present disclosure provides induction blastoid culture medium components useful for inducing and / or maintaining induction blastoids.

[0078] In one embodiment, the first inducible blastoid medium comprises a basal medium and / or additives, and one or more components of the first inducible blastoid medium. In one embodiment, the first inducible blastoid medium (also referred to herein as inducible blastoid medium 1) comprises a serum substitute component, optionally B27 additive and N2 additive, optionally about 0.5% to about 2% B27 additive, optionally about 1% B27 additive, and optionally about 0.5% to about 1% N2 additive, an N2B27 component comprising about 0.5% N2 additive; a MEK / ERK inhibitor component, optionally PD0325901, laboxertinib, GSK1120212, MEK162, PD184352, The formulation includes trametinib, LY3214996, or urixerutinib; 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 lysophosphatidic acid (LPA); and a ROCK inhibitor component, optionally Y27632, fasudil, thiazovibin, or brevistatin. In some embodiments, the inducible blastoid medium 1 may optionally or additionally further contain a serum substitute component selected from one or more of the following: insulin-transferrin-selenium components, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); serum substitutes (SR, Sigma); or knockout serum substitutes (KOSR, Gibco) (for example, in addition to or instead of N2B27).In some embodiments, the inducible blastoid medium 1 further contains: LIF components, 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) components, optionally EGF; GSK3β inhibitors, optionally CHIR99021, BIO, CHIR-98014, LY2090314, or IM-12; Wnt activators, optionally Wnt3a, Wnt agonist 1, or SKL 2001; a histone deacetylase inhibitor (HDAC 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 one or more of SB431542, A83-01, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736. Optionally, Induced Blastoid Medium 1 may contain one or more of N2B27, PD0325901, A83-01, LPA, human LIF, and / or Y27632. In embodiments in which the first inducible blastoid medium contains an EGF component, the first inducible blastoid medium may further contain a fibroblast growth factor (FGF) component, optionally basic fibroblast growth factor (bFGF) or FGF4, at an optionally selected concentration of 10-30 ng / ml.

[0079] In another embodiment, the second inducible blastoid medium comprises a basal medium and / or additives, as well as one or more components of the second inducible blastoid medium. In one embodiment, the second inducible blastoid medium (also referred to herein as inducible blastoid medium 2) comprises a serum substitute, optionally a B27 additive and an N2 additive, optionally about 0.5% to about 2% of the B27 additive, optionally about 1% of the B27 additive, and optionally about 0.5% to about 1% of the N2 additive, an N2B27 component comprising about 0.5% of the N2 additive; a TGFβ-1 inhibitor, optionally A 83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736; HIPPO pathway inhibitors, optionally LPA; and ROCK inhibitor components, optionally Y27632, fasudil, thiazovibin, or brevistatin. In some embodiments, the inducible blastoid medium 2 may optionally or additionally further contain a serum substitute component selected from one or more of the following (e.g., in addition to or instead of N2B27): insulin-transferrin-selenium components, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); serum substitutes (SR, Sigma); or knockout serum substitutes (KOSR, Gibco). In some embodiments, the inducible blastoid medium 2 may further contain one or more of the following: fibroblast growth factor (FGF) components, optionally basic fibroblast growth factor (bFGF) or FGF4; activin A components, optionally human activin A, bovine activin A, or mouse activin A; Wnt activators, optionally Wnt3a, Wnt agonist 1, or SKL2001; and / or GSK3β inhibitors, optionally CHIR99021, BIO, CHIR-98014, LY2090314, or IM-12. Optionally, the inducible blastoid medium 2 may also contain one or more of N2B27, A83-01, LPA, and / or Y27632.In embodiments in which the second induction blastoid medium contains an FGF component, the second induction blastoid medium may further contain an epidermal growth factor (EGF) component, optionally at approximately 50 ng / ml of EGF.

[0080] III. Use of Inducible Blastoids in Cattle Breeding Programs Using the method for preparing bovine-induced blastoids described herein, multiple genetically identical bovine embryos useful in a bovine breeding program can be generated without cloning.

[0081] Germplasmic characterization may be performed to assess market suitability and its role in selection and reproduction systems. Embryos, fetuses, growth products, stem cell lines, or induced blastoids may be biopsied at any stage to isolate several cells (approximately 5-10), and the remaining cells may be kept fresh or cryopreserved for future use in the establishment of cell lines and / or induced blastoids. Alternatively, cell lines may be established from the entire embryo or induced blastoid, and a subset of cells may then be used for genomic and / or epigenetic evaluation. Cell lines may be maintained in active culture while awaiting genomic results, or they may be frozen and thawed later after obtaining genetic and / or predictive performance capability information.

[0082] For genomic or epigenetic analysis, DNA is extracted from biopsied embryonic cells, fetal cells, sperm cells, animal cells, or induced blastoid cells or established cell lines, and optionally amplified to obtain sufficient DNA for genomic analysis. If the amount of collected DNA is insufficient, DNA amplification may be performed using a DNA amplification protocol suitable for samples with small amounts of DNA (e.g., those described in Saadi et al. (2014a) and Saadi et al. (2014b)).

[0083] In one embodiment of the present disclosure, target cells (e.g., embryos, growth products, stem cells, induced blastoids, animals, sperm cells, egg cells) and / or mating pairs are characterized by testing one or more biomarkers, and optionally by scoring and / or selecting desired biomarkers (e.g., genetic, genomic, epigenetic, and / or performance capability traits(s)).

[0084] "Scoring" typically refers to testing one or more biomarkers for multiple traits or characteristics and then creating a score consisting of a set of traits that are weighted equally or differently according to their relative values. Embryos, growth products, stem cells, induced blastoids, animals, sperm cells, egg cells, and / or mating pairs may be scored based on the results of testing one or more biomarkers. A "score" may also refer to a value consisting of hundreds or thousands of genomic markers and / or epigenetic marks that are spread throughout the entire genome and, individually or collectively, constitute a genomic prediction of an individual's breeding value, genetic capacity, or performance capacity. In some embodiments, the score is a genomic score and, optionally, includes a weighted combination of one or more single nucleotide polymorphisms and / or one or more epigenetic marks. Examples of such scores, but not limited to, genomic breeding value, genomic genetic capacity, or genomic predictive performance capacity (gPPA), and, optionally, gPPA may also incorporate effects that may affect performance but are not inherited by future generations (Varona et al., 2018). Optionally, the overall scoring index may be constructed by taking into account specific market, environment, and / or economic conditions, as well as relevant interactions (including relevant interactions with genotype, e.g., genotype due to environmental interactions).

[0085] The homozygosity of cells or mating pairs and their genomic imputations may be performed first when obtaining genomic breeding values ​​from each cell and / or mating pair (Sargolzaei et al., 2014).

[0086] Other inherited genetic and / or non-genetic effects may be incorporated into genomic selection at will (Varona et al., 2018; David et al., 2019).

[0087] Alternatively, genomic breeding values ​​may be obtained without genomic imputation from embryonic cells, induced blastoid cells, fetal cells, sperm cells, egg cells, animal cells, or stem cells, or from mating pairs. In other words, if the density of marker genotypes is sufficient, it is not necessary to infer markers from adjacent markers and suggested lines based on genomic relationships.

[0088] Testing, scoring, and / or selection may also consider the genetic composition of the target cells (e.g., embryos, growth products, stem cells, induced blastoids, animals, sperm cells, egg cells) and / or mating pairs (see Figure 7). For example, Khansefid et al. (2020) report that, "In conclusion, to improve hybrid GP [genomic prediction], a balanced lineage reference is recommended that includes hybrid animals and use a set of SNPs [single nucleotide polymorphisms] that are close to QTLs [quantitative trait loci] and enriched with causative mutations." Thus, in one embodiment, testing, scoring, and / or selection may be carried out considering one or more specific markets and / or one or more genetic (e.g., lineage, family) compositions.

[0089] For example, candidate male and female parent pairs may be characterized as described above to determine the gPPA score of their future offspring. Optionally, the gPPA score may also incorporate one or more of the following effects: non-additive genetic effects, homozygosity effects, and epigenetic marking effects.

[0090] Hayes et al. (2009) describe genome selection as follows: "Genome selection refers to selection decisions based on genomic [estimated] breeding value (GEBV). GEBV is calculated as the sum of the effects of dense genetic markers across the entire genome, or the haplotype effects of these markers, thereby potentially capturing all quantitative trait loci (QTLs) that contribute to trait variability." Thus, "selection" refers to identifying subpopulations of embryos, stem cells, induced blastoids, animals, and / or mating pairs within a population that have the most desirable scores, and selecting one or more individuals from the identified subpopulations for use in the method and / or breeding program. Subpopulations of animals or mating pairs may be selected based on one or more scores, for example, as described above. In one embodiment, selection is made considering one or more specific markets and / or one or more genetic (e.g., lineage, family) composition. Figure 7 shows exemplary steps (testing, screening, scoring, and selection) for characterizing candidate germplasm for its suitability to a target market.

[0091] In one embodiment, cells are scored, and characterizing the cell's genome and / or epigenetic marks includes performing genomic scoring of the cell.

[0092] In one embodiment, mating pairs are scored, and characterizing the genome and / or epigenetic marks of the mating pairs includes performing genomic scoring of the mating pairs.

[0093] In another form of this method, cell selection is performed after evaluation of homozygosity of mating pairs or cells, and / or their genomic imputation, and / or estimation of the genomic breeding value and / or genomic predictive performance capability of each embryo, fetus, induced blastoid, spermatocyte, oocyte, animal, stem cell line, or mating pair.

[0094] In one form of this method, the examination, scoring, and / or selection are carried out to: a) Create subpopulations such as induced blastoids, naive stem cells, haploid embryos, or animals that are concentrated for a specific trait, or b) Create subpopulations such as induced blastoids, naive stem cells, haploid embryos, or animals that do not possess specific traits, or c) Create subpopulations such as induced blastoids, naive stem cells, haploid embryos, or animals that have a set of desirable traits and / or do not have a set of undesirable traits, or d) Create subpopulations such as induced blastoids, naive stem cells, haploid embryos, or animals that do not have one or more harmful haplotypes, or e) Create breeding animals or breeding lines (e.g., induced blastoids, naive stem cells, haploid embryos) that contain a unique combination of alleles, haplotypes, or traits (alleles, haplotypes, or traits usually appear at a "low" frequency, generally less than 50% of the population, but present in at least 0.1% or at least 0.01%). f) Create subpopulations such as induced blastoids, naive stem cells, haploid embryos, and animals that are particularly suited to specific environments or markets.

[0095] In another embodiment of this disclosure, the genetic, genomic, or epigenetic features include one or more productive traits (e.g., milk, fat, protein, fat%, protein%, milk protein variant composition, e.g., A2A2 milk), meat quality traits, growth traits, health traits (e.g., somatic cell score, mastitis resistance, immune response, viability, disease resistance), reproductive traits (e.g., pregnancy rate, conception rate), delivery traits (e.g., ease of delivery, first-fertilization delivery, stillbirth), morphological traits (e.g., polka-podiatosis, mammary and nipple traits, foot and leg traits, body shape). This includes quality, dimensional traits, efficiency traits (e.g., feed efficiency-related, methane efficiency, workability, lifespan, productive lifespan), novel traits (e.g., robotic milking traits, heat tolerance, smooth coat, activity traits, and behavioral traits), composite index traits (e.g., life productivity index (LPI), total productivity index (TPI), lifetime net profit in dollars (NM$)), heterozygosity, and / or absence of various harmful alleles and haplotypes (e.g., haplotypes affecting fertility, dwarfism, single hoof, hypotrichosis, brachydendron, citrullinemia, bovine leukocyte adhesion defects).

[0096] Once one or more mating pairs are selected based on one or more scores or traits (e.g., those mentioned above), one or more embryos may be produced using them. For example, using standard methods known in the art, egg cells and sperm are collected, respectively, from females and males selected based on the gPPA scores of the offspring that make up the highest / most desirable 50%.

[0097] Embryos are produced by in vitro fertilization using germ cells collected from selected mating pairs having the most desirable gPPA score for offspring. Optionally, embryos may be characterized to determine the gPPA score (and / or other desired characteristics) of individual embryos.

[0098] Embryos (or naive stem cells derived therefrom) may be selected based on one or more desired characteristics, scores, etc., and then used to generate multiple genetically identical induced blastoids using the method described herein. These are then implanted into a female surrogate animal to conceive and produce genetically identical offspring. The performance of the offspring can then be determined for one or more traits (e.g., desired milk production traits, meat quality traits, etc., based on suitability for a given environment or market, by arbitrary selection). Alternatively or additionally, phenotypic data from the initial cohort may be incorporated into the analysis to calculate updated scores. If desired, the offspring can then be put into future breeding programs. Alternatively or additionally, induced blastoids may be selected and used in in vitro breeding schemes.

[0099] Additional genetically identical induced blastoids may also be generated from a conserved stock of naive stem cells and / or induced blastoids, if desired, for example, when the performance and / or updated scores of genetically identical offspring are particularly desirable.

[0100] Representative selection and breeding systems that may utilize the disclosed inducible blastoid technology for the production of market-specific gene products, including embryos and offspring, are outlined in Figure 8 for crosses and in Figure 9 for purebreds. In addition to utilizing molecular and advanced reproductive technologies, particularly genome selection, genome prediction performance capabilities, oocyte retrieval, in vitro embryo production, and reconstruction of blastocyst-like inducible blastoids, the system includes an optional genetic modification step. Other breeding systems described (e.g., Hou et al., 2018, Bishop and Van Eenennam, 2020, Mueller and Van Eenennaam, 2022) may also benefit from using the inducible blastoid technology of this disclosure when the production of multiple genetically identical embryos is desired.

[0101] IV. Products and compositions, and their uses One aspect of the present disclosure provides products and compositions useful for the aggregation, formation, and / or maintenance of bovine-induced blastoids.

[0102] Furthermore, this specification provides a culture medium formulation suitable for promoting the aggregation of bovine stem cells, the formation of bovine-induced blastoids, and / or for preparing bovine-induced blastoids.

[0103] In one embodiment, the agglutination medium comprises a basal medium and one or more components specified herein. For example, in one embodiment, the agglutination medium comprises one or more serum substitute components, such as N2B27, and a Rho-related coiled-coil-containing protein kinase (ROCK) inhibitor component.

[0104] In one embodiment, the serum substitute component comprises one or more of the following: a) N2B27 component, b) insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G), c) serum substitute (SR, Sigma), or d) knockout serum substitute (KOSR, Gibco). In one embodiment, the N2B27 component comprises a B27 additive and an N2 additive, optionally about 0.5% to about 2% of the B27 additive, optionally about 1% of the B27 additive, and optionally about 0.5% to about 1% of the N2 additive, optionally about 0.5% of the N2 additive.

[0105] In one embodiment, the ROCK inhibitor component comprises Y27632, fasudil, thiazovibin, or brevistatin. Optionally, the ROCK inhibitor component comprises Y27632 at concentrations of approximately 0.5 μM to 20 μM, approximately 5 μM to 10 μM, or approximately 5 μM or 10 μM.

[0106] In one embodiment, the inducible blastoid medium 1 comprises a basal medium and one or more components specified herein. For example, in one embodiment, the inducible blastoid medium 1 comprises a serum substitute, a MEK inhibitor, a TGFβ-1 inhibitor, a HIPPO pathway inhibitor, and a ROCK inhibitor.

[0107] In one embodiment, the serum substitute component comprises a) the N2B27 component, b) the 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 the B27 additive and the N2 additive, optionally about 0.5%-2% of the B27 additive, optionally about 1% of the B27 additive, and optionally about 0.5%-1% of the N2 additive, optionally about 0.5% of the N2 additive.

[0108] In one embodiment, the MEK / ERK inhibitor component includes PD0325901, laboxertinib, GSK1120212, MEK162, PD184352, trametinib, LY3214996, or urixertinib. Optionally, the MEK / ERK inhibitor component includes PD0325901 or laboxertinib. Optionally, the MEK / ERK inhibitor component contains PD0325901 at concentrations of approximately 0.05 μM to 5 μM, optionally 0.1 μM to 2 μM, or optionally 1 μM. Optionally, the MEK / ERK inhibitor component contains laboxertinib at concentrations of approximately 0.25 μM to 10 μM, optionally 1 μM to 5 μM, or optionally 2.5 μM.

[0109] 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 may include A83-01 at concentrations of approximately 0.1 μM to 5 μM, optionally 0.5 μM to 2 μM, or optionally 1 μM.

[0110] In one embodiment, the HIPPO pathway inhibitor contains lysophosphatidic acid (LPA) at concentrations of approximately 0.1 to 5 μM, approximately 0.5 μM to 2 μM, or approximately 1 μM, which are optional.

[0111] In one embodiment, the ROCK inhibitor component comprises Y27632, fasudil, thiazovibin, or brevistatin. Optionally, the ROCK inhibitor component comprises Y27632 at concentrations of approximately 0.5 μM to 20 μM, approximately 5 μM to 10 μM, or approximately 5 μM or 10 μM.

[0112] In one embodiment, the inducible blastoid medium 1 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 approximately 10 ng / ml; an epidermal growth factor (EGF) component, optionally approximately 50 ng / ml EGF; a Wnt activator, optionally 10–100 ng / mL Wnt3a; and a GSK3β inhibitor, optionally 1–2 μM CHIR990. 21; a histone deacetylase inhibitor (HDAC 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, optionally A83-01, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736. In embodiments in which the first inducible blastoid medium contains an EGF component, the first inducible blastoid medium may further contain a fibroblast growth factor (FGF) component, optionally basic fibroblast growth factor (bFGF) or FGF4, at an optionally selected concentration of 10-30 ng / ml.

[0113] In one embodiment, the LIF component contains human LIF, bovine LIF, porcine LIF, goat LIF, buffalo LIF, or recombinant LIF, optionally at concentrations of approximately 1 ng / ml to approximately 1000 ng / ml, optionally at approximately 5 ng / ml to approximately 100 ng / ml, optionally at approximately 5 ng / ml, approximately 10 ng / ml, approximately 20 ng / ml, or approximately 100 ng / ml.

[0114] In one embodiment, the inducible blastoid medium 2 comprises a basal medium and one or more components specified herein. For example, in one embodiment, the inducible blastoid medium 2 comprises one or more serum substitute components such as N2B27 component, a TGFβ-1 inhibitor component, a HIPPO pathway inhibitor, and a ROCK inhibitor.

[0115] In one embodiment, the serum substitute component comprises one or more of the following: a) N2B27 component, b) insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G), c) serum substitute (SR, Sigma), or d) knockout serum substitute (KOSR, Gibco). In one embodiment, the N2B27 component comprises a B27 additive and an N2 additive, optionally about 0.5% to about 2% of the B27 additive, optionally about 1% of the B27 additive, and optionally about 0.5% to about 1% of the N2 additive, optionally about 0.5% of the N2 additive.

[0116] 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 at an optional concentration of about 0.5 μM to about 2 μM, and optionally about 1 μM.

[0117] In one embodiment, the HIPPO pathway inhibitor contains lysophosphatidic acid (LPA) at an optional concentration of approximately 0.5 μM to approximately 2 μM, and optionally approximately 1 μM.

[0118] In one embodiment, the ROCK inhibitor component comprises Y27632, fasudil, thiazovibin, or brevistatin. Optionally, the ROCK inhibitor component comprises Y27632 at concentrations of approximately 0.5 μM to 20 μM, approximately 5 μM to 10 μM, or approximately 5 μM or 10 μM.

[0119] In one embodiment, the inducible blastoid 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 activin A component, optionally 5–50 ng / ml activin A; a Wnt activator, optionally Wnt3a, Wnt agonist 1, or SKL2001; and / or a GSK3β inhibitor, optionally 1–3 μM CHIR99021. In embodiments in which the inducible blastoid medium 2 contains an FGF component, the inducible blastoid medium 2 may further comprise an epidermal growth factor (EGF) component, optionally at about 50 ng / ml EGF.

[0120] In one embodiment, the activin A component contains human activin A, bovine activin A, or mouse activin A at concentrations of approximately 1 ng / ml to approximately 50 ng / ml, approximately 5 ng / ml to approximately 50 ng / ml, or approximately 10 ng / ml or approximately 20 ng / ml, optionally.

[0121] In one embodiment, the GSK3β inhibitor component includes CHIR99021, BIO, CHIR-98014, LY2090314, or IM-12. Optionally, the GSK3β inhibitor component may include CHIR99021 at concentrations of approximately 0.1 μM to 5 μM, approximately 1 μM to 3 μM, or approximately 1 μM, 2 μM, or 3 μM.

[0122] In one embodiment, the Wnt activator component includes Wnt3a, Wnt agonist 1, or SKL2001. In one embodiment, the Wnt activator component optionally includes Wnt3a at a concentration of about 10 ng / mL to about 100 ng / mL.

[0123] Also provided are kits comprising one or more of the agglutination media, induction blastoid medium 1, and / or induction blastoid medium 2 described herein. In one embodiment, the agglutination media, induction blastoid medium 1, and / or induction blastoid medium 2 are packaged in separate containers. In one embodiment, the kit further comprises a microwell device for preparing induction blastoids.

[0124] The use of the products, compositions, or kits described herein for promoting the aggregation of naive stem cells in culture and / or for the formation of inducible blastoid structures, including bovine inducible blastoids, is also provided.

[0125] For example, in one embodiment, the use of the aggregation medium described herein is provided for promoting the aggregation of dissociated naive stem cells, and optionally bovine naive stem cells.

[0126] In another embodiment, the use of induction blastoid media described herein, for example, induction blastoid medium 1 and induction blastoid medium 2, is provided for culturing aggregated naive stem cells and promoting the formation of induction blastoids. In one embodiment, the induction blastoid media is useful for preparing induction blastoids, optionally bovine induction blastoids.

[0127] The following non-limiting embodiments illustrate the present application.

[0128] Examples Example 1.3 Preparation of a D culture system Naive stem cells, when adhered to feeder cells or a protein matrix, proliferate as flat / dome-shaped colonies. Because cells tend to proliferate via a given support structure (scaffold), generating 3D structures from adherent colonies is inefficient. Various 3D culture systems, such as hanging drop cultures, low-adhesion cell culture dishes, or microwell devices made of synthetic polymers, can be used to efficiently generate blastocyst-like structures from adherent cells. The first goal is to develop a protocol for generating bovine-inducible blastoids from established bovine naive stem cells and established genetically modified bovine naive stem cells. The next step is to adapt the protocol to suit large-scale production of bovine-inducible blastoids for commercial purposes.

[0129] In the following examples, three different 3D culture systems were tested using bovine trophoblast stem cells (TSCs). One reason for using TSCs in these tests is their ease of culture. Because trophoblast stem cells form a blastocoel under appropriate conditions, TSCs can be an ideal model for testing techniques for induced blastoid production. Furthermore, TSCs can be obtained from embryos of various stages and qualities, thus providing a robust testing system.

[0130] To avoid confusion, all protocols described below are based on tests conducted at TSC. Next, the protocol for induced blastoid generation is described in Example 5. Cell dissociation protocol (to provide dissociated cells for use in Examples 2-5) 1. Aspirate the culture medium from the culture dish, Ca 2+ / Mg 2+ Wash the cells three times with DPBS that does not contain [specific ingredient / liquid]. Note: Here, Ca 2+ / Mg 2+ Hanks equilibrium salt solution (HBSS) or Earl equilibrium salt solution (EBSS) that do not contain Ca 2+ and Mg 2+ Other types of buffers / culture media that do not contain this may be used. 2. Add TrypLE to the washed cells and incubate them for 5 minutes in a humidified incubator at 5% CO2 and 38.5°C. Note: Other types of dissociation buffers, such as actase, collagenase IV, and 0.05% trypsin, may be used here. 3. Gently pipette to dissociate the cells into small cell clusters. Transfer them to a conical tube. Ca 2+ / Mg 2+ Add 10 ml of DPBS containing [the substance]. Centrifuge at 300 x g for 5 minutes. Note: Other types of buffers / culture media containing Ca2+ and Mg2+ may be used here, such as Hanks equilibrium salt solution (HBSS), Earl equilibrium salt solution (EBSS), DMEM, DMEM / F12, Advanced DMEM, Advanced DMEM / F12, Neurobasal medium, RPMI1640, α-MEM, Opti-MEM, and other cell culture media. 4. Discard the supernatant and resuspend the cell pellet in 1 ml of fresh N2B27 medium. Note 1: N2B27 medium is a 1:1 mixture of basal medium DMEM / F12 and Neurobasal medium, with 1% B27 additive, 0.5% N2 additive, 1x Glutamax, 1x NEAA, 0.1 mM β-mercaptoethanol, and 50 IU / ml Pen / Strep added. Note 2: N2B27 basal medium can be replaced with DMEM / F12 or Advanced DMEM / F12, and other additives may be used. Centrifuge at 5,300 × g for 5 minutes. 6. Discard the supernatant and resuspend the cell pellet in 1 ml of fresh N2B27 medium.

[0131] Example 2. Hanging Drop System The hanging drop system has classically been used to generate 3D structures from many different types of cells 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 approach, droplets (20 - 30 μl) of dissociated cells containing 100 - 500 cells are made on the lid of a culture dish. Next, the lid is very carefully placed back on the dish to create a hanging drop. This confined environment creates microgravity inside the droplet, causing the cells contained to sediment to the bottom of each droplet. Depending on the cell type, after several hours to several days (24 - 48 hours), cell aggregation begins to form due to the confluent contact conditions induced by microgravity.

[0132] Here, a protocol for generating blastocyst-like structures using the hanging drop system is described. The cell lines for all protocols can include those that are genetically selected or genetically selected and modified.

[0133] Preparation of Hanging Drops In the hanging drop method, dissociated cells were used at a concentration of 1×10 4 cells / ml. 1. Place 20 μl of the dissociated cell suspension (200 cells / drop) on the lid of the dish. 2. Add sterile water to cover the bottom of the dish. Note: Any sterile liquid such as physiological saline, DPBS, HBSS, EBSS, or Milli-Q (ultrapure) water can be used to create a saturated humidity environment. 3. Very carefully turn the lid over (being careful not to break the attached droplet, see Figure 1B) and place it on top of the dish containing the sterile water. 4. Incubate the cells in the hanging drop in a humidified incubator at 5% CO2, 38.5 °C for up to 48 hours until blastocyst-like structures are visible. 5. After 48 hours, transfer the blastocyst-like structures to a normal embryo culture dish.

[0134] Results Aggregation efficiency: Each 20 μL hanging drop contains approximately 10 to 15 aggregated structures.

[0135] The structures produced using this method had diameters ranging from 50 to 150 μm.

[0136] The target diameter for blastocyst-like structures is approximately 100–150 μm (similar to the size of a blastocyst on day 7). Using this method, only one or two structures per 20 μL droplet were within the target size range.

[0137] Another important factor in producing blastocyst-like structures is the presence of a blastocoel. Using the hanging-drop method, approximately 50% of the structures showed a blastocoel.

[0138] Overall, the hanging drop method exhibits relatively low aggregation efficiency (0.03 structures / μL), offers little control over the size of the generated structures, and only a small portion of the structures exhibit the blastocoel.

[0139] Summary: Number of blastocyst-like structures produced: 4 dishes (10 drops / dish) can be prepared by one person / hour. 2 good structures / drop. Total of 80 aggregates / hour (estimated maximum capacity).

[0140] Figures 1D and 1E show comparative images of vesicles stained with Hoechst, prepared using this method, and embryos on day 7.

[0141] As seen in Figures 1D and 1E, the structures created after DNA staining exhibit a cellular tissue pattern that closely matches that which can be observed in a 7-day blastocyst (Figure 1D: TS organoid-blastocyst-like structure, where the absence of ICM is expected; 1E: 7-day blastocyst with ICM, easily observable due to high cell abundance, dotted circle).

[0142] Other observations It should be noted that when the dish lid is inverted to create a "hanging drop," the droplet can easily be disturbed or broken. Significant training is required to obtain reliable results from the hanging drop method. Furthermore, because the hanging drop method can be labor-intensive, it may not be suitable for large-scale production of induced blastoids.

[0143] Finally, the cells are cultured for 48 hours in 20 μl of medium using the hanging-drop method. While not bound by theory, it is known that naive stem cells have a very active metabolism, and the culture conditions are very limited in terms of nutrients available in the medium, which may explain the low yield of blastocyst-like structures.

[0144] Example 3. Low-adhesion dish method The low-adhesion dish method can be used as an alternative to the hanging-drop system. It has been successfully used with many different cell types, including 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-adhesion dish completely inhibits cell adhesion. Because cells cannot adhere to the coated dish, they proliferate as 3D structures in the suspension culture system. The low-adhesion dish method has many potential advantages compared to the hanging-drop method, particularly a significant improvement in processing speed due to the elimination of the need to prepare individual droplets.

[0145] Low-adhesion dish method protocol In the low-adhesion dish method, dissociated cells are divided into 1 × 10⁶ cells. 6 It was used at a concentration of cells / ml. Add 1,500 μl of fresh N2B27 medium to each well of a non-adhesive dish such as Corning, catalog no. 3473; NUNC, catalog no. 174930; or Sciencell, catalog no. 0383. 2.100 μl of dissociated cells (1 × 10⁶ 5 Place the cells (per well) into each well. 3. Culture the cells in a humidified incubator at 5% CO2 and 38.5°C for 48 hours until a blastocyst-like structure is visible. After 4.48 hours of culture, transfer the blastocyst-like structure to an embryo culture dish.

[0146] result Aggregation efficiency: Each well contains approximately 300 good structures / 500 μL (volume of culture medium contained in one well).

[0147] The structures generated using this method ranged in size from 50 to 150 μm in diameter, with 80% of the structures having a diameter of approximately 100 to 150 μm.

[0148] When examining the proportion of structures that possess a blastocoel, 80% of the structures showed a cavity.

[0149] Overall, the low-adhesion dish method showed higher aggregation efficiency (0.38 structures / μL) and higher cavity formation efficiency (80% vs. 50%) compared to the hanging drop method (0.03 structures / μL). However, significant variation in structure size was observed.

[0150] Creating structures of a more uniform size, and more importantly, a size that closely matches the size of a 7-day-old embryo, may require different techniques.

[0151] Example 4. Microwell System While the methods in Examples 2 and 3 are frequently used to generate relatively large organoids (on a mm scale), researchers have developed other methods to generate much smaller structures (in the μm range). Microwell systems have been used to generate cell aggregates, particularly for embryoid body formation (Antonchuk 2013) or adult stem cell spheroid generation (Futrega et al., 2017). Recently, a group studying human embryonic stem cells used a microwell system to generate human induced blastoids (Yu et al., 2021, Liu et al., 2021). It was hypothesized that wells of a predetermined size would provide consistent culture conditions for the cells, resulting in aggregates of uniform size. Therefore, we tested the microwell system for its ability to improve the uniformity of the size of the resulting bovine blastocyst-like structures. protocol The microwell device was prepared by adding 1.1 ml of 0.1% BSA in N2B27 medium to a StemFIT 3D® microwell device (Microfit, catalog number H853400), followed by incubation overnight in a humidified incubator at 38.5°C with 5% CO2. (Figure 3) 2. Prepare cells using the cell dissociation protocol described in Example 1. 3. Dilute the cell pellet to 1 × 10 5 Obtain a concentration of / ml. 4. Add the dissociated cells to a microwell device to obtain approximately 20-60 cells / well. After 24 hours of incubation in a humidified incubator at 5.5% CO2 and 38.5°C, cell aggregates can be observed, and after 48 hours of incubation, structures with blastocoel can be visualized.

[0152] result Aggregation efficiency (Figure 4)

[0153] Each device contains approximately 600 good structures per 1 mL (device volume).

[0154] The structures generated using this method ranged in size from 50 to 150 μm in diameter, with 70% of the structures having a diameter of approximately 100 to 150 μm.

[0155] The blastocoel was observed in approximately 75% of the generated structures.

[0156] Overall, the microwell system exhibits similar aggregation efficiency (0.32 structures / μl) to the low-adhesion culture method (0.38 structures / μl), but with less variability in the size of the generated structures.

[0157] While we do not wish to be bound by theory, the resulting blastocyst-like structures exhibit greater size uniformity than those obtained using the low-adhesion dish method, because the initial cell count and culture space size are similar across all wells.

[0158] The microwell system, compared to the low-adhesion dish method, requires similar production time but yields more homogeneous blastocyst-like structures of a larger size. Another advantage may be related to the nutrient richness to cells, as the volume of the microwells is approximately 5 μL and all wells "share" a total of 1 mL of medium (compared to 20 μL in the hanging-drop method or 500 μL in the low-adhesion method).

[0159] Example 5. Generation of induced blastoids Naive embryonic stem cells can originate from pre-transplant ICM cells or blastomeres of early morula cells and can subsequently 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 blastoids by applying TE and XEN differentiation techniques to naive pluripotent stem cells (Liu et al., 2021 and Yu et al., 2021). Briefly speaking, and without wanting to be constrained by theory, naive stem cells can be induced into the TE lineage by inhibiting the MEK / ERK and TGFβ-1 pathways. Inhibition of the HIPPO pathway can induce polarization of TE cells through activation of the TEAD4 transcription factor, leading to lumen formation into induced blastoids. Subsequently, activation of the MEK / ERK pathway by removing MEK inhibitors can induce priming of naive stem cells and subsequent endodermal differentiation.

[0160] The protocol was as follows: 1. (Day 0) Bovine 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. Note: iPSC-derived naive stem cells, primed embryonic stem cell-derived naive stem cells, embryo-derived proliferated naive stem cells, or other appropriately induced naive stem cells may also be used, and / or the cells may be genetically modified / genome-edited. 2.20 to 30 individual naive stem cells were added to each well of a StemFIT 3D® microwell device (Microfit, catalog number H853400) containing N2B27 medium + 5 to 10 μM of Y27632 (ROCK inhibitor), and cultured for 24 hours at 38.5°C in a humidified incubator with 6.8% CO2 and 5% O2. Note: ROCK inhibitors can reduce cell death induced by mechanical injury (Chen 2010). 3. After 24 hours, the culture medium and non-aggregated cells were removed from the microwell device by gentle pipette aspiration. 4. Induction blastoid medium 1 (N2B27 basal medium + 1 μM PD0325901 (MEK inhibitor) + 1 μM A83-01 (TGFβ-1 inhibitor) + 1 μM LPA (lysophosphatidic acid, HIPPO pathway inhibitor) + 10 ng / ml LIF (leukemia suppressor) + 5-10 μM Y27632 (ROCK inhibitor)) was added. Optional additives may improve efficiency: 0.1-1% ITS-X (insulin-transferrin-selenium-ethanolamine), 50 ng / ml EGF (epidermal growth factor), 1-2 μM CHIR99021 (GSK3β inhibitor), 0.2-0.8 mM VPA (histone deacetylase inhibitor, valproic acid), or 0.1-1 μM SB431542 (TGFβ-1 inhibitor). 5. The microwell devices were incubated at 38.5°C for up to 24 hours in a humidified incubator with 6.8% CO2 and 5% O2. On day 6.2, the culture medium was repeatedly changed using Induced Blastoid Medium 1. 7. On day 3 (approximately 48 hours after induction blastoid medium 1), the medium was replaced with induction blastoid medium 2 (N2B27 basal medium + 1 μM A83-01 + 1 μM LPA + 5-10 μM Y27632). Optional additives may improve efficiency: a second serum substitute, e.g., 0.1-1% ITS-X; 10-30 ng / ml bFGF (basic fibroblast growth factor); 5-50 ng / ml activin A; or 1-3 μM CHIR99021. Samples were incubated in a humidified incubator with 6.8% CO2 and 5% O2 at 38.5°C for up to 24 hours. On day 8.4, the culture medium was repeatedly changed using Induced Blastoid Medium 2.

[0161] Evaluation of induced blastoids (S / C / S staining). Embryos consist of different types of cells depending on their developmental stage. Day 7 embryos mainly contain two types of cells (ICM and TE), while day 9-15 embryos contain three types of cells (ICM, TE, and hypoblast). To detect the presence of each cell type in induced blastoids, morphological evaluation was performed on day 5 induced blastoids and compared with the morphology and S / C / S staining of day 7 and day 11 embryos stained according to a standard protocol.

[0162] The protocol for S / C / S staining of induced blastoids was as follows: 1. Induced blastoid, Ca 2+ / Mg 2+ It was washed three times with DPBS containing [the specified substance]. 2. Induced blastoids were fixed with 2-4% paraformaldehyde (PFA) at low temperature for 15 minutes at room temperature. 3. The sample, Ca 2+ / Mg 2+ Washed three times for 5 minutes each with DPBS that did not contain [unclear]. 4. Place the sample in Ca 2+ / Mg 2+ The samples were permeabilized with 0.5% Triton X in DPBS (without ) for 30 minutes to 1 hour. 5. Place the sample in a blocking solution (Ca 2+ / Mg 2+ Incubated for 1 hour in DPBS (1-3% BSA) without any added substances. 6. The samples were incubated overnight at 4°C in blocking solution with the primary antibody (SOX2 [Invitrogen, 14-9811-82] for ICM, CDX2 [Biogenex, AM382] for TE, and SOX17 [R&D system, AF1924] for hypoblast). 7. Place the sample in DPBST (Ca 2+ / Mg 2+ Washed three times for 5 minutes with 0.05% Twin20 in DPBS (which does not contain [unclear]). 8. The samples were incubated with the secondary antibody in DPBST at room temperature for 1 hour. 9. Place the sample in DPBST(Ca 2+ / Mg 2+Washed three times for 5 minutes with 0.05% Twin20 in DPBS (which does not contain [unclear]). 10. The sample was placed on a glass slide and covered with mounting medium (Vectashield + DAPI).

[0163] result Each cell type within the embryo (ICM, TE, and hypoblast) can be detected by S / C / S staining in embryos at day 11 (Figure 5A).

[0164] SOX2 (ICM marker) and CDX2 (TE marker) are detectable in embryos on days 7 and 11. However, SOX17 (hypoblast marker) begins to be detected from day 9 (not shown) because hypoblast cells begin to appear after blastocyst proliferation, and a very clear signal is obtained from embryos on day 11.

[0165] In summary, S / C / S staining provides good visualization of embryos with high specificity for each desired cell type. This tool may be useful for evaluating induced blastoids.

[0166] The structure of bovine induced blastoids can be further identified by SOX2 and CDX2 staining (Figure 5B). Five days after induction, bovine eve stem cell aggregates express SOX2 and CDX2. Each aggregate lacks SOX2 expression and has a cavity-like structure with fewer cells (arrows in Figure 5B). The size of the induced blastoids is similar to that of embryos on day 7 (Figure 5, scale: 50 μm).

[0167] Example 6. Generation of multiple genetically identical induced blastoids from individual mating. When used in combination with genome selection, other assisted reproductive technologies, and / or in vitro breeding (e.g., Hou et al., 2018), the inducible blastoid technology reported herein will offer further opportunities to improve the genetic acquisition of systems through increased selectivity, shorter generation intervals, and reduced genetic lag. Thus, as shown in Figures 6–9, the inducible blastoid technology has the potential to replace somatic cell nuclear cloning (SCNT) or AI by providing an efficient platform for mass production of genetically modified animals when a specific genome is required, for crossing when a specific breeding composition is desired (e.g., F1, ternary cross, 5 / 8:3 / 8, etc.), for replicating key components and products within conventional and in vitro breeding systems, and for improving the delivery of advanced animal pharmaceutical biologics and therapeutics, and to offer significant value to breeding systems and commercial environments, particularly for animal species with relatively low reproductive capacity and moderate to long generation intervals.

[0168] As shown in Figure 8, using the above-described method for generating induced blastoids, multiple genetically identical induced blastoids are generated from individual matings, as follows. 1. Select one or more candidate male / female parent pairs, optionally, to characterize or evaluate for desired traits, for example, by testing one or more biomarkers (e.g., SNP analysis), and optionally, to further score and / or select them based on one or more selection criteria (e.g., estimated breeding value, genome predictive performance ability, etc.). 2. Egg cells and sperm cells are collected from selected females and males using techniques known in the art. 3. To produce one or more embryos by in vitro fertilization using techniques known in the art. 4. Individual embryos are optionally characterized or evaluated for desired traits, for example, by testing one or more biomarkers, and optionally further scored and / or selected based on one or more selection criteria (e.g., estimated breeding value, genome predictive performance ability, etc.). 5. Induce and proliferate naive stem cells using, for example, the method taught in Patent Cooperation Treaty Application No. PCT / CA2022 / 051664, or the method for proliferating pluripotent stem cells described by Zhao et al. (2021). 6. Characterize or evaluate naive stem cells for desired traits, for example, by testing one or more biomarkers, optionally, further score and / or select them based on one or more selection criteria (e.g., estimated breeding value, genome prediction performance ability, etc.), and / or optionally freeze a subset of naive stem cells for preservation. 7. Generate multiple genetically identical induced blastoids from naive stem cells using the method described herein, for example, as described in Example 5. 8. Characterize or evaluate one or more inducer blastoids for a desired trait, for example, by testing one or more biomarkers, and optionally further score and / or select them based on one or more selection criteria (e.g., estimated breeding value, genome predictive performance ability, etc.).

[0169] Genetically identical induced blastoids are then frozen for preservation and / or implanted into a female surrogate animal to conceive and 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 blastoids are produced from the preserved stock of selected naive stem cells and / or induced blastoids.

[0170] Example 7. Generation of multiple genetically identical embryos, and their use in selection and breeding systems. Given the scope and scale of the global livestock sector, many species have reproductive limitations in one or both sexes. This means that conventional selection and breeding systems have limited ability to evaluate and subsequently supply germ cytoplasm suitable for many specific markets. By enabling the in vitro generation of numerous identical induced blastoids, induced blastoid technology significantly improves the ability to produce offspring with desired performance capabilities when specific genomes (e.g., offspring from a given mating pair, offspring with one or more desirable traits such as resistance to a given disease and / or high methane efficiency, genome-edited offspring, etc.) and / or specific breeding compositions are desired (e.g., F1, ternary cross, 5 / 8:3 / 8, etc.).

[0171] The selected induction blastoid is generated as in Example 6.

[0172] Genetically identical induced blastoids of good quality are frozen for future transplantation or implanted fresh into female surrogate animals (transplant animals) within one or more specific markets, and conception and viable offspring are obtained using conventional embryo transfer protocols.

[0173] Optionally, test offspring for one or more biomarkers (e.g., confirm initial testing for one or more biomarkers). Alternatively or additionally, record performance data of offspring in one or more specific markets (e.g., milk production) and optionally use it to update scores.

[0174] result

[0175] Table 1 shows the improvement in the accuracy of predicted performance of novel induced blastoids for three different traits when induced blastoids from a genetically identical cohort were increased in number, generated, and tested. [Table 1]

[0176] Table 2 shows the accuracy of the predicted mean performance of the induced blastoid cohort for three different traits when induced blastoids from genetically identical cohorts were increased, generated, and tested. [Table 2]

[0177] References Antonchuk J(2013)Formation of embryoid bodies from human pluripotent stem cells using AggreWell(TM) plates. Methods Mol Biol.946)523-533 Bogliotti YS,Wu J,Vilarino M,Okamura D,Soto DA,Zhong C,Sakurai M,Sampaio RV,Suzuki K,Izpisua Belmonte JC et al.(2018)Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts. Proceedings of the National Academy of Sciences of the United States of America 115 2090-2095. Bishop TF and Van Eenennaam AL. (2020) Genome editing approaches to augmented livestock breeding programs. J Exp Biol.223(Suppl_1):jeb207159.doi:10.1242 / jeb.207159. Chen G,Hou Z,Gulbranson DR,Thomson JA(2010)Actin-Myosin Contractility Is Responsible for the Reduced Viability of Dissociated Human Embryonic Stem Cells. Cell stem cell.7 240-248 David I,Canario L,Combes S,and Demars J.2019. Intergenerational Transmission of Characters Through Genetics,Epigenetics,Microbiota,and Learning in Livestock. Front Genet.10:1058.doi:10.3389 / fgene.2019.01058 Dong C,Beltcheva M,Gontarz P,Zhang B,Popli P,Fischer LA,Khan SA,Park KM,Yoon EJ,Xing Xiaoyun et al.(2020)Derivation of trophoblast stem cells from naive human pluripotent stem cells. Elife.9 e52504 Foty R(2011)A simple hanging drop cell culture protocol for generation of 3D spheroids. J Vis Exp.51 2720 Futrega K,Atkinson K,Lott WB,and Doran MR(2017)Spheroid Coculture of Hematopoietic Stem / Progenitor Cells and Monolayer Expanded Mesenchymal Stem / Stromal Cells in Polydimethylsiloxane Microwells Modestly Improves In Vitro Hematopoietic Stem / Progenitor Cell Expansion. Tissue Eng Part C methods.23(4)200-218 Giandomenico SL,Sutcliffe M,and Lancaster MA(2021)Generation and long-term culture of advanced cerebral organoids for studying later stages of neural development. Nat Protoc.16(2)579-602 Guo,G,von Meyenn,F,Rostovskaya,M,Clarke,J,Dietmann,S,Baker,D,Sahakyan,A,Myers,S,Bertone,P,Reik,W,Plath K,and Smith A.(2017). Epigenetic resetting of human pluripotency. Development 144,2748-2763.https: / / doi.org / 10.1242 / dev.146811 Han X,Han J,Ding F,Cao S,Lim S S,Dai Y,Zhang R,Zhang Y,Lim B,Li N.(2011). Generation of induced pluripotent stem cells from bovine embryonic fibroblast cells. Cell Res.21:1509-1512.doi:10.1038 / cr.2011.125 Hayes BJ,Bowman PJ,Chamberlain AJ,and Goddard ME.2009. Genomic selection in dairy cattle:Progress and challenges. J Dairy Sci.92:433-443.doi:10.3168 / jds.2008-1646 Hill JR(2014)Incidence of abnormal offspring from cloning and other assisted reproductive technologies Annu.Rev.Anim. Biosci.2 307-321. Hou Z,An L,Han J,Yuan Y,Chen D,and Tian J.(2018)Revolutionize livestock breeding in the future:an animal embryo-stem cell breeding system in a dish. J Anim Sci and Biotech.2018 9:1 9 1-11. Kagawa H,Javali A,Khoei HH,Sommer TM,Sestini G,Novatchkova M,Reimer YS,Castel G,Bruneau A,Maenhoudt N et al.(2022)Human blastoids model blastocyst development and implantation. Nature 601 600-605. Khansefid M,Goddard ME,Haile-Mariam M,Konstantinov KV,Schrooten C,de Jong G,Jewell EG,O’Connor E,Pryce JE,Daetwyler HD,and MacLeod IM.2020. Improving Genomic Prediction of Crossbred and Purebred Dairy Cattle. Front Genet.11:598580.doi:10.3389 / fgene.2020.598580 Linneberg-Agerholm M,Wong YF,Herrera JAR,Monteiro RS,Anderson KGV and Brickman JM.(2019)Naive human pluripotent stem cells respond to Wnt,Nodal and LIF signalling to produce expandable naive extra-embryonic endoderm. Human development 146(24)dev180620. Liu X,Tan JP,Schroeder J,Aberkane A,Ouyang JF,Mohenska M,Lim SM,Sun YBY,Chen J,Sun G et al.(2021)Modelling human blastocysts by reprogramming fibroblasts into iBlastoids. Nature 591 627-632. Mueller ML and Van Eenennaam AL.(2022). Synergistic power of genomic selection,assisted reproductive technologies,and gene editing to drive genetic improvement of cattle. CABI Agri and Biosci.3:13. Park F.(2007)Lentiviral vectors:are they the future of animal transgenesis? Physiol Genomics.31:159-173. Perez-Gomez A,Gonzalez-Brusi L,Bermejo-Alvarez P,and Ramos-Ibeas P.(2021)Lineage differentiation markers as a proxy for embryo viability in farm ungulates. Front. Vet. Sci.8 680539 Saadi HAS,O’Doherty AM,Gagne D,Fournier E,Grant JR,Sirard MA,and Robert C.(2014a) An integrated platform for bovine DNA methylome analysis suitable for small samples.BMC Genomics.15:451. Saadi HAS,Vigneault C,Sargolzaei M,Gagne D,Fournier E,de Montera B,Chesnais J,Blondin P,and Robert C.(2014b)Impact of whole-genome amplification on the reliability of pre-transfer cattle embryo breeding value estimates.BMC Genomics.15:889 Sargolzaei M,Chesnais JP,and Schenkel FS.2014. A new approach for efficient genotype imputation using information from relatives. BMC Genomics.15:478.doi:10.1186 / 1471-2164-15-478 Shri M,Agrawal H,Rani P,Singh D and Onteru SK(2017)Hanging drop,a best three-dimensional(3D)culture methos for primary buffalo and sheep hapatocytes. Scientific reports.7 1203 Soto DA,Navarro M,Zheng C,Halstead MM,Zhou C,Guiltinan C,Wu J and Ross PJ(2021). Simplification of culture conditions and feeder-free expansion of bovine embryonic stem cells. Scientific Reports 2021 11:1 11 1-15. Springer C,Wolf E,and Simmet K(2021)A new toolbox in experimental embryology - Alternative model organisms for studying preimplantation development. J.Dev. Biol.9 15 Thompson WL,and Takebe T(2020)Generation of multi-cellular human liver organoids from pluripotent stem cells. Methods Cell Biol.159 47-68 US 7067713 B2 Valamehr B,Jonas SJ,Polleux J,Qiao R,Guo S,Gschweng EH,Stiles B,Kam K,Luo T,Witte ON et al.(2008)Hydrophobic surfaces for enhanced differentiation of embryonic stem cell-derived embryoid bodies. PNAS 105(38)14459-14464 Van Vleck LD.1999. Implications of cloning for breed improvement strategies:are traditional methods of animal improvement obsolete? J Anim Sci.77(Suppl 2):111. Varona L,Legarra A,Toro MA,and Vitezica ZG.2018. Non-additive Effects in Genomic Selection. Front Genet.9:78.doi:10.3389 / fgene.2018.00078 Wang S,Wang X,Boone J,Wie J,Yip K.-P,Zhang J,Wang L and Liu R(2017)Application of hanging drop technique for kidney tissue culture. Kidney blood press res.42 220-231. Wang X and Yang P(2008)In vitro differentiation of mouse embryonic stem cells using the hanging drop method. J Vis Exp.17 828 WO 2020 / 168422 A1 Yanagida A,Spindlow D,Nichols J,Dattani A,Smith A and Guo G(2021)Naive stem cell blastocyst model captures human embryo lineage segregation. Cell Stem Cell 28. Yu L,Wei Y,Duan J,Schmitz DA,Sakurai M,Wang L,Wang K,Zhao S,Hon GC and Wu J(2021)Blastocyst-like structures generated from human pluripotent stem cells. Nature 591 620-626. Zhao L,Gao X,Zheng Y,Wang Z,Zhao G,Ren J,Zhang J,Wu J,Wu B,Chen Y,Sun W,Li Y,Su J,Ding Y,Gao Y,Liu M,Bai X,Sun L,Cao G,Tang F,Bao S,Liu P,and Li X.(2021). Establishment of bovine expanded potential stem cells. Proc Natl Acad Sci USA.118:e2018505118.doi: 10.1073 / pnas.2018505118.

Claims

1. A method for preparing bovine-induced blastoids, a) To provide a population of bovine stem cells. b) Culturing the bovine eve stem cells in an aggregation medium under aggregation conditions to produce aggregated bovine eve stem cells. c) Introducing a first induction blastoid medium, wherein optionally, the aggregated medium and non-aggregated cells are removed before introducing the first induction blastoid medium. d) The aggregated bovine stem cells are cultured in the first induction blastoid medium under hypoxic conditions. e) Introducing a second induction blastoid medium, wherein optionally the first induction blastoid medium is removed before introducing the second induction blastoid medium, and f) The aggregated bovine stem cells are cultured in the second induction blastoid medium under hypoxic conditions to obtain the bovine induction blastoid. The method, including the method described above.

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

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

4. The method according to claim 3, wherein the wells of the microwell device have a diameter of about 400 μm to about 600 μm.

5. The method according to claim 3 or claim 4, wherein the bovine stem cell population is added to the microwell device at a concentration of approximately 20 to 60 cells / well, or optionally, approximately 20 to 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 aforementioned agglutinating medium, Serum substitute components, and Rho-related coiled-coil-containing protein kinase (ROCK) inhibitor component, The method according to any one of claims 1 to 6, including the method described in any one of claims 1 to 6.

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

9. The first induction blastoid medium is Serum replacement ingredient, MEK / ERK inhibitor components, TGFβ-1 inhibitor component, HIPPO pathway inhibitor components, and ROCK inhibitor component, The method according to any one of claims 1 to 8, including the method described in any one of claims 1 to 8.

10. The method according to claim 9, The serum substitute component comprises: a) N2B27 component, optionally about 1% B27 additive and about 0.5% N2 additive; b) insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) serum substitute (SR); or d) knockout serum substitute (KOSR). The MEK / ERK inhibitor component comprises PD0325901, laboxerutinib, GSK1120212, MEK162, PD184352, trametinib, LY3214996, or urixerutinib, optionally containing approximately 1 μM of PD0325901. The TGFβ-1 inhibitor component includes A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736, optionally comprising approximately 1 μM of A83-01. The HIPPO pathway inhibitor component comprises lysophosphatidic acid (LPA), optionally about 1 μM of LPA, and / or The method wherein the ROCK inhibitor component comprises Y27632, fasudil, thiazovibin, or brevistatin, optionally containing 5 to 10 μM of Y27632.

11. The first induction blastoid 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 approximately 10 ng / ml; an epidermal growth factor (EGF) component, optionally at approximately 50 ng / ml EGF; a Wnt agonist, optionally Wnt3a, Wnt agonist 1, or SKL2001, optionally at a concentration of 10–100 ng / ml The method according to claim 9 or 10, comprising: Wnt3a L; a GSK3β inhibitor, optionally CHIR99021, BIO, CHIR-98014, LY2090314, or IM-12, optionally 1 to 2 μM of CHIR99021; a histone deacetylase inhibitor, optionally valproic acid (VPA), sodium butyrate, or trichostatin A, optionally 0.2 to 0.8 mM of VPA; and / or a second TGFβ-1 inhibitor, optionally 0.1 to 1 μM of SB431542.

12. The method according to claim 11, wherein the first induction blastoid medium contains an EGF component and further contains a fibroblast growth factor (FGF) component, optionally basic fibroblast growth factor (bFGF) or FGF4, optionally at a concentration of 10 to 30 ng / ml.

13. The second induction blastoid medium described above is Serum replacement ingredient, TGFβ-1 inhibitor component, HIPPO pathway inhibitor components, and ROCK inhibitor component, The method according to any one of claims 1 to 12, including the method described in any one of claims 1 to 12.

14. The serum substitute component comprises: a) N2B27 component, optionally about 1% B27 additive and about 0.5% N2 additive; b) insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) serum substitute (SR); or d) 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 comprising approximately 1 μM of A83-01. The HIPPO pathway inhibitor component comprises lysophosphatidic acid (LPA), optionally about 1 μM of LPA, and / or The method according to claim 13, wherein the ROCK inhibitor component comprises Y27632, fasudil, thiazovibin, or brevistatin, optionally comprising 5 to 10 μM of Y27632.

15. The method according to claim 13 or claim 14, wherein the second inducible blastoid medium further comprises, optionally, a second serum substitute component, 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 at a concentration of 1–3 μM CHIR99021.

16. The method according to claim 15, wherein the second induction blastoid medium contains an FGF component, and further contains an epidermal growth factor component and optionally about 50 ng / ml of EGF.

17. The aforementioned low-oxygen conditions are such that CO2 is approximately 5% to 10%. ２ Approximately 6.8% of CO2 was selected at will. ２ , and about 1% to 5% O ２ Approximately 5% of the O ２ The method according to any one of claims 1 to 16, including the method described in any one of claims 1 to 16.

18. The method according to any one of claims 1 to 17, wherein the first induction blastoid medium and / or the second induction blastoid 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 naive stem cell population is derived from primed pluripotent stem cells or induced pluripotent stem cells, or the bovine naive stem cell population is embryo-derived naive stem cells, optionally derived from a pre-transplant embryo or embryo-derived proliferative pluripotent stem cells, optionally the bovine naive stem cell population is a) To provide a bovine embryo containing bovine stem cell-like cells, wherein the bovine embryo does not contain a zona pellucida (ZP), b) Contacting the bovine embryo with a substrate coated with an extracellular matrix (ECM), wherein the ECM-coated substrate includes a negatively charged substrate surface adjacent to a positively charged biocompatible polymer layer and a negatively charged ECM layer adjacent to the positively charged biocompatible polymer layer, and c) Culturing the bovine embryo in the presence of a growth medium to induce adhesion of the bovine embryo to the ECM-coated substrate and the growth of an inner cell mass (ICM) containing induced bovine eve stem cells, thereby inducing a population of bovine eve stem cells. The method is induced by a method including the method.

20. The method according to any one of claims 1 to 19, wherein the bovine 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 stem cell population for one or more biomarkers, and optionally determining a score based on the determined characteristics.

22. The method according to claim 21, further comprising selecting a population of bovine eve stem cells based on the one or more characteristics and / or scores determined by testing for one or more biomarkers.

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 blastoid, and optionally determining a score based on the determined characteristics.

24. The method according to claim 23, further comprising selecting one or more of the induction blastoids based on the one or more characteristics and / or scores determined by testing one or more biomarkers.

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

26. A method of breeding cattle, a) Select at least one parent cattle from multiple animals. b) Obtaining multiple gametes from at least one parent cattle, c) Creating at least one embryo from the plurality of gametes, d) Inducing a population of naive stem cells from at least one embryo, e) preparing one or more induction blastoids according to the method described in any one of claims 1 to 24, and f) Selecting one or more of the induction blastoids for reproduction, optional in vitro reproduction, and / or for transplantation into a female surrogate animal to produce offspring. The method, including the method described above.

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

28. Agglutination culture medium composition, Serum substitute components, and Rho-related coiled-coil-containing protein kinase (ROCK) inhibitor component, The culture medium composition comprising the above.

29. A culture medium composition according to claim 28, The serum substitute component comprises a) N2B27 component, optionally about 1% of B27 component and about 0.5% of N2 component, b) insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G), c) serum substitute (SR), or d) knockout serum substitute (KOSR), and / or The culture medium composition wherein the ROCK inhibitor component comprises Y27632, fasudil, thiazovibin, or brevistatin, optionally containing 5 to 10 μM of Y27632.

30. Inducible blastoid medium composition, Serum replacement ingredient, MEK / ERK inhibitor components, TGFβ-1 inhibitor component, HIPPO pathway inhibitor components, and ROCK inhibitor component, The induction blastoid medium 1 composition comprising the above.

31. The induction blastoid medium composition according to claim 30, The serum substitute component comprises: a) N2B27 component, optionally approximately 1% of B27 component and approximately 0.5% of N2 component; b) insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) serum substitute (SR); or d) knockout serum substitute (KOSR). The MEK / ERK inhibitor component comprises PD0325901, laboxerutinib, GSK1120212, MEK162, PD184352, trametinib, LY3214996, or urixerutinib, optionally containing approximately 1 μM of PD0325901. The TGFβ-1 inhibitor component includes A83-01, SB431542, LY2109761, SB525334, SB505124, LDN-193189, RepSox, BIBF-0775, TGFβRI-IN-3, or TP0427736, optionally comprising approximately 1 μM of A83-01. The HIPPO pathway inhibitor component comprises lysophosphatidic acid (LPA), optionally about 1 μM of LPA, and / or The induction blastoid medium composition 1 comprises Y27632, fasudil, thiazovibin, or brevistatin, optionally containing 5 to 10 μM of Y27632 as the ROCK inhibitor component.

32. An induction blastoid medium composition according to claim 30 or claim 31, further comprising: The second serum substitute component is an optional 0.1-1% ITS-X. LIF component, optionally selected from human LIF, bovine LIF, goat LIF, pig LIF, buffalo LIF, or recombinant LIF, at an optional concentration of approximately 10 ng / ml. Epidermal growth factor (EGF) component, optionally 50 ng / ml EGF, GSK3β inhibitor, optionally selected from CHIR99021, BIO, CHIR-98014, LY2090314, or IM-12, optionally selected from 1-2 μM of CHIR99021. Wnt activator, optionally Wnt3a, Wnt agonist 1, or SKL2001, optionally 10-100 ng / mL of Wnt3a. Histone deacetylase inhibitors, optionally valproic acid (VPA), sodium butyrate, or trichostatin A, optionally 0.2–0.8 mM VPA, and / or A second TGFβ-1 inhibitor component, SB431542 in an optional concentration of 0.1–1 μM, The induction blastoid medium composition comprising one or more of the following.

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

34. Induced blastoid medium 2 composition, Serum replacement ingredient, TGFβ-1 inhibitor component, HIPPO pathway inhibitor components, and ROCK inhibitor component, The two induction blastoid culture media compositions comprising the above.

35. The induction blastoid medium 2 composition according to claim 34, The serum substitute component comprises: a) N2B27 component, optionally approximately 1% of B27 component and approximately 0.5% of N2 component; b) insulin-transferrin-selenium component, optionally insulin-transferrin-selenium-ethanolamine (ITS-X) or insulin-transferrin-selenium (ITS-G); c) serum substitute (SR); or d) 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 comprising approximately 1 μM of A83-01. The HIPPO pathway inhibitor component comprises lysophosphatidic acid (LPA), optionally about 1 μM of LPA, and / or The ROCK inhibitor component includes Y27632, fasudil, thiazovibin, or brevistatin. The aforementioned induction blastoid medium 2 composition.

36. The induction blastoid medium 2 composition according to claim 34 or claim 35, further comprising, The second serum substitute component is ITS-X, optionally selected, or 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. Activin A component, optionally human activin A, bovine activin A, or mouse activin A, optionally at a concentration of 5 to 50 ng / ml. Wnt activator, optionally Wnt3a, Wnt agonist 1, or SKL2001, and / or GSK3β inhibitor, optionally selected from CHIR99021, BIO, CHIR-98014, LY2090314, or IM-12, optionally selected from 1 to 3 μM of CHIR99021. The induction blastoid medium 2 composition comprising one or more of the following.

37. The induction blastoid medium 2 composition according to claim 36, wherein the induction blastoid medium 2 contains an FGF component, and further contains an epidermal growth factor component and optionally about 50 ng / ml of EGF.

38. Use of the culture medium composition according to any one of claims 28 to 37 for preparing bovine-induced blastoids.

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