Derivation of bovine ground state stem cells using feeder cells

Abstract

This disclosure relates to methods, products, and compositions that can be used to derive bovine primordial stem cells and to establish and maintain bovine primordial stem cell lines. In particular, this disclosure relates to methods for deriving bovine primordial stem cells, optionally using the feeder cell layer microdroplets; and methods for generating bovine primordial stem cells from bovine non-primordial stem cells; and growth and regeneration media specifically formulated for deriving and maintaining bovine primordial stem cells. Bovine primordial stem cells can be used in breeding programs or genetic improvement protocols, for expanding preimplantation embryos with desired genetic characteristics, for deriving primordial germ cells / gametes (including those used in in vitro breeding programs and / or transplanted into surrogates), and for developing and delivering veterinary medical biopharmaceuticals and therapeutics.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 610,130, filed December 14, 2023, the contents of which are incorporated herein by reference in their entirety. field

[0002] This disclosure relates to bovine naïve stem cells, and more specifically to methods and compositions for improving the derivation efficiency, yield, establishment and long-term maintenance of bovine naïve stem cells, and the use of said bovine naïve stem cells. introduce

[0003] Naïve bovine stem cells can differentiate into all types of cells in vivo, including extraembryonic cells such as trophoblast stem cells and extraembryonic endoderm lineage cells. The derivation of bovine naïve stem cells can bring a wide range of benefits to animal and veterinary science: providing an efficient platform for replicating embryos with desired characteristics; large-scale production of genetically modified animals; providing essential technologies for breeding systems, in vitro gamete derivation and production, and regenerative veterinary medicine; and enabling the development and delivery of advanced veterinary medical biopharmaceuticals and therapeutics. The generation of bovine naïve stem cells is reported in PCT patent application (Derivation of naïve bovine stem cells, application number PCT / CA2022 / 051664, filed November 11, 2022), and it is anticipated to be a foundational step in the development of many advanced technologies, including those valuable for livestock breeding systems.

[0004] Derivatization and culture conditions, including supporting factors that promote in vitro cell attachment, proliferation, maintenance of ground-state pluripotency, and self-renewal, are crucial elements contributing to the efficiency and yield of pluripotent primitive stem cell colonies. Supporting factors can be produced by stromal cells (directly attached to the culture dish), commonly referred to as feeder cell systems, or can comprise chemically defined protein matrices as feeder-free systems. Methods for primitive stem cells reported in PCT application PCT / CA2022 / 051664 and methods for non-prime pluripotent bovine stem cells reported by Soto et al. (2021) aim to minimize the risk of cross-contamination because these methods use feeder-free systems without feeder cells. Because the protein matrix and culture medium are chemically defined, feeder-free culture systems produce culture results with less batch-to-batch variability. The resulting minimization of consistency and cross-contamination risks may be desirable in specific situations, such as when deriving, culturing, maintaining, and using primitive stem cell lines for optional international distribution of germplasm, producing genetically modified founder breeding stock, or in xeno-free systems for the development and delivery of advanced medical and veterinary biopharmaceuticals and therapeutics.

[0005] When a heterologous system is not required, mouse embryonic fibroblast (MEF) feeder systems, such as those described by Bogliotti et al. (2018) for non-primitive pluripotent bovine stem cells, can be used. However, culture conditions for non-primitive bovine stem cells (Bogliotti et al., 2018; Han et al., 2011; Soto et al., 2021; WO 2019 / 140260; Zhao et al., 2021) and primitive stem cells from other species (Bayerl et al., 2021; Rostovskaya et al., 2019) have failed to support the self-renewal of bovine primitive stem cell colonies. In particular, MEK / ERK inhibitors (e.g., PD0325901) are considered important for primitive stem cell culture but induce fibroblast transformation into myofibroblasts, which then excessively secrete extracellular matrix (ECM) and may therefore adversely affect the microenvironment of primitive stem cells.

[0006] In addition, allogeneic culture systems, such as those using bovine endometrial matrix or bovine fetal fibroblast (bFF; Zhao et al., 2021) feeder systems, can provide improved support for bovine primitive stem cells while exhibiting a more balanced risk of species cross-contamination.

[0007] Furthermore, greater flexibility in determining the fitness of germplasm for the derivation and maintenance of primordial stem cell lines can benefit breeding systems and related methods that utilize genomic characterization data, such as those described in Hou et al. (2018), Mueller and Van Eenennaam (2022), Goszczynski et al. (2019), and Bogliotti et al. (2018), as well as in PCT applications PCT / CA2022 / 051664 and WO2022251549 (PCT / US2022 / 031210). For example, when using the reported method (PCT application PCT / CA2022 / 051664), day 5 or day 6 embryos can yield primordial embryonic stem cell outgrowth with good derivation efficiency; however, when performing genomic characterization to assess embryo fitness, derivation from day 7 embryos may be preferred. Derivation of high-quality primordial stem cell colonies from day 7 embryos remains challenging.

[0008] The methods described in WO2020168422 (PCT Application No. PCT / CA2020 / 050210) can produce offspring with a predetermined genome. However, when a self-renewing population of ground-state pluripotent stem cells can be generated, such techniques can offer broader benefits, enabling the replication of high-quality single haploid and diploid embryos. Situations where reduced costs and improved efficiency, yield, quality, and / or long-term maintenance of self-renewing primordial stem cell lines can be extremely beneficial in order to fully realize the potential of the resulting techniques include the development of efficient platforms for embryo replication, in vitro gamete derivation and production, and large-scale production of genetically modified animals. Therefore, there is a need for robust and cost-effective methods for deriving and culturing bovine primordial stem cells with improved efficiency, yield, quality, and long-term maintenance and self-renewal of ground-state pluripotency. Overview

[0009] This paper describes a method for deriving bovine primordial stem cell populations from embryos by inducing cell attachment and growth formation to obtain one or more colonies containing bovine primordial stem cells. This paper also describes feeder cell layer droplets and culture media for deriving bovine primordial stem cell populations, as well as methods for preparing said feeder cell layer droplets. The method for deriving bovine primordial stem cell populations described herein can be used to establish self-renewing bovine primordial stem cell lines.

[0010] Therefore, in one aspect, a method for deriving bovine primordial stem cell populations from bovine embryos is provided, the method comprising: a) Provide a feeder cell layer; b) Isolate cells from bovine embryos to obtain a cell population containing bovine primitive stem cell-like cells; c) Optionally, the cell population containing bovine primitive stem cell-like cells is dissociated to obtain one or more cell clusters containing bovine primitive stem cell-like cells; d) Transferring a cell population containing bovine primitive stem cell-like cells or one or more cell clusters containing bovine primitive stem cell-like cells to a feeder cell layer; and e) Culture a cell population or one or more embryonic-derived cell clusters containing bovine primordial stem cell-like cells in the presence of an outgrowth medium to induce the attachment and growth of one or more colonies containing bovine primordial stem cells, wherein the outgrowth medium comprises: i) MEK / ERK inhibitor component, Wnt inhibitor component, PKC inhibitor component, LIF component and optionally ROCK inhibitor component, or ii) Activator A component, Wnt inhibitor component, PKC inhibitor component, RAR inhibitor component, optionally ROCK inhibitor component and optionally LIF component; This leads to the development of bovine primitive stem cell populations.

[0011] In one embodiment, step (b) includes isolating cells from the inner cell mass (ICM) of a bovine embryo to obtain a cell population containing bovine primordial stem cell-like cells.

[0012] In one implementation, step b) includes separating cells by removing the zona pellucida (ZP) of the bovine embryo.

[0013] In one embodiment, the growth medium also contains a CDK8 / 19 inhibitor.

[0014] In one implementation, the density of the feeder cell layer is approximately 1.5 × 10⁻⁶. 4 / cm 2 Up to 7.5×10 4 / cm 2 Preferably about 3.0 × 10 4 / cm 2 Or a density of approximately 0.5 × 10⁻⁶. 4 1 cell / drop to 2.5 × 10⁻⁶ 4 1 cell / drop, preferably about 1.0 × 10⁻⁶ 4 Cells / drop

[0015] In one implementation, the feeder cell layer is feeder cell droplets.

[0016] In one embodiment, the feeder cell layer is prepared at least about 18 hours or about 24 hours prior to step b).

[0017] In one implementation, the feeder cell layer has been adapted to grow out of the culture medium.

[0018] In one embodiment, the feeder cells are mitotically inactivated and are optionally prepared using mitomycin treatment, gamma irradiation, or alcohol fixation.

[0019] In one implementation, the feeder cells are mouse embryonic fibroblast (MEF) cells, bovine fetal fibroblast (bFF) cells, bovine endometrial stromal cells, human fetal myocytes, MEF SNL lineage cells, human fetal fibroblasts, human adult fallopian tube epithelial cells, human dermal fibroblasts, human amniotic mesenchymal cells, human amniotic epithelial cells, mouse bone marrow stromal cells, mouse amniotic fluid cells, human amniotic fluid cells, human foreskin fibroblasts, human amniotic mesenchymal cells, pericellular matrix of decidual mesenchymal cells, human endometrial stromal cells, or rat embryonic fibroblasts.

[0020] In one implementation, the bovine embryo is a bovine embryo that does not contain the zona pellucida (ZP).

[0021] In one embodiment, the isolation of cells from bovine embryos in step b) includes immunosurgery. Optionally, anti-bovine serum antibodies are used for immunosurgery. In one embodiment, the isolated ICMs are placed in microdroplets having a derivatized culture medium containing a ROCK inhibitor.

[0022] In one embodiment, complement serum solution is used for washing and / or incubating cells after immunosurgical treatment. Optionally, the complement serum solution is a solution free of calcium sulfate and / or magnesium sulfate.

[0023] In one embodiment, anti-bovine serum antibody and / or complement serum solution are contained in neurobasal medium.

[0024] In one embodiment, the cell population in step c) is dissociated by gentle pipetting, optionally using a microcapillary syringe, optionally in the presence of a dissociation reagent (optionally TrypLE), to obtain single cells and / or small clusters after immunosurgical treatment. In one embodiment, the dissociation reagent comprises a ROCK inhibitor component.

[0025] This article also describes a method for generating bovine primitive stem cells from bovine non-primitive stem cells. Therefore, one aspect includes a method for generating bovine primitive stem cells from bovine non-primitive stem cells, the method comprising: a) Providing a cell population containing bovine non-primitive stem cells in a culture environment containing feeder cells in a culture medium containing primordial stem cells; and b) Replace the culture medium with the growth medium for at least 4 days or about 4 days, optionally about 5 to about 9 days; The growth culture medium comprises: iii) MEK / ERK inhibitor component, Wnt inhibitor component, PKC inhibitor component, LIF component, and optionally ROCK inhibitor component; or iv) Activator A component, Wnt inhibitor component, PKC inhibitor component, RAR inhibitor component, optionally ROCK inhibitor component and optionally LIF component; This generates bovine primitive stem cells.

[0026] In one embodiment, step (b) includes replacing the culture medium with an epigenetic reset medium containing a MEK / ERK inhibitor component, a LIF component, and an HDAC inhibitor component; culturing the cells; replacing the culture medium with a growth medium; and further culturing the cells for at least 4 days or about 4 days, optionally about 9 to about 11 days.

[0027] In one embodiment, the cells are cultured for at least 40 hours or about 40 hours, optionally about 2 days to about 4 days, before the culture medium is replaced with a growth medium.

[0028] In one embodiment, the growth medium also contains a CDK8 / 19 inhibitor.

[0029] In one implementation, the bovine non-primitive stem cell population comprises primordial pluripotent stem cells.

[0030] In one embodiment, the epigenetic reset medium and / or growth medium further contain an FGF2 component and / or a p38 MAPK inhibitor component.

[0031] In one embodiment, the epigenetic reset medium and / or growth medium contains a ROCK inhibitor component.

[0032] In one embodiment, the feeder cells are mitotically inactivated feeder cells, which are optionally prepared using mitomycin treatment, γ-irradiation, or alcohol fixation, and / or the feeder cells have been adapted to primordial stem cell culture medium prior to step a).

[0033] In one implementation, the method further includes the following prior to step b): i. Culture bovine non-primitive stem cells in primordial stem cell culture medium for at least 3 days or approximately 3 days; ii. Dissociating and transferring bovine non-primitive stem cells into a culture environment containing feeder cells in a culture medium for primordial stem cells; and iii. Culture the cells, optionally for at least 40 hours or about 40 hours or about 2 days.

[0034] In one embodiment, in step ii), the cells are dissociated in the presence of a dissociation agent and / or by mechanical dissociation, wherein the dissociation agent is optionally EDTA.

[0035] In one implementation, steps ii) and iii) are repeated at least once more, and optionally twice more.

[0036] In one implementation, the epigenetic reset medium in step b) is replaced with fresh epigenetic reset medium after at least 10, 20, 30 or 40 hours or about 10, 20, 30 or 40 hours, and / or the growth medium in step b) is replaced with fresh growth medium daily.

[0037] In one implementation, the cells in step b) are at a concentration of 1.5 × 10⁻⁶. 4 cells / cm 2 Up to 2.5×10 4 cells / cm 2 Approximately 2×10 (selectable area) 4 cells / cm 2 To approximately 2.5 × 10 4 cells / cm 2 Administer at the required vaccination density.

[0038] In one embodiment, the method further includes c) passage of primitive stem cells into freshly prepared feeder cells in a growth culture medium, optionally wherein the primitive stem cells are passaged at a density of approximately 1.5 × 10⁻⁶. 4 cells / cm 2 To approximately 2.5 × 10 4 cells / cm 2 Density of vaccination.

[0039] In one implementation, the feeder cells are at approximately 1.5 × 10⁻⁶. 4 cells / cm 2 To approximately 7.5 × 10 4 cells / cm 2 Choose any location approximately 3×10 4 cells / cm 2 Administer at the required vaccination density.

[0040] In one embodiment, bovine non-primitive stem cells are obtained by a method comprising the following steps: i) inducing growth formation according to any one of claims 1 to 17, wherein step e) results in the attachment and growth of one or more colonies containing bovine non-primitive stem cells; ii) isolating the colonies containing bovine non-primitive stem cells to obtain a bovine non-primitive stem cell population; iii) transferring the bovine non-primitive stem cells to a culture environment containing feeder cells in a primordial stem cell culture medium; and iv) culturing the bovine non-primitive stem cells in the primordial stem cell culture medium for at least 4 days or about 4 days.

[0041] In one implementation, steps iii) and iv) are repeated at least once more, and optionally twice more.

[0042] A method for maintaining a bovine primitive stem cell population, comprising: a) Provide a feeder cell layer; b) Provide a cell population containing bovine primitive stem cells; c) Optionally, the cell population containing bovine primitive stem cells is dissociated to obtain one or more cell clusters containing bovine primitive stem cells; d) Transferring a cell population containing bovine primitive stem cells or one or more cell clusters containing bovine primitive stem cells to a feeder cell layer; and e) Culture a cell population or one or more clusters of bovine primordial stem cells in the presence of a maintenance medium to induce the attachment and growth of one or more colonies containing bovine primordial stem cells, wherein the maintenance medium comprises: (i) MEK / ERK inhibitor component, Wnt inhibitor component, PKC inhibitor component, LIF component, and optionally ROCK inhibitor component; or (ii) Activator A component, Wnt inhibitor component, PKC inhibitor component, RAR inhibitor component, optionally ROCK inhibitor component and optionally LIF component; This helps maintain the bovine primitive stem cell population.

[0043] In one embodiment, the maintenance medium also contains a CDK8 / 19 inhibitor.

[0044] In one implementation, the density of the feeder cell layer is approximately 1.5 × 10⁻⁶. 4 / cm 2 Up to 7.5×10 4 / cm 2 Preferably about 3.0 x 10 4 / cm 2 .

[0045] In one implementation, the feeder cell layer is feeder cell droplets.

[0046] In one implementation, the density of the feeder cell layer is approximately 0.5 × 10⁻⁶. 4 1 cell / drop to 2.5 × 10⁻⁶ 4 1 cell / drop, preferably about 1.0 × 10⁻⁶ 4 Cells / drop

[0047] In one embodiment, the feeder cell layer is prepared at least about 18 hours or about 24 hours prior to step b).

[0048] In one embodiment, the feeder cell layer has been adapted to maintain the culture medium.

[0049] In one embodiment, the feeder cells are mitotically inactivated and are optionally prepared using mitomycin treatment, gamma irradiation, or alcohol fixation.

[0050] In one implementation, the feeder cells are mouse embryonic fibroblast (MEF) cells, bovine fetal fibroblast (bFF) cells, bovine endometrial stromal cells, human fetal myocytes, MEF SNL lineage cells, human fetal fibroblasts, human adult fallopian tube epithelial cells, human dermal fibroblasts, human amniotic mesenchymal cells, human amniotic epithelial cells, mouse bone marrow stromal cells, mouse amniotic fluid cells, human amniotic fluid cells, human foreskin fibroblasts, human amniotic mesenchymal cells, pericellular matrix of decidual mesenchymal cells, human endometrial stromal cells, or rat embryonic fibroblasts.

[0051] On the other hand, a method for preparing feeder cell layer microdroplets is provided, the method comprising: a) Bring the growth surface into contact with a certain volume of coating solution; b) Incubate the surface in contact with the coating solution droplets, so that the coating solution layer is deposited on the surface; c) Remove the coating solution and optionally wash the surface; d) Apply a first volume of growth medium (optionally containing feeder cells) onto the coating solution layer deposited on the surface; e) Cover the first volume of growth medium with a layer of hydrophobic fluid, wherein the hydrophobic fluid is optionally mineral oil; f) Adding a second volume of growth medium (optionally containing feeder cells) to a first volume of growth medium, wherein the first volume of growth medium and / or the second volume of growth medium contains feeder cells, thereby preparing microdroplets containing feeder cells; and g) Incubate microdroplets containing feeder cells, so that the feeder cell layer adheres to the coating solution layer deposited on the surface; This allows for the preparation of feeder cell layer microdroplets.

[0052] In one embodiment, the volume of the coating solution is about 20-30 µl, optionally about 25 µl, and / or the coating solution contains gelatin, optionally about 0.05%-0.15% gelatin, optionally about 0.1% gelatin, and the coating solution layer deposited on the surface optionally has a thickness of about 0.33-0.39 cm. 2 Optional, approximately 0.36 cm 2 The area.

[0053] In one embodiment, in step b), the surface in contact with the coating solution droplets is incubated for about 1 hour and / or at about 30-42°C, optionally about 38.5°C, optionally in a humidified environment.

[0054] In one embodiment, the first volume of growth medium and / or the second volume of growth medium is about 20-40 µl, optionally about 30 µl, and / or the combination of the first volume of growth medium and the second volume of growth medium is about 50-70 µl, optionally about 60 µl.

[0055] In one embodiment, the feeder cells are mitotically inactivated and optionally prepared using mitomycin C treatment, gamma irradiation, or alcohol fixation; the feeder cells are MEF cells, bovine fetal fibroblast (bFF) cells, bovine endometrial stromal cells, human fetal myocytes, MEF SNL lineage cells, human fetal fibroblasts, human adult fallopian tube epithelial cells, human dermal fibroblasts, human amniotic mesenchymal cells, human amniotic epithelial cells, mouse bone marrow stromal cells, mouse amniotic fluid cells, human amniotic fluid cells, human foreskin fibroblasts, human amniotic mesenchymal cells, pericellular matrix of decidual-derived mesenchymal cells, human endometrial stromal cells, or rat embryonic fibroblasts; and / or the number of feeder cells in the droplet is approximately 0.5 × 10⁻⁶. 4 Up to 2.5×10 4 Cells, optionally approximately 1.0 × 10⁶ 4 Each cell.

[0056] In one implementation, in step g), the droplets containing the feeder cells are incubated for at least 10 hours, optionally about 1 day.

[0057] One aspect includes feeder cell layer droplets generated according to the methods described herein.

[0058] One aspect of this disclosure includes the use of the feeder cell layer droplets described herein for culturing bovine embryo-derived cells (e.g., isolated ICM cells) to optionally induce the formation of bovine ICM growths and the proliferation of bovine primitive stem cells, and / or deriving bovine primitive stem cells from bovine non-primitive stem cells.

[0059] On the other hand, bovine primordial stem cells or bovine primordial stem cell populations derived using the methods described herein are provided. This includes the use of bovine primordial stem cells derived using the methods described herein in breeding programs or genetic improvement projects; for expanding preimplantation embryos (optionally having the desired genetic characteristics); for deriving primordial germ cells and / or gametes (including those for in vitro breeding programs and / or transplantation into surrogates); and / or for developing and delivering veterinary medical biopharmaceuticals and therapeutics.

[0060] The preceding sections are provided by way of example only and are not intended to limit the scope of this disclosure and the appended claims. In view of the claims, description, and embodiments of this application, those skilled in the art will understand additional objects and advantages associated with the compositions and methods of this disclosure. For example, various aspects and embodiments of this disclosure can be used in a variety of combinations, all of which are expressly considered in this specification. These additional advantages, objects, and embodiments are expressly included within the scope of this disclosure. Publications and other materials used herein to elucidate the background of this disclosure, as well as publications and other materials used in particular to provide additional details on practice, are incorporated by reference and, for convenience, are listed in the appended references section. Attached Figure

[0061] Other objects, features, and advantages of this disclosure will become apparent from the following detailed description, taken in conjunction with the accompanying drawings illustrating illustrative embodiments of the present disclosure, in which: Figure 1 A schematic diagram of an immunosurgical culture dish is shown. A: A culture dish with droplets of DPBS for washing; B: A culture dish with droplets of anti-bovine serum antibody and embryo manipulation medium; C: A culture dish with droplets of complement serum and embryo manipulation medium.

[0062] Figure 2 The image shows the immunosurgical treatment of day 7 embryos. Morphological changes (AC) during immunosurgical treatment and media optimization (DF) during immunosurgical treatment are also shown. A: Embryos before immunosurgical treatment; B: Collapsed embryos after complement treatment; C: ICM and partially digested trophoblastic ectoderm cells after immunosurgical treatment (black arrows); D: In a medium containing Ca... 2+ Mg 2+ and SO4 2- Protein coagulation in standard HEPES buffered medium (black dashed circle), and EF: no protein coagulation in DPBS and neural basal medium, respectively.

[0063] Figure 3 Immunostaining of ICM after immunosurgical treatment is shown.

[0064] Figure 4 The optimal MEF concentration (cells / droplet) for using a microdroplet culture system is shown. Too low: 0.5 × 10⁻⁶ 4 1 cell / drop; ideal: 1.0 × 10⁻⁶ 4 Cells / drop, and too dense: 2.5 × 10⁻⁶ 4 Cells / drop

[0065] Figure 5 The images show growths on a layer-by-layer (LbL) no-feeder layer system (A) and MEF (B and C). A: Embryo-derived ICM growths and endoderm differentiation on the LbL system (black arrows), BC: Trophoblastic ectoderm (TE) differentiation from embryo-derived ICM growths on the MEF (white arrows).

[0066] Figure 6 The derivation and proliferation rates of naïve stem cells in t2iLGöY and PXGL media with and without a feeder layer system are shown.

[0067] Figure 7 The optimization of MEF medium changes is shown (magnification: 40x). A: MEF in MEF medium shows normal fibroblast morphology (white arrow), B: MEF directly exposed to PXGL shows enlargement and elongation (black arrow), C: MEF treated with stepwise adaptation maintains fibroblast morphology (white arrow).

[0068] Figure 8 An exemplary schematic diagram of an ICM dissociation culture dish layout is shown.

[0069] Figure 9 The image shows dissociated ICM cell clusters. Each cluster contains 4 to 5 ICM cells.

[0070] Figure 10 The chart shows the day 0 growths of the complete ICM and the dissociated ICM. A: A primitive colony from day 0 of the complete ICM (black arrow), B: Multiple colonies from day 0 of the dissociated ICM (white arrows).

[0071] Figure 11 Bovine primordial stem cell colonies are shown. AB: Bovine primordial stem cell colonies derived from day 7 embryos with ICM without endoderm differentiation or TE growth (black arrows), C: Bovine primordial stem cell colonies of generation 2 (P2) (black arrows), D: Human primordial embryonic stem cell colonies of the prior art (Guo et al., 2017).

[0072] Figure 12A morphological comparison of bovine primordial embryonic stem cells and bovine expanded stem cells (EPSCs) with a feeder layer system is shown. Bovine primordial stem cells in PXGL medium (A and B) as described herein, and bovine EPSCs in bovine expanded potential stem cell medium (C and D) as described by Zhao et al. (2021).

[0073] Figure 13 The expression of the primitive state-specific marker SUSD2 (FITC-A) in bovine primitive stem cells and bovine expanded potential stem cells is shown. Passage 3-5 (P3-P5) bovine primitive stem cells cultured in PXGL medium showed SUSD2 expression levels of 32-45% (top panel); P3-P5 bovine EPSCs showed SUSD2 expression levels below 10% (bottom panel).

[0074] Figure 14 The images show bovine primordial pluripotent stem cells derived from embryonic growths that never exhibited characteristics of primitive stem cells. A: Embryonic growths with primordial ICM colonies at day 7. B: Heterogeneous populations of primordial pluripotent stem cell colonies (black arrows) with extraembryonic cells (white arrows) at passage 2. CD: Homogeneous primordial pluripotent stem cell colonies (black arrows) at passages 5 and 6, respectively. E: Bovine primordial pluripotent stem cells cultured without a feeder layer. F: Colony reconstitution of bovine primordial pluripotent stem cells on feeder cells after culture without a feeder layer.

[0075] Figure 15 This demonstrates the recovery of bovine primordial pluripotent stem cells after thawing.

[0076] Figure 16 The changes in MEF fibrosis are shown depending on the type and composition of the culture medium.

[0077] Figure 17 The morphological changes during the direct reset process and up to the 4th generation (P4) are shown.

[0078] Figure 18 This shows epigenetic reprogramming and morphological changes in the third generation (P3).

[0079] Figure 19 The enhanced morphology of bovine naïve stem cell colonies was shown by adding FGF2 to selected passages.

[0080] Figure 20 The cell membrane damage and cell viability after various single-cell dissociation methods are shown.

[0081] Figure 21 (A and B) show bovine primitive stem cells characterized by primitive state-specific markers at passage 2 (P2) and passage 5 (P5) (scale bar: 50 µm).

[0082] Figure 22 A schematic flowchart is shown, illustrating the efficient establishment of bovine primitive stem cells and selected industrial application examples.

[0083] Figure 23 The derivation of bovine primordial stem cells using AXRG medium and a feeder layer system is shown. ICM growth from day 7 embryos (A). P1 colonies manually subcultured (B). P4 colonies enzymatically subcultured (C). Successful establishment of bovine primordial stem cells at P5 (D).

[0084] Figure 24 Population-level expression of pluripotency marker (SOX2) and primitive state marker (SUSD2) in bovine primitive stem cells cultured long-term in AXRG and AXRGL media is shown.

[0085] Figure 25 Bovine primordial stem cells with and without LIF are shown. Bovine primordial stem cells in AXRG medium that have never been exposed to LIF show flattened colonies at P9 (black arrow), while cells supplemented with LIF from P7 onwards maintain a dome-shaped morphology at P9. Bovine primordial stem cells at P9 in AXRG medium (A). Bovine primordial stem cells at P9 in AXRG (AXRGL) medium supplemented with LIF from P7 onwards (B).

[0086] Figure 26 The images show that bovine primordial stem cells in AXRGL maintained a dome-shaped morphology during passage to P15, and lost this morphology after LIF removal from the culture medium (black arrows). Bovine primordial stem cells at P15 in AXRGL medium (A). Bovine primordial stem cells at P15 after LIF removal (B). Bovine primordial stem cells at P22 in AXRG medium (C).

[0087] Figure 27 The images show the recovery of bovine naïve stem cells from AXRG after thawing. Day 1 (A and B) and Day 2 (C and D) after thawing.

[0088] Figure 28 The viability of bovine primordial stem cells in AXRG and PXGL after thawing was demonstrated. Bovine primordial stem cells showed 88.11% and 79.35% viability after thawing in AXRG (P8) and PXGL (P4) media, respectively.

[0089] Figure 29The morphology and self-renewal capacity of bovine primordial stem cells (BSCs) were shown when supplemented with a CDK8 / 19 inhibitor. (A) The morphology of BSCs in AXRGL medium and AXRGL medium supplemented with different levels of the CDK8 / 19 inhibitor CCT251545 is shown. BSC colonies treated with high concentrations of CCT251545 showed a looser colony shape with indistinct and irregular boundaries. Also at higher concentrations, BSC colonies showed an asymmetrical shape rather than a spherical dome shape. Colonies maintained in AXRGL + 2.5 nM CCT251545 showed morphological characteristics similar to those of subsamples in control AXRGL medium. (B) Cells supplemented with 2.5 nM CCT251545 showed a significant improvement in self-renewal. When CCT251545 was introduced into BSCs, the population doubling time was shortened to the level of early passage. (C) Cells supplemented with CCT251545 exhibited stable population doubling time and no undesirable morphological changes. In contrast, the control group showed increased population doubling time and loss of primitive state-specific colony shape in subsequent passages. Description of various implementation schemes

[0090] The following is a detailed description provided to assist those skilled in the art in practicing this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. The terminology used in this description is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. All publications, patent applications, patents, figures, and other references mentioned herein are expressly incorporated herein by reference in their entirety.

[0091] Furthermore, the definitions and embodiments described in certain sections are intended to apply to other embodiments described herein that are understood to be applicable by those skilled in the art. For example, different aspects of this disclosure are defined in more detail in the following paragraphs. Each aspect so defined may be combined with any other one or more aspects unless expressly indicated to the contrary. In particular, any feature described herein may be combined with any other one or more features described herein.

[0092] I. General Definition As used herein, the following terms may have the meanings assigned to them below unless otherwise stated. However, it should be understood that other meanings known or understood by one of ordinary skill in the art are also possible and are within the scope of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0093] Where a numerical range is provided, it should be understood that, unless the context clearly specifies otherwise, every intermediate value (accurate to one-tenth of the lower limit unit) between the upper and lower limits of the range, as well as any other referred value or intermediate value within the range, is included in the specification. A range from any lower limit to any upper limit is contemplated. The upper and lower limits of these smaller ranges (which may be independently included within the smaller ranges) are also covered in the specification, subject to any specifically excluded limits within the range. When the range includes one or two limits, the range excluding any one or both of the included limits is also included in the specification.

[0094] It should be noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly specifies otherwise.

[0095] All numerical values ​​in the detailed description herein and in the claims are modified by the terms “about” or “approximately” and take into account experimental errors and variations expected by one of ordinary skill in the art.

[0096] As used herein in the specification and claims, the phrase “and / or” should be understood to mean “any one or two” of the elements so connected, that is, elements that exist together in some cases and separately in others. Multiple elements listed by “and / or” should be interpreted in the same way, that is, “one or more” of the elements so connected. Other elements besides those specifically identified by the “and / or” clause may optionally exist, whether or not such other elements are related to those specifically identified.

[0097] As used in the specification and claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted as inclusive, that is, including several elements or at least one of the elements in the list, but also including several elements or more than one of the elements in the list, and optionally additional unlisted items. Only terms that explicitly define the opposite (e.g., “only one” or “exact one” or, as used in the claims, “consisting of”) will refer to several elements or exactly one of the elements in the list. In general, the term “or” as used herein may be interpreted as indicating an exclusive choice (i.e., “one or the other, but not both”) only when it is preceded by an exclusive term (e.g., “any one,” “one,” “only one,” or “exact one”).

[0098] In the claims and the foregoing description, all transitional phrases, such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” etc., should be understood as open-ended, meaning including but not limited to. Only the transitional phrases “composed of” and “mainly composed of” should be closed or semi-closed transitional phrases, respectively.

[0099] As used in the specification and claims, the phrase "at least one" relating to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list, but does not necessarily include at least one of each element specifically listed in the list, and does not exclude any combination of elements in the list. This definition also allows for the optional presence of elements other than those specifically identified in the list referred to by the phrase "at least one," regardless of whether such elements are related to those specifically identified elements.

[0100] As used herein, the term “about” means ±10%-15%, ±5%-10%, or optionally ±about 5% of the value indicated.

[0101] It should be understood that in some methods described herein that include more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are stated, unless the context otherwise requires.

[0102] It should also be understood that any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this disclosure.

[0103] II. Methods This document describes methods for deriving bovine primordial stem cell populations from bovine embryos by inducing embryo-derived formations, and methods for deriving bovine primordial stem cells from said embryo-derived growths or from bovine non-primordial stem cells. As illustrated in the examples, the inventors have demonstrated methods for preparing feeder cell layer droplets, their use in the formation of derived bovine embryo-derived growths, and methods for optionally using said feeder cell layer droplets to derive bovine primordial stem cells therefrom, and methods for generating bovine primordial stem cells from bovine non-primordial stem cells, as well as specially formulated growth and regeneration media. The feeder cell layer droplets and growth media support the in vitro or extracellular attachment and proliferation of embryo-derived cells isolated from bovine embryos (e.g., morula or blastocyst stage (e.g., bovine day 6 or 7 embryos), and support subsequent culture of embryo-derived cells for the derivation and maintenance of bovine primordial stem cells. The regeneration media described herein support the regeneration of bovine non-primordial stem cells to generate bovine primordial stem cells. This document also describes methods and compositions for maintaining bovine primordial stem cells (e.g., maintaining colony replication and self-renewal rate of bovine primordial stem cell lines). Therefore, the materials and methods described herein can be used to derive and maintain bovine primordial stem cells and establish bovine primordial stem cell lines, optionally for breeding programs, genetic modification programs, for expanding preimplantation embryos with desired genetic characteristics and / or generating induced blastocyst-like structures, for deriving primordial germ cells and / or gametes (including for use in in vitro breeding programs and / or transplantation into surrogate bodies), and for developing and delivering veterinary medical biopharmaceuticals and therapeutics.

[0104] As used herein, the term "primitive stem cells" refers to stem cells that are capable of being derived and maintained in an undifferentiated, self-renewing state without the need for exogenous expression of pluripotency factors, and are capable of developing into a complete organism and / or retaining the ability to generate a full range of extraembryonic tissues, adult tissues, and / or cell types. Primitive stem cells exhibit molecular characteristics substantially similar to ICM cells of morula and early blastocysts, and female primitive stem cells possess X chromosome characteristics similar to those of preimplantation embryos. Bovine primitive stem cells can be identified by, for example, round, dome-shaped cell colony morphology, and the co-expression of one or more pluripotency markers (e.g., SOX2, OCT4, or NANOG) and one or more primitive factors SUSD2 and / or TFCP2L1 and / or KLF4 and / or other selected pluripotency and primitive-specific factors such as those described by Messimer et al. (2019). It is currently unclear whether primitive stem cells observed in vitro exist in vivo, but they can be derived from fully undifferentiated embryonic-derived cells (referred to herein as "primitive stem cell-like cells") under certain cell culture conditions.

[0105] Using the methods described herein, primitive stem cells (e.g., bovine primitive stem cells) can be directly derived from fully undifferentiated diploid or haploid tissues (e.g., intact embryos or ICM of preimplantation embryos). Primitive stem cells can also be derived from non-primitive stem cells, such as growths that do not exhibit primitive stem cell characteristics, induced pluripotent stem cells, expanded (or extended) pluripotent stem cells, or primordial pluripotent stem cells, for example using resetting-based methods.

[0106] As used in this article, “non-primitive stem cells” refers to cells that have acquired / retained the expression of pluripotent stem cell markers such as SOX2, OCT4, NANOG, SSEA4 and / or other pluripotency markers, but do not exhibit primitive-specific colony morphology or express SUSD2, TFCP2L1, KLF4 or other primitive-specific factors such as those described by Messmer et al. (2019).

[0107] Most cells in a culture (except for matrix-derived cells) require a support layer for attachment and in vitro proliferation, such as in a tissue culture dish. This support layer can be made from matrix cells (attached directly to the culture dish), commonly referred to as a feeder cell system. For culturing stem cells, mouse embryonic fibroblast (MEF) feeder layer systems are frequently used. As illustrated in the examples herein, the optimal ratio of feeder cell growth area to culture medium volume for establishing the growth can be achieved by applying a first volume of growth medium to the growth surface, followed by a mineral oil capping layer, and then adding a second volume of growth medium to the original droplets, thereby producing “higher” droplets with a reduced contact area and optimized droplet volume.

[0108] Therefore, in one aspect, a method for preparing feeder cell layer droplets is provided. In one embodiment, the method includes: a) Bring the surface into contact with a certain volume of coating solution (optionally gelatin); b) Incubate the surface in contact with the coating solution droplets, so that the coating solution layer is deposited on the surface; c) Remove the coating solution and optionally wash the surface; d) Apply a first volume of growth medium (optionally containing feeder cells) onto the coating solution layer deposited on the surface; e) Cover the first volume of growth medium with a layer of hydrophobic fluid, wherein the hydrophobic fluid is optionally mineral oil; f) Adding a second volume of growth medium (optionally containing feeder cells) to a first volume of growth medium, wherein the first volume of growth medium and / or the second volume of growth medium contains feeder cells, thereby preparing microdroplets containing feeder cells; and g) Incubate microdroplets containing feeder cells so that the feeder cell layer adheres to the coating solution layer deposited on the surface; This allows for the preparation of feeder cell layer microdroplets.

[0109] To achieve the optimal ratio of feeder cell growth area or surface area to culture medium volume in droplets, a droplet no larger than 1 cm can be used. 2 Or, optionally, approximately 0.33 cm 2 To approximately 0.39 cm 2 Or, optionally, approximately 0.36 cm 2 The surface area and total volume are approximately 60 µL. Approximately 0.33 cm³ 2 To approximately 0.39 cm 2 Or approximately 0.36 cm 2 The surface area can be achieved by applying a certain amount of coating solution and / or a first volume of about 25-35 µL or about 30 µL of growth medium. Therefore, in one embodiment, the first volume of growth medium is about 25-35 µL or about 30 µL.

[0110] Suitable growth media include media suitable for or generally used for culturing feeder cells, growth media disclosed herein (e.g., AXRG, AXRGL, or PXGL media), or combinations of feeder cell media and growth media (e.g., AXRG, AXRGL, and / or PXGL media) (e.g., a mixture of about 1:1). Optionally, the growth medium includes a CDK8 / 19 inhibitor. In one embodiment, the growth medium is a medium suitable for or generally used for culturing feeder cells. For example, if MEF or bFF is used, the growth medium may be MEF medium. In one embodiment, the growth medium is a mixture of a medium generally used for culturing feeder cells (e.g., MEF medium) and a growth medium (e.g., PXGL, AXRGL, or AXRG), for example, a mixture of about 1:1.

[0111] The first volume of growth medium is covered with a biocompatible hydrophobic fluid layer, such as mineral oil or paraffin oil (e.g., Ovoil™).

[0112] A second volume of growth medium is then added to the microdroplet to achieve a final volume of approximately 50-70 µl, optionally approximately 60 µL. Therefore, the second volume of growth medium can be, for example, approximately 25-35 µL, or approximately 30 µL.

[0113] The choice of feeder cells will depend on the application and may include, for example, but not limited to, mouse embryonic fibroblasts (MEF), bovine fetal fibroblasts (bFF), bovine endometrial stromal cells, human fetal myocytes, MEF SNL lineage cells, human fetal fibroblasts, human adult fallopian tube epithelial cells, human dermal fibroblasts, human amniotic mesenchymal cells, human amniotic epithelial cells, mouse bone marrow stromal cells, mouse amniotic fluid cells, human amniotic fluid cells, human foreskin fibroblasts, human amniotic mesenchymal cells, human endometrial stromal cells, rat embryonic fibroblasts, and pericellular matrix of decidual-derived mesenchymal cells. In one embodiment, the feeder cell is mouse embryonic fibroblasts (MEF).

[0114] In one embodiment, the feeder cells are mitotically inactivated feeder cells. Suitable methods for inactivating feeder cells are known in the art and include, for example, mitomycin treatment, gamma irradiation, and alcohol fixation. Thus, in one embodiment, the feeder cells are mitomycin-treated, gamma-irradiated, or alcohol-fixed cells.

[0115] Feed cells at a density suitable for bovine growth product (e.g., 0.5 x 10⁻⁶). 4 Up to 2.5 x 10 4 1 cell / drop or optionally about 1.0 x 10 4 (1 cell / droplet) inoculation. Feeder cells can be introduced into the microdroplets using a first volume of culture medium and / or a second volume of culture medium. Thus, in one embodiment, the first volume of culture medium contains 0.5 x 10⁻⁶ cells / droplet. 4 Up to 2.5 x 10 4 Cells, optionally about 1.0 x 10 4 10 cells. In another embodiment, the second volume of culture medium contains 0.5 x 10 cells. 4 Up to 2.5 x 10 4 Cells, optionally about 1.0 x 10 4 10 cells. In another embodiment, the first volume of culture medium contains approximately 0.25 x 10 cells. 4 One cell to approximately 2.0 x 10 4 Cells, optionally about 0.5 x 10 4 One cell, and the second volume of culture medium contains approximately 0.25 x 10 cells. 4 One cell to approximately 2.0 x 10 4 Cells, optionally about 0.5 x 10 4 Each cell.

[0116] The droplets containing feeder cells are then incubated at a specific temperature for a specified period of time to allow the feeder cells to adhere to or attach to the growth surface. In one embodiment, the incubation temperature is from about 38°C to about 39°C, optionally about 38.5°C. In one embodiment, the incubation period is from about 6 hours to about 3 days, optionally 12 to 36 hours or about one day. In one embodiment, the droplets containing feeder cells are incubated for up to about 7 days (e.g., from about 2 days to about 4 days or about 3 days) and then co-cultured with whole embryos, ICM cells, primordial stem cell-like cells, and stem cells such as bovine embryonic stem cells, bovine primordial stem cells, bovine pluripotent stem cells, bovine expanded (or extended) pluripotent stem cells, or bovine induced pluripotent stem cells.

[0117] Coating the growth surface with a coating solution, such as gelatin, can facilitate the adhesion and / or health of the feeder cell layer. Therefore, in one embodiment, the growth surface of the microdroplets is first coated with a coating solution layer. In one embodiment, the coating solution contains gelatin, optionally at a concentration of about 0.05% to 0.5%, optionally 0.1% to 0.2%, or optionally 0.1%. Type A gelatin or Type B gelatin can be used. In one embodiment, Type A gelatin is used. Suitable gelatin is available from commercially available suppliers and may be derived from tissue or cell cultures of pigs, cattle, birds (poultry), or fish. Optionally or additionally, the coating solution may contain collagen, laminin, fibronectin, fibronectin, geltrex, Matrigel, or a biologically equivalent cell-derived alternative. The growth surface is contacted with the coating solution for a certain period of time under conditions sufficient to allow the coating material (e.g., gelatin) to deposit on the surface. For example, the growth surface can be incubated for about 30 minutes to about 24 hours (e.g., about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, or about 16 hours) in a temperature range of about 4°C to about 42°C (e.g., at room temperature, about 35°C, about 37°C, or about 38.5°C). Optionally, the growth surface is in contact with a humidified environment.

[0118] As understood in the art, the term "incubate" or "incubating" refers to maintaining, for example, a substance, material, composition, etc., at a specific temperature or temperature range for a certain period of time.

[0119] As illustrated in the embodiments herein, bovine primitive stem cells can be derived by: isolating cells from bovine embryos, optionally from the intracellular matrix (ICM) of the bovine embryo, and optionally dissociating the ICM to obtain one or more ICM cell clusters containing bovine primitive stem cell-like cells; transferring each ICM cell cluster to a culture environment (e.g., microdroplets) containing a feeder cell layer, and culturing the clusters in a growth medium (e.g., AXRGL, AXRG, or PXGLY) to induce the attachment and growth of colonies containing bovine primitive stem cell-like cells; and culturing the bovine primitive stem cell-like cells to obtain bovine primitive stem cells, thereby deriving bovine primitive stem cells.

[0120] Therefore, in one respect, a method for obtaining bovine primitive stem cell populations derived from bovine embryos is provided, the method comprising: a) Provide a feeder cell layer; b) Isolate cells from bovine embryos, optionally from the ICM of bovine embryos, to obtain a cell population containing bovine primitive stem cell-like cells; c) Optionally, the cell population containing bovine primitive stem cell-like cells is dissociated to obtain one or more cell clusters containing bovine primitive stem cell-like cells; d) Transfer a cell population containing bovine primitive stem cell-like cells or one or more cell clusters containing bovine primitive stem cell-like cells to a feeder cell layer; e) In the presence of a growth medium, culture a cell population containing bovine primitive stem cell-like cells or one or more cell clusters containing bovine primitive stem cell-like cells to induce attachment and growth of one or more colonies containing bovine primitive stem cells. This led to the acquisition of bovine primitive stem cell populations.

[0121] Primitive stem cells (e.g., bovine primordial stem cells) can be derived directly from fully undifferentiated tissues, such as one or more blastomeres of an early cleavage-stage embryo (2-4 cell embryo), intact embryos, or ICM cells of a preimplantation embryo, such as a morula (stage 4), blastocyst (stage 5), expanding blastocyst (stage 6), expanded blastocyst (stage 7), hatching blastocyst (stage 8), or hatched blastocyst (stage 9). In one embodiment, bovine primordial stem cells may be derived from bovine embryos of 2-8 days, optionally 2, 3, 4, 5, 6, 7, or 8 days, or 5-7 days. In one embodiment, the bovine embryo is an early cleavage-stage embryo (2-4 cell embryo); a morula (stage 4); a blastocyst (stage 5); an expanding blastocyst (stage 6); an expanded blastocyst (stage 7); a hatching blastocyst (stage 8); or a hatched blastocyst (stage 9). In one embodiment, the bovine embryo is a 3- to 7-day-old embryo, optionally a 5- to 7-day-old embryo, or a 6- or 7-day-old embryo. In one embodiment, the embryo-derived cells are ICM-derived cells. In one embodiment, the bovine embryo is a bovine embryo without the zona pellucida. In one embodiment, the embryo is a preimplantation embryo. In one embodiment, the embryo is a previously frozen and / or biopsied embryo. In one embodiment, the embryo is a haploid embryo.

[0122] In all respects described herein, embryos, primordial stem cells, non-primordial stem cells, etc., have been tested against one or more biomarkers, and optionally scored and / or selected based on tests against one or more biomarkers. As used herein, “testing against one or more biomarkers” includes testing for genetic, genomic, and / or epigenetic characteristics, including but not limited to one or more specific alleles, single nucleotide polymorphisms (SNPs), haplotypes, copy numbers, homozygosity, genomic insertions and / or deletions, histone modifications, protamine modifications, DNA methylation, gene or mRNA expression levels, protein expression or modifications, and the presence or absence of metabolite analysis. Testing against one or more biomarkers can be performed at any step of the methods described herein. For example, embryos, growths, primordial stem cells, non-primordial stem cells, induced blastocysts, sperm, oocytes, and / or animals (single female and male animals, or optionally mated pairs) can be tested.

[0123] As used herein, “trait” or “characteristic” refers to a specific feature of an animal, embryo, growth, stem cell, sperm, oocyte, or induced blastocyst that may be influenced by or determined by one or more genetic factors (e.g., allele variants, epigenetic markers, homozygosity, copy number, and combinations thereof) and / or environmental factors.

[0124] In all aspects described in this article, bovine embryos, primitive stem cells, and non-primitive stem cells have been genetically modified.

[0125] "Genetically modified cells" refer to cells in which the genomic DNA has been manipulated to express one or more exogenous genes and / or to introduce one or more mutations into endogenous genes or intergenic regions that affect the expression or functional activity of one or more endogenous genes or gene products. Successful examples of gene modification in livestock include the introduction of transgenes via microinjection (US 7,067,713) and lentiviral infection (Park, 2007), as well as more recent genome editing (Bishop and Van Eenennaam, 2020 reviewed for livestock and Mueller and Van Eenennaam, 2022 reviewed for cattle), which utilizes transfection and 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. Common targets of bovine gene modification are milk protein genes, such as β-lactoglobulin, β-casein, myostatin, horned / hornless genes, prolactin receptors that impart smooth coat to improve heat resistance, and various genes involved in disease susceptibility or resilience (Bishop and Van Eenennam, 2020; and Mueller and Van Eenennam, 2022).

[0126] Similarly, a “genetically modified embryo” refers to an embryo in which the genomic DNA of the cells in the embryo has been manipulated to express one or more exogenous genes and / or to introduce one or more mutations into endogenous genes or intergenic regions that affect the expression or functional activity of one or more endogenous genes or gene products.

[0127] As illustrated in the embodiments, the method described herein for embryo-derived growth formation improves the yield of bovine embryonic growths containing cells with ICM-like characteristics (58% vs. 8-25%, using day 7 embryos) compared to standard methods for deriving human embryonic stem cells, and produces growths containing cell colonies exhibiting primordial stem cell characteristics, as well as growths containing cell colonies exhibiting non-primordial stem cell characteristics. Therefore, in one embodiment, at least 25% of the growth contains ICM-like cells. In one embodiment, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or more than 55% of the growth contains ICM-like cells. As shown herein, using the method described herein, the growth produces colonies containing bovine primordial stem cells at a higher frequency than previously described. Using the method described herein, approximately 50% of these colonies maintain a primordial state across multiple passages. Therefore, in one embodiment, at least 25% or approximately 25% or more of the growth produces bovine primordial stem cells.

[0128] Appropriately, the feeder cell layer is prepared at least 18 hours before derivation.

[0129] As illustrated herein, prior to derivatization, the feeder cell layer may be adapted to a growth medium (e.g., AXRGL, AXRG, and / or PXGL media), optionally wherein the growth medium contains a CDK8 / 19 inhibitor. Therefore, in one embodiment, the feeder cell layer is adapted from the feeder cell medium to the growth medium in a stepwise manner, for example by replacing the growth medium with an increasing proportion of growth medium over time. In one embodiment, approximately 18 hours before adding bovine embryo-derived cells to the droplets, the growth medium in the droplets is replaced with a 1:1 MEF:growth medium, and / or approximately 4 hours or less before adding bovine embryo-derived cells to the droplets, the growth medium in the droplets is replaced with growth medium.

[0130] Suitable feeder cells include mouse embryonic fibroblasts (MEF), bovine fetal fibroblasts (bFF), bovine endometrial stromal cells, human fetal myocytes, MEF SNL lineage cells, human fetal fibroblasts, human adult fallopian tube epithelial cells, human dermal fibroblasts, human amniotic mesenchymal cells, human amniotic epithelial cells, mouse bone marrow stromal cells, mouse amniotic fluid cells, human amniotic fluid cells, human foreskin fibroblasts, human amniotic mesenchymal cells, human endometrial stromal cells, rat embryonic fibroblasts, and the pericellular matrix of decidual-derived mesenchymal cells. In one embodiment, the feeder cell is mouse embryonic fibroblasts (MEF).

[0131] The optimal feeder cell density can be, for example, approximately 1.5 × 10⁻⁶. 4 / cm2 Up to 7.5×10 4 / cm 2 Preferably about 3.0 × 10 4 / cm 2 , or 0.5×10 4 1 cell / drop to 2.5 × 10⁻⁶ 4 1 cell / drop, preferably about 1.0 × 10⁻⁶ 4 Cells / drop

[0132] The zona pellucida (ZP) prevents embryonic cells from attaching to the culture medium. Therefore, in one embodiment, the ZP of the embryo is removed before the embryonic cells are separated. In fact, in one embodiment, cells are separated from bovine embryos by removing the zona pellucida. Embryos free of ZP can be provided or obtained using any suitable method. For example, ZP can be thinned and / or ruptured using enzymatic, chemical, and / or mechanical methods, and subsequently separated from the embryo by mechanical manipulation to obtain a ZP-free embryo. Suitable enzymatic or chemical methods for thinning and / or rupturing ZP include, for example, the use of proteases such as streptokinase, collagenase, or acidified Tyrode's solution. Suitable mechanical and non-contact methods for rupturing ZP include, for example, the use of a microblade, micropipette, microneedle, or laser. The ruptured ZP can be separated from the embryo, said separation being, for example, by agitation, such as blowing, vortexing, or direct manipulation using a micropipette or microneedle. The embryos from which ZP-free bovine embryos can be obtained can be fresh or previously frozen, and optionally can be obtained from biopsy-frozen embryos. In one embodiment, the embryos are genetically tested embryos.

[0133] As described and illustrated in the embodiments herein, immunosurgical treatment can be used to facilitate the isolation of ICM cells to derive bovine primitive stem cells. Therefore, in one embodiment, immunosurgical treatment is used to isolate ICM cells. When a heterologous system is desired, laser-assisted techniques can be used to isolate ICM cells (Turetsky et al., 2008). In one embodiment, laser-assisted dissection is used to isolate ICM cells.

[0134] As used in this article, the term "immunosurgery" refers to the method of separating the inner cell mass from the trophectoderm (TE) of a blastocyst-stage embryo using antibodies.

[0135] As described in Example 1, cells can be isolated from the ICM of a ZP-free day 7 embryo by incubating ZP-free embryos with anti-bovine serum antibody, followed by incubation with complement serum solution. The trophoblast cells and cell debris are then removed by mechanical pipetting, and the remaining ICM is optionally treated with a calcium-free solution. 2+ Mg 2+ and SO4 2- Culture media (e.g., those without Ca) 2+ and Mg 2+ Embryos were washed with DPBS, neural basal medium, Ca / Mg-free HBSS, Ca / Mg-free EBSS, or physiological saline, and inoculated into growth medium, optionally into feeder cell layer droplets. In one embodiment, the embryos were incubated with about 25% anti-bovine serum antibody solution at about 38.5°C in a humidified 5% CO2 incubator for about 1 hour. In one embodiment, the embryos were incubated with about 25% complement solution, optionally guinea pig complement serum, human complement serum, rabbit complement serum, hamster complement serum, canine complement serum, mouse complement serum, porcine complement serum, sheep complement serum, goat complement serum, llama complement serum, monkey complement serum, or rat complement serum at about 38.5°C in a humidified 5% CO2 incubator for about 1 hour. In one embodiment, the complement serum was guinea pig complement serum. As shown in the examples, when using Ca / Mg-free... 2+ Mg 2+ and SO4 2- When anti-bovine serum antibodies and complement serum were diluted in a culture medium containing Ca2+, higher immunosurgical processing efficiency was observed, and the time required to complete TE cell digestion was reduced. Therefore, in one embodiment, anti-bovine serum antibodies and / or complement serum are diluted in a Ca2+-free culture medium. 2+ Mg 2+ and SO4 2- Dilute in a culture medium (e.g., neural basal medium).

[0136] As illustrated in the examples, derivatization efficiency is improved when ICM cells are dissociated to obtain smaller ICM cell clusters. Therefore, in one embodiment, ICM (or other embryo-derived) cells are dissociated before transfer to a feeder cell layer. ICM and other embryo-derived cells can be dissociated by gentle pipetting with a small-bore pipette, micropipette tip, microcapillary, or microcapillary syringe, optionally in the presence of a dissociation reagent. Suitable small-bore pipettes, microcapillaries, etc., may have an inner diameter ranging from about 10 µm to about 15 µm to obtain single cells, or an inner diameter ranging from about 20 µm to about 35 µm to obtain cell clusters. Microcapillaries can be prepared using methods such as those described herein or methods known to those skilled in the art. Suitable dissociation reagents and conditions include, but are not limited to, TrypLE, 0.25% trypsin / EDTA, 0.05% trypsin / EDTA, and / or Accutase, optionally maintained for about 2 to about 10 minutes prior to pipetting. Alternatively, in the methods described herein, EGTA, citrate, or other biocompatible chelating agents can be used instead of EDTA. Those skilled in the art will understand that Ca... 2+ Mg 2+ and SO4 2- The presence of [a substance] will interfere with dissociation. Therefore, the dissociation reagent will [dissociate in the absence of Ca]. 2+ Mg 2+ and SO4 2- Dilute in buffer solution.

[0137] In one embodiment, embryonic cells are dissociated in the presence of one or more Rho-kinase (ROCK) inhibitor components, including but not limited to Y27632, Thiazovin, fasudil, and Blebbistatin. CEPT mixtures containing ROCK inhibitor components can also be used as ROCK inhibitor components.

[0138] In one implementation, a ROCK inhibitor is included when the cells are manipulated and / or subjected to stress. For example, a ROCK inhibitor may be added to the culture medium at the onset of growth and / or during immunosurgical treatment, resetting, passage (mechanical or enzymatic), freezing, and / or thawing.

[0139] As illustrated herein, the attachment and growth formation of bovine embryos are influenced by the composition of the culture medium used to culture the embryos and / or the cells derived from the embryos. As shown in Example 3, a culture medium designed for use in feeder-free cultures may not be particularly suitable for feeder-based systems. Therefore, bovine primordial stem cells can be cultured in a growth medium (also referred to herein as “growth medium”) such as “AXRGL”, “AXRG”, “PXGL”, “PXGLY”, or the “enhanced growth” medium described herein. In one embodiment, the growth medium includes a CDK8 / 19 inhibitor.

[0140] As illustrated in Example 6 of this document, the resetting method can be used to convert bovine non-primitive stem cells into bovine primitive stem cells. As used herein, the term "resetting" refers to reversing cells in a higher developmental state (e.g., primordial pluripotent stem cells, induced pluripotent stem cells, expanded pluripotent stem cells) back to a primordial developmental state.

[0141] It is understood that pluripotency goes through at least two stages: the primitive state and the primordial state. Primitive state stem cells are considered the developmental baseline, exhibiting unlimited developmental potential, homogeneity, and high differentiation capacity, and forming dense, dome-shaped colonies. In contrast, primordial state pluripotent stem cells and induced pluripotent stem cells do not exhibit the primitive state morphology; instead, they are flat and monolayered, exhibiting repressive chromatin characteristics and lineage priming, do not show unlimited developmental potential, and cannot be used for further passage of primitive state cells.

[0142] Resetting cellular potential is considered an alternative method for deriving primitive stem cells. For example, Guo et al. (2017) reported the derivation of human primitive stem cell cultures after resetting human induced pluripotent stem cells and human primordial pluripotent stem cells. Thus, in one embodiment, bovine primitive stem cells are derived from induced pluripotent stem cells. In another embodiment, bovine primitive stem cells are derived from primordial pluripotent stem cells.

[0143] Nascent pluripotent stem cells can be derived, for example, from embryonic-derived extrusions (obtained using the methods described above) that do not exhibit a primitive morphology, using various methods including those described herein. To isolate and establish nascent pluripotent stem cell lines from extrusions that do not exhibit a primitive morphology and / or contain a mixture of extraembryonic cells, selective dissociation can be applied. Extrusions that do not exhibit a primitive morphology can be selectively dissociated into smaller fragments using EDTA solution (Beers et al., 2012) or other suitable dissociation agents, thereby allowing the removal of extraembryonic cells during a subculture process (“EDTA-subculture”), and yielding clusters of nascent pluripotent stem cells. Other sources of non-primitive stem cells may include those derived using methods described, for example, those described by Soto et al. (2021), Bigliotti et al. (2018), Zhao et al. (2021), and Han et al. (2011).

[0144] Therefore, in one aspect, a method for generating bovine primitive stem cells from bovine non-primitive stem cells (optionally primordial stem cells) is provided, the method comprising: a) Providing a cell population containing bovine non-primitive stem cells in a culture environment containing feeder cells in a culture medium containing primordial stem cells; and b): I. Replace the culture medium with an epigenetic reset medium containing a MEK / ERK inhibitor component, a LIF component, and an HDAC inhibitor component; culture the cells optionally for at least 10, 20, 30, or 40 hours, or about 10, 20, 30, or 40 hours, optionally about 2 days to about 4 days; replace the culture medium with a growth medium; and culture the cells for at least 4 days, or about 4 days, optionally about 9 to about 11 days; or II. Replace the culture medium with the growth medium for at least 4 days or about 4 days, optionally about 5 to about 9 days; This generates bovine primitive stem cells.

[0145] In one embodiment, the growth culture medium in I. and II. comprises: i) MEK / ERK inhibitor components, Wnt inhibitor components, PKC inhibitor components, LIF components, and ROCK inhibitor components; or ii) Activator A component, Wnt inhibitor component, PKC inhibitor component, RAR inhibitor component, ROCK inhibitor component and optionally LIF component; The growth medium in I. and II. may also contain CDK8 / 19 inhibitors.

[0146] Epigenetic reset medium and / or growth medium may further contain FGF2 components and / or p38 MAPK inhibitor components. Optionally or additionally, epigenetic reset medium and / or growth medium may further contain ROCK inhibitor components.

[0147] If the number of non-primitive stem cells is limited and / or the non-primitive stem cell population also contains additional cells such as extraembryonic cells (e.g., when using “non-passivated” ICM colonies derived using the method described herein), the method may further include, prior to step b): i. Culture bovine non-primitive stem cells in primordial stem cell culture medium for at least 3 days or approximately 3 days; ii. Dissociating and transferring bovine non-primitive stem cells into a culture environment containing feeder cells in a culture medium containing primordial stem cells; and iii. Culture the cells, optionally for at least 10, 20, 30 or 40 hours or about 10, 20, 30 or 40 hours, optionally about 2 days.

[0148] If necessary, for example to increase the number or percentage of non-primitive stem cells in the population, steps ii) and iii) may be repeated at least once more, and optionally twice or more.

[0149] As described herein, in step ii), the cells may be dissociated in the presence of a dissociation agent such as EDTA and / or by mechanical dissociation.

[0150] Optionally, the epigenetic reset medium in step b) may be replaced with fresh epigenetic reset medium after at least 10, 20, 30 or 40 hours or about 10, 20, 30 or 40 hours, and / or the growth medium in step b) may be replaced with fresh growth medium daily.

[0151] In one implementation, the cells in step b) are at a concentration of 1.5 × 10⁻⁶. 4 cells / cm 2 Up to 2.5×10 4 cells / cm 2 Approximately 2×10 (selectable area) 4 cells / cm 2 To approximately 2.5 × 10 4 cells / cm 2 Inoculation density of the slab.

[0152] In one embodiment, the method further includes c) passage of primitive stem cells into freshly prepared feeder cells in a growth culture medium, optionally wherein the primitive stem cells are passaged at a density of approximately 1.5 × 10⁻⁶. 4 cells / cm 2 To approximately 2.5 × 10 4cells / cm 2 Density paving.

[0153] II. Product and Substance Compositions In one aspect of this disclosure, products and material compositions that can be used for the derivation and culture of bovine primitive stem cells are provided.

[0154] As illustrated herein, bovine primordial stem cells are derived or generated by culturing in a variety of culture medium compositions, including a growth medium (also referred to herein as a “growth medium”, such as “PXGL”, “PXGLY”, “AXRG” and “AXRGL” media as described by Bredenkamp et al. (2019b), a reset medium (also referred herein as a “reset medium”) (e.g., “epigenetic reset” medium) and a maintenance medium (also referred herein as a “maintenance medium”).

[0155] Various culture media may contain, for example, basal media and one or more small molecules, growth factors, and / or nutrients. Technicians can readily determine suitable basal media, which include, but are not limited to, DMEM / F12 (Duborough Modified Eagle Medium F12), advanced DMEM / F12, neural basal media, and mixtures thereof. Technicians can readily determine suitable supplements, which include, but are not limited to, minimum essential medium (MEM), non-essential amino acids (NEAA), L-glutamine (e.g., 1-2 mM), Glutamax, ascorbic acid, insulin, BSA (component V), β-mercaptoethanol, penicillin / streptomycin, and / or gentamicin. In one embodiment, the basal media comprises a 1:1 mixture of DMEM / F12 and neural basal media. In one embodiment, the basal media further comprises 1X Glutamax + 1X NEAA + 1 mg / ml BSA + 50 ug / ml ascorbic acid + 0.1 mM β-mercaptoethanol + 50 IU / ml penicillin / streptomycin.

[0156] The various growth media described in this article include “PXGL”, “PXGLY”, “AXRG”, and “AXRGL”. Commercially available media such as RSeT can also be used.

[0157] As used herein, “PXGL” medium comprises: a basal medium (e.g., DMEM / F12: neural basal medium (1:1 mixture)); a serum substitute component (e.g., N2B27 serum); a MEK / ERK inhibitor component, optionally PD0325901, Ravoxertinib, GSK1120212, MEK162, PD184352, Trametinib, LY3214996, or Ulixertinib, preferably 1 µM PD0325901; and a Wnt inhibitor component, optionally XAV-939, IWR-1, or IWP-2, preferably 2 µM. XAV-939; a PKC inhibitor component, optionally Gö6983, Gö6976, LY317615, LY333531, PKC412, GSK690693, sotrastaurin, staurosporine, or bisindomaleimide, preferably 4 µM Gö6983; and 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 LIF. The “PXGLY” medium also contains a ROCK inhibitor component, optionally Y27632, fasudil, thiazovivin, or blebbistatin, optionally 5-10 µM Y27632. A CEPT mixture containing a ROCK inhibitor component can also be used as a ROCK inhibitor component.

[0158] As used herein, “AXRG” medium comprises: a basal medium (e.g., DMEM / F12: neural basal medium (1:1 mixture)); a serum substitute component (e.g., N2B27 serum); a Wnt inhibitor component, optionally XAV-939, IWR-1, or IWP-2, preferably 2 µM XAV-939; a PKC inhibitor component, optionally Gö6983, Gö6976, LY317615, LY333531, PKC412, GSK690693, sotrastuxin, astrococcus, or bisindomaleimide, preferably 2 µM Gö6983; and one or more RAR inhibitor components, optionally BMS493, BMS-189453, BMS-195614, AGN 193109, AGN193491, AGN 193618, AGN 194202, AGN The medium contains 194301, AGN 194574, Ro 41-5253, ER 50891, ATRA, MM 11253, phenylacetylene analog 6, phenylacetylene analog 7, quinolone derivative analog 15, quinolone derivative analog 16, preferably 1 μM BMS493; and activator A, optionally human activator A, preferably 5 ng / ml human activator A. The “AXRGL” medium also contains 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 LIF.

[0159] In one embodiment, a CDK8 / 19 inhibitor component is added to any one of PXGL, PXGLY, AXRG, and AXRGL. As used herein, the term "CDK8 / 19 inhibitor" refers to an inhibitor of the mediator kinases CDK8 and CDK19. CDK8 / 19 inhibitors are well known in the art. In one embodiment, the CDK19 inhibitor is CCT251545. In other embodiments, the CDK8 / 19 inhibitor is CCT251921, 16-disehydrocorticostatin A (dCA), 15w, MSC2530818, JH-XVI-178, AS2863619, BI-1347, JH-XI-10-02 (D), CDK8 / 19-IN-1, CDK8 / 19-IN-2, Senexin B, Senexin C, Senexin A, BRD6989, corticostatin A, or SEL120-34A.

[0160] In another embodiment, the ROCK inhibitor component is added to any one of PXGL, PXGLY, AXRG, and AXRGL.

[0161] Enhanced growth medium (for pristine culture or resetting) comprises growth medium (e.g., PXGL, PXGLY, AXRG, or AXRGL) and also contains an FGF2 component, optionally human FGF2, bovine FGF2, mouse FGF2, monkey FGF2, porcine FGF2, chicken FGF2, goat FGF2, buffalo FGF2, and recombinant FGF2, optionally at a concentration of about 10 ng / ml FGF2. Optionally or additionally, the enhanced growth medium may also contain a p38 MAPK inhibitor component, optionally SB203580, BIRB796, LY2228820, SB202190, VX-702, Neflamapimod, TAK 715, SB239063, Talmapimod, Pamapimod, PF-07265803, or Losmapimod, preferably 1 µM SB203580.

[0162] This article describes examples of nascent stem cell culture media, including AFX medium, which is similar to that described by Rostovskaya et al. (2019). Nascent stem cell culture media, such as AFX, may contain, for example, a basal medium (e.g., a mixture of DMEM / F12: neural basal medium (1:1), 1X Glutamax and 1X NEAA, and optionally 1 mg / ml BSA, 50 ug / ml ascorbic acid, 0.1 mM β-mercaptoethanol and / or 50 IU / ml penicillin / streptomycin or other suitable antimicrobial agents such as gentamicin); a serum replacement component (e.g., 0.5X N2 supplement + 1X B27 supplement); a basic fibroblast growth factor (bFGF) component, optionally 10-30 ng / ml bFGF, preferably 20 ng / ml bFGF; a TGF-β signaling component, such as TGF-β or activin A component, optionally human activin A, bovine activin A or mouse activin A, optionally at a concentration of 5-50 ng / ml, preferably 20 ng / ml. ml activator A; and a Wnt inhibitor component, optionally XAV-939, IWR-1, or IWP-2, preferably 2 µM XAV-939. In one embodiment, the nascent stem cell culture medium or AFX further contains a ROCK inhibitor component, optionally Y27632, fasudil, Thiazovivin, or Blebbistatin, optionally 5-10 µM Y27632. CEPT mixtures containing a ROCK inhibitor component can also be used as the ROCK inhibitor component. Commercially available nascent stem cell culture media with added Wnt inhibitors, such as mTeSR, TeSR-E8 (Stem Cell Technologies), or Essential 8, can be used as described above.TM Culture medium (Gibco). Essential 6 TM The culture medium (Gibco) can be used as a basal medium.

[0163] This article describes examples of resetting culture media, including “epigenetic resetting” media. Epigenetic reset medium may contain a basal medium (e.g., a 1:1 mixture of DMEM / F12:neural basal medium, 1X Glutamax, 1X NEAA, 50 µg / ml BSA, 50 µg / ml ascorbic acid, 0.1 mM β-mercaptoethanol, and 50 IU / ml penicillin / streptomycin or other suitable antimicrobial agents such as gentamicin); a serum replacement component (0.5X N2 supplement + 1X B27 supplement); a MEK / ERK inhibitor component, optionally PD0325901, Ravoxertinib, GSK1120212, MEK162, PD184352, Trametinib, LY3214996, or Uritinib, preferably 1 µM PD3025901; and 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. LIF; and a histone deacetylase inhibitor (HDAC inhibitor) component, optionally valproic acid (VPA), sodium butyrate, or trichostatin A, preferably 1 mM valproic acid. In one embodiment, the epigenetic reset medium also contains a ROCK inhibitor component, optionally Y27632, fasudil, thiazovivin, or Blebbistatin, optionally 5-10 µM Y27632. A CEPT mixture containing a ROCK inhibitor component can also be used as a ROCK inhibitor component. In one embodiment, the epigenetic reset medium also contains an FGF2 component and / or a p38 MAPK inhibitor component.

[0164] This document describes examples of maintenance media that may contain a growth medium, such as the PXGL, PXGLY, AXRG, or AXRGL media described herein, and also contain a CDK8 / CDK19 inhibitor component, such as about 2.5 nMCCT251545. In one embodiment, an example of a maintenance medium is AXRGL medium that also contains CCT251545.

[0165] Typically, bovine primordial stem cells are cultured and maintained in a medium similar to the medium from which they are derived. For example, if bovine primordial stem cells are derived in “PXGL” (or “PXGLY”), they are typically maintained in PXGL with or without one or more of the following components: a ROCK inhibitor component, an FGF2 component, a p38 MAPK inhibitor component, and / or a CDK8 / 19 inhibitor component (or other desired components). Similarly, if bovine primordial stem cells are derived in “AXRG” (or “AXRGL”), they are typically maintained in AXRG with or without one or more of the following components: a LIF component, an FGF2 component, a p38 MAPK inhibitor component, and / or a CDK8 / 19 inhibitor component (or other desired components).

[0166] It should be understood that each of the PXGL, PXGLY, AXRG, AXRGL, and AXRGL with CDK8 / 19 inhibitor media described herein can be used in both the growth and maintenance phases, with the medium selected based on colony viability and condition. Below are examples of media that can be used as cells progress from the growth phase to the maintenance phase: · PXGLY PXGLY · AXRGL AXRGL · AXRG AXRGL · AXRG AXRGL plus CDK8 / 19 inhibitor.

[0167] It should also be understood that the growth culture medium described in this article can be used to maintain naïve stem cells until a certain passage.

[0168] Any suitable dissociation buffer may be used in the methods described herein, including but not limited to TrypLE, 0.25% trypsin / EDTA, 0.05% trypsin / EDTA and / or Accutase.

[0169] The various protocols described herein can use a variety of buffers or saline solutions, such as phosphate-buffered saline (PBS) or Duchenne phosphate-buffered saline (DPBS), (if specified) free of Ca2+. 2+ Mg 2+ and / or SO4 2- Optional implementations may use any suitable buffer or saline solution (without the specified components), such as Hanks' balanced salt solution (HBSS), Earle's balanced salt solution (EBSS), or normal saline.

[0170] As used herein, the term “physiological pH” means a pH of about 7.1 to about 7.6, optionally about 7.15 to about 7.45, about 7.2 to about 7.4, about 7.25 to about 7.35 or about 7.3.

[0171] As is known in the art, pH is affected by the concentration of carbon dioxide (CO2) in the environment. Typical concentrations used for tissue culture range from about 5% CO2 to about 10% CO2, optionally about 5% CO2 or about 6.8% CO2.

[0172] As used herein, the term "hypoxic conditions" refers to conditions where the oxygen (O2) concentration is lower than the atmospheric oxygen concentration (i.e., lower than about 20.95% oxygen). For example, when cells are incubated under hypoxic conditions, the cells are incubated in a reduced oxygen environment (e.g., about 1% to about 15% oxygen, optionally about 1% to 10% oxygen, or about 1% to about 5% oxygen).

[0173] III. Applications The products, compositions, or kits described herein are also provided for use in supporting the attachment of bovine embryo-derived cells in culture, the growth of cells with ICM-like characteristics, and the production of cell colonies exhibiting characteristics of primitive stem cells and / or cell colonies exhibiting characteristics of non-primitive stem cells, and / or for the derivation of bovine primitive stem cells from bovine embryo-derived growths and from bovine non-primitive stem cells.

[0174] On the other hand, this includes the use of bovine primordial stem cells derived using the methods described herein in breeding programs or genetic improvement projects; or for the expansion of preimplantation embryos with desired genetic characteristics, the derivation of primordial germ cells and / or gametes (including those used in in vitro breeding programs and / or transplantation into surrogates), and / or for the development and delivery of veterinary medical biopharmaceuticals and therapeutics. As used herein, references to “breeding programs or genetic improvement projects” include in vivo programs in which gametes and progeny originate from and / or are generated from animals, in vitro programs in which at least one generation of gametes and ‘progeny’ are generated in vitro, and combinations thereof.

[0175] The following non-limiting embodiments are illustrative of this application: Example Example 1. Immunosurgery (based on the method described in Solter et al., 1975) improved on day 7 embryos Derivation of the original state background When intact day 7 embryos were used for growth establishment, compared with growth establishment from day 5 embryos, delayed attachment of transferred clusters, lower ICM growth rate, and higher TE proliferation were observed (Table 1). Therefore, there is a need to develop protocols that produce superior ICM growth establishment rates and quality.

[0176] Table 1: Attachment and proliferation rates during embryonic development in a feeder-free system.

[0177]

[0178] The immunosurgical treatment plan is as follows: 1. Preparation of a 25% anti-bovine serum antibody solution and a 25% complement serum (from guinea pig) diluted in an embryo manipulation medium, wherein the embryo manipulation medium contains 1) Ca 2+ Mg 2+ and SO4 2- 1) Standard HEPES buffered medium; 2) Ca-free 2+ Mg 2+ or SO4 2- 3) DPBS; or neural basal medium.

[0179] 2. Use Figure 1 The layout described herein involves preparing immunosurgical droplets using the solution from step 1.

[0180] 3. Use calcium-free 2+ and Mg 2+ Day 7 embryos washed with DPBS (without ZP) 6 times Figure 1 A).

[0181] 4. Wash away excess calcium-free material from day 7 embryos that do not contain ZP with 25% anti-bovine serum antibody solution. 2+ and Mg 2+ The embryos were incubated twice with DPBS, and then at 38.5 °C in a humidified 5% CO2 incubator, the ZP-free embryos were incubated in 25% anti-bovine serum antibody solution for 1 hour. Figure 1 B).

[0182] 5. After incubation for 1 hour, wash the embryos 3 times with embryo manipulation medium, and then transfer the embryos to 25% guinea pig complement serum solution.

[0183] 6. Wash the embryos twice with 25% guinea pig complement serum solution, then incubate the ZP-free embryos in 25% complement serum solution for 1 hour at 38.5°C in a humidified 5% CO2 incubator. Figure 1 C).

[0184] 7. After incubation for 1 hour, remove the digested trophoblast cells / debris by mechanical pipetting.

[0185] 8. After mechanically removing the trophoblast cells, wash the ICM three times with embryo manipulation medium.

[0186] 9. Inoculate the embryos into growth culture droplets containing t2iLGöY medium.

[0187] result Day 7 embryo ( Figure 2 A) Collapses approximately 20 to 30 minutes after exposure to complement serum. Figure 2 B). Subsequently, the digested trophoblast cells are easily removed by mechanical pipetting. Figure 2 C). Following immunosurgical treatment and subsequent pipetting, pure ICM was successfully obtained. Figure 3 As shown, SOX2 (ICM marker) positive staining and CDX2 (TE marker) positive staining were observed after immunosurgical treatment. Figure 3 (Top right image) Unstained. Results also showed that this protocol did not affect cell viability, as demonstrated by staining in the absence of annexin V or propidium iodide (PI). Figure 3 (See the image below).

[0188] Protein coagulation is frequently observed when anti-bovine serum antibodies and complement serum from guinea pigs are diluted in solutions containing calcium sulfate or magnesium sulfate. Figure 2 D, dashed circle). Besides coagulation, the time required to complete TE cell digestion is highly variable between embryos, ranging from 0.5 hours to 2 hours. Without being bound by theory, it is speculated that the reduced protein utilization in the culture medium due to coagulation is partly responsible for the lower efficiency of the complement-induced immune response. No protein coagulation was observed under conditions without calcium sulfate or magnesium sulfate. Figure 2 E;DPBS, Figure 2 F; Neural Basis Medium, which also showed higher immunosurgical processing efficiency, as observed by a reduction in the time required to complete the digestion of trophectoderm cells. Furthermore, the time required to digest TE cells varied less when using a MgSO4-free medium, meaning this step of the protocol is now completed within 30 to 60 minutes. Therefore, the protocol optionally uses Neural Basis Medium as the basis for diluting anti-bovine serum antibodies and complement serum, as well as for several washes.

[0189] An optimized immunosurgical treatment protocol, using a feeder-free system and t2iLGöY medium, significantly increased the proportion of transfer clusters exhibiting ICM growth (Table 2). This improvement can be partly explained by the lower TE growth expression compared to when immunosurgical treatment was not used.

[0190] Table 2: Attachment and proliferation rates with and without immunosurgical treatment.

[0191]

[0192] Example 2. Derivation of a growth containing primitive stem cells on feeder cells background When using other methods developed for deriving human embryonic stem cells, bovine ICM colonies can be derived with an efficiency of approximately 50%; however, almost all of the grown-outs do not exhibit characteristics of the primitive state after the first passage. It is noted that differentiation always tends towards the endoderm lineage, such as... Figure 5 As shown in Figure A. Reubinoff et al. (2000) previously reported that suboptimal culture conditions for generating ES cells from human blastocysts often lead to cell differentiation, and that using mouse embryonic fibroblasts (MEF) as a feeder cell layer can overcome this deficiency. The feeder layer system is a classic approach that not only provides effective cell attachment for embryonic cells but also provides several soluble factors to support the cells (Eiselleova et al., 2008). The culture conditions for bovine primordial stem cells remain unclear, therefore feeder cells may be advantageous in overcoming suboptimal culture conditions, particularly for the initiation of primordial stem cell culture. Furthermore, to improve the quality, yield, and biosafety of primordial stem cell colonies, the use of allogeneic systems, such as feeder cells derived from bovine endometrial stroma or bovine fetal fibroblasts, may be preferred for culturing bovine primordial stem cells. Once fully established, naïve stem cell lines can be converted to a feeder-free system to partially simplify workflows for downstream applications, such as the expansion of preimplantation embryos with desired genetic characteristics, the generation of induced blastocysts (a non-cloning method for bovine embryo replication, U.S. Provisional Patent Application No. 63 / 456,624, filed April 3, 2023), the generation and maintenance of cells more tolerant to genetic manipulation, the derivation and replication of germ cells, and the robust generation of somatic tissues for regenerative medicine. The transition to a feeder-free system is also expected to minimize the risk of cross-species contamination.

[0193] plan Mitomycin C treatment can be used to generate mitotically inactivated mouse embryonic fibroblast feeder cells, as described, for example, in Jozefczuk et al. (2012). Alternatively, gamma irradiation can be used to generate mitotically inactivated feeder cells, as described, for example, in Jiang et al. (2016). Other suitable feeder cells may include alcohol-fixed cells prepared as described, for example, in Xu et al. (2022) and / or Ren et al. (2022).

[0194] Preparation of feeder cell layer: 1. Prepare a 0.1% gelatin coating solution.

[0195] 2. Apply 25 µl of coating solution droplets onto the surface of a 35 mm cell culture dish to generate microdroplets.

[0196] 3. Incubate the petri dish at 38.5°C in a humidified 5% CO2 incubator for 1 hour.

[0197] 4. Remove the coating solution and optionally rinse the surface gently.

[0198] 5. Apply 20 µl of fresh MEF medium to the coated surface.

[0199] 6. Apply a mineral oil coating to the coated surface and MEF medium.

[0200] 7. Thaw and inactivate the MEF according to the standard procedure.

[0201] 8. Disperse the cell pellet with 1 ml of fresh MEF medium.

[0202] 9. Take 15 µl of cell suspension and mix it with 15 µl of 0.4% trypan blue solution for live / dead cell counting.

[0203] 10. Use a blood cell counter to count the number of clear (live) cells.

[0204] 11. Dilute the cells to 5 x 10⁻⁶. 5 The concentration was set at 1 x 10⁻⁶ ml, and 1 x 10⁻⁶ ml was used. 4 (20 µl) was inoculated into gelatin-coated droplets containing 40 µl of fresh MEF medium (total 60 µl of medium).

[0205] 12. Prepare the feeder layer at least 18 hours before the derivation step.

[0206] result Feeder cell density is a factor in the success of feeder cell systems (Heng et al., 2004). When using unique droplet culture systems to generate growths (including bovine growths), it is essential to validate the optimal feeder cell density before generating the growths. This is because MEF cells tend to form a higher density of cells in the center of the droplet. The growth area of ​​the droplets used in this study was approximately 0.36 cm². 2 This corresponds to 30 + 30 µL. To achieve this, first add 20-30 µL, followed by a mineral oil or paraffin oil capping layer, and then add another 30-40 µL to the original droplets to obtain a final volume of approximately 60 µL. The goal is to produce “higher” droplets, reducing contact with the culture dish. This represents the optimal droplet volume for growth establishment. Experiments have been conducted from 0.5 x 10⁻⁶ droplets. 4 1 cell / drop to 2.5 x 10 4 Several different MEF concentrations in the range of cells / droplet ( Figure 4 The ideal feeder cell density is 1.0 x 10⁻⁶. 4 Cells / drop

[0207] When using culture wells, the ideal feeder cell density is 1.5 x 10⁻⁶. 4 / cm 2 Up to 7.5 x 10 4 / cm 2 Optional 3.0 x 10 4 / cm 2 For example, for a typical 12-well plate (growth area: 4 cm²) 2 ), 1.2 x 10 5 / pores represent the ideal cell density.

[0208] When MEF was used as the feeder layer, embryo attachment rate reached 100%. However, TE growth in the colony increased significantly while ICM growth rate decreased considerably (Table 3). In other words, attached ICMs rapidly differentiated into TEs, such as... Figure 5 As shown in B and C, soluble factors secreted by feeder cells facilitate attachment but also limit ICM growth.

[0209] Table 3: Attachment and proliferation rates via MEF.

[0210]

[0211] Example 3. Optimization of culture medium using MEF background Preliminary results indicate that the culture conditions optimized for the feeder-free system (i.e., t2iLGöY) were not optimized for the feeder system. It is well known that feeder cells secrete several soluble factors (Eiselleova et al., 2008; Talbot et al., 2012; Valenta et al., 2016), therefore, the pristine culture medium without a feeder layer may not be suitable for feeder systems. Therefore, we tested the compatibility of two different pristine stem cell culture media in feeder cell conditions and MEF medium.

[0212] plan A. Composition of MEF medium • DMEM / F12 + 10% FBS + 1% NEAA + 1% Glutamax + 50 IU / ml Pen / Strep + 50 µM β-mercaptoethanol.

[0213] B. Composition of PXGL medium • DMEM / F12: Neural basal medium (1:1 mixture) + N2B27 serum + 1 µM PD0325901 + 2 µM XAV-939 + 4 µM Gö6983 + 10 ng / ml LIF.

[0214] Composition of C. t2iLGö medium • DMEM / F12: Neural basal medium (1:1 mixture) + N2B27 serum + 1µM CHIR99021 + 1 µMPD0325901 + 4 µM Gö6983 + 10 ng / ml LIF.

[0215] result In the feederless system (layer-by-layer system, LbL), PXGL medium showed significantly lower ICM growth compared to growth using t2iLGöY medium. Figure 6 However, when using feeder cells, PXGL medium showed improved ICM growth rate, suggesting that PXGL medium may be superior to t2iLGöY medium when using a feeder system.

[0216] Maintaining healthy MEFs is extremely important in any program using feeder cells. However, it has been noted that when MEF cells are directly exposed to PXGL medium (i.e., abruptly changing from MEF medium to pristine medium), the MEF cells exhibit an enlarged or elongated morphology. Figure 7 To prevent this morphological change, a stepwise adaptation from MEF conditions to pristine cell culture conditions was tested. In short, 18 hours before derivatization or passage, the MEF medium was replaced with a 1:1 MEF:PXGL mixture, followed by 100% PXGL medium. MEF directly exposed to PXGL medium showed enlarged morphology after 48 hours, while MEF treated with stepwise medium adaptation maintained the same morphology as MEF cultured in MEF medium for up to 72 hours. Figure 7 ).

[0217] Example 4. Dissociation background Although cells from early embryos and ICMs may be expected to have single-cell colony-forming capacity, previous methods have involved culturing intact embryos and ICMs without any dissociation process to minimize damage to embryo-derived cells used for primordial colony derivation. Takahashi et al. (2022) recently reported that manipulating stem cells in a dissociation agent rather than a culture medium during passage reduced DNA damage and apoptosis. Furthermore, these researchers reported that prolonged exposure to TrypLE (up to 1 hour) did not affect cell viability. Therefore, to increase the chances of obtaining multiple colonies from the same embryo, it was decided to test and compare methods of culturing intact ICMs that dissociate into small cell clusters versus allowing only a single colony to form from a single ICM.

[0218] plan A. ICM dissociation 1. For example Figure 8 The culture dish shown is for preparing dissociation.

[0219] 2. After immunosurgical treatment, the ICM was transferred to the first droplet in the dissociation culture dish and washed by moving it to the second and third droplets.

[0220] 3. Incubate at 38.5 °C in TrypLE (the third droplet) for 10 minutes.

[0221] 4. Gently blow with a microcapillary syringe to separate ICMs into single cells or small clusters.

[0222] 5. Transfer the cells to the 4th droplet, and then carefully transfer them to the 5th droplet to completely remove the TrypLE solution.

[0223] 6. Seed the cell clusters onto the prepared MEF.

[0224] B. The microcapillaries used for ICM dissociation are prepared as follows: 1. Use a micropipette puller to pull microcapillaries.

[0225] 2. Use a microcontroller to cut microcapillaries at an outer diameter of 40-50 µm.

[0226] 3. Reduce the inner diameter of the microcapillary to 10-15 µm (for single-cell dissociation) or 20-35 µm (for cell cluster dissociation), and fire polish the tip.

[0227] 4. Cut the microcapillaries in the coarsest region to obtain a final length of 2-3 cm.

[0228] 5. Tightly assemble the microcapillary with the micro-injector.

[0229] result The intracellular matrix (ICM) of bovine day 7 embryos contains approximately 30 to 40 cells, which can be dissociated into smaller cell clusters after immunosurgical treatment using mild enzymatic treatment and pipetting via a small-bore pipette or microcapillary tube. It is speculated that separating a single ICM into multiple clusters increases the yield of colonies containing cells with ICM-like characteristics (including primordial stem cell-like cells). After incubation in TrypLE for 10 minutes, followed by gentle pipetting via microcapillary tube, 6 to 7 clusters of approximately 4 to 5 cells / cluster were obtained from a single ICM. Figure 9 ).

[0230] When the ICM dissociated into smaller clusters, ICM growth between clusters was improved to some extent: 58% of the cells were dissociated, compared to 42% for the intact ICM. A key result was that dissociating the ICM increased the number of colonies containing cells with ICM-like characteristics during the growth phase. For the intact ICM group, 20 embryos produced 20 attached clusters (100% attachment rate); while for the dissociated ICM group (100% attachment rate), 20 embryos produced approximately 120–140 attached clusters (Table 4 and...). Figure 10 When the entire early embryo is dissected into smaller clusters, a similar increase in the number of colonies containing cells with ICM-like features can be expected.

[0231] Table 4: Attachment and proliferation rates of ICM dissociation (n = 20 embryos per treatment group).

[0232]

[0233] Example 5. Establishing stem cell colonies from whole embryos and embryo-derived growths such as ICM growths. Bovine primordial stem cells, primordial stem cell-like cells, etc., can be passaged using mechanical or enzymatic methods as described below. Typically, mechanical methods are used for the initial 1-2 passages following the aforementioned immunosurgical treatment and ICM or embryonic cell dissociation protocol, and enzymatic methods can be used thereafter. However, appropriate methods can be selected for each single passage, as determined by a person skilled in the art.

[0234] plan A. When using mechanical methods to passage bovine primitive stem cells or primitive stem cell-like cells. 1. PXGLY medium was prepared by adding 10 µM Y27632 to 10 ml PXGL medium.

[0235] 2. Prepare microdroplets with MEF according to the description in "Procedure: Preparation of Feeder Cell Layer".

[0236] 3. 18 hours before passage of primitive stem cells, ICM colonies, etc., replace the MEF medium in the droplets with MEF:PXGL medium (1:1 mixture).

[0237] 4. If the embryo-derived explant culture shows TE growth, mechanically remove it to make the ICM colony accessible.

[0238] 5. The primitive stem cell colonies and ICM colonies were separated into 2 to 6 clusters by mechanical dissociation, and the separated clusters were transferred into new microdroplets containing PXGLY medium.

[0239] 6. After 24 hours, replace the culture medium with PXGL.

[0240] B. When using enzymatic methods to passage bovine primitive stem cells or primitive stem cell-like cells. 1. PXGLY medium was prepared by adding 10 µM Y27632 to 10 ml PXGL medium.

[0241] 2. Prepare microdroplets with MEF according to the description in "Procedure: Preparation of Feeder Cell Layer".

[0242] 3. 18 hours before passage of primitive stem cells, ICM colonies, etc., replace the MEF medium in the droplets with MEF:PXGL medium (1:1 mixture).

[0243] 4. If the embryo-derived growth shows TE growth, mechanically remove it to make the ICM colony accessible.

[0244] 5. Remove the culture medium from the droplets containing primitive stem cells, ICM colonies, etc.

[0245] 6. Use 60 µl of Ca-free... 2 + and Mg 2+ Wash the microdroplets three times with DPBS.

[0246] 7. Add 60 µl TrypLE and incubate for 3 minutes to dissociate ICM.

[0247] 8. ICM colonies are separated into single cells or small cell clusters by gentle blowing.

[0248] 9. Transfer the cells to a 1.5 ml tube and add 500 µl of Ca2+. 2 + and Mg 2+ DPBS.

[0249] 10. Centrifuge at 300 ×g for 5 min.

[0250] 11. Discard the supernatant and gently pipette 1 ml of PXGL medium to break up the precipitate.

[0251] 12. Centrifuge at 300 ×g for 5 min.

[0252] 13. Discard the supernatant and break up the precipitate with PXGLY medium from fresh droplets.

[0253] 14. Inoculate the cells into new droplets.

[0254] 15. After 24 hours, replace the culture medium with PXGL.

[0255] result Colonies containing primordial stem cell-like cells with a diameter of 25–100 µm can be successfully obtained using the following combination of conditions: treatment of day 7 embryos by immunosurgical intervention followed by mild enzymatic ICM dissociation and culture on MEFs adapted to stepwise culture medium. Figure 11 (A, B). Colonies derived using these conditions exhibit clearer and brighter boundaries, denser clumps, and display the distinctive 3D shape typical of human primordial stem cell colonies. Compared to methods for deriving human primordial stem cells for bovine cells, this characteristic primordial state of colonies was observed more frequently using the disclosed methods and feeding conditions. Furthermore, no signs of endoderm differentiation were observed up to the 3rd passage when using the disclosed feeder cell method. The disclosed feeder system and PXGL medium have shown to be a highly effective combination for preventing endoderm differentiation in bovine primordial stem cells.

[0256] The suitability of PXGL for bovine primitive stem cell cultures was tested by comparing results with those for bovine expanded potential stem cells (non-primitive), as described by Zhao et al. (2021). Stem cells were generated as described above or according to the methods described in Zhao, and SUSD2 expression was characterized as described in Example 6, “J. Characterization of bovine primitive stem cells” below. Results were... Figure 12 and 13 As shown in the diagram. Bovine primitive stem cells generated under the PXGL conditions described above exhibit primitive-state-specific characteristics: a typical dome-shaped morphology and SUSD2 expression. (See diagram for reference.) Figure 13 As shown, SUSD2 was expressed in >30% of cells at P3, and its expression was maintained or increased in subsequent P4 and P5 generations. Conversely, bovine expanded pluripotent stem cells generated and maintained under bovine EPSC conditions described by Zhao et al. (2021) showed significantly lower SUSD2 expression compared to bovine naïve stem cells and failed to exhibit the typical dome-shaped colony morphology. Figure 13As shown, SUSD2 is expressed in <10% of cells, and even fewer cells express SUSD2 in subsequent passages.

[0257] Example 6. Recovery of primitive pluripotency from bovine non-primitive growths background To improve the yield, quality, and long-term maintenance of primitive stem cell lines, and to target the emergence of "non-passivated" embryo-derived colonies ("non-primitive" stem cells) that do not exhibit primitive stem cell morphology, Figure 14 -A), a solution was designed. Non-passivated embryo-derived colonies exhibited a flattened shape specific to primordial pluripotency, meaning they could not be used for further passage of primitive cells. Primordial pluripotent stem cells are generally thought to originate from the epiblast after implantation. After 7 days of culture, these “non-passivated” growths showed similarities to day 14 embryos and likely contained epiblast cell populations (Pérez-Gomez et al., 2021). Therefore, it was hypothesized that bovine primordial pluripotent stem cells could originate from growths from days 5 to 7, exhibiting the primordial pluripotent stem cell morphology. If successful, the established primordial pluripotent stem cells could be reset to a primitive stem cell state using a method based on the protocol described by Guo et al. (2017).

[0258] In the current protocol, colonies are selected from embryonic growths on days 5–7, and more specifically from those exhibiting atypical primitive stem cell morphology, but more typically from growths of higher developmental states (e.g., primordial morphology). The selected cells are then subjected to primordial embryonic stem cell conditions (Bogliotti, 2018). After establishing bovine primordial pluripotent stem cell lines, the goal is to restore primitive pluripotency through an optimized “reset protocol.”

[0259] Guo et al. (2017) initially reported a technique called “reset” that uses histone deacetylase (HDAC) inhibitors to reverse primordial state ES cells to a primitive state, and referred to in this paper as reset via epigenetic modification. Furthermore, Bayerl et al. (2021) reported a one-step technique that reverses primordial stem cells to a primitive developmental state by directly replacing the primordial culture medium with the primitive culture medium.

[0260] Because deriving primordial stem cells from embryos is highly challenging (requiring a continuous supply of embryos, extensive embryological knowledge, and specific techniques for embryonic growth derivation), pluripotency resetting (Guo et al., 2017; Brenkenkamp et al., 2019b) offers an alternative and cost-effective method for deriving bovine primordial stem cells. This paper describes an optimized resetting technique for converting bovine primordial pluripotent stem cells into primordial stem cells. The efficiency of the resetting method was assessed using primordial stem cell-specific markers.

[0261] Compared to media used for nascent stem cells (e.g., AFX), bovine naïve stem cell culture medium (PXGL) contains MEK1 / 2 inhibitors, which can induce fibroblast differentiation. MEF cultured in PXGL medium showed differentiation into myofibroblasts, including excessive production of extracellular matrix and the appearance of α-smooth muscle actin. Figure 16 According to fibroblast pathology (Tai et al., 2021), myofibroblasts are considered key cellular mediators of fibrosis. Because primordial stem cells possess more epithelial-like characteristics (Pham et al., 2022), the fibrosis-inducing environment can affect the quality of primordial stem cells. Based on this, the addition of low concentrations of FGF2 or p38 MAPK inhibitors to PXGL medium was tested.

[0262] plan A. Composition of bovine primordial stem cell culture medium (AFX medium) • DMEM / F12: Neural basal medium mixture (1:1) + 0.5X N2 supplement + 1X B27 supplement + 1X Glutamax + 1X NEAA + 1 mg / ml BSA + 50 ug / ml ascorbic acid + 0.1 mM β-mercaptoethanol + 50 IU / ml penicillin / streptomycin + 20 ng / ml bFGF + 20 ng / ml activin A + 2 µM XAV-939.

[0263] B. Isolation of bovine primordial pluripotent stem cells 1. Bovine embryonic growths derived under primitive stem cell conditions.

[0264] 2. On days 5 to 7, select the growths that exhibit the initial stage morphology. Figure 14 -A), and mechanically remove TE cell colonies to expose the ICM of the colonies.

[0265] 3. Mechanically remove feeder cells around ICM colonies by gently scraping with the tip of a pipette.

[0266] 4. Use a 125 µm well pipette to scrape the ICM colonies to separate them into small clusters, and transfer the ICM cell clusters to microdroplets onto feeder cells (passage 1) freshly prepared under primordial pluripotent stem cell conditions.

[0267] C. Establishment of bovine primordial pluripotent stem cells 1. Culture the cells from "Step 4" of the "Protocol: Isolation of Bovine Primordial Pluripotent Stem Cells".

[0268] 2. Change the culture medium daily.

[0269] 3. On day 7 after starting culture under nascent pluripotent stem cell conditions, passage the cells using an enzyme-based method, namely “EDTA-subculture” (steps 4 to 9).

[0270] 4. Remove the culture medium from the culture droplet.

[0271] 5. Use 50 µl of Ca-free... 2 + and Mg 2+ Wash the culture medium with DPBS drops 3 times.

[0272] 6. Use 50 µl of Ca-free... 2 + and Mg 2+ Wash the culture medium twice with 0.5 mM EDTA in DPBS.

[0273] 7. Add 50 µl of Ca-free solution. 2 + and Mg 2+ 0.5 mM EDTA in DPBS and incubated at 38.5 °C for 2 minutes.

[0274] 8. Remove the EDTA solution very carefully.

[0275] 9. Add 50 µl of fresh AFX medium directly to the culture surface.

[0276] 10. Transfer the cell clusters to a 600 µl tube.

[0277] 11. Dilute the cell suspension with fresh AFX medium according to the required dilution ratio.

[0278] 12. Note: Add 50 µl of fresh culture medium to this passage to dilute the cells at a ratio of 1:2.

[0279] 13. Seed the cells onto freshly prepared MEF (passage 2) in droplets.

[0280] 14. Note: When passaged, transfer 50 µl of cell suspension into each droplet.

[0281] 15. Culture the cell culture at 38.5°C in a humidified 5% CO2 incubator.

[0282] 16. Repeat the "EDTA-subculture" process. 17. Transfer the cell clusters from the two droplets into the same 600 µl tube.

[0283] 18. Seed cells into one well of a 12-well plate onto freshly prepared MEF.

[0284] 19. Optional: Growth factor-reduced substrate gels or Geltrex coated plates can be used for feederless culture here.

[0285] 20. Culture the cells at 38.5°C in a humidified 5% CO2 incubator.

[0286] 21. Repeat EDTA-subculture every 3-4 days, starting with a dilution ratio of 1:6 to 1:10, and further dilutions as described herein.

[0287] a. Steps 4-7: 50 µl -> 300 µl b. Step 9: 50 µl -> 1 ml c. Step 10: 600 µl tube -> 15 ml conical tube D. Cryopreservation / thawing of bovine primordial pluripotent stem cells can be performed using standard techniques.

[0288] E. Composition of 2x frozen medium • 80% AFX medium + 20% DMSO (Sigma, D2650) + 10 µM Y27632.

[0289] F. Composition of epigenetic resetting medium • DMEM / F12: Neural basal medium mixture (1:1) + 0.5X N2 supplement + 1X B27 supplement + 1X Glutamax + 1X NEAA + 50 µg / ml BSA + 50 µg / ml ascorbic acid + 0.1 mM β-mercaptoethanol + 50 IU / ml penicillin / streptomycin + 1 µM PD3025901 + 10 ng / ml LIF + 1 mM valproic acid.

[0290] G. Epigenetic Reset 1. In cell culture dishes, use 1 x 10 4 Up to 2 x 10 4 / cm 2Inoculation density for culturing bovine primordial pluripotent stem cells.

[0291] 2. Prepare for reset (steps 3 to 9 below) 3. The culture medium is extracted by gentle aspiration.

[0292] 4. Use 1 ml of Ca-free solution 2+ and Mg 2+ Wash three times with DPBS.

[0293] 5. Use 300 µl of Ca-free... 2+ and Mg 2+ Wash with 0.5 mM EDTA in DPBS. Other implementation methods may use alternative chelating agents, such as EGTA.

[0294] 6. Add 300 µl of Ca-free solution to the well. 2+ and Mg 2+ Add 0.5 mM EDTA to DPBS and incubate at 38.5 °C for 5 minutes.

[0295] 7. Carefully remove the EDTA solution and add 1 ml of fresh AFX medium + 10 µM Y27632 to lift the cell clusters.

[0296] 8. Transfer the cells to a 15 ml conical tube and pipette to separate single cells.

[0297] 9. Count the number of cells and use 1 x 10⁻⁶ cells. 4 Up to 2 x 10 4 / cm 2 Cells were seeded onto freshly prepared MEF and treated with 1 ml AFX medium + 10 µM Y27632.

[0298] 10. Two days after cell plating, change the culture medium to epigenetic reset medium.

[0299] 11. Two days after the first culture medium change (step 10), replace the culture medium with fresh epigenetic reset medium.

[0300] 12. One day after completing step 11, replace the medium with fresh PXGL medium, and replace the medium with fresh PXGL medium daily until day 9.

[0301] H. Direct Reset 1. Prepare for resetting using steps 1 through 9 of the epigenetic resetting protocol.

[0302] 2. Two days after cell plating, replace the culture medium directly with PXGL medium containing or without 10 ng / ml FGF2 and / or p38MAPK inhibitor (1µM SB203580 or 1µM BIRB796).

[0303] 3. Change the culture medium daily until day 5-9.

[0304] I. Establishment of Reset Bovine Primitive Stem Cells 1. The program begins with the last step of each reset scheme (epigenetic and direct reset schemes).

[0305] 2. Perform single-cell dissociation (steps 3-8 below).

[0306] 3. The culture medium is extracted by gentle aspiration.

[0307] 4. Use 1 ml of Ca-free solution 2+ and Mg 2+ Wash three times with DPBS.

[0308] 5. Add 300 µl TrypLE to the well and incubate at 38.5 °C for 3 minutes.

[0309] 6. Perform blowing and dissociation to separate into single cells.

[0310] 7. Transfer the cell suspension to a 15 ml conical tube.

[0311] 8. Wash with DMEM / F12 + 0.1% BSA (or other suitable washing medium, such as one containing Ca). 2+ and Mg 2+ DPBS containing Ca 2+ and Mg 2+ Fill the tube to 10 ml with HBSS, DMEM, DMEM / F12, ADMEM, ADMEM / F12 or neural basal medium.

[0312] 9. Centrifuge at 300 xg for 5 minutes. Discard the supernatant.

[0313] 10. Disperse the precipitate with 1 ml of fresh PXGL medium + 10 µM Y27632. Centrifuge at 300 xg for 5 minutes.

[0314] 11. Discard the supernatant and break up the precipitate with 1 ml of fresh PXGL medium + 10 µM Y27632.

[0315] 12. Count the number of cells using a hematology counter, and use a scale of 1.5 x 10⁻⁶. 4 Up to 2.5 x 104 Reset cells / cm 2 Optional 2 x 10 4 Up to 2.5 x 10 4 Reset cells / cm 2 Inoculate onto freshly prepared MEF and use fresh PXGL medium + 10 µM Y27632. For 12-well plates, use 6 x 10... 4 Up to 10 x 10 4 Cells / well, using 1 ml fresh PXGL medium + 10 µM Y27632. For 35 mm culture dishes, 1.3 x 10⁻⁶ cells / well. 5 Up to 2.2 x 10 5 Cells / plate, using 2 ml of fresh PXGL medium + 10 µM Y27632.

[0316] 13. Change the culture medium daily.

[0317] 14. Subculture the cells every 3-4 days.

[0318] J. Characteristics of bovine primitive stem cells 1. Bovine primitive stem cells can be characterized by live staining of primitive state-specific cell surface markers, including Sushi domain 2 (SUSD2) and primitive state-specific nuclear protein TFCP2L1, as reported by Bredenkamp et al. (2019a).

[0319] 2. Replace the culture medium with fresh PXGL medium containing a working concentration of SUSD2 antibody conjugated with the fluorescent dye.

[0320] 3. Incubate the cells at 38.5°C in a humidified 5% O2 / 6.8% CO2 incubator for 1 hour.

[0321] 4. Rinse the wells 5 times with fresh PXGL medium, and then fill the wells with fresh PXGL medium.

[0322] 5. Observe under a fluorescence microscope.

[0323] result Bovine primordial pluripotent stem cells can be successfully derived from growths exhibiting atypical primitive morphology. Figure 14 Under naïve stem cell culture conditions, some growths on days 6 to 7 showed flattened ICM-like colonies, resembling the typical morphology of naïve pluripotent stem cells. Figure 14 -A). In early passages, flattened ICM-like cells ( Figure 14 -B, black arrow) heterogeneous growth, accompanied by trophoblastic ectoderm and endoderm lineage cells ( Figure 5 A, black arrow). Homogeneous pluripotent cell colonies begin to appear in the 4th to 5th generations. Figure 14 -C, black arrow). Homogeneous primordial pluripotent cell morphology was observed after the 6th generation. Figure 14 -D).

[0324] Bovine primordial pluripotent stem cells cultured on feeder cells can be converted into a feeder-free system. Figure 14 -E). When grown on a feeder-free system, bovine primordial pluripotent stem cells grow in a proliferating monolayer pattern; however, when grown on feeder cells, the cells again form distinct colonies ( Figure 14 -F).

[0325] Bovine primordial pluripotent stem cells were successfully recovered from cryopreserved samples. Figure 15 Small primordial pluripotent cell colonies ( Figure 15 (The black arrow) is visible 48 hours after thawing and forms distinct colonies 96 hours after thawing.

[0326] The resetting protocol induced significant morphological changes in primordial pluripotent stem cells during and after the process. Figure 17 and 18 The direct reset protocol produced significant changes in cell morphology within 24 hours. Figure 17 (See above image). After 2-3 passages following the reset process, directly reset cells exhibited small, dense, primitive-like colonies. Figure 17 (See figure below). Cells treated with the epigenetic reset protocol showed slower morphological changes during the 8-10 day period of the reset protocol. Figure 18 (See above image). Morphological changes begin in the center of the colony, accompanied by intense cell death, followed by slow growth of the colony during epigenetic resetting. After 2-3 passages, epigenetically reset cells exhibit small, dense, primitive-like colonies. Overall, reset cells from both protocols show similar morphological characteristics, but epigenetic resetting displays brighter and more characteristic 3D-shaped primitive colonies.

[0327] After adding FGF2 or p38 MAPK inhibitors, the reset cells exhibited denser and smoother primitive-like colonies. Figure 19 Furthermore, it can be maintained under more stable conditions, such as greater stability in lifespan, proliferation rate, and cell death. In addition, the addition of FGF2 or p38 MAPK inhibitors promoted improvements in feeder quality, which was negatively impacted by MEK 1 / 2 inhibitors in PXGL medium. MEF cultured in enhanced growth medium exhibited a spindle-shaped morphology and significantly reduced ECM secretion. Figure 16 ).

[0328] Regenerated bovine stem cells express well-characterized primitive stem cell-specific markers (Bredenkamp et al., 2019a) Figure 21 Dense and bright bull reset colonies show a positive SUSD2 signal. Figure 21 A), while flattened colonies (typical primordial pluripotent stem cell colonies) did not show any SUSD2 signal. Figure 21 -A, dashed circle). All pluripotent stem cell colonies expressed the pluripotency marker SOX2, but flattened colonies from early passages after repotting did not express TFCP2L1, a primitive-state specific nuclear protein. Figure 21 -B). Based on the expression patterns of primitive stem cell-specific markers, regenerated bovine stem cells exhibited significant characteristics of primitive stem cells.

[0329] Methods for establishing bovine primitive stem cells with high derivatization efficiency, yield, quality, and long-term maintenance, along with selected examples of industrial applications. Figure 22 Described in the text.

[0330] Example 7. Establishment of stem cell colonies from embryo-derived, for example, ICM growths using AXRG / AXRGL medium. plan A. Stem cell colonies can be established and maintained by using AXGR or AXGRL medium instead of PXGL medium, according to the method described in Example 5.

[0331] • AXRG composition: DMEM / F12: neural basal medium (1:1 mixture) + N2B27 serum + 5 ng / ml activin A + 2 µM XAV-939 + 1 µM BMS493 + 2 µM Gö6983 • AXRGL composition: AXRG + 10 ng / ml LIF result Bovine primitive stem cells can be derived and cultured using AXRG medium. Figure 23 After the 8th or 9th generation, although SUSD2 and SOX2 maintain high and consistent expression levels, colonies may become flattened and begin to lose their dome-shaped morphology. Figure 24 Flattened colonies may be an indicator of early differentiation; however, the addition of bovine LIF (AXRGL) to AXRG medium prevented this morphological change. Figure 25 In early passages after establishment, SUSD2 expression levels in bovine naïve stem cells in AXRG medium exceeded 95%, and this high expression level was maintained in multiple passages in both AXRG and AXRGL cultured cells. Figure 24 When the cattle LIF is removed, the settlement loses its dome-shaped form again. Figure 26 Using AXRGL medium or AXRG medium followed by AXRGL, bovine primitive stem cell lines can be derived and remain stable over more than 20 passages, exhibiting high expression levels of primitive and pluripotent markers. Figure 24 ).

[0332] Example 8. Cryopreservation of bovine primitive stem cells plan A. Cryopreservation / thawing of bovine primitive stem cells can be performed using standard techniques.

[0333] • Composition of 2x frozen culture medium: 80% virgin stem cell culture medium + 20% DMSO (Sigma, D2650) + 10 µM Y27632 • The culture medium for primitive stem cells can be PXGL, AXRG, or AXRGL.

[0334] result Frozen bovine primordial stem cells established in AXRG and PXGL media fully recovered within 2 days after thawing. Figure 27 The cells exhibited a doubling time of 22–24 h, comparable to the proliferation observed in unfrozen cells. The viability of blast-formed stem cells after thawing was 88.11% and 79.35% in AXRG (P8) and PXGL (P4) media, respectively. Figure 28 ).

[0335] Example 9. Optimizing the Replication and Self-Renewal Rate of Stem Cell Line Colonies As shown in Example 7, bovine primitive stem cells can be successfully established under AXRG and AXRGL conditions. After approximately 12 passages, the selected bovine primitive stem cell lines have shown longer population doubling times, accompanied by degradation of primitive-specific colony morphology.

[0336] For some bovine primitive stem cell lines, population doubling time may become more variable and / or increase during long-term culture (e.g., beyond p12 or p14). To reduce excessive variability during long-term culture, the roles of various factors and their pathways have been investigated, and, as demonstrated below, factors known to inhibit CDK8 and CDK19 kinases (CDK8 / 19) have shown to be beneficial.

[0337] plan For the long-term culture and self-renewal of both susceptible and unsustainable bovine primordial stem cell lines, the following steps can be used. Cell lines can be established and maintained in AXRGL or other suitable growth media described herein, as described in Example 7. Most notably, susceptible cell lines can benefit when the medium used for growth and / or maintenance also contains a CDK8 / 19 inhibitor component (e.g., 2.5 nM CCT251545). A. When passaged using mechanical methods, bovine primordial stem cells or primordial stem cell-like cells, the following protocol can be used: 1. Maintenance medium was prepared by adding 2.5 nM CCT251545 to 10 ml AXRGL medium.

[0338] 2. Prepare microdroplets with MEF according to the description in "Procedure: Preparation of Feeder Cell Layer".

[0339] 3. 18 hours before passage of primitive stem cells, embryo-derived colonies, ICM colonies, etc., replace the MEF medium in the droplets with MEF:maintenance medium (1:1 mixture).

[0340] 4. If the growth shows TE growth, mechanically remove it to make the ICM colony accessible.

[0341] 5. The ICM colonies were separated into 2 to 6 clusters by mechanical dissociation, and the separated clusters were transferred into new droplets with maintenance culture medium.

[0342] 6. After 24 hours, replace the culture medium with fresh maintenance medium.

[0343] 7. Cryopreservation / thawing of bovine primitive stem cells can be performed using standard techniques.

[0344] B. When using enzymatic methods to passage bovine primitive stem cells or primitive stem cell-like cells.

[0345] 1. Maintenance medium was prepared by adding 2.5 nM CCT251545 to 10 ml AXRGL medium. The ROCK inhibitor (10 µM Y27632) was then added to the prepared maintenance medium.

[0346] 2. Prepare microdroplets with MEF according to the description in "Procedure: Preparation of Feeder Cell Layer".

[0347] 3. 18 hours before passage of primitive stem cells, embryo-derived colonies, ICM colonies, etc., replace the MEF medium in the droplets with MEF:maintenance medium (1:1 mixture).

[0348] 4. If the growth shows TE growth, mechanically remove it to make the ICM colony accessible.

[0349] 5. Remove the culture medium from the culture droplet.

[0350] 6. Wash the culture medium three times with 60 µl of DPBS free of Ca2+ and Mg2+.

[0351] 7. Add 60 µl TrypLE and incubate for 3 minutes.

[0352] 8. ICM colonies are separated into single cells or small cell clusters by gentle blowing.

[0353] 9. Transfer the cells to a 1.5 ml tube and add 500 µl of DPBS containing Ca2+ and Mg2+.

[0354] 10. Centrifuge at 300 ×g for 5 min.

[0355] 11. Discard the supernatant and gently agitate the precipitate with 1 ml of maintenance culture medium.

[0356] 12. Centrifuge at 300 ×g for 5 min.

[0357] 13. Discard the supernatant and break up the precipitate with maintenance medium from fresh droplets.

[0358] 14. Inoculate the cells into new droplets.

[0359] 15. Cryopreservation / thawing of bovine primordial stem cells can be performed using standard techniques.

[0360] result According to the methods described in the above embodiments, stem cell colonies can be established and maintained using a culture medium supplemented with CDK8 and CDK19 kinase inhibitors, such as AXRGL + CCT251545. Preliminary studies have demonstrated that, for these susceptible cell lines, undesirable trends in doubling time and colony morphology are reversed when the culture medium is supplemented with Wnt signaling and CDK8 / 19 inhibitors (CCT251545). However, the concentrations of Wnt signaling and CDK8 / 19 inhibitors are important for maintaining pristine, specific colony morphologies.

[0361] To evaluate the effects of different levels of CCT251545 on the morphology of bovine naïve stem cell lines, cell lines were established in AXRGL as described in Example 7, and for p8 to p12, subsamples were maintained in control AXRGL and AXRGL supplemented with one of six different levels of CCT251545. Figure 29As shown in (A), bovine primordial stem cell colonies exhibited less defined colony shapes with irregular and indistinct boundaries when the culture medium contained 5 nM or higher levels of CCT251545. Furthermore, at higher concentrations of CCT251545, the colonies displayed asymmetrical shapes, contrasting with the characteristic spherical dome-shaped formation of primordial colonies. On the other hand, when maintained at AXRGL + 2.5 nM CCT251545, the colonies exhibited morphological characteristics comparable to those of subsamples maintained to p12 under AXRGL control conditions. Figure 29 (A)).

[0362] For subsamples of colonies cultured in AXRGL + CCT251545 medium, the doubling time decreased and, by p15–p18, was equivalent to the doubling time of all treatments at p10–p12. Figure 29 (B)). For the control group without CCT251545, the doubling time remained significantly higher than before p13. Furthermore, bovine primordial stem cell colonies under AXRGL + CCT251545 conditions maintained primordial-specific colony morphology and had a higher passage number, contrary to the characteristic morphological degradation exhibited by susceptible colonies undergoing increased doubling time under control conditions. Figure 29 (C)). Similar results were observed in the control group and the AXRGL + CCT251545 treatment group when using frozen-thawed stem cell lines.

[0363] References:

[0364]

[0365]

Claims

1. A method for deriving bovine primordial stem cell populations from bovine embryos, the method comprising: a) Provide a feeder cell layer; b) Isolate cells from bovine embryos to obtain a cell population containing bovine primitive stem cell-like cells; c) Optionally, the cell population containing bovine primitive stem cell-like cells is dissociated to obtain one or more cell clusters containing bovine primitive stem cell-like cells; d) Transferring the cell population containing bovine primitive stem cell-like cells or one or more cell clusters containing bovine primitive stem cell-like cells to the feeder cell layer; and e) Culture the cell population containing bovine primordial stem cell-like cells or one or more embryonic-derived cell clusters in the presence of an extrinsic culture medium to induce attachment and growth of one or more colonies containing bovine primordial stem cells, the extrinsic culture medium comprising: i) MEK / ERK inhibitor component, Wnt inhibitor component, PKC inhibitor component, LIF component and optionally ROCK inhibitor component, or ii) Activator A component, Wnt inhibitor component, PKC inhibitor component, RAR inhibitor component, optionally ROCK inhibitor component and optionally LIF component; This led to the acquisition of bovine primitive stem cell populations.

2. The method of claim 1, wherein step b) comprises isolating cells from the inner cell mass (ICM) of a bovine embryo to obtain the cell population containing bovine primordial stem cell-like cells.

3. The method of claim 1, wherein step b) comprises separating cells by removing the zona pellucida (ZP) of the bovine embryo.

4. The method according to any one of claims 1 to 3, wherein the growth medium further comprises a CDK8 / 19 inhibitor.

5. The method according to any one of claims 1 to 4, wherein the density of the feeder cell layer is about 1.5 x 10⁻⁶. 4 / cm 2 Up to 7.5 x 10 4 / cm 2 Preferably about 3.0 x 10 4 / cm 2 .

6. The method according to any one of claims 1 to 4, wherein the feeder cell layer is a feeder cell droplet.

7. The method according to claim 6, wherein the density of the feeder cell layer is about 0.5 × 10⁻⁶. 4 1 cell / drop to 2.5 × 10⁻⁶ 4 1 cell / drop, preferably about 1.0 × 10⁶ 4 Cells / drop 8. The method according to any one of claims 1 to 7, wherein the feeder cell layer is prepared at least about 18 hours or about 24 hours prior to step b).

9. The method according to any one of claims 1 to 8, wherein the feeder cell layer has been adapted to the growth culture medium.

10. The method according to any one of claims 1 to 9, wherein the feeder cells are mitotically inactivated and optionally prepared using mitomycin treatment, γ-irradiation, or alcohol fixation.

11. The method according to any one of claims 1 to 10, wherein the feeder cells are mouse embryonic fibroblasts (MEF), bovine fetal fibroblasts (bFF), bovine endometrial stromal cells, human fetal myocytes, MEF SNL lineage cells, human fetal fibroblasts, human adult fallopian tube epithelial cells, human dermal fibroblasts, human amniotic mesenchymal cells, human amniotic epithelial cells, mouse bone marrow stromal cells, mouse amniotic fluid cells, human amniotic fluid cells, human foreskin fibroblasts, human amniotic mesenchymal cells, pericellular matrix of decidual mesenchymal cells, human endometrial stromal cells, or rat embryonic fibroblasts.

12. The method according to any one of claims 1 to 11, wherein the separation of cells from bovine embryos in step b) comprises immunosurgical treatment.

13. The method of claim 12, wherein the anti-bovine serum antibody is used in the immunosurgical treatment.

14. The method of claim 13, wherein, after immunosurgical treatment, complement serum solution is used for washing and / or incubating cells.

15. The method of claim 14, wherein the complement serum solution is a solution free of calcium sulfate and / or magnesium sulfate.

16. The method according to any one of claims 13 to 15, wherein the anti-bovine serum antibody and / or the complement serum solution are contained in a neural basal culture medium.

17. The method according to any one of claims 12 to 16, wherein the cell population in step c) is dissociated by gentle pipetting, the dissociation optionally being performed with a microcapillary syringe, optionally in the presence of a dissociation reagent, to obtain single cells and / or clusters after immunosurgical treatment, the dissociation reagent optionally being TrypLE.

18. A method for generating bovine primitive stem cells from bovine non-primitive stem cells, the method comprising: a) Provide a cell population containing bovine non-primitive stem cells in a culture environment containing feeder cells in a culture medium containing primordial stem cells; and b) Replace the culture medium with a growth medium for at least 4 days or about 4 days, optionally about 5 to about 9 days; The growth culture medium comprises: i) MEK / ERK inhibitor component, Wnt inhibitor component, PKC inhibitor component, LIF component, and optionally ROCK inhibitor component; or ii) Activator A component, Wnt inhibitor component, PKC inhibitor component, RAR inhibitor component, optionally ROCK inhibitor component and optionally LIF component; This generates bovine primitive stem cells.

19. The method of claim 18, wherein step (b) comprises replacing the culture medium with an epigenetic reset culture medium containing a MEK / ERK inhibitor component, a LIF component, and an HDAC inhibitor component; culturing the cells; replacing the culture medium with a growth medium; and further culturing the cells for at least 4 days or about 4 days, optionally about 9 to about 11 days.

20. The method of claim 19, wherein the cells are cultured for at least 40 hours or about 40 hours, optionally about 2 days to about 4 days, before the culture medium is replaced with a growth medium.

21. The method according to any one of claims 18 to 20, wherein the growth medium further comprises a CDK8 / 19 inhibitor.

22. The method according to any one of claims 18 to 21, wherein the bovine non-primitive stem cell population comprises primordial pluripotent stem cells.

23. The method according to any one of claims 18 to 22, wherein the epigenetic reset medium and / or the growth medium further comprises an FGF2 component and / or a p38 MAPK inhibitor component.

24. The method according to any one of claims 18 to 23, wherein the epigenetic reset medium and / or the growth medium comprises a ROCK inhibitor component.

25. The method according to any one of claims 18 to 24, wherein the feeder cells are mitotically inactivated feeder cells, which are optionally prepared by treatment with mitomycin C, γ-irradiation or alcohol fixation and / or the feeder cells have been adapted to primordial stem cell culture medium prior to step a).

26. The method according to any one of claims 18 to 25, wherein the method further comprises, prior to step b): i. Culture the bovine non-primitive stem cells in the primordial stem cell culture medium for at least 3 days or about 3 days; ii. Dissociate and transfer the bovine non-primitive stem cells into a culture environment containing feeder cells in a culture medium containing primordial stem cells; and iii. Selectively culture the cells for at least 10, 20, 30 or 40 hours or about 10, 20, 30 or 40 hours or about 2 days.

27. The method of claim 26, wherein in step ii), the cells are dissociated in the presence of a dissociation reagent and / or by mechanical dissociation, the dissociation reagent optionally being EDTA.

28. The method according to claim 26 or 27, wherein steps ii) and iii) are repeated at least once, and optionally twice.

29. The method according to any one of claims 18 to 28, wherein the epigenetic reset medium in step b) is replaced with fresh epigenetic reset medium after at least 10, 20, 30 or 40 hours or about 10, 20, 30 or 40 hours, and / or the growth medium in step b) is replaced with fresh growth medium daily.

30. The method according to any one of claims 18 to 29, wherein the cells in step b) are at a density of 1.5 × 10⁻⁶. 4 cells / cm 2 Up to 2.5×10 4 cells / cm 2 Approximately 2×10 (selectable area) 4 cells / cm 2 To approximately 2.5 × 10 4 cells / cm 2 Administer at the required vaccination density.

31. The method according to any one of claims 18 to 30, further comprising c) passage the primitive stem cells into freshly prepared feeder cells in a growth culture medium, optionally wherein the primitive stem cells are at a density of about 1.5 x 10⁻⁶. 4 cells / cm 2 To approximately 2.5 × 10 4 cells / cm 2 Density of vaccination.

32. The method according to any one of claims 18 to 31, wherein the feeder cells are at a density of about 1.5 x 10⁻⁶. 4 cells / cm 2 To approximately 7.5 x 10 4 cells / cm 2 Optional, approximately 3 x 10 4 cells / cm 2 Administer at the required vaccination density.

33. The method according to any one of claims 18 to 32, wherein the bovine non-primitive stem cells are obtained by a method comprising the following steps: i) inducing growth formation according to steps a)-e) of any one of claims 1 to 17, wherein step e) results in the attachment and growth of one or more colonies containing bovine non-primitive stem cells; ii) separating the colonies containing bovine non-primitive stem cells to obtain a bovine non-primitive stem cell population; iii) transferring the bovine non-primitive stem cells to a culture environment containing feeder cells in a culture medium containing primordial stem cells; and iv) culturing the bovine non-primitive stem cells in a culture medium containing primordial stem cells for at least 4 days or about 4 days.

34. The method of claim 33, wherein steps iii) and iv) are repeated at least once more, and optionally twice more.

35. A method for maintaining a bovine primitive stem cell population, the method comprising: a) Provide a feeder cell layer; b) Provide a cell population containing bovine primitive stem cells; c) Optionally, the cell population containing bovine primitive stem cells is dissociated to obtain one or more cell clusters containing bovine primitive stem cells; d) Transferring the cell population containing bovine primitive stem cells or one or more cell clusters containing bovine primitive stem cells to the feeder cell layer; and e) Culturing the cell population or one or more clusters of bovine primordial stem cells in the presence of a maintenance medium to induce the attachment and growth of one or more colonies containing bovine primordial stem cells, wherein the maintenance medium comprises: i) MEK / ERK inhibitor component, Wnt inhibitor component, PKC inhibitor component, LIF component, and optionally ROCK inhibitor component; or ii) Activator A component, Wnt inhibitor component, PKC inhibitor component, RAR inhibitor component, optionally ROCK inhibitor component and optionally LIF component; This helps maintain the bovine primitive stem cell population.

36. The method of claim 35, wherein the maintenance medium further comprises a CDK8 / 19 inhibitor.

37. The method according to claim 35 or 36, wherein the density of the feeder cell layer is about 1.5 x 10⁻⁶. 4 / cm 2 Up to 7.5 x 10 4 / cm 2 Preferably about 3.0 x 10 4 / cm 2 .

38. The method according to claim 35 or 36, wherein the feeder cell layer is a feeder cell droplet.

39. The method of claim 38, wherein the density of the feeder cell layer is about 0.5 × 10⁻⁶. 4 1 cell / drop to 2.5 × 10⁻⁶ 4 1 cell / drop, preferably about 1.0 × 10⁻⁶ 4 Cells / drop 40. The method according to any one of claims 35 to 39, wherein the feeder cell layer is prepared at least about 18 hours or about 24 hours prior to step b).

41. The method according to any one of claims 35 to 40, wherein the feeder cell layer has been adapted to the maintenance culture medium.

42. The method according to any one of claims 35 to 41, wherein the feeder cells are mitotically inactivated and optionally prepared using mitomycin treatment, γ-irradiation, or alcohol fixation.

43. The method according to any one of claims 35 to 42, wherein the feeder cells are mouse embryonic fibroblasts (MEF), bovine fetal fibroblasts (bFF), bovine endometrial stromal cells, human fetal myocytes, MEF SNL lineage cells, human fetal fibroblasts, human adult fallopian tube epithelial cells, human dermal fibroblasts, human amniotic mesenchymal cells, human amniotic epithelial cells, mouse bone marrow stromal cells, mouse amniotic fluid cells, human amniotic fluid cells, human foreskin fibroblasts, human amniotic mesenchymal cells, pericellular matrix of decidual mesenchymal cells, human endometrial stromal cells, or rat embryonic fibroblasts.

44. A bovine primitive stem cell, said bovine primitive stem cell being produced using the method of any one of claims 1 to 43.

45. Use of bovine primitive stem cells according to claim 44 in breeding programs or genetic improvement projects.

46. ​​The use of bovine primordial stem cells according to claim 44, for expanding preimplantation embryos, said preimplantation embryos optionally having desired genetic characteristics; deriving primordial germ cells and / or gametes, said primordial germ cells and / or gametes optionally for in vitro breeding programs and / or transplantation into surrogates; and / or developing and delivering veterinary medical biopharmaceuticals or therapeutics.

47. A method for preparing feeder cell layer microdroplets, the method comprising: a) Bring the growth surface into contact with a certain volume of coating solution; b) Incubate the surface in contact with the coating solution droplets, such that the coating solution layer is deposited on the surface; c) Remove the coating solution and optionally wash the surface; d) Apply a first volume of growth medium onto the coating solution layer deposited on the surface, wherein the first volume of growth medium optionally contains feeder cells; e) Cover the first volume of growth medium with a layer of hydrophobic fluid, wherein the hydrophobic fluid is optionally mineral oil; f) Add a second volume of growth medium to the first volume of growth medium, the second volume of growth medium optionally containing feeder cells, wherein the first volume of growth medium and / or the second volume of growth medium contains feeder cells, thereby preparing microdroplets containing feeder cells. and g) Incubate the droplets containing feeder cells so that the feeder cell layer adheres to the coating solution layer deposited on the surface; This allows for the preparation of feeder cell layer microdroplets.

48. The method of claim 47, wherein the volume of the coating solution is about 20-30 µl, optionally about 25 µl, and / or the coating solution comprises gelatin, optionally about 0.05%-0.15% gelatin, optionally about 0.1% gelatin, and optionally the coating solution layer deposited on the surface has a thickness of about 0.33-0.39 cm. 2 Optional, approximately 0.36 cm 2 The area.

49. The method according to claim 47 or 48, wherein in step b), the surface in contact with the coating solution droplets is optionally incubated in a humidified environment for about 1 hour and / or at about 30-42°C, optionally about 38.5°C.

50. The method according to any one of claims 47 to 49, wherein the first volume of growth medium and / or the second volume of growth medium is about 20-40 µl, optionally about 30 µl, and / or the combination of the first volume of growth medium and the second volume of growth medium is about 50-70 µl, optionally about 60 µl.

51. The method according to any one of claims 47 to 50, wherein the feeder cells are mitotically inactivated and optionally prepared using mitomycin C treatment, γ-irradiation, or alcohol fixation; the feeder cells are MEF cells, bovine fetal fibroblasts (bFF), bovine endometrial stromal cells, human fetal myocytes, MEF SNL lineage cells, human fetal fibroblasts, human adult fallopian tube epithelial cells, human dermal fibroblasts, human amniotic mesenchymal cells, human amniotic epithelial cells, mouse bone marrow stromal cells, mouse amniotic fluid cells, human amniotic fluid cells, human foreskin fibroblasts, human amniotic mesenchymal cells, pericellular matrix of decidual mesenchymal cells, human endometrial stromal cells, or rat embryonic fibroblasts; and / or the number of feeder cells in the droplets is about 0.5 × 10⁻⁶. 4 Up to 2.5×10 4 Cells, optionally approximately 1.0 × 10⁶ 4 Each cell.

52. The method according to any one of claims 47 to 51, wherein in step g), the droplets containing the feeder cells are incubated for at least 10 hours, optionally about 1 day.

53. The method according to any one of claims 1-3, 35 and 36, wherein the feeder cell layer is prepared by any one of claims 47 to 52.

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