Derivation of bovine naïve stem cells using feeder cells

Abstract

The disclosure relates to methods, products and compositions useful for the derivation of bovine naïve stem cells and establishment and maintenance of bovine naïve stem cell lines. In particular, the disclosure relates to methods of deriving bovine naïve stem cells, optionally using said feeder cell layer micro-drops, and methods of generating bovine naïve stem cells from bovine non-naïve stem cells, as well as outgrowth media and resetting media specially formulated for deriving and maintaining bovine naïve stem cells. The bovine naïve stem cells may be used in a breeding program or genetic improvement scheme, for the multiplication of preimplantation embryos with desirable genetic characteristics, deriving primordial germ cells / gametes including for in vitro breeding programs and / or transplantation into surrogates, and developing and delivering veterinary medical biologicals and therapeutics.

Description

TITLE: DERIVATION OF BOVINE NAIVE STEM CELLS USING FEEDER CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of 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] The present disclosure relates to bovine naive stem cells and more specifically to associated methods and compositions that improve the derivation efficiency, yield, establishment, and long-term maintenance of bovine naive stem cells and their use.INTRODUCTION

[0003] Naive stem cells can differentiate into all types of cells in the body, including extraembryonic cells such as trophoblast stem cells and extraembryonic endodermal lineage cells. The derivation of bovine naive stem cells may facilitate a broad array of benefits for animal and veterinary sciences by providing an efficient platform for replicating embryos with desirable characteristics; producing genetically modified animals at scale; delivering important constituent technologies for breeding systems, in vitro gamete derivation and production, and regenerative veterinary medicine; and enabling the development and delivery of advanced veterinary medical biologies and therapeutics. The production of bovine naive stem cells has been reported in a PCT patent application (Derivation of naive bovine stem cells, Application No. PCT / CA2022 / 051664, filed November 11 , 2022) and is expected to be a foundational step for the development of many advanced technologies including those with value for domestic animal breeding systems.

[0004] Derivation and culture conditions including supporting factors which facilitate in vitro cell attachment, proliferation, maintenance of ground-state pluripotency, and self-renewal are important elements contributing to the efficiency and yield of pluripotent naive stem cell colonies. Supporting factors may be produced by stromal cells (directly attached to the dish), commonly known as a feeder cell system, or may comprise a chemically defined protein matrix as a feeder-free system. Methods reported in PCT Application No. PCT / CA2022 / 051664 for naive stem cells and Soto et al. (2021) for non-naive primed pluripotent bovine stem cells aim to minimize risks for cross contamination as these methods use a feeder-free system and employ no feeder cells. Feeder-free culture systems may yield culture results with less variability from batch to batch as the protein matrix and media are chemically defined. The resulting consistency and minimization of risk for cross contamination may be desired in specific instances, such as when deriving, culturing, maintaining, and using naive stem cell lines optionally to distribute germplasm internationally, for the production of genetically modified founder breeding animals or in xeno-free systems for developing and delivering advanced medical and veterinary biologies and therapeutics.

[0005] When xeno-free systems are not required, a mouse embryonic fibroblast (MEF) feeder system, for example, may be used, such as is described by Bogliotti et al. (2018) for non-naTve primed pluripotent bovine stem cells. However, culture conditions for bovine non-naive stem cells (Bogliotti et al., 2018; Han et al., 2011 ; Soto et al., 2021 ; WO 2019 / 140260; Zhao et al. , 2021) and naive stem cells of other species (Bayerl et al., 2021 ; Rostovskaya et al., 2019) have failed to support self-renewal of bovine naive stem cell colonies. In particular, a MEK / ERK inhibitor (for example PD0325901) is thought to be important for naive stem cell culture but induces transformation of fibroblasts toward myofibroblasts which in turn over-secrete extracellular matrix (ECM) and therefore may adversely affect the microenvironment of the naive stem cells.

[0006] Also, allogeneic culture systems such as those employing a bovine endometrial stromal or bovine fetal fibroblast (bFF; Zhao et al., 2021 ) feeder system may provide improved support for bovine naive stem cells while also presenting a more balanced risk profile for cross-species contamination.

[0007] Additionally, greater flexibility when making decisions about the suitability of germplasm for naive stem cell line derivation and maintenance may be beneficial for breeding systems and associated methods which utilize genomic characterization data such as are described by Hou et al. (2018), Mueller and Van Eenennaam (2022), Goszczynski et al. (2019), and Bogliotti et al. (2018), and in PCT Application No. PCT / CA2022 / 051664, and WO2022251549 (PCT / US2022 / 031210). For example, when using reported methods (PCT Application No. PCT / CA2022 / 051664,) naive embryonic stem cell outgrowths can be obtained with good derivation efficiency using either day-5 or day-6 embryos; however, when conducting genomic characterization to assess thesuitability of embryos, derivation from day-7 embryos may be preferred. It has remained challenging to derive high quality naive stem cell colonies from day-7 embryos.

[0008] Offspring with predetermined genomes may be produced using methods described in W02020168422 (PCT Application No. PCT / CA2020 / 050210), however technologies such as this can deliver benefits more widely when self-renewing populations of ground-state pluripotent stem cells may be produced thus enabling the replication of elite singular haploid and diploid embryos. Instances when lowered costs and improved derivation efficiency, yield, quality, and I or long-term maintenance of selfrenewing naive stem cell lines may be extremely beneficial in order to fully realize the potential of the resulting technology include developing of an efficient platform for the replication of embryos, in vitro gamete derivation and production, and producing genetically modified animals at scale. A need, therefore, exists for robust and cost- effective methods of deriving and culturing bovine naive stem cells with improved efficiency, yield, quality, and long-term maintenance and self-renewal of ground-state pluripotency.SUMMARY

[0009] Described herein are methods for deriving a population of bovine naive stem cells from an embryo by inducing embryo-derived cell attachment and outgrowth formation to obtain one or more colonies comprising bovine naive stem cells. Also described herein are feeder cell layer micro-drops and media for use in deriving a population of bovine naive stem cells, and methods for preparing such feeder cell layer micro-drops. The methods of deriving a population of bovine naive stem cells described herein can be used to establish a self-renewing bovine naive stem cell line.

[0010] Accordingly, in one aspect a method of deriving a population of bovine naive stem cells from a bovine embryo, the method comprising: a) providing a feeder cell layer; b) isolating cells of a bovine embryo to obtain a population of cells comprising bovine naive stem cell-like cells; c) optionally dissociating the population of cells comprising bovine naive stem celllike cells to obtain one or more clusters of cells comprising bovine naive stem cell-like cells;d) transferring the population of cells comprising bovine naive stem cell-like cells or the one or more clusters of cells comprising bovine naive stem cell-like cells to the feeder cell layer; and e) culturing the population of cells comprising bovine naive stem cell-like cells or the one or more clusters of embryo-derived cells in the presence of outgrowth medium, the outgrowth medium comprising: i) a MEK / ERK inhibitor component, a Wnt inhibitor component, a PKC inhibitor component, a LIF component, and optionally a ROCK inhibitor component, or ii) an Activin A component, a Wnt inhibitor component, a PKC inhibitor component, a RAR inhibitor component, optionally a ROCK inhibitor component and optionally a LIF component, to induce attachment and outgrowth of one or more colonies comprising bovine naive stem cells; thereby deriving a population of bovine naive stem cells.

[0011] In an embodiment, step (b) comprises isolating cells from the inner cell mass (ICM) of a bovine embryo to obtain a population of cells comprising bovine naive stem cell-like cells.

[0012] In an embodiment, step b) comprises isolating cells by removing the Zona Pellucida (ZP) of the bovine embryo.

[0013] In an embodiment, the outgrowth medium further comprises a CDK8 / 19 inhibitor.

[0014] In an embodiment, the feeder cell layer is at a density of about 1 .5 to 7.5 x 104 / cm2, preferably about 3.0 x 104 / cm2or at a density of about 0.5 x 104to 2.5 x 104cells I drop, preferably about 1 .0 x 104cells I drop.

[0015] In an embodiment, the feeder cell layer is a feeder cell micro-drop.

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

[0017] In an embodiment, the feeder cell layer has been adapted to the outgrowth medium.

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

[0019] In an embodiment, the feeder cells are mouse embryonic fibroblast (MEF) cells, bovine fetal fibroblasts (bFF), bovine endometrial stromal cells, human fetal muscle cells, MEF SNL line cells, human fetal fibroblasts, human adult fallopian tubal epithelial cells, human dermal fibroblasts, human amniotic mesenchymal cells, human amniotic epithelial cells, mouse bone marrow stromal cells, murine amniocytes, human amniocytes, human foreskin fibroblasts, human amniotic mesenchymal cells, pericellular matrix of decidua-derived mesenchymal cells, human endometrial stromal cells, or rat embryo fibroblast cells.

[0020] In an embodiment, the bovine embryo is a Zona Pellucida (ZP)-free bovine embryo.

[0021] In an embodiment, isolating cells from the bovine embryo in step b) comprises immunosurgery. Optionally, an anti-bovine serum antibody is used for the immunosurgery. In an embodiment, isolated ICM is put into a microdrop with derivation media containing ROCK inhibitor

[0022] In an embodiment, complement serum solution is used for washing and / or incubating the cells following the immunosurgery. Optionally, the complement serum solution is a calcium sulfate free and / or magnesium sulfate free solution.

[0023] In an embodiment, the anti-bovine serum antibody and / or the complement serum solution are comprised in a neurobasal medium.

[0024] In an embodiment, the population of cells in step c) is dissociated by gentle pipetting, optionally with a microcapillary syringe, optionally in the presence of a dissociation reagent, optionally TrypLE to obtain single cells and / or small clusters following the immunosurgery. In an embodiment, the dissociation reagent comprises a ROCK inhibitor component.

[0025] Also described herein are methods of generating bovine naive stem cells from bovine non-naive stem cells. Accordingly, an aspect includes a method of generating bovine naive stem cells from bovine non-naive stem cells, the method comprising: a) providing a population of cells comprising bovine non-naive stem cells in a culture environment comprising feeder cells in primed stem cell medium; andb) replacing the medium with outgrowth medium for at least or about 4 days, optionally about 5 to about 9 days; wherein the outgrowth medium comprises: iii) a MEK / ERK inhibitor component, a Wnt inhibitor component, a PKC inhibitor component, a LIF component and optionally a ROCK inhibitor component; or iv) an Activin A component, a Wnt inhibitor component, a PKC inhibitor component, a RAR inhibitor component, optionally a ROCK inhibitor component and optionally a LIF component; thereby generating bovine naive stem cells.

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

[0027] In an embodiment, the cells are cultured prior to replacing the medium with outgrowth medium for at least or about 40 hours, optionally about 2 days to about 4 days.

[0028] In an embodiment, the outgrowth medium further comprises a CDK8 / 19 inhibitor.

[0029] In an embodiment, the population of bovine non-naive stem cells comprises primed pluripotent stem cells.

[0030] In an embodiment, the epigenetic resetting medium and / or the outgrowth medium further comprises an FGF2 component and / or a p38 MAPK inhibitor component.

[0031] In an embodiment, the epigenetic resetting medium and / or the outgrowth medium comprises a ROCK inhibitor component.

[0032] In an embodiment, the feeder cells are mitotically inactivated feeder cells, optionally prepared using mitomycin treatment, gamma irradiation, or alcohol fixation, and / or the feeder cells have been adapted to primed stem cell medium prior to step a).

[0033] In an embodiment, the method further comprises, prior to step b):i. culturing the bovine non-naive stem cells in the primed stem cell medium for at least or about 3 days; ii. dissociating and transferring the bovine non-naive stem cells to a culture environment comprising feeder cells in primed stem cell medium; and iii. culturing the cells, optionally for at least or about 40 hours or about 2 days.

[0034] In an embodiment, the cells are dissociated in step ii) in the presence of a dissolution reagent, optionally EDTA, and / or by mechanical dissociation.

[0035] In an embodiment, steps ii) and iii) are repeated at least one additional time, optionally two additional times.

[0036] In an embodiment, the epigenetic resetting medium in step b) is replaced with fresh epigenetic resetting medium after at least or about 10, 20, 30 or 40 hours, and / or the outgrowth medium in step b) is replaced with fresh outgrowth medium daily.

[0037] In an embodiment, the cells of step b) are seeded at a seeding density of 1 .5 to 2.5 x 104cells / cm2, optionally about 2 x 104to about 2.5 x 104cells / cm2.

[0038] In an embodiment, the method further comprises c) passaging the naive stem cells onto freshly prepared feeder cells in outgrowth medium, optionally wherein the naive stem cells are seeded at a density of about 1 .5 x 104to about 2.5 x 104cells I cm2.

[0039] In an embodiment, feeder cells are seeded at a seeding density of about 1.5 to about 7.5 x 104cells / cm2, optionally about 3 x 104cells / cm2.

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

[0041] In an embodiment, steps iii) and iv) are repeated at least one additional time, optionally two additional times.

[0042] A method of maintaining a population of bovine naive stem cells, the method comprising: a) providing a feeder cell layer; b) providing a population of cells comprising bovine naive stem cells; c) optionally dissociating the population of cells comprising bovine naive stem cells to obtain one or more clusters of cells comprising bovine naive stem cells; d) transferring the population of cells comprising bovine naive stem cells or the one or more clusters of cells comprising bovine naive stem cells to the feeder cell layer; and e) culturing the population of cells comprising bovine naive stem cells or the one or more clusters of bovine naive stem cells in the presence of maintenance medium, the maintenance medium comprising:(i) a MEK / ERK inhibitor component, a Wnt inhibitor component, a PKC inhibitor component, a LIF component, optionally a ROCK inhibitor component; or(ii) an Activin A component, a Wnt inhibitor component, a PKC inhibitor component, a RAR inhibitor component, optionally a ROCK inhibitor component, and optionally a LIF component; to induce attachment and outgrowth of one or more colonies comprising bovine naive stem cells; thereby maintaining a population of bovine naive stem cells.

[0043] In an embodiment, the maintenance medium further comprises a CDK8 / 19 inhibitor

[0044] In an embodiment, the feeder cell layer is at a density of about 1 .5 to 7.5 x 104 / cm2, preferably about 3.0 x 104 / cm2.

[0045] In an embodiment, the feeder cell layer is a feeder cell micro-drop.

[0046] In an embodiment, the feeder cell layer is at a density of about 0.5 x 104to2.5 x 104cells I drop, preferably about 1.0 x 104cells I drop.

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

[0048] In an embodiment, wherein the feeder cell layer has been adapted to the maintenance medium.

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

[0050] In an embodiment, the the feeder cells are mouse embryonic fibroblast (MEF) cells, bovine fetal fibroblasts (bFF), bovine endometrial stromal cells, human fetal muscle cells, MEF SNL line cells, human fetal fibroblasts, human adult fallopian tubal epithelial cells, human dermal fibroblasts, human amniotic mesenchymal cells, human amniotic epithelial cells, mouse bone marrow stromal cells, murine amniocytes, human amniocytes, human foreskin fibroblasts, human amniotic mesenchymal cells, pericellular matrix of decidua-derived mesenchymal cells, human endometrial stromal cells, or rat embryo fibroblast cells.

[0051] In another aspect there is provided a method of preparing a feeder cell layer micro-drop, the method comprising: a) contacting a growth surface with a volume of coating solution; b) incubating the surface in contact with the drop of coating solution such that a layer of coating solution is deposited on the surface; c) removing the coating solution and optionally washing the surface; d) applying a first volume of growth medium, optionally comprising feeder cells, to the layer of coating solution deposited on the surface; e) overlaying a layer of a hydrophobic fluid, optionally mineral oil, over the first volume of growth medium; f) adding a second volume of growth medium, optionally comprising feeder cells, to the first volume of growth medium, wherein the first volume of growth medium and / or the second volume of growth medium comprises feeder cells, thereby preparing a micro-drop comprising feeder cells; and g) incubating the micro-drop comprising feeder cells such that a layer of the feeder cells attaches to the layer of coating solution deposited on the surface; thereby preparing a feeder cell layer micro-drop.

[0052] In an embodiment, the volume of coating solution is about 20-30 pl, optionally about 25 pl, and / or the coating solution comprises gelatin, optionally about 0.05%-0.15% gelatin, optionally about 0.1 % gelatin, optionally the layer of coating solution deposited on the surface has an area of about 0.33-0.39 cm2, optionally about 0.36 cm2.

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

[0054] In an embodiment, the first volume of growth medium and / or second volume of growth medium is about 20-40 pl, optionally about 30 pl, and / or wherein the first volume of growth medium and second volume of growth medium combined is about 50-70 pl, optionally about 60 pl.

[0055] In an embodiment, the feeder cells are mitotically inactivated, optionally prepared using mitomycin treatment, gamma irradiation, or alcohol fixation; the feeder cells are MEF cells, bovine fetal fibroblast (bFF) cells, bovine endometrial stromal cells, human fetal muscle cells, MEF SNL line cells, human fetal fibroblasts, human adult fallopian tubal epithelial cells, human dermal fibroblasts, human amniotic mesenchymal cells, human amniotic epithelial cells, mouse bone marrow stromal cells, murine amniocytes, human amniocytes, human foreskin fibroblasts, human amniotic mesenchymal cells, pericellular matrix of decidua-derived mesenchymal cells, human endometrial stromal cells, or rat embryo fibroblast cells; and / or the number of feeder cells in the micro-drop is about 0.5 x 104to 2.5 x 104cells, optionally about 1 .0 x 104cells.

[0056] In an embodiment, the micro-drop comprising feeder cells in step g) is incubated for at least 10 hours, optionally about 1 day.

[0057] An aspect includes a feeder cell layer micro-drop produced according to the methods described herein.

[0058] An aspect of the disclosure includes use of a feeder cell layer micro-drop described herein for culturing bovine embryo-derived cells, for example isolated ICM cells, optionally to induce bovine ICM outgrowth formation and the proliferation of bovine naive stem cells, and / or for the derivation of bovine naive stem cells from bovine non-naive stem cells.

[0059] In another aspect there is provided a bovine naive stem cell, or population of bovine naive stem cells, derived using the methods described herein. A further aspectincludes use of a bovine naive stem cell derived using the methods described herein in a breeding scheme or genetic improvement program; and for multiplying preimplantation embryos, optionally having desirable genetic characteristics; deriving primordial germ cells and / or gametes including for in vitro breeding programs and / or transplantation into surrogates, and / or developing and delivering veterinary medical biologicals and therapeutics.

[0060] The preceding section is provided by way of example only and is not intended to be limiting on the scope of the present disclosure and appended claims. Additional objects and advantages associated with the compositions and methods of the present disclosure will be appreciated by one of ordinary skill in the art in light of the instant claims, description, and examples. For example, the various aspects and embodiments of the disclosure may be utilized in numerous combinations, all of which are expressly contemplated by the present description. These additional advantages objects and embodiments are expressly included within the scope of the present disclosure. The publications and other materials used herein to illuminate the background of the disclosure, and in particular cases, to provide additional details respecting the practice, are incorporated by reference, and for convenience are listed in the appended reference section.DRAWINGS

[0061] Further objects, features and advantages of the disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the disclosure, in which:

[0062] Fig. 1 shows a schematic of immunosurgery dishes. A: Dish with DPBS drops for washing, B: Dish for anti-bovine serum antibody and embryo manipulation media drops, C: Dish for complement serum and embryo manipulation media drops.

[0063] Fig. 2 shows immunosurgery of day-7 embryos. The morphological change during immunosurgery (A-C) and the optimization of immunosurgery medium (D-F). A: Embryos before immunosurgery, B: Embryos collapsed after complement treatment, C: ICM and partially digested trophectoderm cells (black arrow) after immunosurgery, D: Protein coagulation (black dotted circle) in a standard HEPES buffered medium containing Ca2+, Mg2+, and SO42; and E-F: no protein coagulation in DPBS and Neurobasal medium, respectively.

[0064] Fig. 3 shows immunostaining of ICM after immunosurgery.

[0065] Fig. 4 shows MEF concentration (# cells / drop) optimization with micro-drop culture system. Too low: 0.5 x 104cells / drop; Ideal: 1.0 x 104cells / drop and too dense: 2.5 x 104cells / drop.

[0066] Fig. 5 shows outgrowth on Layer by Layer Feeder Free (A) and MEF (B and C). A: embryo-derived ICM outgrowth and endodermal differentiation (black arrow) on Layer by Layer (LbL), B-C: trophectoderm (TE) differentiation (white arrow) from embryo- derived ICM outgrowth on MEF.

[0067] Fig. 6 shows naive stem cell derivation and proliferation rates for t2iLG6Y and PXGL medium without and with a feeder system.

[0068] Fig. 7 shows optimization of medium change for MEF (Magnification: 40 x). A: MEF in MEF medium showed normal fibroblastic morphology (white arrow), B: MEF exposed directly to PXGL showed enlargement and elongation (black arrow), C: MEF exposed to a stepwise adaptation maintained fibroblastic morphology (white arrow).

[0069] Fig. 8 shows an exemplary schematic of the layout of an ICM dissociation dish.

[0070] Fig. 9 shows dissociated ICM cell clusters. Each cluster contains 4 to 5 ICM cells.

[0071] Fig. 10 shows day-0 outgrowth of entire ICM and dissociated ICM. A: One naive colony (black arrow) at day 0 from entire ICM, B: Multiple colonies (white arrows) at day 0 from dissociated ICM.

[0072] Fig. 11 shows bovine naive stem cells colonies. A-B: Bovine naive stem cell colonies (black arrows) derived from ICM of day-7 embryo without endodermal differentiation or TE growth, C: Bovine naive stem cell colonies (black arrows) at passage 2 (P2), D: Prior art human naive embryonic stem cell colonies (Guo et al., 2017).

[0073] Fig. 12 shows a morphological comparison of bovine naive embryonic stem cells and bovine expanded stem cells (EPSC) with feeder systems. Bovine naive stem cells in PXGL media as described herein (A and B) and bovine EPSC in bovine expanded potential stem cell media as described in Zhao et al., 2021 (C and D).

[0074] Fig. 13 shows expression of naive specific marker SUSD2 (FITC-A) in bovine naive stem cells and bovine expanded potential stem cells. Passages 3 to 5 (P3-P5) bovine naive stem cell with PXGL media showed 32-45% of SUSD2 expression level (upper panel); P3-P5 bovine EPSC showed less than 10% of SUSD2 expression level (lower panel).

[0075] Fig. 14 shows derivation of bovine primed pluripotent stem cell from embryo-derived outgrowths that do not exhibit naive stem cell characteristics. A: Embryo outgrowth with primed ICM colony at day 7. B: Heterogeneous population of primed pluripotent stem cell colonies (black arrows) with extraembryonic cells (white arrowhead) at passage 2. C-D: Homogeneous primed pluripotent stem cell colonies (black arrowhead) at passage 5 and 6, respectively. E: Bovine primed pluripotent stem cells on feeder free culture. F: Colony reformation of bovine primed pluripotent stem cells on the feeder cells after feeder free culture.

[0076] Fig. 15 shows post-thaw recovery of bovine primed pluripotent stem cells.

[0077] Fig. 16 shows the fibrotic change of MEF depending on culture medium type and composition.

[0078] Fig. 17 shows morphological changes during direct resetting and to passage 4 (P4).

[0079] Fig. 18 shows morphological changes of epigenetic resetting and to passage 3 (P3).

[0080] Fig. 19 shows the enhanced morphology of bovine naive stem cells colonies by addition of FGF2 at selected passages.

[0081] Fig. 20 shows the cell membrane damage and the cell viability after various single cell dissociation methods.

[0082] Fig. 21 (A and B) shows characterization of bovine naive stem cells with naive specific markers at passages 2 (P2) and 5 (P5) (scale bar: 50 pm).

[0083] Fig. 22 shows a schematic flow chart for high efficiency bovine naive stem cell establishment and selected examples of industrial uses.

[0084] Fig. 23 shows derivation of bovine naive stem cells using AXRG media and a feeder system. Outgrowth from ICM of day-7 embryo (A). Manually sub-cultured P1 colonies (B). Enzymatically sub-cultured P4 colonies (C). Successfully established bovine naive stem cells at P5 (D).

[0085] Fig. 24 shows population level expression of pluripotent marker (SOX2) and naive marker (SUSD2) in bovine naive stem cell in AXRG and AXRGL media over longterm culture.

[0086] Fig. 25 shows bovine naive stem cells with and without LIF. Bovine naive stem cells in AXRG media which have never been exposed to LIF displayed flat colonies at P9 (black arrow) while cells supplemented with LIF from P7 maintained a dome shape morphology at P9. Bovine naive stem cells in AXRG media at P9 (A). Bovine naive stem cells at P9 in AXRG supplemented with LIF (AXRGL) from P7 (B).

[0087] Fig. 26 shows bovine naive stem cells in AXRGL maintained a dome shape morphology over P15 and lost this morphology following withdrawal of LI F from the media (black arrow). Bovine naive stem cells in AXRGL media at P15 (A). Bovine naive stem cells at P15 following withdrawal of LIF (B). Bovine naive stem cells in AXRG media at P22 (C).

[0088] Fig. 27 shows post-thaw recovery of bovine naive stem cells in AXRG. Day 1 after thawing (A and B) and day 2 after thawing (C and D).

[0089] Fig. 28 shows viability of bovine naive stem cells in AXRG and PXGL after thawing. Bovine naive stem cells exhibited 88.11 % and 79.35% of post-thaw viability in AXRG (P8) and PXGL (P4) media, respectively.

[0090] Fig. 29 shows morphology and self-renewal capacity of bovine naive stem cells when supplemented with a CDK8 / 19 inhibitor. (A) shows morphology exhibited by bovine naive stem cells in AXRGL media and AXRGL supplemented with varying levels of a CDK8 / 19 inhibitor, CCT251545. Bovine naive stem cell colonies subjected to the high concentrations of CCT251545 displayed a more loose colony shape with unclear and irregular borders. Also at higher concentrations, bovine naive stem cell colonies exhibited an asymmetrical shape rather than a spherical dome-shape. Colonies maintained in AXRGL + 2.5 nM CCT251545 showed similar morphological features as the subsample in control AXRGL media. (B) Cells supplemented with 2.5 nM CCT251545 showed significant improvement in self-renewal. When CCT251545 was introduced to bovine naive cells, population doubling time was shortened to levels of earlier passages. (C) Cells supplemented with CCT251545 demonstrated stable population doubling times and no undesirable morphological changes. In contrast, at later passages the control group showed an increasing population doubling time and a loss of naive specific colony shape.DESCRIPTION OF VARIOUS EMBODIMENTS

[0091] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

[0092] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature described herein may be combined with any other feature or features described herein.I. General Definitions

[0093] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0094] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the description. Ranges from any lower limit to any upper limit are contemplated. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the description, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the description.

[0095] It must 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 dictates otherwise.

[0096] All numerical values within the detailed description and the claims herein are modified by “about” or “approximately” the indicated value, and take into account experimental error and variations that would be expected by a person having ordinary skill in the art.

[0097] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e. , elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements specifically identified.

[0098] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of" or, when used in the claims, "consisting of' will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0099] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of” and "consisting essentially of” shall be closed or semi-closed transitional phrases, respectively.

[0100] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements,but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.

[0101] The term “about” as used herein means plus or minus 10%-15%, 5-10%, or optionally about 5% of the number to which reference is being made.

[0102] It should be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0103] It should also be understood that any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure.II. Methods

[0104] Described herein are methods for deriving a population of bovine naive stem cells from a bovine embryo by inducing embryo-derived formation, as well as the derivation of bovine naive stem cells from said embryo-derived outgrowth or from bovine non-naive stem cells. As set out in the Examples, the inventors have demonstrated methods of preparing feeder cell layer micro-drops, and their use in deriving bovine embryo-derived outgrowth formations, as well as methods of deriving bovine naive stem cells therefrom, optionally using said feeder cell layer micro-drops, and methods of generating bovine naive stem cells from bovine non-naive stem cells, as well as specially formulated outgrowth media and resetting media. The feeder cell layer micro-drops and outgrowth media support ex vivo or in vitro attachment and proliferation of embryo-derived cells isolated from bovine embryos, such as morula or blastocyst stage (e.g. bovine day- 6 or day-7) embryos, as well as supporting subsequent culture of embryo-derived cells for the derivation and maintenance of bovine naive stem cells. The resetting media described herein support the resetting of bovine non-naive stem cells to generate bovine naive stem cells. Also described herein are methods and compositions for maintaining bovine naive stem cells, for example maintaining the colony replication and self-renewal rate of bovine naive stem cell lines. The materials and methods described herein aretherefore useful for deriving and maintaining bovine naive stem cells and establishing bovine naive stem cell lines, optionally for use in breeding programs, genetic improvement schemes, for the multiplication of preimplantation embryos with desirable genetic characteristics and / or the production of iblastoid structures, deriving primordial germ cells and / or gametes including for in vitro breeding programs, and / or transplantation into surrogates and developing and delivering veterinary medical biologicals and therapeutics.

[0105] As used herein, the term “naive stem cell” refers to stem cells which are capable of being derived and maintained in an undifferentiated state of self-renewal without the need for exogenously expressed pluripotency factors and are capable of developing into a complete organism and / or may retain the capacity to give rise to the full complement of extraembryonic tissues, adult tissues, and / or cell types. Naive stem cells exhibit molecular characteristics that are substantially similar to morula and ICM cells of early blastocysts and female naive stem cells have an X chromosome signature similar to preimplantation embryos. Bovine naive stem cells may be identified for example by round, dome-shaped cell colony morphology, and co-expression of one or more pluripotency markers such as SOX2, OCT4, or NANOG, and one or more of the naive factors SUSD2 and / or TFCP2L1 and / or KLF4 and / or other selected pluripotency and naive-specific factors such as those described by Messmer et al. (2019). It is unclear at this time whether naive stem cells as are observed in vitro exist in vivo, but can be derived from sufficiently undifferentiated embryo-derived cells, referred to herein as “naive stem cell-like cells”, under certain cell culture conditions.

[0106] Naive stem cells, such as bovine naive stem cells, may be derived directly from sufficiently undifferentiated diploid or haploid tissues, such as for example an entire embryo or the ICM of a preimplantation embryo, using the methods described herein. Naive stem cells also may be derived from non-naive stem cells, such as for example outgrowths that do not exhibit naive stem cell characteristics, induced pluripotent stem cells, expanded (or extended) pluripotent stem cells, or primed pluripotent stem cells for example using resetting-based methods.

[0107] As used herein, “non-naive stem cells” refers to cells which attain / retain expression of pluripotent stem cell markers such as SOX2, OCT4, NANOG, SSEA4 and / or other pluripotent markers, but do not exhibit naive-specific colony morphology or express SUSD2, TFCP2L1 , KLF4, or other naive-specific factors such as those described by Messmer et al. (2019).

[0108] Most cells in culture (except stromal derived cells) require a supporting layer to attach and proliferate in vitro such as in a tissue culture dish. This supporting layer can be made from stromal cells (directly attached on the dish), commonly known as a feeder cell system. For the culture of stem cells, a mouse embryonic fibroblast (MEF) feeder system is frequently used. As shown in the Examples herein, optimal ratios of feeder cell growth area vs. media volume for outgrowth establishment can be achieved by applying a first volume of growth media to the growth surface, followed by a mineral oil overlay, and then a second volume of growth media is added inside the original micro-drop, thereby creating a “taller” micro-drop, with reduced contact area and optimized micro-drop volume.

[0109] Accordingly, in one aspect there is provided a method of preparing a feeder cell layer micro-drop. In one embodiment, the method comprises: a) contacting a surface with a volume of coating solution, optionally gelatin; b) incubating the surface in contact with the drop of coating solution such that a layer of coating solution is deposited on the surface; c) removing the coating solution and optionally washing the surface; d) applying a first volume of growth medium, optionally comprising feeder cells, to the layer of coating solution deposited on the surface; e) overlaying a layer of hydrophobic fluid, optionally mineral oil, over the first volume of growth medium; f) adding a second volume of growth medium, optionally comprising feeder cells, to the first volume of growth medium, wherein the first volume of growth medium and / or the second volume of growth medium comprises feeder cells, thereby preparing a micro-drop comprising feeder cells; and g) incubating the micro-drop comprising feeder cells such that a layer of feeder cells attaches to the layer of coating solution deposited on the surface; thereby preparing a feeder cell layer micro-drop.

[0110] For the formation of a micro-drop with optimal feeder cell growth area or surface area vs. media volume, a surface area of not larger than 1 cm2, or optionally about 0.33 to about 0.39 cm2, or optionally about 0.36 cm2, and a total volume of about 60 pL may be used. A surface area of about 0.33 to about 0.39 cm2, or about 0.36 cm2, can beachieved by applying an amount of coating solution and / or first volume of growth medium of about 25-35 pL, or about 30 pL. Accordingly, in an embodiment, the first volume of growth medium is about 25-35 pL, or about 30 pL.

[0111] Suitable growth media include media suitable or typically used to culture the feeder cells, the outgrowth media disclosed herein e.g. AXRG, AXRGL, or PXGL media, or a combination (for example about 1 :1 mixture) of feeder cell medium and an outgrowth medium such as AXRG, AXRGL, and I or PXGL media. Optionally, the outgrowth medium includes a CDK8 / 19 inhibitor. In an embodiment, the growth media are media suitable or typically used to culture the feeder cells. For example, if MEFs or bFFs are used, the growth media may be MEF media. In an embodiment, the growth media is a mixture, for example about 1 :1 mixture, of the medium typically used to culture the feeder cells (for example MEF medium) and the outgrowth medium (for example PXGL, AXRGL, or AXRG).

[0112] The first volume of growth medium is overlayed with a layer of a biocompatible hydrophobic fluid such as mineral oil or paraffin oil (e.g. Ovoil™).

[0113] A second volume of growth medium is then added to the micro-drop to achieve a final volume of about 50-70 pl, optionally about 60 pL. Accordingly, the second volume of growth medium may be for example about 25-35 pL, or about 30 pL.

[0114] The choice of feeder cells will depend on the application, and may include for example and without limitation, mouse embryonic fibroblasts (MEFs) bovine fetal fibroblast (bFF) cells, bovine endometrial stromal cells, human fetal muscle cells, MEF SNL line cells, human fetal fibroblasts, human adult fallopian tubal epithelial cells, human dermal fibroblasts, human amniotic mesenchymal cells, human amniotic epithelial cells, mouse bone marrow stromal cells, murine amniocytes, human amniocytes, human foreskin fibroblasts, human amniotic mesenchymal cells, human endometrial stromal cells, rat embryo fibroblast cells, and pericellular matrix of decidua-derived mesenchymal cells. In an embodiment, the feeder cells are mouse embryonic fibroblasts (MEFs).

[0115] In an embodiment, the feeder cells are mitotically inactivated feeder cells. Suitable methods for inactivating the feeder cells are known in the art and include for example mitomycin treatment, gamma irradiation, and alcohol fixation. Accordingly, in an embodiment, the feeder cells are mitomycin treated, gamma irradiated, or alcohol fixed cells.

[0116] The feeder cells are seeded at a density suitable for bovine outgrowth derivation, for example 0.5 x 104to 2.5 x 104cells I drop or optionally about 1 .0 x 104cells I drop. The feeder cells can be introduced into the micro-drop with the first volume of culture medium, and / or the second volume of culture medium. Accordingly, in one embodiment, the first volume of culture medium comprises 0.5 x 104to 2.5 x 104cells, optionally about 1.0 x 104cells. In another embodiment, the second volume of culture medium comprises 0.5 x 104to 2.5 x 104cells, optionally about 1 .0 x 104cells. In a further embodiment, the first volume of culture medium comprises about 0.25 x 104cells to about 2.0 x 104cells, optionally about 0.5 x 104cells, and the second volume of culture medium about 0.25 x 104cells to about 2.0 x 104cells, optionally about 0.5 x 104cells.

[0117] The micro-drop comprising feeder cells is then incubated at a temperature and for a period of time to allow adherence or attachment of the feeder cells to the growth surface. In an embodiment, the incubation temperature is about 38°C to about 39°C, optionally about 38.5°C. In one embodiment, the period of time is about 6 hours to about3 days, optionally 12 to 36 hours or about one day. In an embodiment, the micro-drop comprising feeder cells is incubated up to about 7 days, for example about 2 days to about4 days or about 3 days prior to co-culture with whole embryo, ICM cells, naive stem celllike cells, and stem cells such as bovine embryonic stem cells, bovine naive stem cells, bovine pluripotent stem cells, bovine expanded (or extended) pluripotent stem cells, or bovine induced pluripotent stem cells.

[0118] Coating the growth surface with a coating solution such as gelatin may aid in the adherence and / or health of the feeder cell layer. Accordingly, in an embodiment, the growth surface of the micro-drop is first coated with a layer of coating solution. In an embodiment, the coating solution comprises gelatin, optionally at a concentration of about 0.05% to 0.5%, optionally 0.1 % to 0.2%, or optionally 0.1 %. Gelatin type A or gelatin type B may be used. In an embodiment, gelatin type A is used. Suitable gelatin is available from commercial providers and may have been derived from porcine, bovine, avian (poultry), or piscine tissues or cell culture. Alternatively or additionally, the coating solution may comprise Collagen, Laminin, Fibronectin, Vitronectin, Geltrex, Matrigel, or bioidentical cell-derived alternatives. The growth surface is contacted with the coating solution for a time and under conditions sufficient to allow for the deposition of a coating (e.g. gelatin) onto the surface. For example, the growth surface may be incubated at a temperature ranging from about 4°C to about 42°C, for example at room temperature, atabout 35°C, at about 37°C, or at about 38.5°C, and for a time ranging from about 30 minutes to about 24 hours, for example about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, or about 16 hours. Optionally the growth surface is contacted in a humidified environment.

[0119] As understood in the art, the terms “incubate” or “incubating” means to maintain for example a substance, material, composition, etc. at a particular temperature, or within a temperature range, for a period of time.

[0120] As shown in the Examples herein, bovine naive stem cells can be derived by isolating cells from a bovine embryo, optionally from the ICM of a bovine embryo, and optionally dissociating the ICM to obtain one or more clusters of ICM cells comprising bovine naive stem cell-like cells, transferring individual clusters of ICM cells to a culture environment (for example, a micro-drop) comprising a feeder cell layer, and culturing the clusters in outgrowth medium such as AXRGL, AXRG, or PXGLY to induce attachment and outgrowth of colonies comprising bovine naive stem cell-like cells, and culturing the bovine naive stem cell-like cells to obtain bovine naive stem cells, thereby deriving bovine naive stem cells.

[0121] Accordingly, in one aspect there is provided a method of deriving a population of bovine naive stem cells from a bovine embryo, the method comprising: a) providing a feeder cell layer; b) isolating cells, optionally from the ICM, of a bovine embryo to obtain a population of cells comprising bovine naive stem cell-like cells; c) optionally dissociating the population of cells comprising bovine naive stem celllike cells to obtain one or more clusters of cells comprising bovine naive stem celllike cells; d) transferring the population of cells comprising bovine naive stem cell-like cells, or the one or more clusters of cells comprising bovine naive stem cell-like cells to the feeder cell layer; e) culturing the population of cells comprising bovine naive stem cell-like cells or one or more clusters of cells comprising bovine naive stem cell-like cells in the presence of outgrowth medium to induce attachment and outgrowth of one or more colonies comprising bovine naive stem cells;thereby deriving a population of bovine naive stem cells.

[0122] Naive stem cells, such as bovine naive stem cells, may be derived directly from sufficiently undifferentiated tissues, such as for example one or more blastomeres of an early cleavage embryo (2-4 cell embryo), entire embryo or the cells of the ICM of a preimplantation embryo, for example 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, bovine naive stem cells may be derived from a 2- to 8-day bovine embryo, optionally a 2-, 3-, 4-, 5-, 6-, 7-, or 8- day bovine embryo, or a 5- to 7- day bovine embryo. In an embodiment, the bovine embryo is an early cleavage 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 an embodiment, the bovine embryo is a 3- to 7-day embryo, optionally a 5- to 7- day embryo or 6- or 7-day embryo. In an embodiment, the embryo-derived cells are ICM-derived cells. In an embodiment, the bovine embryo is a Zona Pellucida-free bovine embryo. In one embodiment, the embryo is a preimplantation embryo. In one embodiment, the embryo is an embryo that has previously been frozen and / or biopsied. In an embodiment, the embryo is a haploid embryo.

[0123] In various aspects described herein, the embryo, naive stem cell, non-naive stem cell, etc. has been tested for one or more biomarkers, and optionally scored and / or selected based on testing for one or more biomarkers. As used herein, “testing for one or more biomarkers” includes testing for genetic, genomic, and / or epigenetic characteristics, including, but not limited to, the presence or absence of one or more specific alleles, single nucleotide polymorphisms (SNPs), haplotypes, copy number, homozygosity, genomic insertions and / or deletions, histone modifications, protamine modifications, DNA methylation, gene or mRNA expression levels, protein expression or modifications, and metabolite analysis. Testing for one or more biomarkers may be carried out at any step of the methods described herein. For example, testing may be carried out on embryos, outgrowths, naive stem cells, non-naive stem cells, iblastoids, sperm, oocytes, and / or animals (individual dams and sires, or optionally a mating pair).

[0124] As used herein, a “trait” or “characteristic” refers to a specific feature of an animal, embryo, outgrowth, stem cell, sperm, oocyte, or iblastoid which may be influencedor determined by one or more genetic factors (e.g. allelic variants, epigenetic marks, homozygosity, copy number, and combinations there-of) and / or environmental factors.

[0125] In various aspects described herein, the bovine embryos, naive stem cells, non-naive stem cells, etc. have been genetically modified.

[0126] A “genetically modified cell” refers to a cell where genomic DNA of the cell has been manipulated to express one or more exogenous genes and / or to introduce mutation(s) within endogenous genes or intergenic regions which affects expression or functional activity of one or more endogenous genes or gene products. Examples of successful genetic modifications in livestock have included the introduction of transgenes by microinjection (U.S. 7,067,713) and lentiviral infection (Park, 2007) and most recently genome editing (reviewed by Bishop and Van Eenennaam, 2020 for livestock and by Mueller and Van Eenennaam, 2022 for cattle) using transfection and genome editors, such as Zinc Finger Nucleases, transcription activator like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeat / CRISPR associated gene (CRISPR / Cas) system. Common bovine targets for genetic modification are milk protein genes such as p-lactoglobulin, p-casein, myostatin, horned / polled, prolactin receptor conferring a slick haircoat for improving heat tolerance, and various genes involved in disease susceptibility or resilience (Bishop and Van Eenennam, 2020 and Mueller and Van Eenennaam, 2022).

[0127] Likewise, a “genetically modified embryo” refers to an embryo where the genomic DNA of cells in the embryo has been manipulated to express one or more exogenous genes and / or to introduce mutation(s) within endogenous genes or intergenic regions which affects expression or functional activity of one or more endogenous genes or gene products.

[0128] As shown in the Examples, the methods for embryo-derived outgrowth formation described herein provide improved yield for bovine embryos of outgrowths comprising cells having ICM-like characteristics compared to standard methods used for deriving human embryonic stem cells (58% vs 8-25% using day-7 embryos) and give rise to outgrowths comprising colonies of cells exhibiting naive stem cell characteristics, as well as outgrowths comprising colonies of cells exhibiting non-naive stem cell characteristics. Accordingly, in an embodiment, at least 25% of outgrowths comprise ICM- like cells. In an embodiment, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55% or more than 55% of outgrowths comprise ICM-like cells. Asshown herein, the outgrowths give rise to colonies comprising bovine naive stem cells using the methods described herein at greater frequency than previously described. Approximately 50% of these colonies maintain naive status over multiple passages using the methods described herein. Accordingly, in an embodiment, at least or about 25%, or more than 25% of the outgrowths give rise to bovine naive stem cells.

[0129] The feeder cell layer is suitably prepared at least 18 hours before derivation.

[0130] As shown herein, the feeder cell layer may be adapted to outgrowth medium such as AXRGL, AXRG, and / or PXGL medium, optionally wherein the outgrowth medium includes a CDK8 / 19 inhibitor, before starting the derivation. Accordingly, in an embodiment, the feeder cell layer is adapted to outgrowth medium from feeder cell medium in a step-wise manner, for example by replacing the growth medium with increasing ratios of outgrowth medium over time. In an embodiment, the growth medium in the micro-drop is replaced with 1 :1 MEF:outgrowth media about 18 hours before bovine embryo-derived cells are added to the micro-drop, and / or replaced with outgrowth media about four hours or less before bovine embryo-derived cells are added to the micro-drop.

[0131] The feeder cells are suitably mouse embryonic fibroblasts (MEFs) bovine fetal fibroblast (bFF) cells, bovine endometrial stromal cells, human fetal muscle cells, MEF SNL line cells, human fetal fibroblasts, human adult fallopian tubal epithelial cells, human dermal fibroblasts, human amniotic mesenchymal cells, human amniotic epithelial cells, mouse bone marrow stromal cells, murine amniocytes, human amniocytes, human foreskin fibroblasts, human amniotic mesenchymal cells, human endometrial stromal cells, rat embryo fibroblast cells, and pericellular matrix of decidua-derived mesenchymal cells. In an embodiment, the feeder cells are mouse embryonic fibroblasts (MEFs).

[0132] The optimal feeder cell density may be for example about 1 .5 to 7.5 x 104 / cm2, preferably about 3.0 x 104 / cm2, or 0.5 x 104to 2.5 x 104cells I drop, preferably about 1.0 x 104cells / drop.

[0133] The Zona Pellucida (ZP) prevents attachment of embryonic cells to culture substrates. Accordingly, in an embodiment, the ZP of the embryo is removed prior to isolating the cells of the embryo. Indeed, in one embodiment, cells are isolated from a bovine embryo by removing the Zona Pellucida. ZP-free embryos can be provided or obtained using any suitable method. For example, the ZP may be thinned and / or ruptured using enzymatic, chemical, and / or mechanical means, and subsequently separated fromthe embryo by mechanical manipulation to obtain a ZP-free embryo. Suitable enzymatic or chemical means for thinning and / or rupturing the ZP include for example the use of proteases such as pronase, collagenase, or acidified Tyrode’s solution. Suitable mechanical and non-contact methods for rupturing the ZP include for example the use of a microblade, micropipette, microneedle, or laser. The ruptured ZP may be separated from the embryo for example by agitation such as pipetting, vortexing, or direct manipulation using a micropipette or microneedle. Embryos from which the ZP-free bovine embryo is obtained may be fresh or previously frozen, and optionally may be obtained from biopsied-frozen embryos. In an embodiment the embryo is a genetically tested embryo.

[0134] As described and shown in the Examples herein, immunosurgery can be used to facilitate isolation of cells of the ICM for derivation of bovine naive stem cells. Accordingly, in an embodiment, the ICM cells are isolated using immunosurgery. When xeno-free systems are desired, ICM cells may be isolated using laser-assisted techniques (Turetsky et al., 2008). In an embodiment, the ICM cells are isolated using laser-assisted dissection.

[0135] As used herein, the term “immunosurgery” refers to a method of separating the inner cell mass from the trophectoderm (TE) of a blastocyst stage embryo using antibodies.

[0136] As described in Example 1 , cells can be isolated from the ICM of ZP-free day-7 embryos by incubating the ZP-free embryos with anti-bovine serum antibody followed by incubation with complement serum solution. Trophectoderm cells and cellular debris are then removed by mechanical pipetting and the remaining ICM is optionally washed with a Ca2+, Mg2+, and SO42' free medium (e.g. DPBS without Ca2+and Mg2+, Neurobasal medium, HBSS without Ca / Mg, EBSS without Ca / Mg, or normal saline) and seeded in outgrowth medium, optionally into a feeder cell layer micro-drop. In an embodiment, the embryos are incubated with about 25% anti-bovine serum antibody solution for about 1 hour in a humidified 5% CO2 incubator at about 38.5 °C. In an embodiment, the embryos are 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, for about 1hour in a humidified 5% CO2 incubator at about 38.5 °C. In an embodiment, the complement serum is guinea pig complement serum. As shown in the Examples, when Ca2+, Mg2+, and SO42'-free medium was used to dilute the anti-bovine serum antibody and the complement serum, greater immunosurgery efficiency was observed and the time required to complete the TE cell digestion was reduced. Accordingly, in an embodiment, the anti-bovine serum antibody and / or the complement serum is diluted in a Ca2+, Mg2+, and SO42'-free medium, for example neurobasal medium.

[0137] As shown in the Examples, derivation efficiency is improved when the ICM cells are dissociated to obtain smaller clusters of ICM cells. Accordingly, in an embodiment, the ICM (or other embryo-derived) cells are dissociated prior to transferring to the feeder cell layer. ICM and other embryo-derived cells may 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 10pm to about 15pm to obtain single cells, or about 20pm to about 35pm to obtain cell clusters. Microcapillaries may be made using for example the methods described herein or as is known by those skilled in the art. Suitable dissociation reagents and conditions include, without limitation, TrypLE, 0.25% Trypsin / EDTA, 0.05% Trypsin / EDTA, and I or Accutase, optionally for about 2 mins to about 10 mins prior to pipetting. Alternatively, EGTA, citrate, or other biocompatible chelating agents can be used in place of EDTA in the methods described herein. As will be understood by the skilled person, the presence of Ca2+, Mg2+and SO42' may interfere with dissociation. Accordingly, the dissociation reagent will be diluted in a Ca2+, Mg2+and SO42' -free buffer.

[0138] In an embodiment, the embryo-derived cells are dissociated in the presence of one or more Rho-kinase (ROCK) inhibitor components, including but not limited to Y27632, Thiazovivin, Fasudil, and Blebbistatin. CEPT cocktail, which comprises a ROCK inhibitor component, may also be used as a ROCK inhibitor component.

[0139] In an embodiment, a ROCK inhibitor is included when cells are manipulated and / or stressed. For example, a ROCK inhibitor can be added to the medium at the beginning of outgrowth and / or during immunosurgery, resetting, passaging (mechanical or enzymatic), freezing and / or thawing.

[0140] As shown herein, the attachment and outgrowth formation from bovine embryos is influenced by the composition of the medium in which the embryos and I orembryo-derived cells are cultured. As shown in Example 3, media designed for feeder- free culture may not be particularly suitable for feeder systems. Accordingly, bovine naive stem cells may be cultured in a medium for outgrowth (also referred to herein as “outgrowth medium”) for example “AXRGL”, “AXRG”, “PXGL”, “PXGLY”, or an “enhanced outgrowth” medium described herein. In an embodiment, the outgrowth medium includes a CDK8 / 19 inhibitor.

[0141] As shown in Example 6 herein, a resetting method can be used to convert bovine non-naive stem cells to bovine naive stem cells. As used herein, the term “resetting” refers to reversing cells of a more advanced developmental state (e.g. primed pluripotent stem cells, induced pluripotent stem cells, expanded pluripotent stem cells) to a naive developmental state.

[0142] It is understood that pluripotency proceeds through at least two phases: naive and primed. Naive stem cells are thought to be the developmental ground state, showing unrestricted developmental potential, a uniform and high differentiation ability, and forming compact dome-like colonies. In contrast, primed pluripotent stem cells and induced pluripotent stem cells do not exhibit naive morphology but are flat and monolayer, display repressive chromatin features and lineage priming, do not exhibit unrestricted developmental potential, and are unusable for further naive cell passages.

[0143] The resetting of cell potency is recognized as an alternative method for deriving naive stem cells. For example, Guo et al. (2017) reported deriving naive human stem cell cultures following resetting of induced human pluripotent stem cells and primed human pluripotent stem cells. Accordingly, in an embodiment, bovine naive stem cells are derived from induced pluripotent stem cell. In another embodiment, bovine naive stem cells are derived from primed pluripotent stem cells.

[0144] Primed pluripotent stem cells may be derived for example, from those embryo-derived outgrowths that do not exhibit a naive morphology (obtained using the methods described above) by using various methods including those described herein. To isolate and establish primed pluripotent stem cell lines from the outgrowths that do not exhibit naive morphology and / or which include a mixture of extraembryonic cells, selective dissociation can be applied. Outgrowths that do not exhibit naive morphology can selectively be dissociated into small clumps with EDTA solution (Beers et al., 2012) or other suitable dissociation reagent, so extraembryonic cells can be eliminated during the subculture procedure (“EDTA-subculture”) and primed pluripotent stem cells clusterscan be obtained. Other sources of non-naive stem cells may include those derived using methods described by Soto et al. (2021), Bigliotti et al. (2018), Zhao et al. (2021), Han et al. (2011 ), for example.

[0145] Accordingly, in one aspect there is provided a method of generating bovine naive stem cells from bovine non-naive stem cells, optionally primed stem cells, the method comprising: a) providing a population of cells comprising bovine non-naive stem cells in a culture environment comprising feeder cells in primed stem cell medium; and b) either:I. replacing the medium with epigenetic resetting medium comprising a MEK / ERK inhibitor component, a LIF component, and an HDAC inhibitor component; culturing the cells optionally for at least or about 10, 20, 30 or 40 hours, optionally about 2 days to about 4 days; replacing the medium with outgrowth medium; and culturing the cells for at least or about 4 days, optionally about 9 to about 11 days; orII. replacing the medium with outgrowth medium for at least or about 4 days, optionally about 5 to about 9 days; thereby generating bovine naive stem cells.

[0146] In an embodiment, the outgrowth medium in I. and II. comprises: i) a MEK / ERK inhibitor component, a Wnt inhibitor component, a PKC inhibitor component, a LIF component and a ROCK inhibitor component; or ii) an Activin A component, a Wnt inhibitor component, a PKC inhibitor component, a RAR inhibitor component, a ROCK inhibitor component and optionally a LIF component;

[0147] The outgrowth medium in I. and II. may further comprise a CDK8 / 19 inhibitor.

[0148] The epigenetic resetting medium and / or the outgrowth medium may further comprise an FGF2 component and / or a p38 MAPK inhibitor component. Alternatively or additionally, the epigenetic resetting medium and / or the outgrowth medium may further comprise a ROCK inhibitor component.

[0149] Where the number of non-naive stem cells is limited and / or the population of non-naive stem cells further comprises additional cells such as extraembryonic cells (for example when using “non-passageable” ICM colonies derived using the methods described herein) the method may further comprise, prior to step b): i. culturing the bovine non-naive stem cells in the primed stem cell medium for at least or about 3 days; ii. dissociating and transferring the bovine non-naive stem cells to a culture environment comprising feeder cells in primed stem cell medium; and iii. culturing the cells, optionally for at least or about 10, 20, 30 or 40 hours, optionally about 2 days.

[0150] If desired, for example to increase cell numbers or the percentage of non- naive stem cells in the population, steps ii) and iii) may be repeated at least one additional time, optionally two or more additional times.

[0151] As described herein, the cells may be dissociated in step ii) in the presence of a dissociation reagent, for example EDTA, and / or by mechanical dissociation.

[0152] Optionally, the epigenetic resetting medium in step b) may be replaced with fresh epigenetic resetting medium, optionally after at least or about 10, 20, 30 or 40 hours, and / or the outgrowth medium in step b) may be replaced with fresh outgrowth medium daily.

[0153] In an embodiment, the cells of step b) are plated at a seeding density of 1 .5 to 2.5 x 104cells / cm2, optionally about 2 x 104to about 2.5 x 104cells / cm2.

[0154] In an embodiment, the method further comprises c) passaging the naive stem cells onto freshly prepared feeder cells in outgrowth medium, optionally wherein the naive stem cells are plated at a density of about 1 .5 x 104to about 2.5 x 104cells I cm2.II. Products and Compositions of Matter

[0155] In one aspect of the disclosure there are provided products and compositions of matter useful for the derivation and culture of bovine naive stem cells.

[0156] As shown herein, bovine naive stem cells are derived or generated by culture in various media compositions, including media for outgrowth (also referred to herein as “outgrowth medium” (e.g. “PXGL” as described by Bredenkamp et al. (2019b), for example, “PXGLY”, “AXRG”, and “AXRGL” media), media for resetting (also referredto herein as “resetting media”) (e.g. “Epigenetic resetting” media), and media for maintenance (also referred to herein as “maintenance media”).

[0157] The various media may comprise for example a base medium, and one or more small molecules, growth factors, and / or nutrients. Suitable base media can be readily determined by the skilled person and include without limitation DMEM / F12 (Dulbecco’s Modified Eagle’s Medium F12), advanced DMEM / F12, Neurobasal medium, and mixtures thereof. Suitable supplements can be readily determined by the skilled person and include without limitation Minimum Essential Medium (MEM), non-essential amino acids (NEAA), L-glutamine (e.g. 1-2 mM), Glutamax, ascorbic acid, insulin, BSA (fraction V), beta-mercaptoethanol, penicillin / streptomycin, and / or gentamycin. In an embodiment, the base medium comprises 1 :1 DMEM / F12 and Neurobasal medium. In an embodiment, the base medium further comprises 1X Glutamax + 1X NEAA + 1 mg / ml BSA + 50 ug / ml Ascorbic acid + 0.1 mM p-mercaptoethanol + 50 lll / ml Pen / Strep.

[0158] Various media for outgrowth described herein includes “PXGL”, “PXGLY”, “AXRG”, and “AXRGL”. Commercial media such as RSeT can also be used.

[0159] As used herein, “PXGL” medium comprises a base medium (e.g. DMEM / F12:Neurobasal medium (1 :1 mixture)); a serum replacement component (e.g. N2B27 serum); a MEK / ERK inhibitor component, optionally PD0325901 , Ravoxertinib, GSK1120212, MEK162, PD184352, Trametinib, LY3214996, or Ulixertinib, preferably 1 pM PD0325901 ; a Wnt inhibitor component, optionally XAV-939, IWR-1 , or IWP-2, preferably 2 pM XAV-939; a PKC inhibitor component, optionally G66983, G66976, LY317615, LY333531 , PKC412, GSK690693, Sotrastaurin, Staurosporine, or Bisindolylmaleimide, preferably 4 pM G66983; and a LIF component, optionally human LIF, bovine LIF, caprine LIF, porcine LIF, buffalo LIF, or recombinant LIF, optionally at a concentration of about 10 ng / ml LIF. “PXGLY” medium further comprises a ROCK inhibitor component, optionally Y27632, fasudil, Thiazovivin, Blebbistatin, optionally 5-10 pM Y27632. CEPT cocktail, which comprises a ROCK inhibitor component, may also be used as a ROCK inhibitor component.

[0160] As used herein, “AXRG” medium comprises a base medium (e.g. DMEM / F12:Neurobasal medium (1 :1 mixture)); a serum replacement component (e.g. N2B27 serum); a Wnt inhibitor component, optionally XAV-939, IWR-1 , or IWP-2, preferably 2 pM XAV-939; a PKC inhibitor component, optionally G66983, G66976, LY317615, LY333531 , PKC412, GSK690693, Sotrastaurin, Staurosporine, orBisindolylmaleimide, preferably 2 pM G66983; one or more RAR inhibitor components, optionally BMS493, BMS-189453, BMS-195614, AGN 193109, AGN 193491 , AGN 193618, AGN 194202, AGN 194301 , AGN 194574, Ro 41-5253, ER 50891 , ATRA, MM 11253, Phenylacetylene analogs 6, Phenylacetylene analogs 7, Quinolone derivatives analog 15, Quinolone derivatives analog 16, preferably 1 uM BMS493; and Activin A, optionally human Activin A, preferably 5 ng / ml human Activin A. “AXRGL” medium further comprises a LIF component, optionally human LIF, bovine LIF, caprine LIF, porcine LIF, buffalo LIF, or recombinant LIF, optionally at a concentration of about 10 ng / ml LIF.

[0161] In an embodiment, a CDK8 / 19 inhibitor component is added to any of PXGL, PXGLY, AXRG and AXRGL. As used herein, the term “CDK8 / 19 inhibitor” refers to an inhibitor of 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-didehydro-cortistatin 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, Cortistatin A or SEL120- 34A.

[0162] In another embodiment, a ROCK inhibitor component is added to any of PXGL, PXGLY, AXRG and AXRGL.

[0163] Enhanced outgrowth media (for naive culture or resetting), comprises outgrowth media, for example PXGL, PXGLY, AXRG, or AXRGL media and further comprises an FGF2 component, optionally human FGF2, bovine FGF2, mouse FGF2, monkey FGF2, porcine FGF2, chicken FGF2, caprine FGF2, buffalo FGF2 and recombinant FGF2, optionally at a concentration of about 10 ng / ml FGF2. Alternatively, or additionally, enhanced outgrowth media may further comprise a p38 MAPK inhibitor component, optionally SB203580, BIRB796, LY2228820, SB202190, VX-702, Neflamapimod, TAK 715, SB239063, Talmapimod, Pamapimod, PF-07265803 or Losmapimod, preferably 1 pM SB203580.

[0164] Examples of primed stem cell media are described herein, including AFX medium, similar to Rostovskaya et al (2019). Primed stem cell media such as AFX may comprise for example a base medium (e.g. DMEM / F12 : Neurobasal medium mixture (1 :1 ), 1X Glutamax, and 1X NEAA, and optionally 1 mg / ml BSA, 50 ug / ml Ascorbic acid, 0.1 mM p-mercaptoethanol, and / or 50 lU / ml Pen / Strep or other suitable antimicrobial agent such as Gentamycin); 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-p signaling component, for example TGF-p or an Activin A component, optionally human Activin A, bovine Activin A, or murine Activin A, optionally at a concentration of 5-50 ng / ml, preferably 20 ng / ml Activin A; and a Wnt inhibitor component, optionally XAV-939, IWR-1 , or IWP-2, preferably 2 pM XAV- 939. In an embodiment, the primed stem cell media or AFX further comprises a ROCK inhibitor component, optionally Y27632, fasudil, Thiazovivin, or Blebbistatin, optionally 5- 10 pM Y27632. CEPT cocktail, which comprises a ROCK inhibitor component, may also be used as a ROCK inhibitor component. Commercial primed stem cell media such as mTeSR, TeSR-E8 (Stem Cell Technologies) or Essential 8™ medium (Gibco) can be used after addition of a Wnt inhibitor as described above. Essential 6™ medium (Gibco) can be used as a basal medium.

[0165] Examples of resetting media are described herein, including “epigenetic resetting” medium. Epigenetic resetting media may comprise a base medium (e.g. DMEM / F12: Neurobasal medium mixture (1 :1 ), 1X Glutamax, 1X NEAA, 50 pg / ml BSA, 50 pg / ml Ascorbic acid, 0.1 mM p-mercaptoethanol, and 50 lll / ml Pen / Strep or other suitable antimicrobial agent / s such as Gentamycin); 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 Ulixertinib, preferably 1 pM PD3025901 ; a LIF component, optionally human LIF, bovine LIF, caprine 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 an embodiment, the epigenetic resetting medium further comprises a ROCK inhibitor component, optionally Y27632, fasudil, Thiazovivin, or Blebbistatin, optionally 5-10 pM Y27632. CEPT cocktail, which comprises a ROCK inhibitor component, may also be used as a ROCK inhibitor component. In an embodiment, the epigenetic resetting medium further comprises an FGF2 component and / or a p38 MAPK inhibitor component.

[0166] Examples of media for maintenance are described herein which can comprise an media used for outgrowth, for example PXGL, PXGLY, AXRG, or AXRGL media described herein, and further comprise a CDK8 / CDK19 inhibitor component, forexample, about 2.5 nM CCT251545. In one embodiment, an example of a maintenance media isAXRGL media further comprising CCT251545.

[0167] Typically, bovine naive stem cells are cultured and maintained in media similar to the medium in which they were derived. For example, if bovine naive stem cells were derived in “PXGL” (or “PXGLY”), they are typically maintained in PXGL with or without one or more of 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 naive stem cells were derived in “AXRG” (or “AXRGL”), they are typically maintained in AXRG with or without one or more of a LIF component, an FGF2 component, a p38 MAPK inhibitor component, and / or a CDK8 / 19 inhibitor component (or other desired components).

[0168] It is appreciated that each of the PXGL, PXGLY, AXRG, AXRGL and AXRGL plus CDK8 / 19 inhibitor media described herein can be used during outgrowth and maintenance phases, with the media chosen based on the vigor and condition of the colonies. The following are examples of media that can be used as the cells progress from the outgrowth to the maintenance phase:lus CDK8 / 19 inhibitor.

[0169] It is further appreciated that naive stem cells may be maintained until a certain passage using outgrowth media as described herein.

[0170] Any suitable dissociation buffers may be used in the methods described herein, including and without limitation, TrypLE, 0.25% Trypsin / EDTA, 0.05% T rypsin / EDTA, and I or Accutase,

[0171] The various protocols described herein may use various buffer or saline solutions, for example phosphate buffered saline (PBS) or Dulbecco’s Phosphate Buffered Saline (DPBS), without Ca2+, Mg2+, and / or SO42' (where indicated). Alternative embodiments may use any suitable buffer or saline solution (without the indicated components), for example Hanks’ balanced salt solution (HBSS), Earle’s balanced salt solution (EBSS), or normal saline.

[0172] 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.

[0173] As understood in the art, pH is influenced by the carbon dioxide (CO2) concentration 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.

[0174] As used herein, the term “hypoxic conditions” means conditions with an oxygen (O2) concentration lower than atmospheric oxygen concentrations, namely lower than about 20.95% oxygen. For example, when cells are incubated under hypoxic conditions, the cells are incubated in a reduced oxygen environment, for example between about 1 % and about 15% oxygen, optionally between about 1 % and 10% oxygen, or between about 1 % and about 5% oxygen.III. Uses

[0175] Also provided is the use of the products, compositions or kits described herein for supporting the attachment of bovine embryo-derived cells in culture, outgrowth of cells having ICM-like characteristics, and which give rise to colonies of cells exhibiting naive stem cell characteristics and / or colonies of cells exhibiting non-naive stem cell characteristics, and / or for derivation of bovine naive stem cells from bovine embryo- derived outgrowths and from bovine non-naive stem cells.

[0176] A further aspect includes use of a bovine naive stem cell derived using the methods described herein in a breeding scheme or genetic improvement program, or for multiplying preimplantation embryos having desirable genetic characteristics, deriving primordial germ cells and / or gametes including for in vitro breeding programs and / or transplantation into surrogates, and / or developing and delivering veterinary medical biologicals and therapeutics. As used herein, the reference to a “breeding scheme or genetic improvement program” includes in vivo programs where gametes and progeny originate and / or are generated by animals in vitro programs where at least one generation of gametes and 'progeny' are generated ex vivo, and combinations thereof.

[0177] The following non-limiting examples are illustrative of the present application:EXAMPLESExample 1. Immunosurgery (based on the methods described in Solter et al., 1975) to improve the naive derivation with dav-7 embryosBackground

[0178] When entire day-7 embryos have been used for outgrowth establishment, delayed attachment, low ICM growth rates, and higher TE proliferation among transferred clusters have been observed as compared to outgrowth establishment from day-5 embryos (Table 1 ). Thus, it was necessary to develop protocols which yield superior rates of establishment and quality of ICM outgrowths.Table 1 : Attachment and proliferation rates by stage of embryo development in a feeder- free system.Medium Attach % TE % ICM %Day-5 embryo t2ILGoY 97% 74% 53%Day-7 embryo t2ILGoY 97% 91% 25%

[0179] The protocol for immunosurgery was as follows:1. Prepare 25% anti-bovine serum antibody solution and 25% complement serum (from guinea pig) diluted in embryo manipulation medium comprising 1 ) standard HEPES buffered medium containing Ca2+, Mg2+, and SO42-; 2) DPBS without Ca2+, Mg2+, or SO42-; or 3) Neurobasal medium.2. Prepare immunosurgery drops with the solutions from step 1 using the layout described in Figure 1.3. Wash ZP-free day-7 embryos with DPBS without Ca2+and Mg2+, 6 times (Figure 1A).4. Rinse excess DPBS without Ca2+and Mg2+from ZP-free day-7 embryos two times with 25% anti-bovine serum antibody solution, then incubate ZP-free embryos in 25% anti-bovine serum antibody solution for 1 hour in humidified 5% CO2 incubator at 38.5 °C (Figure 1 B).5. After 1 hour incubation, wash embryos with embryo manipulation medium 3 times, then transfer embryos into 25% guinea pig complement serum solution.6. Wash embryos two times with 25% guinea pig complement serum solution, then incubate ZP-free embryos in 25% complement serum solution for 1 hour in humidified 5% CO2 incubator at 38.5 °C (Figure 1C).7. After 1 hour incubation, remove digested trophectoderm cells / debris by mechanical pipetting.8. After mechanical removal of trophectoderm cells, wash ICM 3 times with embryo manipulation medium.9. Seed embryo into outgrowth culture drop containing t2iLG6Y medium.Results

[0180] Day-7 embryos (Figure 2A) collapsed after approximately 20 to 30 minutes of exposure to the complement serum (Figure 2B). Thereafter, digested trophectoderm cells were easily removed by mechanical pipetting (Figure 2C). Pure ICM was successfully obtained after immunosurgery followed by pipetting. As shown in Figure 3, positive staining for SOX2 (ICM marker) and no staining for CDX2 (TE marker, Figure 3, top right panel) were observed following immunosurgery. The results also show that the protocol does not affect cell viability as demonstrated by the absence of Annexin V or Propidium Iodide (PI) staining (Figure 3, bottom panel).

[0181] When anti-bovine serum antibody and complement serum from guinea pig were diluted into a solution containing calcium sulfate or magnesium sulfate, protein coagulation was frequently observed (Figure 2D, dotted circle). In addition to coagulation, the time required to complete digestion of TE cells was highly variable between embryos and ranged from 0.5 hr to 2 hrs. Without being bound by theory, it was hypothesized that the reduced protein availability in the medium due to coagulation was in part responsible for a less effective immunological reaction induced by the complement. Protein coagulation was not observed under calcium sulfate-free or magnesium sulfate-free conditions (Figure 2E; DPBS, Figure 2F; neurobasal medium) which also showed greater immunosurgery efficiency as observed by the reduced time required to complete trophectoderm cell digestion. Moreover, when MgSCU-free medium was used, times required for digestion of TE cells were less variable meaning that this step of the protocol is now completed in 30 to 60 minutes. As a consequence, the protocol optionally uses neurobasal medium as the base for diluting anti-bovine serum antibody and complement serum and for the several washes.

[0182] With an optimized immunosurgery protocol, it was possible to significantly increase the proportion of transferred clusters exhibiting ICM growth using a feeder-free system and t2iLG6Y medium (Table 2). This improvement can be explained in part by lower expression of TE growth than when immunosurgery was not used.Table 2: Attachment and proliferation rates with and without immunosurgery.Origin Medium Attach % TE % ICM %Day-7 embryo t2iLG6 97% 91% 25%Day-7 immunosurgery t2iLG6 100% 73% 58%Example 2. Derivation of outgrowths comprising naive stem cells on feeder cellsBackground

[0183] When using other methods developed for deriving human embryonic stem cells, bovine ICM colonies can be derived with approximately 50% efficiency, however virtually all outgrowths failed to exhibit characteristics of naive status after the first passage. It was noticed that the differentiation was always directed towards the endodermal lineage as illustrated in Figure 5A. Reubinoff et al. (2000) have previously reported that suboptimal culture conditions for the production of ES cells from human blastocyts freguently leads to differentiation of the cells, and that the use of mouse embryonic fibroblasts (MEF) as a feeder cell layer may overcome this deficiency. A feeder system is a classical method for providing not only efficient cell attachment for embryonic cells, but also provides several soluble factors to support cells (Eiselleova et al., 2008). Bovine naive stem cell culture conditions still are not well defined, so feeder cells may be beneficial for overcoming suboptimal culture conditions especially for the initiation of naive stem cell culture. Moreover, for increased guality, yield, and biosecurity of naive stem cell colonies, it may be preferable to use an allogeneic system such as using bovine endometrial stromal or bovine fetal fibroblast derived feeder cells for bovine naive stem cell cultures. When fully established, naive stem cell lines may be switched to a feeder- free system in part to simplify the workflow for downstream applications such as the multiplication of preimplantation embryos with desirable genetic characteristics, production of iblastoids (Non-cloning methods of bovine embryo replication, United States Provisional Patent Application No. 63 / 456,624, filed April 3, 2023), generation andmaintenance of cells that better tolerate genetic manipulation, derivation and replication of germ cells, and the robust generation of somatic tissues for regenerative medicine. The transition to a feeder-free system also would be expected to minimize risks associated with cross-species contamination.Protocols

[0184] Mitotically inactivated mouse embryonic fibroblast feeder cells were produced using mitomycin treatment as described for example in Jozefczuk et al. (2012). Alternatively, gamma irradiation can be used to produce 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).Preparation of feeder cell layer:1 . Prepare 0.1 % gelatin coating solution.2. Apply 25 pl drops of coating solution to the surface of a 35mm cell culture dish to create micro-drops.3. Incubate the dish in a humidified 5% CO2 incubator at 38.5°C for 1 hour.4. Remove coating solution and optionally gently rinse the surface.5. Apply 20 pl of fresh MEF medium to the coated surface.6. Apply a mineral oil overlay to the coated surface and MEF medium.7. Thaw inactivated MEFs according to standard protocols.8. Break cell pellet with 1 ml of fresh MEF medium.9. T ake 15 pl of cell suspension and mix with 15 pl of 0.4% of trypan blue solution for live / dead cell counting.10. Count the number of transparent (live) cells using hemacytometer.11 . Dilute cells to a concentration of 5 x 105 / ml and seed 1 x 104(20 pl) into gelatin coated micro-drop containing 40 pl of fresh MEF medium (total 60 pl of culture medium).12. Prepare feeder layer at least 18 hours before derivation step.Results

[0185] The density of feeder cells is one factor for a successful feeder cell system (Heng et al., 2004). When the unique micro-drop culture system is used for outgrowth derivation including for deriving bovine outgrowth, the optimal feeder cell density must be verified prior to outgrowth derivation. The reason is that MEF cells tend to form a higher density of cells towards the middle of the micro-drop. The growth area of an outgrowth micro-drop used herein is about 0.36 cm2, which corresponds to 30 + 30 pL. To achieve this, 20-30 pL is first added, followed by a mineral oil or paraffin oil overlay and then another 30-40 pL is added inside the original micro-drop to attain a final volume of about 60 pL. The goal is to create a “taller” micro-drop with reduced contact with the petri dish. This represents the optimal micro-drop volume for outgrowth establishment. Several different MEF concentrations ranging from 0.5 x 104to 2.5 x 104cells I drop have been tested (Figure 4). The ideal feeder cells density was 1 .0 x 104cells I drop.

[0186] When culture wells are used, the ideal feeder cells density is 1.5 to 7.5 x 104 / cm2, optionally, 3.0 x 104 / cm2. For example, for typical 12 well plates (growth area: 4 cm2), 1 .2 x 105 / well represents the ideal cell density.

[0187] When using MEF as the feeder layer, embryo attachment rates reached 100%. However, TE growth significantly increased and ICM growth rate was greatly reduced among colonies (Table 3). In other words, attached ICMs were rapidly differentiated into TE as shown in figure 5B and C. The soluble factors secreted by feeder cells contributed to attachment but also curtailed ICM growth.Table 3: Attachment and proliferation rates with MEFs.Origin Medium Attach % TE % ICM %Day-7 immunosurgery t2iLG6Y 100% 100% 8%Example 3. Optimization of culture media with MEFBackground

[0188] Based on preliminary results, it was concluded that culture conditions optimized for a feeder-free system (i.e. t2iLG6Y) were not optimized for a 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), consequently the feeder-free naive culture medium may not be suitable for feeder systems. Therefore, we tested the compatibility oftwo different naive stem cell culture media in feeder cell conditions and MEF culture medium.ProtocolsA. Composition of MEF culture medium• DMEM / F12 + 10% FBS + 1 % NEAA + 1 % Glutamax + 50 lU / ml Pen / Strep + 50 pM p-mercaptoethanol.B. Composition of PXGL medium• DMEM / F12:Neurobasal medium (1 :1 mixture) + N2B27 serum + 1 pM PD0325901 + 2 pM XAV-939 + 4 pM G66983 + 10 ng / ml LIF.C. Composition of t2iLGo medium• DMEM / F12:Neurobasal medium (1 :1 mixture) + N2B27 serum + 1 pM CHIR99021 + 1 pM PD0325901 + 4 pM G66983 + 10 ng / ml LIF.Results

[0189] In a feeder free system (layer by layer, LbL), PXGL medium showed significantly lower ICM growth than growth when t2iLG6Y medium was used (Figure 6). However, PXGL medium showed improved ICM growth rates when feeder cells were used suggesting that PXGL medium may be superior to t2iLG6Y medium when using a feeder system.

[0190] In any protocol using feeder cells, maintaining healthy MEF is of prime importance. However, it was noticed that when directly exposing MEF cells to PXGL medium (i.e. , abrupt change from MEF medium to the naive medium), MEF cells were displaying an enlarged or elongated morphology (Figure 7). To prevent this morphology change, a stepwise adaptation to the naive cell culture conditions from the MEF conditions was tested. Briefly, 18 hours before starting the derivation or passaging, MEF culture medium was replaced by MEFPXGL media (1 :1 mixture), before making a change to 100% PXGL medium. MEF exposed directly to PXGL medium exhibited enlarged morphology after 48 hours, whereas MEF exposed to the stepwise medium adaptation maintained the same morphology as MEF cultured in MEF medium for up to 72 hours (Figure 7).Example 4. dissociationBackground

[0191] Although cells of the early embryo and ICM may be expected to have single cell clonogenicity, entire embryos and ICM were cultured previously without any dissociation process to minimize damage to embryo-derived cells used for naive colony derivation. Takahashi et al. (2022) recently reported that manipulating stem cells in a dissociation reagent rather than culture medium during passaging reduced DNA damage and apoptosis. Furthermore, these researchers reported that extended exposure to TrypLE (up to 1 hour) did not affect cell viability. Therefore, to increase the chance of obtaining multiple colonies from the same embryo, it was decided to test and compare the dissociation of ICM into small cell clusters and an entire ICM culture method which allows only a single colony to form from one ICM.ProtocolsA. ICM dissociation1 . Prepare dissociation dish as shown in Figure 8.2. After immunosurgery, transfer ICM into drop 1 of dissociation dish and wash it by moving it to drops 2 and 3.3. Incubate 10 minutes in TrypLE (drop 3) at 38.5 °C.4. Do gentle pipetting with a microcapillary syringe to break ICM into single cells or small clusters.5. Transfer cells to drop 4 and then drop 5 carefully to completely remove TrypLE solution.6. Seed the cell clusters onto prepared MEFs.B. Microcapillaries for ICM dissociation were made as follows:1 . The microcapillary was pulled using a micropipette puller2. The microcapillary was cut at 40-50 pm outer diameter using a microforge3. The inner diameter of the microcapillary was reduced to 10-15 pm (for single cell dissociation) or 20-35 pm (for cell cluster dissociation) and the tip was fire polished4. The microcapillary was cut in the thickest region to achieve a final length of 2-3 cm5. The microcapillary was tightly assembled with the microliter syringe.Results

[0192] The ICM of bovine day-7 embryos contain approximately 30 to 40 cells, which may be dissociated into smaller cell clusters following immunosurgery by using a mild enzyme treatment and pipetting through a small bore pipette or microcapillary. Separating a single ICM into multiple clusters was hypothesized to improve the yield of colonies comprising cells having ICM-like characteristics (comprising naive stem cell-like cells). Six to 7 clusters with approximately 4 to 5 cells / cluster were obtained from individual ICMs after 10 minutes of incubation in TrypLE (Figure 9) followed by gentle pipetting with a microcapillary.

[0193] When ICM were dissociated into smaller clusters, somewhat improved ICM growth among clusters was obtained: 58% from dissociated cells compared with 42% from entire ICM. The key outcome is that by dissociating the ICM, the number of colonies comprising cells having ICM-like characteristics at the outgrowth stage was increased. For the Entire ICM group, 20 embryos yielded 20 attached clusters (100% attachment rate); whereas forthe Dissociated ICM group (100% attachment rate), 20 embryos yielded approximately 120-140 attached clusters (Table 4 and Figure 10). Similar improvements in the number of colonies comprising cells having ICM-like characteristics may be expected when whole early stage embryos are dissociated into smaller clusters.Table 4: Attachment and proliferation rates with ICM dissociation (n = 20 embryos per treatment group).Origin Medium Attach % TE % ICM %Entire ICM PXGL 100% 75% 42%Dissociated ICM PXGL 100% 75% 58%Example 5. Establishment of stem cell colonies from whole embryos, embryo-derived outgrowths e.q. ICM outgrowths

[0194] Bovine naive stem cells, naive stem cell-like cells, etc. can be passaged using mechanical or enzymatic methods as described below. Typically, following the immunosurgery and ICM or embryo-derived cells dissociation protocols described above, mechanical methods are used for the first 1-2 passages and enzymatic methods may beused thereafter. However, the appropriate choice of method can be made for individual passages as determined by the skilled person.ProtocolsA. When using mechanical method for passaging of bovine naive stem cells or naive stem cell-like cells1. Prepare PXGLY medium by adding 10 pM Y27632 to 10 ml PXGL medium.2. Prepare micro-drop with MEF as described in ‘Protocol: Preparation of feeder cells layer1.3. Change the MEF medium in the micro-drop to MEFPXGL media (1 :1 mixture) 18 hours before passaging naive stem cells, ICM colony, etc.4. If the embryo-derived outgrowth culture is showing TE growth, remove it mechanically to make ICM colony accessible.5. Divide the naive stem cell colony, ICM colony etc. into 2 to 6 clusters by mechanical dissociation and transfer the divided clusters into the new micro-drop with PXGLY medium.6. After 24 hours, change medium to PXGL.B. When using enzymatic method for passaging of bovine naive stem cells or naive stem cell-like cells1. Prepare PXGLY medium by adding 10 pM Y27632 to 10 ml PXGL medium.2. Prepare micro-drop with MEF as described in ‘Protocol: Preparation of feeder cell layer1.3. Change the MEF medium in the micro-drop to MEFPXGL media (1 :1 mixture) 18 hours before passaging naive stem cells, ICM colony etc.4. If the embryo-derived outgrowth is showing TE growth, remove it mechanically to make ICM colony accessible.5. Remove culture medium from the microdrop containing naive stem cells, ICM colony etc..6. Wash the microdrop with 60 pl DPBS without Ca2+ and Mg2+, 3 times.7. Add 60 pl TrypLE, incubate 3 minutes to dissociate ICM.8. Divide the ICM colony into single cells or small cell clusters with gentle pipetting.9. T ransfer cells into 1 .5 ml tube and add 500 pl DPBS with Ca2+and Mg2+.10. Centrifuge at 300 xg, 5 min.11 . Discard supernatant and break pellet using gentle pipetting of 1 ml PXGL medium.12. Centrifuge at 300 xg, 5 min.13. Discard supernatant and break pellet with PXGLY medium from new micro-drop.14. Seed cells into new micro-drop.15. After 24 hours, change medium to PXGL.Results

[0195] Colonies comprising naive stem cell-like cells having a diameter of 25-100 pm can be obtained successfully using the following combination of conditions: day-7 embryos treated by immunosurgery, followed by mild enzymatic ICM dissociation and culture on stepwise media-adapted MEF (Figure 11 A, B). Colonies derived using these conditions have clearer and brighter borders, more compacted mass, and show a distinct 3D shape typical of human naive stem cell colonies. Such colonies displaying a characteristic naive status were observed more frequently using the disclosed methods and feeder conditions than when methods for deriving human naive stem cells are used for bovine cells. In addition, no signs of endodermal differentiation were observed to passage 3 when the disclosed feeder cell methods were used. The disclosed feeder system and PXGL medium appear to be a very effective combination for preventing endodermal differentiation of bovine naive stem cells.

[0196] The suitability of PXGL for bovine naive stem cell cultures was tested by comparing with results for bovine expanded potential stem cells (non-naive) as described by Zhao et al. (2021). Stem cells were generated as described above or according to the methods described in Zhao and characterized for SUSD2 expression as described below in Example 6, “J. Characterization of bovine naive stem cells”. Results are shown in Figures 12 and 13. Bovine naive stem cells generated under PXGL conditions described above exhibited naive specific characteristics: classical dome shape morphology and SUSD2 expression. As shown in Figure 13, SUSD2 was expressed in >30% of cells at P3, and expression was maintained or increased in subsequent passages P4 and P5.Conversely, bovine expanded potential stem cells generated and maintained under bovine EPSCs conditions as described in Zhao et al., 2021 , displayed a significantly lower expression of SUSD2 and failed to display typical dome shape colony morphology compared to bovine naive stem cells. As shown in Figure 13, SUSD2 was expressed in <10% of cells, and fewer cells expressed SUSD2 in subsequent passages.Example 6. Recovery of naive pluripotency from bovine non-naive outgrowthBackground

[0197] To improve the yield, quality, and long-term maintenance of naive stem cell lines, a solution was devised for the occurrence of “non-passageable” embryo-derived colonies that do not exhibit naive stem cell morphology (“non-naive” stem cells) (Figure 14-A). Non-passageable embryo-derived colonies exhibit the primed pluripotent-specific flattened shape, which means that they are unusable for further naive cells passage. It is generally assumed that primed pluripotent stem cells can be derived from postimplantation epiblast. After 7 days of culture, these “non-passagable” outgrowths appear to be comparable to the embryonic day-14 and may contain epiblast population (Perez- Gomez et al., 2021). Therefore, it was hypothesized that bovine primed pluripotent stem cells could be derived from day-5 to day-7 outgrowths which presented primed pluripotent stem cells morphology. If successful, the established primed pluripotent stem cells could then be reset to a naive stem cell status using an approach based on the protocol described by Guo et al. (2017).

[0198] In the current protocol, colonies were selected from day 5-7 embryo outgrowths and more specifically from those that exhibited a morphology that is atypical for naive stem cells, but is more typical of a more advanced developmental status (e.g. primed morphology). The selected cells then were subjected to primed embryonic stem cell conditions (Bogliotti, 2018). After establishing bovine primed pluripotent stem cell lines, the goal was to recover naive pluripotency through an optimized ‘resetting protocol’.

[0199] Guo et al. (2017) have initially reported a technique called “Resetting” to reverse primed into naive ES cells using a Histone Deacetylase (HDAC) inhibitor, and is referred to herein as resetting though epigenetic modification. Additionally, Bayerl et al. (2021) have reported a one-step technique for reversing primed stem cells to a naive developmental status by directly replacing primed culture medium to naive medium.

[0200] Because the derivation of naive stem cells from embryos is quite challenging, requiring a constant supply of embryos, deep knowledge of embryology, and specific techniques for embryo outgrowth derivation, the resetting of pluripotency (Guo et al., 2017; Bredenkamp et al., 2019b) may present an alternative and cost-effective approach for deriving bovine naive stem cells. Herein, an optimized resetting technique to convert bovine primed pluripotent stem cells to naive stem cells is described. Efficiency of the resetting method was evaluated using naive stem cell specific markers.

[0201] Compared to media for primed stem cells (e.g. AFX), bovine naive stem cell media (PXGL) contains a MEK1 / 2 inhibitor, which can induce differentiation of fibroblasts. MEFs cultured in PXGL media show differentiation into myofibroblasts, including overproduction of extracellular matrix and appearance of alpha smooth muscle actin (Figure 16). According to fibroblast pathology (Tai et al., 2021), myofibroblasts are considered as the key cellular mediator of fibrosis. Because naive stem cells possess more epithelial-like characteristics (Pham et al., 2022), fibrosis-inducing environments may affect the quality of naive stem cells. Based on this, the addition into PXGL media of low concentrations of FGF2 or a p38 MAPK inhibitor was tested.ProtocolsA. Composition of bovine primed stem cell medium (AFX medium)• DMEM / F12 : Neurobasal 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 p-mercaptoethanol + 50 lU / ml Pen / Strep + 20 ng / ml bFGF + 20 ng / ml Activin A + 2 pM XAV-939.B. Isolate bovine primed pluripotent stem cells1 . Derive bovine embryo outgrowth under naive stem cell condition.2. At day 5 to 7, select outgrowths which show a primed morphology (Figure 14-A) and mechanically remove TE cell colonies to expose the ICM of the colony.3. Mechanically remove feeder cells around ICM colonies by lightly scratching with a pipette tip4. Scrape ICM colonies with 125 pm bore pipette to break into small clusters and transfer ICM cell clusters onto freshly prepared feeder cells under primed pluripotent stem cell conditions in the micro-drop (passage 1).C. Establishment of bovine primed pluripotent stem cells1 . Culture cells from ‘Step 4’ of ‘Protocol: Isolate bovine primed pluripotent stem cells’.2. Change medium every day.3. At day 7 after initiating culture under primed pluripotent stem cell conditions, passage cells using an enzyme-based approach i.e. ‘EDTA-subculture’ (steps 4 to 9).4. Remove culture medium from the culture drop.5. Wash culture drop with 50 pl DPBS without Ca2+and Mg2+, 3 times.6. Wash culture drop with 50 pl 0.5 mM EDTA in DPBS without Ca2+and Mg2+, 2 times.7. Add 50 pl 0.5 mM EDTA in DPBS without Ca2+and Mg2+, incubate 2 min at 38.5 °C.8. Remove EDTA solution very carefully.9. Add 50 pl fresh AFX medium directly onto the culture surface.10. Transfer cells clusters into 600 pl tube.11 . Dilute cell suspension with fresh AFX medium according to desired dilution ratio.12. Note: add 50 pl fresh medium at this passage to dilute cells by a 1 :2 ratio13. Seed cells onto freshly prepared MEF in the micro-drop (passage 2).14. Note: transfer 50 pl cell suspension into each drop at the passage.15. Culture cell cultures in 5% CO2, humidified incubator at 38.5 °C.16. Repeat ‘EDTA-subculture’17. Transfer cell clusters from two drops into the same 600 pl tube.18. Seed cells onto freshly prepared MEF in one well of a 12-well plate.19. Optional: growth factor reduced Matrigel or Geltrex coated plate can be used here for feeder-free culture.20. Culture cells in 5% CO2, humidified incubator at 38.5 °C.21. Repeat EDTA-subculture every 3-4 days beginning with 1 :6 to 1 :10 dilution ratio and diluting further as described herein.a. Step 4-7: 50 pl -> 300 pl b. Step 9: 50 pl -> 1 ml c. Step 10: 600 pl tube -> 15 ml conical tubeD. Cryopreservation / thawing of bovine primed pluripotent stem cells can be carried out using standard techniques.E. Composition of 2x freezing medium• 80% AFX medium + 20 % DMSO (Sigma, D2650) + 10 pM Y27632.F. Composition of epigenetic resetting medium• DMEM / F12: Neurobasal medium mixture (1 :1) + 0.5X N2 supplement + 1X B27 supplement + 1X Glutamax + 1X NEAA + 50 pg / ml BSA + 50 pg / ml Ascorbic acid + 0.1 mM p-mercaptoethanol + 50 lll / ml Pen / Strep + 1 pM PD3025901 + 10 ng / ml LIF + 1 mM Valproic acid.G. Epigenetic resetting1. Culture bovine primed pluripotent stem cells in cell cultureware with seeding density at 1 x 104to 2 x 104 / cm2.2. Prepare resetting (steps 3 to 9 below)3. Remove medium by gentle aspiration.4. Wash with 1 ml DPBS without Ca2+and Mg2+, 3 times.5. Wash with 300 pl 0.5 mM EDTA in DPBS without Ca2+and Mg2+. Other embodiments may use alternate chelating agents such as EGTA.6. Add 300 pl 0.5 mM EDTA in DPBS without Ca2+and Mg2+into well and incubate for 5 min at 38.5 °C.7. Carefully remove EDTA solution and add 1 ml of fresh AFX medium + 10 pM Y27632 to lift cell clusters8. Transfer cells into 15 ml conical tube and do pipetting to isolate single cells.9. Count the number of cells and seed 1 x 104to 2 x 104 / cm2cells onto freshly prepared MEFs with 1 ml of AFX medium +10 pM Y27632.10. Two days after cell plating, change medium to epigenetic resetting medium.11.Two days after first medium change (step 10), change medium to fresh epigenetic resetting medium.12. One day after completing step 11 , change medium to fresh PXGL medium and change medium to fresh PXGL daily until day 9.H. Direct resetting1 . Prepare resetting by step 1 to 9 from the protocol for Epigenetic resettting.2. Two days after cell plating, change medium directly to PXGL medium, with or without 10 ng / ml FGF2 and / or a p38 MAPK inhibitor (1 pM SB203580 or 1 pM BIRB796).3. Change medium daily until day 5-9.I. Establishment of reset bovine naive stem cells1 . Protocol begins from the last step of each resetting protocol (epigenetic and direct).2. Conduct single cell dissociation (Step 3-8 below)3. Remove medium by gentle aspiration.4. Wash with 1 ml DPBS without Ca2+and Mg2+, 3 times.5. Add 300 pl TrypLE into the well and incubate for 3 min at 38.5 °C.6. Do pipetting to dissociate to single cells.7. Transfer cell suspension into 15 ml conical tube.8. Fill the tube to 10 ml with DMEM / F12 + 0.1 % BSA (or other suitable washing medium, for example DPBS with Ca2+and Mg2+, HBSS Ca2+and Mg2+, DMEM, DMEM / F12, ADMEM, ADMEM / F12 or Neurobasal medium).9. Centrifuge at 300 xg for 5 min. Discard supernatant.10. Break pellet with 1 ml of fresh PXGL medium + 10 pM Y27632. Centrifuge at 300 xg for 5 min.11. Discard supernatant and break pellet with 1 ml of fresh PXGL medium + 10 pM Y27632.12. Count the number of cells using hemacytometer, and seed 1.5 to 2.5 x 104, optionally 2 to 2.5 x 104reset cells I cm2onto freshly prepared MEF with fresh PXGL medium + 10 pM Y27632. For a 12 well plate, 6 to 10 x 104cells I well with1 ml of fresh PXGL medium + 10 pM Y27632. For a 35 mm dish, 1.3 to 2.2 x 105cells / dish with 2 ml of fresh PXGL medium + 10 pM Y27632.13. Change medium daily.14. Subculture cells every 3-4 days.J. Characterization of bovine naive stem cells1 . Bovine naive stem cells can be characterized by live staining of naive specific cellsurface marker, Sushi Containing Domain2 (SUSD2) and naive specific nuclear protein, TFCP2L1 reported by Bredenkamp et al. (2019a).2. Change medium to fresh PXGL medium containing working concentration SUSD2 antibody conjugated with fluorochrome.3. Incubate cells in the humidified 5% 02 / 6.8% CO2 incubator at 38.5°C for 1 hour4. Rinse wells with fresh PXGL medium, 5 times and then fill wells with fresh PXGL medium.5. Observe under fluorescence microscope.Results

[0202] Bovine primed pluripotent stem cells can be derived successfully from outgrowths showing an atypical naive morphology (Figure 14). Under naive stem cell culture conditions, some day-6 to day-7 outgrowths showed a flattened ICM-like colony similar to typical primed pluripotent stem cell morphology (Figure 14-A). At early passages, flattened ICM-like cells (Figure 14-B, black arrow) were growing heterogeneously with trophectodermal and endodermal lineage cells (Figure 5A, black arrow). At passage 4 to 5, homogeneous primed pluripotent cell colonies started to appear (Figure 14-C, black arrow). After passage 6, homogeneous primed pluripotent cell morphology was observed (Figure 14-D).

[0203] The culture of bovine primed pluripotent stem cells on feeder cells can be converted to a feeder free system (Figure 14-E). When on the feeder-free system, bovine primed pluripotent stem cells grow as a proliferative monolayer pattern; however, cells form distinct colonies again when grown on feeder cells (Figure 14-F).

[0204] Bovine primed pluripotent stem cells are successfully recovered from cryopreservation (Figure 15). Small colonies of primed pluripotent cells (Figure 15, black arrow) are visible 48 hours after thawing and form distinct colonies 96 hours after thawing.

[0205] The resetting protocol induced a significant morphological change of primed pluripotent stem cells during and after the procedure (Figure 17 and 18). The direct resetting protocol yields a dramatic change of cell morphology within 24 hours (Figure 17, upper panel). After 2-3 passages following the resetting process, directly reset cells show small and compact naive-like colonies (Figure 17, lower panel). Cells treated via the epigenetic resetting protocol show a slower morphological change during 8-10 days of the resetting protocol (Figure 18, upper panel). The change of morphology is initiated from the centre of the colony with intensive cell death, then the colony grows slowly during the epigenetic resetting protocol. After 2-3 passages, epigenetically reset cells show small- and compact naive like colonies. Generally, reset cells from the two protocols present similar morphological characteristics, but epigenetic resetting shows brighter and more characteristic 3D shaped naive colonies.

[0206] With the addition of FGF2 or p38 MAPK inhibitor, reset cells present more compact and smooth naive-like colonies (Figure 19), and can be maintained under more stable conditions such as longevity, proliferation speed, cell death. Furthermore, the addition of FGF2 or p38 MAPK inhibitor promote an improvement in the quality of feeder layer which was negatively affected by MEK 1 / 2 inhibitor in PXGL media. MEFs cultured in enhanced outgrowth media show spindle-shaped morphology and significantly reduced ECM secretion (Figure 16).

[0207] Reset bovine stem cells express well characterized naive stem cell specific markers (Bredenkamp et al., 2019a) (Figure 21 ). Compacted and bright bovine reset colonies show a positive signal for SUSD2 (Figure 21 A), but flattened colonies which are typical of primed pluripotent stem cell colonies fail to show any signal for SUSD2. (Figure 21 -A, dotted circles). All pluripotent stem cell colonies express the pluripotent marker SOX2, but flattened colonies at early passages after resetting don’t express TFCP2L1 which is a naive-specific nuclear protein (Figure 21-B). Based on the expression pattern of naive stem cell-specific markers, reset bovine stem cells show distinctive features of naive stem cells.

[0208] Methods for establishing bovine naive stem cells with high derivation efficiency, yield, quality, and long-term maintenance and selected examples of industrial use are depicted in Figure 22.Example 7. Establishment of stem cell colonies from embryo-derived e.q. ICM outgrowths using AXRG / AXRGL mediumProtocolsA. Stem cell colonies can be established and maintained according to the methods described in Example 5 using AXGR or AXGRL medium in place of PXGL medium.• Composition of AXRG: DMEM / F12:Neurobasal medium (1 :1 mixture) + N2B27 serum + 5 ng / ml Activin A + 2 pM XAV-939 + 1 pM BMS493 + 2 pM G66983• Composition of AXRGL: AXRG + 10 ng / ml LIFResults

[0209] Bovine naive stem cells may be derived and cultured with AXRG media (Figure 23). Beyond passage 8 or 9, colonies may flatten and begin to lose dome shape morphology despite retaining high and consistent expression levels for SUSD2 and SOX2 (Figure 24). Flat colonies may be an indicator of early differentiation, however adding bovine LIF to AXRG media (AXRGL) prevented this morphological change (Figure 25). Expression level of SUSD2 by bovine naive stem cells in AXRG media exceeds 95% at early passage after establishment and this high expression level is maintained over multiple passages in both the AXRG and AXRGL cultured cells (Figure 24). When bovine LIF is removed, colonies again lose their dome shape morphology (Figure 26). Using AXRGL media or AXRG media followed by AXRGL, bovine naive stem cell lines may be derived and remain stable with high expression levels of naive and pluripotent markers over more than 20 passages (Figure 24).Example 8. Cryopreservation of bovine naive stem cellsProtocolsA. Cryopreservation / thawing of bovine naive stem cells can be carried out using standard techniques.Composition of 2x freezing medium: 80% Naive stem cells media + 20% DMSO (Sigma, D2650) + 10 pM Y27632Naive stem cells media may be PXGL, AXRG or AXRGL.Results

[0210] Frozen bovine naive stem cells established in AXRG and PXGL media were fully recovered within 2 days after thawing (Figure 27) and exhibited a doubling time of 22-24 h, which is comparable to proliferation observed for non-frozen cells. Viability of naive stem cells after thawing was 88.11 % and 79.35% for AXRG (P8) and PXGL (P4) media, respectively (Figure 28).Example 9. Optimizing stem cell line colony replication and self-renewal ra te

[0211] As demonstrated in Example 7, bovine naive stem cells may be successfully established in AXRG and AXRGL conditions. Beyond passage 12 or so, select bovine naive stem cell lines have demonstrated longer population doubling times accompanied by degradation of naive-specific colony morphology.

[0212] For some lines of bovine naive stem cells, population doubling times may become more variable and / or increase during long-term culture (e.g. beyond p12 orp14). To reduce excessive variability during long-term culture, a variety of factors and their pathways were investigated for their effects and as demonstrated below, factors known to have inhibitory effects on CDK8 and CDK19 kinases (CDK8 / 19) demonstrated benefits.ProtocolsFor long-term culture and self-renewal of susceptible and non-susceptible bovine naive stem cell lines the following steps may be used. Lines may be established and maintained in AXRGL as described in Example 7 or other suitable outgrowth media described herein. Most notably, susceptible lines can benefit when media for outgrowth and / or maintenance further comprise a CDK8 / 19 inhibitor component, e.g. 2.5 nM CCT251545. A. When using a mechanical method for passaging of bovine naive stem cells or naive stem celllike cells the following protocol may be used:1. Prepare maintenance medium by adding 2.5 nM CCT251545 to 10 ml AXRGL medium.2. Prepare micro-drop with MEF as described in ‘Protocol: Preparation of feeder cells layer1.3. Change the MEF medium in the micro-drop to MEF: maintenance media (1 :1 mixture) 18 hours before passaging naive stem cells, embryo-derived colony, ICM colony, etc.4. If the outgrowth is showing TE growth, remove it mechanically to make ICM colony accessible.5. Divide the ICM colony into 2 to 6 clusters by mechanical dissociation and transfer the divided clusters into a new micro-drop with maintenance medium.6. After 24 hours, replace medium with fresh maintenance medium.7. Cryopreservation / thawing of bovine naive stem cells can be carried out using standard techniques.B. When using an enzymatic method for passaging of bovine naive stem cells or naive stem cell-like cells.1. Prepare maintenance medium by adding 2.5 nM CCT251545 to 10 ml AXRGL medium. Add ROCK inhibitor (10 pM Y27632) to prepared maintenance medium.2. Prepare micro-drop with MEF as described in ‘Protocol: Preparation of feeder cell layer1.3. Change the MEF medium in the micro-drop to MEF: maintenance media (1 :1 mixture) 18 hours before passaging naive stem cells, embryo-derived colony, ICM colony etc.4. If the outgrowth is showing TE growth, remove it mechanically to make ICM colony accessible.5. Remove culture medium from the culture drop.6. Wash culture drop with 60 pl DPBS without Ca2+ and Mg2+, 3 times.7. Add 60 pl TrypLE, incubate 3 min.8. Divide the ICM colony into single cells or small cell clusters with gentle pipetting.9. T ransfer cells into 1 .5 ml tube and add 500 pl DPBS with Ca2+ and Mg2+.10. Centrifuge at 300 xg, 5 min.11. Discard supernatant and break pellet using gentle pipetting of 1 ml maintenance medium.12. Centrifuge at 300 xg, 5 min.13. Discard supernatant and break pellet with maintenance medium from new microdrop.14. Seed cells into new micro-drop.15. Cryopreservation / thawing of bovine naive stem cells can be carried out using standard techniques.Results

[0213] Stem cell colonies can be established and maintained according to the methods described in the above Examples using media supplemented with an inhibitor of CDK8 and CDK19 kinases such as AXRGL + CCT251545. Preliminary work demonstrated that for these susceptible lines undesirable trends in doubling times and colony morphology were reversed when media was supplemented with an inhibitor of Wnt signaling and CDK8 / 19 (CCT251545). However, concentration of the Wnt signaling and CDK8 / 19 inhibitor was important for maintaining naive-specific colony morphology.

[0214] To evaluate the impact of differing levels of CCT251545 on morphology of bovine naive stem cell lines, lines were established in AXRGL as described in Example 7 and for p8 through p12, subsamples were maintained in control AXRGL and AXRGL supplemented conditions with one of 6 different levels of CCT251545. As illustrated in Figure 29 (A), when media contained levels of CCT251545 of 5nM and higher, bovine naive stem cell colonies exhibited less-well defined colony shape with irregular and unclear borders. Also, with higher concentrations of CCT251545, colonies demonstrated asymmetrical shape in contrast to the spherical dome-shape that is characteristic of naive colonies. On the other hand, when maintained in AXRGL + 2.5 nM CCT251545 conditions, colonies exhibited morphological features comparable to the subsample maintained in AXRGL control conditions through p12 (Figure 29 (A)).

[0215] For the subsample of colonies cultured in AXRGL + CCT251545 media, doubling times declined and by p15-p18 were equivalent to doubling times of all treatments at p10 to p12 (Figure 29 (B)). Whereas for the control fraction which included no CCT251545, doubling times remained notably higher than pre-p13 levels. Additionally, bovine naive stem cell colonies in AXRGL + CCT251545 conditions maintained naive- specific colony morphology with higher numbers of passages as opposed to the degradation of characteristic shape exhibited by susceptible colonies in control conditionsexperiencing increases in doubling times (Figure 29 (C)). Similar results for control and AXRGL + CCT251545 treatment groups were observed when frozen-thawed stem cell lines were used.References:Bayerl J, Ayyash M, Shani T, Manor YS, Gafni O, Massarwa R, Kalma Y, Aguilera- Castrejon A, Zerbib M, Amir H (2021) Principles of signaling pathway modulation for enhancing human naive pluripotency induction. 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Claims

CLAIMS:1 . A method of deriving a population of bovine naive stem cells from a bovine embryo, the method comprising: a) providing a feeder cell layer; b) isolating cells of a bovine embryo to obtain a population of cells comprising bovine naive stem cell-like cells; c) optionally dissociating the population of cells comprising bovine naive stem celllike cells to obtain one or more clusters of cells comprising bovine naive stem cell-like cells; d) transferring the population of cells comprising bovine naive stem cell-like cells or the one or more clusters of cells comprising bovine naive stem cell-like cells to the feeder cell layer; and e) culturing the population of cells comprising bovine naive stem cell-like cells or the one or more clusters of embryo-derived cells in the presence of outgrowth medium, the outgrowth medium comprising: i) a MEK / ERK inhibitor component, a Wnt inhibitor component, a PKC inhibitor component, a LIF component, and optionally a ROCK inhibitor component, or ii) an Activin A component, a Wnt inhibitor component, a PKC inhibitor component, a RAR inhibitor component, optionally a ROCK inhibitor component and optionally a LIF component, to induce attachment and outgrowth of one or more colonies comprising bovine naive stem cells; thereby deriving a population of bovine naive stem cells.

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

3. The method of claim 1 , wherein step b) comprises isolating cells by removing the Zona Pellucida (ZP) of the bovine embryo.

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

5. The method of any one of claims 1 to 4, wherein the feeder cell layer is at a density of about 1 .5 to 7.5 x 104 / cm2, preferably about 3.0 x 104 / cm2.

6. The method of any one of claims 1 to 4, wherein the feeder cell layer is a feeder cell micro-drop.

7. The method of claim 6, wherein the feeder cell layer is at a density of about 0.5 x 104to 2.5 x 104cells I drop, preferably about 1 .0 x 104cells I drop.

8. The method of any one of claims 1 to 7, wherein the feeder cell layer is prepared at least about 18 hours or about 24 hours before step b).

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

10. The method of any one of claims 1 to 9, wherein the feeder cells are mitotically inactivated, optionally prepared using mitomycin treatment, gamma irradiation, or alcohol fixation.11 . The method of any one of claims 1 to 10, wherein the feeder cells are mouse embryonic fibroblast (MEF) cells, bovine fetal fibroblasts (bFF), bovine endometrial stromal cells, human fetal muscle cells, MEF SNL line cells, human fetal fibroblasts, human adult fallopian tubal epithelial cells, human dermal fibroblasts, human amniotic mesenchymal cells, human amniotic epithelial cells, mouse bone marrow stromal cells, murine amniocytes, human amniocytes, human foreskin fibroblasts, human amniotic mesenchymal cells, pericellular matrix of decidua-derived mesenchymal cells, human endometrial stromal cells, or rat embryo fibroblast cells.

12. The method of any one of claims 1 to 11 , wherein isolating cells from the bovine embryo in step b) comprises immunosurgery.

13. The method of claim 12, wherein an anti-bovine serum antibody is used for the immunosurgery.

14. The method of claim 13, wherein a complement serum solution is used for washing and / or incubating the cells following the immunosurgery.

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

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

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

18. A method of generating bovine naive stem cells from bovine non-naive stem cells, the method comprising: a) providing a population of cells comprising bovine non-naive stem cells in a culture environment comprising feeder cells in primed stem cell medium; and b) replacing the medium with outgrowth medium for at least or about 4 days, optionally about 5 to about 9 days; wherein the outgrowth medium comprises: i) a MEK / ERK inhibitor component, a Wnt inhibitor component, a PKC inhibitor component, a LIF component and optionally a ROCK inhibitor component; or ii) an Activin A component, a Wnt inhibitor component, a PKC inhibitor component, a RAR inhibitor component, optionally a ROCK inhibitor component and optionally a LIF component; thereby generating bovine naive stem cells.

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

20. The method of claim 19, wherein the cells are cultured prior to replacing the medium with outgrowth medium for at least or about 40 hours, optionally about 2 days to about 4 days.21 . The method of any one of claims 18 to 20, wherein the outgrowth medium further comprises a CDK8 / 19 inhibitor.

22. The method of any one of claims 18 to 21 , wherein the population of bovine non- naive stem cells comprises primed pluripotent stem cells.

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

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

25. The method of any one of claims 18 to 24, wherein the feeder cells are mitotically inactivated feeder cells, optionally prepared using mitomycin treatment, gamma irradiation, or alcohol fixation and / or the feeder cells have been adapted to primed stem cell medium prior to step a).

26. The method of any one of claims 18 to 25, further comprising, prior to step b): i. culturing the bovine non-naive stem cells in the primed stem cell medium for at least or about 3 days; ii. dissociating and transferring the bovine non-naive stem cells to a culture environment comprising feeder cells in primed stem cell medium; and iii. culturing the cells, optionally for at least or about 10, 20, 30 or 40 hours or about 2 days.

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

28. The method of claim 26 or 27, wherein steps ii) and iii) are repeated at least one additional time, optionally two additional times.

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

30. The method of any one of claims 18 to 29, wherein the cells of step b) are seeded at a seeding density of 1 .5 to 2.5 x 104cells / cm2, optionally about 2 x 104to about 2.5 x 104cells / cm2.31 . The method of any one of claims 18 to 30, further comprising c) passaging the naive stem cells onto freshly prepared feeder cells in outgrowth medium, optionally wherein the naive stem cells are seeded at a density of about 1 .5 x 104to about 2.5 x 104cells I cm2.

32. The method of any one of claims 18 to 31 , wherein the feeder cells are seeded at a seeding density of about 1 .5 to about 7.5 x 104cells / cm2, optionally about 3 x 104cells / cm2.

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

34. The method of claim 33, wherein steps iii) and iv) are repeated at least one additional time, optionally two additional times.

35. A method of maintaining a population of bovine naive stem cells, the method comprising: a) providing a feeder cell layer; b) providing a population of cells comprising bovine naive stem cells; c) optionally dissociating the population of cells comprising bovine naive stem cells to obtain one or more clusters of cells comprising bovine naive stem cells; d) transferring the population of cells comprising bovine naive stem cells or the one or more clusters of cells comprising bovine naive stem cells to the feeder cell layer; and e) culturing the population of cells comprising bovine naive stem cells or the one or more clusters of bovine naive stem cells in the presence of maintenance medium, the maintenance medium comprising: i) a MEK / ERK inhibitor component, a Wnt inhibitor component, a PKC inhibitor component, a LIF component, optionally a ROCK inhibitor component; or ii) an Activin A component, a Wnt inhibitor component, a PKC inhibitor component, a RAR inhibitor component, optionally a ROCK inhibitor component, and optionally a LIF component; to induce attachment and outgrowth of one or more colonies comprising bovine naive stem cells; thereby maintaining a population of bovine naive stem cells.

36. The method of claim 35, wherein the maintenance medium further comprises a CDK8 / 19 inhibitor37. The method of claim 35 or 36, wherein the feeder cell layer is at a density of about 1.5 to 7.5 x 104 / cm2, preferably about 3.0 x 104 / cm2.

38. The method of claim 35 or 36, wherein the feeder cell layer is a feeder cell microdrop.

39. The method of claim 38, wherein the feeder cell layer is at a density of about 0.5 x 104to 2.5 x 104cells I drop, preferably about 1 .0 x 104cells I drop.

40. The method of any one of claims 35 to 39, wherein the feeder cell layer is prepared at least about 18 hours or about 24 hours before step b).41 . The method of any one of claims 35 to 40, wherein the feeder cell layer has been adapted to the maintenance medium.

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

43. The method of any one of claims 35 to 42, wherein the feeder cells are mouse embryonic fibroblast (MEF) cells, bovine fetal fibroblasts (bFF), bovine endometrial stromal cells, human fetal muscle cells, MEF SNL line cells, human fetal fibroblasts, human adult fallopian tubal epithelial cells, human dermal fibroblasts, human amniotic mesenchymal cells, human amniotic epithelial cells, mouse bone marrow stromal cells, murine amniocytes, human amniocytes, human foreskin fibroblasts, human amniotic mesenchymal cells, pericellular matrix of decidua-derived mesenchymal cells, human endometrial stromal cells, or rat embryo fibroblast cells.

44. A bovine naive stem cell produced using the method of any one of claims 1 to 43.

45. A use of the bovine naive stem cell of claim 44 in a breeding scheme or genetic improvement program.

46. A use of the bovine naive stem cell of claim 44 for multiplying preimplantation embryos, optionally having desirable genetic characteristics; deriving primordial germ cells and / or gametes, optionally for in vitro breeding programs and / or transplantation into surrogates, and / or developing and delivering veterinary medical biologicals or therapeutics.

47. A method of preparing a feeder cell layer micro-drop, the method comprising: a) contacting a growth surface with a volume of coating solution;b) incubating the surface in contact with the drop of coating solution such that a layer of coating solution is deposited on the surface; c) removing the coating solution and optionally washing the surface; d) applying a first volume of growth medium, optionally comprising feeder cells, to the layer of coating solution deposited on the surface; e) overlaying a layer of a hydrophobic fluid, optionally mineral oil, over the first volume of growth medium; f) adding a second volume of growth medium, optionally comprising feeder cells, to the first volume of growth medium, wherein the first volume of growth medium and / or the second volume of growth medium comprises feeder cells, thereby preparing a micro-drop comprising feeder cells; and g) incubating the micro-drop comprising feeder cells such that a layer of the feeder cells attaches to the layer of coating solution deposited on the surface; thereby preparing a feeder cell layer micro-drop.

48. The method of claim 47, wherein the volume of coating solution is about 20-30 pl, optionally about 25 pl, and / or the coating solution comprises gelatin, optionally about 0.05%-0.15% gelatin, optionally about 0.1 % gelatin, optionally the layer of coating solution deposited on the surface has an area of about 0.33-0.39 cm2, optionally about 0.36 cm2.

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

50. The method of any one of claims 47 to 49, wherein the first volume of growth medium and / or second volume of growth medium is about 20-40 pl, optionally about 30 pl, and / or wherein the first volume of growth medium and second volume of growth medium combined is about 50-70 pl, optionally about 60 pl.51 . The method of any one of claims 47 to 50, wherein the feeder cells are mitotically inactivated, optionally prepared using mitomycin treatment, gamma irradiation, or alcohol fixation; the feeder cells are MEF cells, bovine fetal fibroblast (bFF) cells,bovine endometrial stromal cells, human fetal muscle cells, MEF SNL line cells, human fetal fibroblasts, human adult fallopian tubal epithelial cells, human dermal fibroblasts, human amniotic mesenchymal cells, human amniotic epithelial cells, mouse bone marrow stromal cells, murine amniocytes, human amniocytes, human foreskin fibroblasts, human amniotic mesenchymal cells, pericellular matrix of decidua-derived mesenchymal cells, human endometrial stromal cells, or rat embryo fibroblast cells; and / or the number of feeder cells in the micro-drop is about 0.5 x 104to 2.5 x 104cells, optionally about 1 .0 x 104cells.

52. The method of any one of claims 47 to 51 , wherein the micro-drop comprising feeder cells in step g) is incubated for at least 10 hours, optionally about 1 day.

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