Ungulate pluripotent stem cell culture medium

Abstract

The present invention relates to an ungulate pluripotent stem cell culture medium, the use thereof and a method to culture ungulate pluripotent stem cells. In particular, the present invention relates to a culture medium comprising a Wnt pathway inhibitor and a SMAD2 / 3 activator, wherein said culture medium comprises less than or equal to 1,000 mg / L protein.

Description

[0001] UNGULATE PLURIPOTENT STEM CELL CULTURE MEDIUM

[0002] Technical field of the invention

[0003] The present invention relates to an ungulate pluripotent stem cell culture medium, the use thereof and a method to culture ungulate pluripotent stem cells. In particular, the present invention relates to a culture medium comprising a Wnt pathway inhibitor and a SMAD2 / 3 activator, wherein said culture medium comprises less than or equal to 1,000 mg / L protein.

[0004] Background of the invention

[0005] Cell-based production is replacing animal-based production, with technologies such as cultivated meat production emerging as a promising solution to address the growing concerns related to conventional livestock farming, including environmental impact, animal welfare, and food security. Cultivated meat, also known as lab-grown or cell-based meat, involves the production of meat products through the cultivation and differentiation of animal cells in vitro. This innovative approach within cellular agriculture offers a sustainable and ethical alternative to traditional animal agriculture.

[0006] In the context of cultivated meat production, the utilization of pluripotent stem cells from livestock animals is a novel approach for reaching price-parity with conventional animal-sourced products, such as conventionally farmed meat. Pluripotent stem cells possess the remarkable ability to differentiate into all specialized cell types of the adult organism, including muscle, fat, liver, heart, and blood cells. In culturing pluripotent stem cells, it becomes possible to produce cultivated meat products that closely mimic the taste, texture, and nutritional composition of conventionally farmed meat from animal slaughter.

[0007] The field of cellular agriculture is dominated by the use of multipotent or unipotent adult stem cells harvested from animals ( / .e. myosatellite cells or fibroblasts) which are poorly suited for large scale production due to their mortality, requirement for repeated biopsy sampling, slow division rates and typically use of scaffolds. The use of pluripotent stem cells offers several advantages for cultivated meat production. Pluripotent stem cells are immortal and therefore inexhaustible, exhibit faster growth rates enabling shorter cultivation times, can be cultured in suspension at higher cell densities and without scaffolds. Additionally, the versatility of pluripotent stem cells in differentiating into any cell type provides the potential for producing a wider range of biological products with diverse compositions and applications than those from multipotent or unipotent cells. Thus, this invention also has applications for the production of any other biological product traditionally extracted from ungulates, such as medicines, health supplements and cosmetic products.

[0008] However, the isolation of pluripotent stem cells from the embryos of livestock animals, or the production via reprogramming for induced pluripotent stem cells (iPSCs), is a challenging task with limited examples to date. All pluripotent stem cells are inherently sensitive to environmental cues and any deviations in culture conditions can lead to a loss of pluripotency, reduced rates of cell division, or unintended / uncontrolled differentiation. Published and reported culturing systems for bovine or porcine pluripotent stem cells rely on genetic modification to overexpress pluripotency proteins (Habekost et al, 2019), support cells, such as embryonic fibroblasts, (Bogliotti et al, 2018; Zhi et al, 2021), complex and protein-rich mediums containing ill-defined supplements or expensive growthfactors, such as FGF2 (Choi et al, 2019; Soto et al, 2021), all of which are undesirable and pose a challenge for achieving consistent and cost-effective upscaled production.

[0009] There is a need for novel culturing systems that specifically maintains ungulate pluripotent stem cells without genetic modification, preferably without supportcells and simultaneously maintain expression of key pluripotency genes as well as a need for developing simpler culturing medium for pluripotent stem cells from ungulates, such as livestock animals.

[0010] WO23177181 Al relates to a medium composition comprising LDN-193189 for culturing porcine pluripotent stem cells. The medium furthermore comprises protein-rich compounds such as Knockout serum replacement.

[0011] WO2019245278 Al relates to a medium composition comprising CHIR99021 for culturing porcine pluripotent stem cells. The medium furthermore comprises protein-rich compounds such as Knockout serum replacement.

[0012] W02019140260 Al relates to a medium comprising FGF2, IWR-1, Activin A, and protein-rich compounds such as N2 and B27 supplement.

[0013] Hence, an improved culture medium for culturing ungulate pluripotent stem cells would be advantageous, and in particular a simpler and thereby a more cost- effective culture medium would be advantageous. A culture medium for ungulate pluripotent stem cells that promotes superior growth, pluripotency, aggregate formation, and suspension culture compared to a more complex medium would also be advantageous. A fully defined, low-protein medium is also highly advantageous for regulatory approval, as it contains fewer components and excludes animal-derived ingredients, thereby reducing variability and simplifying compliance with safety and ethical standards.

[0014] Summary of the invention

[0015] An object of the present invention relates to providing an ungulate pluripotent stem cell culture medium that can be used to culture ungulate pluripotent stem cells for an extended period of time whilst retaining the pluripotency of said stem cells. In particular, it is an object of the present invention to provide a culture medium that solves the above-mentioned problems of the prior art with complex and costly culture mediums.

[0016] The present invention introduces novel culturing mediums for growing pluripotent stem cells from ungulates to produce any cell-based product, such as cultivated meat, exemplified using porcine pluripotent stem cells and bovine pluripotent stem cells. By utilizing a simple and cost-effective culture medium, this invention enables efficient and improved growth and maintenance of pluripotent stem cells from ungulates, thereby enhancing scalability and addressing the challenges faced by any industry relying on cell-based production, such as the cellular agriculture industry.

[0017] The unique aspects of this invention lie in its ability to use a minimum combination of factors that eliminate the need for genetic modification to maintain pluripotency of pluripotent stem cells from ungulates. By carefully controlling the culture conditions modulating specific signalling pathways, this invention offers precise regulation of pluripotency. Furthermore, the inclusion of suspension culture options using bioreactor systems enhances scalability and cell densities, addressing the challenges faced by the industry.

[0018] Compared to existing methods, this invention represents a significant advancement in cell-based production, such as cultivated meat production. It offers improved efficiency, reduced costs, and enhanced scalability, while promoting sustainability and ethical practices. By harnessing the potential of pluripotent stem cells from ungulates, this invention paves the way for a more sustainable and commercially viable approach to meat production.

[0019] Thus, an aspect of the present invention relates to an ungulate pluripotent stem cell culture medium comprising a Wnt pathway inhibitor and a SMAD2 / 3 activator, wherein said culture medium comprises less than or equal to 1,000 mg / L protein.

[0020] Another aspect of the invention relates to the use of the ungulate pluripotent stem cell culture medium according to the present invention for culturing ungulate pluripotent stem cells.

[0021] Yet another aspect of the present invention relates to a method for culturing ungulate pluripotent stem cells, said method comprising the steps: a) mixing a base medium with a Wnt pathway inhibitor and a SMAD2 / 3 activator to obtain a culture medium, b) adding the culture medium of step a) to ungulate pluripotent stem cells, and c) culturing the ungulate pluripotent stem cells in a culture vessel, wherein said culture medium comprises less than or equal to 1,000 mg / L protein.

[0022] Brief description of the figures

[0023] Figure 1: Morphology images of porcine embryonic stem cells (pESCs) (MT002 and MT003 cell lines) in feeder-dependent culture, in medium comprising FGF2, IWR-1 and Activin A (termed FF3 medium), and in medium comprising IWR-1 and Activin A (termed FF2 medium). Figure 2: Immunofluorescence staining of three key pluripotency markers (Oct4, Nanog and Sox2) and DAPI of porcine embryonic stem cells (MT002 cell line) after five passages in FF3 medium.

[0024] Figure 3: Immunofluorescence staining of three key pluripotency markers (Oct4, Nanog and Sox2) and DAPI of porcine embryonic stem cells (MT002 cell line) after five passages in FF2 medium.

[0025] Figure 4: Immunofluorescence staining of three key pluripotency markers (Oct4, Nanog and Sox2) and DAPI of porcine embryonic stem cells (MT003 cell line) after five passages in FF3 medium.

[0026] Figure 5: Immunofluorescence staining of three key pluripotency markers (Oct4, Nanog and Sox2) and DAPI of porcine embryonic stem cells (MT003 cell line) after five passages in FF2 medium.

[0027] Figure 6: Comparison of pluripotency levels (% triple positive cells of total DAPI) of pESC (MT002 and MT003 cell line) in FF2, FF3 or commercial hiPSC medium (E8).

[0028] Figure 7: Homogenous aggregate formation of MT003 cell line in suspension culture using either FF2 or FF3 medium after single-cell inoculation (DIVO), 24 hours (DIV1) and 48 hours (DIV2).

[0029] Figure 8: Pluripotency quality after 45 passages in FF3. Immunofluorescence staining of three key pluripotency markers (Oct4, Nanog and Sox2) and DAPI of porcine embryonic stem cells (MT003 cell line) after 45 passages in FF3 medium. Figure 9: Pluripotency quality in Low Protein Medium and High Protein Medium containing FGF2, IWR-1 and Activin A. Immunofluorescence staining of three key pluripotency markers (Oct4, Nanog and Sox2) and DAPI of porcine embryonic stem cells (MT003 cell line) in Low protein Medium and High Protein Medium (FF3) after five passages.

[0030] Figure 10: Pluripotency quality in Low Protein Medium (FF3) of bovine embryonic stem cells (bESCs). A) Immunofluorescence staining of two key pluripotency markers (Oct4, Sox2) and DAPI in two bovine ESC lines in Low Protein Medium (FF3) compared to porcine ESCs (pESCs), and fibroblasts. B) Immunofluorescence staining of the key pluripotency marker, Nanog, and DAPI in two bovine ESC lines in Low Protein Medium (FF3).

[0031] Figure 11: Comparison of bovine ESC proliferation in High-Protein FF3 versus Low-Protein FF3 medium across two cell lines. Figure 12: A) Comparison of bESC proliferation in Low-Protein FF3 medium versus Low-Protein FF2 medium. B) Immunofluorescence staining of three key pluripotency markers (Oct4, Nanog and Sox2) and DAPI in bESC lines in Low Protein FF2 Medium.

[0032] Figure 13: Titration of FGF2 in FF3 Low-Protein Medium. Immunofluorescence staining of key pluripotency marker, Oct4, and DAPI in porcine ESCs.

[0033] Figure 14: Titration of IWR-l-endo in FF3 Low-Protein Medium.

[0034] Immunofluorescence staining of key pluripotency marker, Oct4, and DAPI in porcine ESCs.

[0035] Figure 15: Titration of Activin A in FF3 Low-Protein Medium. Immunofluorescence staining of key pluripotency marker, Oct4, and DAPI in porcine ESCs.

[0036] Figure 16: Comparison of several SMAD2 / 3 activators with no SMAD 2 / 3 activator in Low-Protein Medium comprising FGF2, IWR-1 and a SMAD2 / 3 activator. Immunofluorescence staining of key pluripotency marker, Oct4, and DAPI in porcine ESCs.

[0037] Figure 17: Comparison of several WNT pathway inhibitors with no WNT pathway inhibitor in Low-Protein Medium comprising FGF2, Activin A and a Wnt pathway inhibitor. Immunofluorescence staining of key pluripotency marker, Oct4, and DAPI in porcine ESCs.

[0038] Figure 18: Comparison of several base media in Low-Protein FF3 Medium. Immunofluorescence staining of key pluripotency marker, Oct4, and DAPI in porcine ESCs.

[0039] Figure 19: Comparison of pESC proliferation in Low-Protein FF3 Medium containing transferrin versus Low-Protein FF3 Medium without transferrin.

[0040] Figure 20: Pluripotency quality in Low-Protein FF3 Medium containing transferrin versus Low-Protein FF3 Medium without transferrin. Immunofluorescence staining of key pluripotency marker, Oct4, and DAPI in pESCs.

[0041] Figure 21: Comparison of porcine ESC proliferation in High-Protein FF3 versus Low-Protein FF3 medium in suspension culture over time.

[0042] Figure 22: Superior aggregate formation of pESC line MT002 in Low-Protein FF3 versus High-Protein FF3 medium in suspension after 4 Days in vitro (DIV, 96 hours).

[0043] Figure 23: Pluripotency quality in High-Protein FF3 versus Low-Protein FF3 medium in suspension. Immunofluorescence staining of key pluripotency marker, Oct4, and DAPI in porcine ESCs. The present invention will now be described in more detail in the following.

[0044] Detailed description of the invention

[0045] Definitions

[0046] Prior to discussing the present invention in further details, the following terms and conventions will first be defined :

[0047] Ungulate:

[0048] An ungulate is any mammal with hooves. In the present context, an "ungulate" is preferably hoofed mammals belonging to domesticated or domestication- susceptible species commonly associated with agricultural, livestock, or commercial production. This includes, but is not limited to, animals within the orders Artiodactyla, mammals with a foot structure that is paraxonic (e.g., cattle, pigs, sheep, goats, deer, alpacas, and llamas) and Perissodactyla (e.g., horses and donkeys). The group includes, but is not limited to, porcine, bovine, bubaline, cameline, giraffa, rhinocerotic, ovine, caprine, equine, and cervine animals.

[0049] Stem cell:

[0050] The term "stem cell" as used herein refers to a cell characterized by its capacity for both differentiation and proliferation, specifically its ability to self-renew. This cell type retains its potential for differentiation while encompassing various categories, including but not limited to pluripotent stem cells, multipotent stem cells, unipotent stem cells, and similar variants, categorized based on their distinct levels of differentiation potential.

[0051] Pluripotent Stem Cell:

[0052] The term "pluripotent stem cell" (PSC), as employed within this document, pertains to a stem cell that can be cultured in vitro, has the capacity for selfrenewal and possesses the ability to differentiate into various cell lineages found throughout the body from the three primary germ layers— ectoderm, mesoderm, and endoderm. This term also encompasses lineage-restricted undifferentiated stem cells that cannot make every terminal cell type of an organism. Pluripotent stem cells can be derived from sources such as fertilized eggs, somatic nuclear transfer embryos, germ stem cells, and similar entities. Instances of pluripotent stem cells comprise embryonic stem cells (ESC), embryonic germ cells (EG cells), induced pluripotent stem cells (iPSC), parthenote embryos and related forms. Pluripotent stem cells are considered immortal, unlike all other classes of stem cells. For the purposes of this application pluripotent stem cell also includes totipotent stem cells which have an expanded differentiation cell repertoire, being able to generate extraembryonic tissue.

[0053] Induced Pluripotent Stem Cell:

[0054] The term "induced pluripotent stem cell" (iPSC), also referred to as iPS cells or iPSCs, denotes a type of pluripotent stem cell that was generated from non- pluripotent cells with a nucleus. This process involves activating or increasing the expression of pluripotency and pluripotency-associated genes and reprogramming the non-pluripotent cells into pluripotent stem cells.

[0055] Embryonic Stem Cell:

[0056] The term "embryonic stem cell," as employed herein, signifies a pluripotent stem cell derived from the inner cell mass of a blastocyst. Pluripotent embryonic stem cells may also originate from parthenote embryos. Additionally, embryonic stem cells can be obtained from a single blastomere or by cultivating an inner cell mass without compromising the embryo's integrity.

[0057] Lineage-Restricted Stem Cell:

[0058] Lineage-restricted undifferentiated stem cells are pluripotent stem cells that do not have the capability to form a certain cell type(s), lineage(s), or developmental region(s) due to intervention to silence temporarily a gene through methods such as RNA interference, or permanently inactivate a gene through genetic modification with CRISPR or other gene editing methods.

[0059] Culture medium:

[0060] The terms 'culture media' and 'culture medium' are used interchangeably and refer to a solid or liquid substance employed to facilitate the growth of cells, including stem cells. Specifically, 'culture media' as described herein denotes a liquid substance capable of preserving stem cells in an undifferentiated state. This culture medium is typically water-based and comprises a combination of essential components such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins, including cytokines, growth factors, and hormones. These constituents are vital for cell proliferation and maintaining stem cells in an undifferentiated condition. For example, a culture media can be a synthetic culture media such as, for example, minimum essential media a (MEM-a) (HyClone Thermo Scientific, Waltham, MA, USA), DMEM / Ham's F-12, GlutaMAX (Life Technologies), Neurobasal Medium (Life Technologies), KO-DMEM (Life Technologies), DMEM / Ham's F-12 (Life Technologies), supplemented with the additives typically used in the field such as ITS supplement, N-2 supplement or B- 27™ supplement with or without vitamin A (all Life Technologies).

[0061] Pluripotency:

[0062] Stem cells that are pluripotent can potentially give rise to various cell lineages and tissues found throughout the body, while still retaining the capacity for selfrenewal. They are characterised by the expression and repression of specific genes simultaneously. Organisms of the Mammalia Class are known to have similar gene expression within their pluripotent stem cells, and co-express the following genes, proteins and markers: POU5F1 (Oct4), NANOG and SOX2. Many other genes are also expressed by mammalian PSCs including but not limited to: LIN28A, SSEA3, SSEA4, TRA-1-60, CD9, DMT3B, KLF4, and C-Myc.

[0063] Wnt pathway inhibitor:

[0064] A Wnt pathway inhibitor is any compound that can inhibit the canonical Wnt pathway. The term "Wnt pathway inhibitor" refers to any inhibitor of the pathways by which binding of the Wnt protein to extracellular receptors is either translated into the nucleus and results in transcriptional activation of a variety of genes, or otherwise results in biochemical changes that influence cell behaviour. The Wnt protein signalling pathways involve a variety of proteins including Frizzled, Disheveled, Axin, APC, GSK30, 0-catenin, LEF / TCF transcription factors, etc. The Wnt pathway inhibitor includes but is not limited to IWR-l-endo (CAS No. 1127442-82-3), IWR-1 (CAS No. 1127442-82-3), IWR-exo-1 (CAS No. 1127442- 87-8), IWR-2 (PubMED CID: 52944858), ICG-001 (CAS No. 780757-88-2), Cerberus, Wnt-C59 (CAS No. 1243243-89-1), IWP-1 (CAS No. 2074607-48-8), IWP-2 (CAS No. 686770-61-6), IWP-3 (CAS No. 687561-60-0), IWP-4 (CAS No. 686772-17-8), AZD5055, DKK-1, KY02111 (CAS No. 1118807-13-8), KYA1797K (CAS No. 1956356-56-1), JW67 (CAS No. 442644-28-2), JW74 (CAS No. 863405- 60-1), LGK974 (CAS No. 1243244-14-5), Niclosamide (CAS No. 50-65-7), Notum, NSC668036 (CAS No. 144678-63-7), CGP049090 (CAS No. 35082-49-6), BC2059 (CAS No. 1227637-23-1), ETC-159 (CAS No. 1638250-96-0), ETC-131, sFRPl, SFRP2, Shizokao ID (CAS No. 142279-42-3), XAV939 (CAS No. 284028-89-3), and WIFI.

[0065] SMAD2 / 3 activator:

[0066] A SMAD2 / 3 activator is any compound that activates SMAD2 and / or SMAD3. The SMAD abbreviation refers to the homologies to the Caenorhabditis elegans SMA ("small" worm phenotype) and MAD family ("Mothers Against Decapentaplegic") of genes in Drosophila. SMAD2 and SMAD3 are receptor-regulated SMADS and are involved in direct signalling from the TGF-p receptor. The SMAD2 / 3 activator includes but is not limited to activin A, activin B, transforming growth factor beta 1 (TGF-pi), TGF-P2, TGF-g3, Growth Differentiation Factor 1 (GDF1), GDF3, GDF8, GDF9a, GDF10, GDF11, Inducer of Definitive Endoderm 1 (IDE1), IDE2, Nodal, PD0332991 (CAS No. 827022-32-2), JNK inhibitor IX (CAS No. 312917-14- 9), LY-294002 (CAS No. 934389-88-5), JNJ-42041935 (CAS No. 1193383-09-3), and BRD-K42644990. Typically, SMAD2 / 3 activation is mediated via anaplastic lymphoma kinases such as ALK4, ALK5, ALK7.

[0067] Cell-based:

[0068] In the present context, "cell-based" refers to a method, process, or system that employs living ungulate cells as the primary biological units for in vitro cultivation or production. Such processes involve the growth, maintenance, and / or manipulation of these cells under controlled conditions to achieve cell proliferation and / or the generation of target biological materials. The cells used may be primary cells or established cell lines, and may be genetically modified or unmodified, depending on the application.

[0069] Protein:

[0070] In the present context, the term "protein" refers to polymers of greater than 50 amino acid residues bound together by amide linkages. The term "total protein" refers to the complete protein content in the culture medium. Said protein content can be measured by e.g., immunohistochemistry, flowcytometry, western blot, Enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, High- performance liquid chromatography (HPLC), and Liquid chromatography-mass spectrometry (LC / MS).

[0071] Peptidomimetic:

[0072] The term 'peptidomimetic' refers to a peptide-like molecule, a modified peptide or any other molecule that biologically mimics the action or activity of some other protein or peptide. Peptidomimetic or protein mimetic are referred to interchangeably.

[0073] Selenium:

[0074] Selenium is a chemical element with the symbol Se and atomic number 34.

[0075] Selenium is an essential trace element for normal cell growth and development in vivo and in vitro. It is incorporated into enzymes that protect cells by reducing peroxides, organic hydroperoxides, and peroxynitrites to non-harmful species.

[0076] Selenium is added to cell culture media as selenium or as a compound comprising selenium, such as selenium dioxide, or sodium selenite.

[0077] Animal origin:

[0078] In the present context, the term "animal origin" refers to any compound or substance derivable from animals which are living organisms that feed on organic matter.

[0079] Fatty acid:

[0080] In chemistry, particularly in biochemistry, a fatty acid is a carboxylic acid with an aliphatic chain, which is either saturated or unsaturated. In the present context, the fatty acids include but are not limited to free fatty acids, linoleic acid, linolenic acid, and oleic acid. The fatty acid is preferably linoleic acid or linolenic acid.

[0081] Activator of the Fibroblast Growth Factor receptor (FGFR):

[0082] The term "activator of the Fibroblast Growth Factor receptor (FGFR)" refers to any activator or inducer of FGFR, i.e. any compound that can induce downstream signaling from the FGFR. The activators of the FGFR include but are not limited to Fibroblast Growth Factor 1 (FGF1), FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF22, and DJ-1. Base medium:

[0083] In the present context, the term "base medium" refers to any cell culture medium that comprises essential components that ensure that the cells can proliferate and survive. Base medium denotes a liquid substance which is typically water-based and comprises a combination of essential components such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins, including cytokines, growth factors, and hormones. These constituents are vital for cell proliferation and survival of the cells. The base medium includes but is not limited to Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Ham's Nutrient Mixture F-12 (F-12), Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F-12), RPMI 1640, and Minimum Essential Medium Eagle - Alpha Modification (Alpha MEM). The preferred base medium is DMEM / Ham's F-12 which comprises the following components: L-Tyrosine disodium salt di hydrate

[0084] Serum:

[0085] The term "serum" refers to any animal-derived sera and includes but is not limited to fetal bovine serum (FBS).

[0086] Antibiotic:

[0087] Antibiotic refers to any type of antimicrobial substance that is active against bacteria. Antibiotic includes but is not limited to penicillin and streptomycin.

[0088] Growth factor:

[0089] In cell biology, particularly in the context of stem cell biology, a 'growth factor' refers to a naturally occurring regulatory molecule, usually a protein and / or hormone and / or peptide mimetic, that stimulates cellular growth, proliferation, maintenance of cell state and / or differentiation. These factors bind specific receptors on the surface of target cells, triggering a series of intracellular signalling pathways that influence gene expression and cellular behaviour. Growth factors are essential in the maintenance, expansion, and directed differentiation of stem cells in culture, providing critical signals that mimic the in vivo environment to sustain stem cell viability and function. Growth factors include but are not limited to epidermal growth factor (EGF), fibroblast growth factor (FGF), and transforming growth factor-beta (TGF-P). Each growth factor has specific roles and effects, depending on the type of stem cells and the desired outcome in the culture system.

[0090] Feeder cells:

[0091] The term "feeder cells' refer to mammalian cells that have become mitotically inactive, often achieved by subjecting the cells to gamma irradiation or incubating them in the presence of mitomycin-C. Non-exhaustive illustrations of such cells encompass gamma-irradiated mouse embryonic fibroblasts (MEFs).

[0092] Culture or cell culture: In the present context, the terms "culture" or "cell culture" are used interchangeably and denotes the propagation of cells in the presence of supportive media, nutrients, growth factors, support cells, or any chemical or biological substance required to achieve the desired quantity of cells or cell type.

[0093] Expression:

[0094] In the present context, the term "expression" or "express" pertains to the process wherein polynucleotides are transcribed into mRNA, and / or the subsequent process in which the transcribed mRNA is translated into peptides, polypeptides, or proteins. In cases where the polynucleotide originates from genomic DNA, expression may encompass the splicing of mRNA within a eukaryotic cell.

[0095] Doubling time:

[0096] In the present context, the doubling time is the time it takes for a cell population to double in size.

[0097] 3D culture:

[0098] The term "3 dimensional (3D)" pertains to a cellular cultivation method wherein cells are cultured within a liquid suspension without attachment to solid or semisolid surfaces. A 3D cell culture represents an artificially engineered setting in which biological cells are allowed to proliferate or interact with neighbouring cells. This technique facilitates the in vitro expansion of cells in multiple dimensions, closely mimicking their growth patterns in an in vivo environment. "3D culture" and "suspension culture" are referred to interchangeably.

[0099] 2D culture:

[0100] The term "2 dimensional (2D)" pertains to a cellular cultivation method wherein cells are cultured by attachment (adherence) to a solid or semi-solid surface. This includes culture in suspension on scaffolds such as microcarriers.

[0101] Culture vessel:

[0102] The terms "vessel" or "culture vessel" are used interchangeable and denote any container within which PSCs (Pluripotent Stem Cells) can be cultured in suspension or by adhering to the container's material. These containers can vary in size, for suspension culture ranging from milliliters, such as non-adherent plates or Erlenmeyer flasks, to thousands of liters, such as bioreactors. For adherent conditions, from small <lcm2of surface area to thousands of cm2and includes microcarriers that cells adhere to. Culture vessel include but are not limited to a petri dish, a culture flask, a multiwell plate, a shaker flask or bioreactor, a spinner flask or bioreactor, and a scaffold-based culture.

[0103] Cryopreserva tion :

[0104] In the present context, "cryopreservation" refers to the addition of a compound (a cryoprotective) to a cell suspension before freezing. The cryoprotective safeguards the cells during the processes of freezing and thawing. Non-exhaustive examples of such compounds encompass DMSO, glycerol, and polyethylene glycol (PEG)."

[0105] Product claims

[0106] An object of the present invention relates to providing an ungulate pluripotent stem cell culture medium that can be used to culture ungulate pluripotent stem cells for an extended period of time whilst retaining the pluripotency of said stem cells. In particular, it is an object of the present invention to provide a culture medium that is more simple and thereby more cost-effective than the culture mediums described in the prior art. Another object is to provide an ungulate pluripotent stem cell culture medium that improves growth, pluripotency, and aggregate formation of ungulate pluripotent stem cells. Thus, an aspect of the present invention relates to an ungulate pluripotent stem cell culture medium comprising a Wnt pathway inhibitor and a SMAD2 / 3 activator, wherein said culture medium comprises less than or equal to 1,000 mg / L protein.

[0107] An alternative aspect of the present invention relates to a culture medium for culturing ungulate pluripotent stem cells, said culture medium comprises a Wnt pathway inhibitor and a SMAD2 / 3 activator, wherein said culture medium comprises less than or equal to 1,000 mg / L protein.

[0108] Another alternative aspect of the present invention relates to an ungulate pluripotent stem cell medium composition comprising a Wnt pathway inhibitor and a SMAD2 / 3 activator, wherein said culture medium comprises less than or equal to 1,000 mg / L protein. Yet another aspect of the invention relates to an ungulate pluripotent stem cell culture medium comprising a Wnt inhibitor and a SMAD2 / 3 activator, wherein said culture medium comprises less than or equal to 1,000 mg / L protein.

[0109] Examples 1-6, and 8-13 demonstrate that the culture medium of the present invention can be used to culture porcine pluripotent stem cells. Thus, an alternative aspect of the present invention relates to a porcine pluripotent stem cell culture medium comprising a Wnt pathway inhibitor and a SMAD2 / 3 activator, wherein said culture medium comprises less than or equal to 1,000 mg / L protein.

[0110] Another alternative aspect of the invention relates to an ungulate pluripotent stem cell culture medium consisting of a base medium, such as DMEM / Ham's F-12, insulin, transferrin, selenium and / or a compound comprising selenium, ascorbic acid, NaHCOs, an activator of the Fibroblast Growth Factor receptor (FGFR), a Wnt pathway inhibitor and a SMAD2 / 3 activator, wherein said culture medium comprises less than or equal to 1,000 mg / L protein.

[0111] Yet another aspect relates to an ungulate pluripotent stem cell culture medium consisting of a base medium, such as DMEM / Ham's F-12, insulin, selenium and / or a compound comprising selenium, ascorbic acid, NaHCOs, an activator of the Fibroblast Growth Factor receptor (FGFR), a Wnt pathway inhibitor and a SMAD2 / 3 activator, wherein said culture medium comprises less than or equal to 1,000 mg / L protein.

[0112] Still another alternative aspect relates to an ungulate pluripotent stem cell culture medium consisting of a base medium, such as DMEM / Ham's F-12, insulin, transferrin, selenium and / or a compound comprising selenium, ascorbic acid, NaHCC , a Wnt pathway inhibitor and a SMAD2 / 3 activator, wherein said culture medium comprises less than or equal to 1,000 mg / L protein.

[0113] Another alternative aspect relates to an ungulate pluripotent stem cell culture medium consisting of a base medium, such as DMEM / Ham's F-12, insulin, selenium and / or a compound comprising selenium, ascorbic acid, NaHCOs, a Wnt pathway inhibitor and a SMAD2 / 3 activator, wherein said culture medium comprises less than or equal to 1,000 mg / L protein. The present invention differs from the prior art by having a significantly reduced total protein content in the final culture medium. Culture mediums of prior art often comprise between 6,000 mg / L to 10,000 mg / L of total protein. Thus, in an embodiment, the culture medium comprises less than or equal to 800 mg / L protein, such as less than or equal to 700 mg / L protein, more preferably less than or equal to 500 mg / L protein, such as less than or equal to 200 mg / L protein, most preferably less than or equal to 150 mg / L protein, such as less than or equal to 100 mg / L protein, such as less than or equal to 60 mg / L protein, such as less than or equal to 40 mg / L protein, such as less than or equal to 30 mg / L protein, such as less than or equal to 20 mg / L protein, such as 0 mg / L protein.

[0114] In an embodiment, the culture medium comprises a protein level selected from the range of 1,000 mg / L to 0 mg / L protein, such as 800 mg / L to 5 mg / L protein, such as 700 mg / L to 10 mg / L protein, preferably 500 mg / L to 15 mg / L protein, more preferably 200 mg / L to 20 mg / L protein, most preferably 150 mg / L to 20 mg / L protein.

[0115] The culture medium of the present invention may be completely devoid of constituents of animal origin. Hence, in an embodiment, the culture medium does not comprise a component of animal origin. The component of animal origin could be a serum, such as fetal bovine serum (FBS). Thus, in an embodiment, the culture medium does not comprise serum, such as fetal bovine serum (FBS). In a preferred embodiment, the culture medium does not comprise fetal bovine serum (FBS).

[0116] Another parament in which the present invention differs from prior art is the content of fatty acids. Thus, in an embodiment, the culture medium comprises less than or equal to 25 mg / L fatty acids, such as less than or equal to 22 mg / L fatty acids, such as less than or equal to 20 mg / L fatty acids, such as less than or equal to 15 mg / L fatty acids, preferably less than or equal to 10 mg / L fatty acids, such as less than or equal to 5 mg / L fatty acids, such as less than or equal to 3 mg / L fatty acids, more preferably less than or equal to 1 mg / L fatty acids, most preferably the culture medium is essentially free of fatty acids. In an embodiment, the fatty acid is linoleic acid and / or linolenic acid. In Examples 1-4 and 12-13, the inventors demonstrate that Fibroblast Growth Factor 2 (FGF2) is not an essential component to culture ungulate pluripotent stem cells whilst maintaining the pluripotency of said stem cells. Thus, in an embodiment, the culture medium does not comprise Fibroblast Growth Factor 2 (FGF2). In another embodiment, said culture medium does not comprise an activator of the Fibroblast Growth Factor receptor (FGFR), such as Fibroblast Growth Factor 1 (FGF1), FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF22, or DJ-1. In yet another embodiment, the Fibroblast Growth Factor receptor (FGFR) is FGFR1, FGFR2, FGFR3, or FGFR4. However, the inventors also demonstrate that FGF2 (in FF3 medium) appears to result in a minor increase in the percentage of triple positive pESCs, although it is not required for maintaining pluripotency (Example 2). Thus, in an embodiment, the culture medium further comprises an activator of the Fibroblast Growth Factor receptor (FGFR), such as Fibroblast Growth Factor 1 (FGF1), FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF22, or DJ-1. In another embodiment, the Fibroblast Growth Factor receptor (FGFR) is FGFR1, FGFR2, FGFR3, or FGFR4. In yet another embodiment, said activator of the Fibroblast Growth Factor receptor (FGFR) is Fibroblast Growth Factor 2 (FGF2). In a preferred embodiment, the culture medium further comprises Fibroblast Growth Factor 2 (FGF2). In another embodiment, said culture medium comprises less than 200 ng / mL Fibroblast Growth Factor 2 (FGF2), preferably in the range of 0-100 ng / mL FGF2, more preferably 0-50 ng / mL FGF2, most preferably 50 ng / mL FGF2.

[0117] The Wnt pathway inhibitor could be a protein, a peptide, a peptidomimetic, or a small molecule. Thus, in an embodiment, the Wnt pathway inhibitor is selected from the group consisting of a protein, a peptide, a peptidomimetic, and a small molecule, preferably a small molecule. In an embodiment, the Wnt pathway inhibitor inhibits the canonical Wnt pathway. In an embodiment, the Wnt pathway inhibitor is selected from the group consisting of IWR-l-endo (CAS No. 1127442- 82-3), IWR-1 (CAS No. 1127442-82-3), IWR-exo-1 (CAS No. 1127442-87-8), IWR-2 (PubMED CID: 52944858), ICG-001 (CAS No. 780757-88-2), Cerberus, Wnt-C59 (CAS No. 1243243-89-1), IWP-1 (CAS No. 2074607-48-8), IWP-2 (CAS No. 686770-61-6), IWP-3 (CAS No. 687561-60-0), IWP-4 (CAS No. 686772-17- 8), AZD5055, DKK-1, KY02111 (CAS No. 1118807-13-8), KYA1797K (CAS No. 1956356-56-1), JW67 (CAS No. 442644-28-2), JW74 (CAS No. 863405-60-1), LGK974 (CAS No. 1243244-14-5), Niclosamide (CAS No. 50-65-7), Notum, NSC668036 (CAS No. 144678-63-7), CGP049090 (CAS No. 35082-49-6), BC2059 (CAS No. 1227637-23-1), ETC-159 (CAS No. 1638250-96-0), ETC-131, sFRPl, SFRP2, Shizokao ID (CAS No. 142279-42-3), XAV939 (CAS No. 284028-89-3), and WIFI, or a mixture thereof, preferably IWR-l-endo. The present inventors have used IWR-l-endo (Examples 1-9, and 11-13) and IWR-1, XAV-939, IWP-2, and Wnt-C59 (Example 10) as the Wnt pathway inhibitor in the culture medium of the present invention. Thus, in a preferred embodiment, the Wnt pathway inhibitor is selected from the group consisting of IWR-l-endo, IWR-1, XAV-939, IWP-2, and Wnt-C59, or mixtures thereof, preferably IWR-l-endo. In an embodiment, the culture medium comprises 0.1-20 pM of the Wnt pathway inhibitor, such as 0.5-15 pM of the Wnt pathway inhibitor, such as 0.7-10 pM of the Wnt pathway inhibitor, preferably 1-5 pM of the Wnt pathway inhibitor, such as 1-4 pM of the Wnt pathway inhibitor, more preferably 2-3 pM of the Wnt pathway inhibitor, most preferably 2.5 pM of the Wnt pathway inhibitor.

[0118] The SMAD2 / 3 activator could be a protein, a peptide, a peptidomimetic, or a small molecule. Thus, in an embodiment, the SMAD2 / 3 activator is selected from the group consisting of a protein, a peptide, a peptidomimetic, and a small molecule, preferably a protein. In an embodiment, the SMAD2 / 3 activator is selected from the group consisting of activin A, activin B, transforming growth factor beta 1 (TGF-pi), TGF-P2, TGF-g3, Growth Differentiation Factor 1 (GDF1), GDF3, GDF8, GDF9a, GDF10, GDF11, Inducer of Definitive Endoderm 1 (IDE1), IDE2, Nodal, PD0332991 (CAS No. 827022-32-2), JNK inhibitor IX (CAS No. 312917-14-9), LY- 294002 (CAS No. 934389-88-5), JNJ-42041935 (CAS No. 1193383-09-3), and BRD-K42644990, or a mixture thereof. In an embodiment, the SMAD2 / 3 activator activates SMAD2 and / or SMAD3 using Anaplastic lymphoma kinase 4 (ALK4), Anaplastic lymphoma kinase 5 (ALK5) and / or Anaplastic lymphoma kinase 7 (ALK7). The present inventors have used activin A (Examples 1-13) or TGF-pi, GDF8, and GDF11 (Example 9) as the SMAD2 / 3 activator in the culture medium of the present invention. Thus, in a preferred embodiment, the SMAD2 / 3 activator is activin A, activin B, GDF1, GDF3, GDF8, GDF11, TGF-gl, TGF-g2, or TGF-03. Activin A, GDF8, and GDF11 appeared to be more effective than TGF-pi (Example 9). Without being bound by theory, the inventors hypothesize that this is because TGF-pi signals through both ALK-5 (activating SMAD2 / 3) and ALK-1 (activating SMAD1 / 5 / 8). Hence, SMAD2 / 3 activators that target SMAD2 and / or SMAD3 without activating SMAD1 / 5 / 8 might be preferred. In a more preferred embodiment, the SMAD2 / 3 activator is activin A, GDF8, or GDF11. In the most preferred embodiment, the SMAD2 / 3 activator is activin A.

[0119] The concentration of the SMAD2 / 3 activator was optimized in Example 3 and Example 8. Thus, in an embodiment, the culture medium comprises 5-100 ng / mL of the SMAD2 / 3 activator, such as 10-100 ng / mL of the SMAD2 / 3 activator, preferably 5-50 ng / mL of the SMAD2 / 3 activator, more preferably 10-30 ng / mL of the SMAD2 / 3 activator, most preferably 25 ng / mL of the SMAD2 / 3 activator.

[0120] In another embodiment, the ratio between the Wnt pathway inhibitor and the SMAD2 / 3 activator (w / w) is selected from the range of 1: 10 to 1: 100, such as 1: 15 to 1:80, such as 1:20 to 1: 50, such as 1:20 to 1: 30, preferably 1:25.

[0121] In an embodiment, the culture medium does not comprise a compound of formula

[0122] The compound of formula (I) is called LDN-193189 (CAS No. 1062368-24-4).

[0123] In an embodiment, the culture medium does not comprise a compound of formula (II) :

[0124] The compound of formula (II) is called CHIR99021 (CAS No. CAS 252917-06-9).

[0125] In an embodiment, the culture medium does not comprise a retinoic acid receptor inhibitor.

[0126] The culture medium used in the examples comprises a base medium. Thus, in an embodiment, the culture medium further comprises a base medium, such as Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Ham's Nutrient Mixture F-12 (F-12), Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F-12), RPMI 1640, Minimum Essential Medium Eagle - Alpha Modification (Alpha MEM), or a mixture thereof, preferably DMEM / Ham's F-12, MEM, F-12, or RPMI 1640, more preferably DMEM / Ham's F-12. In another embodiment, the base medium is selected from the group consisting of Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Ham's Nutrient Mixture F-12 (F-12), Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F-12), RPMI 1640, and Minimum Essential Medium Eagle - Alpha Modification (Alpha MEM), or a mixture thereof, preferably DMEM / Ham's F-12, MEM, F-12, or RPMI 1640. In a preferred embodiment, the base medium is DMEM / Ham's F-12. In yet another embodiment, the DMEM / Ham's F-12 comprises: Aspartic acid Magnesium Sulfate (MgSO4) (anhyd.)

[0127] In examples 6-13, the inventors tested a low protein culture medium comprising DMEM / Ham's F-12, insulin, transferrin, selenium, ascorbic acid, NaHCOs, FGF2, IWR-1 and Activin A. Thus, in an embodiment, the culture medium further comprises selenium and / or a compound comprising selenium, such as SeOs2-, such as sodium selenite, such as selenium dioxide. In another embodiment, the culture medium further comprises insulin, insulin-like Growth Factor-1 (IGF-1), or insulin-like growth factor 2 (IGF-2), or a mixture thereof. In yet another embodiment, the culture medium further comprises ascorbic acid. In still another embodiment, the culture medium further comprises a pH buffer, such as NaHCOs.

[0128] In an embodiment, the culture medium further comprises transferrin. In another embodiment, the culture medium further comprises albumin, such as bovine serum albumin (BSA). In yet another embodiment, the culture medium further comprises bovine serum albumin (BSA). The inventors have demonstrated that the protein supplements transferrin and albumin may improve the culture medium, but it is not required constituents in the ungulate pluripotent stem cell culture medium to culture porcine pluripotent stem cells (Example 12). Thus, in an embodiment, the culture medium does not comprise transferrin. In another embodiment, the culture medium does not comprise albumin, such as bovine serum albumin (BSA).

[0129] In an embodiment, the culture medium further comprises a base medium, such as Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F- 12), insulin, transferrin, ascorbic acid, an activator of the Fibroblast Growth Factor receptor (FGFR), albumin, NaHCC , and selenium and / or a compound comprising selenium. In another embodiment, the culture medium further comprises a base medium, such as Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F-12), insulin, transferrin, ascorbic acid, an activator of the Fibroblast Growth Factor receptor (FGFR), and selenium and / or a compound comprising selenium. In yet another embodiment, the culture medium further comprises a base medium, such as Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F-12), insulin, transferrin, ascorbic acid, and selenium and / or a compound comprising selenium. In still another embodiment, the culture medium further comprises a base medium, such as Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F-12), insulin, ascorbic acid, an activator of the Fibroblast Growth Factor receptor (FGFR), and selenium and / or a compound comprising selenium.

[0130] To avoid microbial contamination, antibiotics can be added to the culture medium. Thus, in an embodiment, the culture medium further comprises an antibiotic, such as penicillin and / or streptomycin. The culture medium of the present invention can optionally comprise different types of supplements. Said supplements are not necessary to culture ungulate pluripotent stem cells, as demonstrated in the examples, but the supplements might improve the culture medium for different cell lines. Thus, in an embodiment, the culture medium further comprises L- glutamine or a source thereof, such as L-alanyl-L-glutamine dipeptide or GlutaMax™. In another embodiment, the culture medium further comprises GlutaMax™. In yet another embodiment, the culture medium further comprises N- 2 supplement. In still another embodiment, said N-2 supplement comprises transferrin, insulin, progesterone, putrescine and SeOs2-. In an embodiment, the culture medium does not comprise more than 0.1 % (vol / vol) of any one of the N-2 supplement, bovine serum albumin (BSA), and Glutamax™. In another embodiment, the culture medium does not comprise fetal bovine serum (FBS), Knockout™ Serum Replacement, N-2 supplement, and / or B- 27™ supplement. In another embodiment, the B-27™ supplement is without vitamin A.

[0131] The culture medium may also comprise non-essential amino acids (NEAA). Thus, in an embodiment, the culture medium further comprises non-essential amino acids selected from the group consisting of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, serine, arginine, cysteine, glutamine, and tyrosine, or a mixture thereof, preferably glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine, or a mixture thereof. In a preferred embodiment, the culture medium comprises a mixture of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine.

[0132] The culture medium can comprise growth factors, such as transforming growth factor beta 1 (TGF-pi), TGF-[32, TGF-[33, insulin-like Growth Factor-1 (IGF-1), or insulin-like growth factor 2 (IGF-2). Thus, in an embodiment, the culture medium comprises no more than three growth factors. In another embodiment, the culture medium comprises no more than two growth factors. In another embodiment, the growth factor is selected from the group consisting of Activin A, transforming growth factor beta 1 (TGF-pi), TGF-[32, TGF-[33, insulin, insulin-like Growth Factor-1 (IGF-1), insulin-like growth factor 2 (IGF-2), Fibroblast Growth Factor 1 (FGF1), FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, and FGF22.

[0133] In an embodiment, the culture medium comprises no more than three proteins, such as no more than two proteins. In another embodiment, the protein is selected from the group consisting of Activin A, transforming growth factor beta 1 (TGF-pi), TGF-P2, TGF-[33, insulin, insulin-like Growth Factor-1 (IGF-1), insulinlike growth factor 2 (IGF-2), Fibroblast Growth Factor 1 (FGF1), FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF22, Albumin, such as bovine serum albumin (BSA), transferrin, catalase, superoxide dismutase, apolipoprotein, laminin, fibronectin, GDF1, GDF3, GDF8, GDF9a, GDF10 and GDF11. The ungulate pluripotent stem cells cultured in examples 1-6 and 8-13 are porcine pluripotent stem cells. Thus, in an embodiment, the culture medium is a porcine pluripotent stem cell culture medium.

[0134] An alternative aspect relates to a kit for culturing ungulate pluripotent stem cells, said kit comprising: a) ungulate pluripotent stem cells; and b) a culture medium according to the present invention.

[0135] Use of the culture medium

[0136] An aspect relates to the use of the ungulate pluripotent stem cell culture medium according to the present invention for culturing ungulate pluripotent stem cells. In an embodiment, the ungulate pluripotent stem cells belong to the Artiodactyla order, preferably terrestrial artiodactyl. In an embodiment, the Artiodactyla order has a paraxonic foot structure. A paraxonic foot structure means that the weight is distributed on the third and the fourth toe on all legs. In another embodiment, the ungulate pluripotent stem cells belong to the Suina order or Ruminantia order. In yet another embodiment, the ungulate pluripotent stem cells belong to the Suidae order or Bovidae order.

[0137] In an embodiment, the ungulate pluripotent stem cells are selected from the group consisting of porcine, bovine, bubaline, cameline, giraffa, rhinocerotic, ovine, caprine, equine, and cervine pluripotent stem cells, preferably porcine, bovine, or ovine pluripotent stem cells, more preferably porcine or bovine pluripotent stem cells, most preferably porcine pluripotent stem cells. As demonstrated in examples 6 and 8-13, the inventors cultured pESCs in a culture medium with a low protein content. Thus, in a preferred embodiment, the ungulate pluripotent stem cells are porcine pluripotent stem cells or bovine pluripotent stem cells, preferably porcine pluripotent stem cells.

[0138] The inventors demonstrated in Example 1, that the pESCs displayed a morphology characteristic of pluripotent stem cells and the morphology was not affected by the presence of feeder cells. Also, the pluripotency quality of the pESCs were investigated without feeder cells in Example 2, 3, 5 and 6 and the cells displayed a high pluripotency despite the omission of feeder cells. Thus, in an embodiment, the cell culture does not comprise feeder cells, such as mouse embryonic fibroblasts (MEFs).

[0139] In example 2, 3, 5-7 the pluripotency of the pESCs and bESCs were evaluated based on the expression of Oct4 (POU5F1), Sox2 and Nanog. A high percentage of the pESCs and bESCs were triple positive for the pluripotency markers when cultured in culture medium of the present invention. Thus, in an embodiment, the cultured ungulate pluripotent stem cells express POU5F1, NANOG and SOX2. In another embodiment, at least 75 %, such as 80 %, such as 85 %, preferably at least 90 % of the cultured ungulate pluripotent stem cells express POU5F1, NANOG and SOX2. The absence of FGF2 would reduce or eliminate Erk translocation to the nucleus, Erk phosphorylation, presence of ETV4 / 5, and / or SPRY2 / 4 gene activation. Thus, in an embodiment, the cultured ungulate pluripotent stem cells display absence of Erk translocation to the nucleus, and / or absence of Erk-phosphorylation, and / or absence of ETV4 / 5 and / or absence of SPRY2 / 4 gene activation. The presence of a Wnt pathway inhibitor would lead to Beta-catenin phosphorylation, Beta-catenin translocation to the nucleus, SP gene (SP 1-9) activation, and / or AXIN2 gene activation. Hence, in another embodiment, the cultured ungulate pluripotent stem cells display presence of Beta-catenin phosphorylation, and / or absence of Beta-catenin translocation to the nucleus, and / or absence of SP gene (SP 1-9) activation, and / or absence of AXIN2 gene activation. The presence of a SMAD2 / 3 activator would lead to SMAD2 / 3 phosphorylation and / or SMAD2 / 3 translocation to the nucleus. Thus, in yet another embodiment, the cultured ungulate pluripotent stem cells display presence of SMAD2 / 3 phosphorylation, and / or presence of SMAD2 / 3 translocation to the nucleus.

[0140] In Example 4, the pESCs line, MT003, was successfully cultured in suspension in both FF2 and FF3 medium and formed homogenous and proliferative aggregates after only 24 hours. Thus, in an embodiment, the doubling time of the ungulate pluripotent stem cells is less than 30 hours, such as less than 28 hours, preferably less than 25 hours.

[0141] The pESCs were cultured in 2D in the culture medium of the present invention in Example 1. Thus, in an embodiment, the ungulate pluripotent stem cells are cultured in 2D. However, the pESCs could also be cultured in suspension, in 3D, as demonstrated in Example 4. Thus, in an embodiment, the ungulate pluripotent stem cells are cultured in 3D. In another embodiment, the ungulate pluripotent stem cells are cultured in suspension. In another embodiment, the ungulate pluripotent stem cells are cultured in suspension without a scaffold, such as a microcarrier.

[0142] In an embodiment, the cultured ungulate pluripotent stem cells or derivatives thereof are used for the production of cultivated meat products.

[0143] In another embodiment, the cultured ungulate pluripotent stem cells or derivatives thereof are used in animal breeding programs.

[0144] In yet another embodiment, the cultured ungulate pluripotent stem cells or derivatives thereof are used for the production of therapeutic compounds. In still another embodiment, the cultured ungulate pluripotent stem cells or derivatives thereof are used for toxicological screening, drug discovery, or disease modelling.

[0145] Method for culturing ungulate pluripotent stem cells

[0146] Yet another aspect of the present invention relates to a method for culturing ungulate pluripotent stem cells, said method comprising the steps: a) mixing a base medium with a Wnt pathway inhibitor and a SMAD2 / 3 activator to obtain a culture medium, b) adding the culture medium of step a) to ungulate pluripotent stem cells, and c) culturing the ungulate pluripotent stem cells in a culture vessel, wherein said culture medium comprises less than or equal to 1,000 mg / L protein. Another aspect of the present invention relates to a method for culturing ungulate pluripotent stem cells, said method comprising the steps: a) mixing a Wnt pathway inhibitor and a SMAD2 / 3 activator to obtain a culture medium according to the present disclosure, b) adding the culture medium of step a) to ungulate pluripotent stem cells, and c) culturing the ungulate pluripotent stem cells in a culture vessel.

[0147] In an embodiment, the temperature in step c) is in the range of 35 to 42°C, such as 37 °C to 40 °C, preferably 38 °C. In another embodiment, the CO2 level in step c) is in the range of 3 to 7 %, preferably 5 %. In yet another embodiment, the relative humidity in step c) is in the range of 85 to 95 %.

[0148] In an embodiment, the culture vessel is selected from the group consisting of a petri dish, a culture flask, a multiwell plate, a shaker flask or bioreactor, a spinner flask or bioreactor, and a scaffold-based culture. In a preferred embodiment, the culture vessel is a bioreactor.

[0149] In an embodiment, the base medium is selected from the group consisting of Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Ham's Nutrient Mixture F-12 (F-12), Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F-12), RPMI 1640, and Minimum Essential Medium Eagle - Alpha Modification (Alpha MEM), or a mixture thereof, preferably DMEM / Ham's F-12, MEM, F-12, or RPMI 1640, more preferably DMEM / Ham's F-12. In another embodiment, the culture medium further comprises a base medium selected from the group consisting of Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Ham's Nutrient Mixture F-12 (F-12), Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F- 12), RPMI 1640, and Minimum Essential Medium Eagle - Alpha Modification (Alpha MEM), or a mixture thereof, preferably DMEM / Ham's F-12, MEM, F-12, or RPMI 1640, more preferably DMEM / Ham's F-12.

[0150] The present inventors have demonstrated in the examples that the culture medium of the present invention can be used to culture pESCs in 2D or 3D. Thus, in an embodiment, the ungulate pluripotent stem cells are cultured in 2D. In another embodiment, the ungulate pluripotent stem cells are cultured in 3D.

[0151] The ungulate pluripotent stem cells of the present invention can be frozen and defrosted when needed (data not shown). Thus, in an embodiment, the ungulate pluripotent stem cells are defrosted prior to step b). In another embodiment, the ungulate pluripotent stem cells are cryopreserved after step c).

[0152] The pESCs established in Example 1 could be used to generate cultivated meat. In order to do so, the pluripotent stem cells of the present invention should differentiate into specialized cell types of the adult organism, including muscle, fat, liver, heart, and blood cells, which are essential components of meat tissue. Thus, in an embodiment, the ungulate pluripotent stem cells are differentiated after step c).

[0153] It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

[0154] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0155] The invention will now be described in further details in the following non-limiting examples.

[0156] Examples

[0157] Example 1 - Establishing porcine embryonic stem cell lines

[0158] Aim of study

[0159] The aim of this study was to establish porcine embryonic stem cell lines and investigate the morphology of said cell lines.

[0160] Materials and methods

[0161] Establishing porcine embryonic stem cell lines

[0162] Flushing of pre-implanted embryos after insemination is a regular procedure commonly carried out in conventional porcine breeding. These pre-implanted embryos were utilized for the derivation of porcine embryonic stem cells (pESCs). To dissociate the inner cell mass of the pre-implanted embryos, from which the pESCs originate, enzymatic treatment was applied (TrypLE Express, Gibco) whereafter the cells were seeded onto a cell culture plate in a medium comprising FGF2 (10 ng / mL, Miltenyi, also termed bFGF), Activin A (25 ng / mL, Miltenyi Biotec) and IWR-l-endo (2.5 pM, Tocris) on inactivated mouse embryonic fibroblasts (MEFs). The pESCs were passaged in clumps every 4-5 days using EDTA (0.5 mM, Invitrogen).

[0163] Adherent culturing of porcine embryonic stem cells (pESCs) in feeder-free and defined medium conditions To enable adherent culture of pESCs in feeder-free and defined medium conditions, pESCs were transferred onto cell culture plates (Sarstedt) with suitable coating such as Geltrex (Gibco), Matrigel (Corning) or Laminin 521 (BioLamina). The cell culture medium was composed of DMEM / Ham's F-12 medium (86%, Gibco), Glutamax™ (1%, Gibco), NEAA (non-essential amino acids, 1%, Gibco), PenStrep (Penicillin / streptomycin, lOU / mL, Gibco), Ascorbic acid (50 pg / mL, Merck), B-27™ supplement without vitamin A (1%), N-2 supplement (0.5%), Knockout™ Serum Replacement (4%), bovine serum albumin (6.25%), and supplemented with Activin A (25 ng / mL, Miltenyi Biotec) and IWR-l-endo (2.5 pM, Tocris). Additionally, pESCs could be cultured with (termed FF3 medium) or without (termed FF2 medium) the presence of FGF2 (50 ng / mL, Miltenyi). The pESCs were passaged in clumps every 4-5 days using EDTA (0.5 mM, Invitrogen) without the use of ROCK inhibitors.

[0164] Morphology

[0165] The brightfield images presented in Example 1 was acquired through Leica DMil inverted microscope with a lOx PHI objective. The pESCs lines (MT002 and MT003) displayed a cellular morphology characteristic of pluripotent stem cells (clear borders; small round cells without spaces between them; and large nuclei)

[0166] Results

[0167] Two porcine embryonic stem cell lines called MT002 and MT003 were established, and the pESCs grew in distinct colonies. The pESCs lines (MT002 and MT003) displayed a cellular morphology characteristic of pluripotent stem cells (clear borders; small round cells without spaces between them; and large nuclei) both on feeders and in feeder-free cultures (FF3 and FF2) (Figure 1). The presence of FGF2 in the medium (FF3) does not appear to affect the morphology of the pESCs.

[0168] Conclusion

[0169] The present inventors established two porcine embryonic stem cell lines (MT002 and MT003). The pESCs displayed a morphology characteristic of pluripotent stem cells and the morphology was not affected by the presence of feeders or FGF2. Thus, the culture medium comprising a Wnt pathway inhibitor (IWR-l-endo) and a SMAD2 / 3 activator (Activin A) could be used to culture porcine pluripotent stem cells. Example 2 - the pluripotency quality of the porcine embryonic stem cells

[0170] Aim of study

[0171] The aim of this study was to investigate whether the pESC lines, MT002 and MT003 were in fact pluripotent by assessing the expression of pluripotency markers.

[0172] Materials and methods

[0173] Immunofluorescence staining

[0174] Cells were washed once with D-PBS and fixed in 4% paraformaldehyde (Thermo Scientific) for 15 min. Cells were then washed with D-PBS and treated with blocking buffer (D-PBS + 5% donkey serum (VWR) + 0.1% Triton X (Sigma- Aldrich)) for lh at RT. Primary antibodies were diluted in blocking buffer and incubated overnight at 4°C. Subsequently, cells were washed twice with D-PBS and incubated with secondary antibodies and DAPI (Invitrogen, lpg / mL) for 2h. Cells were then washed twice with D-PBS before imaging with a Leica SP5 Confocal microscope. The following primary antibodies were used: Oct3 / 4 (Santa Cruz, sc5279; R&D systems, AF1759), Nanog (Abeam, ab80892), and SOX2 (R&D systems, AF2018). The following secondary antibodies were used: Alexa Fluor 488 anti-mouse (Thermo, A21202), Alexa Fluor 555 anti-rabbit (Thermo, A31572), and Alexa Fluor 647 anti-goat (Thermo, A32733).

[0175] Quantification

[0176] The percentage of triple (Oct4 / Nanog / Sox2) positive cells out of total DAPI positive cells was determined using FIJI image processing software.

[0177] Results

[0178] The expression of Oct4, Nanog and Sox2 was chosen as pluripotency markers, since the retention of pluripotency of ESCs depends on the expression of transcription factors, i.e., Sox2, Oct4 and Nanog. The MT002 pESC line express the three pluripotency markers (Oct4, Nanog, Sox2) in FF3 medium (Figure 2) and in FF2 medium (Figure 3). The MT003 pESCs also expressed the pluripotency markers in both FF3 (Figure 4) and FF2 medium (Figure 5). Thus, FGF2 is not a necessary constituent of a culture medium to culture pluripotent stem cells and maintaining the pluripotency of said cells. The triple (Oct4 / Nanog / Sox2) positive cells of total DAPI were quantified using the FIJI image processing software based on the intensity of the immunofluorescence staining (Figure 6). In addition to culturing the MT002 and MT003 pESCs in FF2 and FF3 medium, the MT003 pESCs were also cultured in a commercial pluripotent stem cell (PSC) medium called E8 (Essential 8 Medium, Gibco). As observed in figures 2-5, both the MT002 and MT003 pESCs displayed a high expression of Oct, Nanog and Sox2 in FF3 and FF2 medium with the percentages of triple positive cells ranging from 75 % to 95 % of total DAPI. The presence of FGF2 (in FF3 medium) appears to result in a minor increase in the percentage of triple positive cells in the MT002 and MT003 pESCs. However, in the absence of FGF2 (in FF2 medium) the cells are still able to maintain pluripotency above 75%. FGF2 is therefore not a required constituent of a culture medium for pluripotent stem cells.

[0179] Importantly, the commercial E8 medium could not be used to culture the porcine MT003 ESCs, since only 0% of the MT003 pESCs retained the expression of all three pluripotency markers suggesting that said cells were no longer pluripotent. These observations indicate that the culture mediums are species specific, but it is likely that a culture medium for porcine pluripotent stem cells could also be used to culture other ungulate pluripotent stem cells.

[0180] Conclusion

[0181] The FF2 and FF3 mediums can be used to culture porcine embryonic stem cells (pESCs), possibly also other ungulate pluripotent stem cells, since the pESCs express a high level of pluripotency markers (Oct4 / Nanog / Sox2), suggesting that said cells remain undifferentiated and retain their pluripotency. While the presence of FGF2 (in FF3 medium) appears to result in a minor increase in the percentage of triple positive pESCs , it is not required for maintaining pluripotency. Thus, FGF2 is not an essential constituent of a culture medium for culturing pluripotent stem cells.

[0182] Example 3 - Optimization of the ungulate pluripotent stem cell culture medium

[0183] Aim of study

[0184] The aim of this study was to investigate the optimal concentrations of a Wnt pathway inhibitor (IWR-l-endo), a SMAD2 / 3 activator (Activin A) and an activator of the Fibroblast Growth Factor receptor (FGFR) (FGF2) in an ungulate pluripotent stem cell culture medium.

[0185] Materials and methods pESCs (MT003) were cultured in feeder-free and defined medium conditions on cell culture plates (Sarstedt) with suitable coating such as Geltrex (Gibco). The cell culture medium was composed of DMEM / Ham's F-12 medium (86%), Glutamax™ (1%, Gibco), NEAA (non-essential amino acids, 1%, Gibco), PenStrep (lOU / mL, Gibco), Ascorbic acid (50 pg / mL, Merck), B-27™ supplement without vitamin A (1%), N-2 supplement (0.5%), Knockout™ Serum Replacement (4%), bovine serum albumin (6.25%), and supplemented with Activin A (0, 10, 25 or 100 ng / mL, Miltenyi Biotec) and IWR-l-endo (0.1, 1, 2.5, 10 pM, Tocris) and FGF2 (0, 10, 50, 200 ng / mL, Miltenyi). The pESCs were passaged in clumps every 4-5 days using EDTA (0.5 mM, Invitrogen) without the use of ROCK inhibitors. Immunofluorescence staining and subsequent quantification was performed as described in Example 2.

[0186] Results

[0187] The concentration of FGF2 and Activin A were varied between 0-200 ng / mL and 0- 100 ng / mL, respectively, whereas the concentration of IWR-l-endo was kept constant at 2.5 pM (Table 1).

[0188] Table 1: Overview of pluripotency levels and survival after five passages across titration of FGF2 (0, 10, 50 200 ng / mL), Activin A (0, 10, 25, 100 ng / mL), IWR-1 (0.1, 1, 2.5 pM)

[0189] The " "-tick indicates that the pluripotent stem cell culture had >75 % of triple positive (Oct4 / Nanog / Sox2) cells (top row) or survived past 5 passages (bottom row), whereas the "X"-tick indicates that the pluripotent stem cell culture did not have >75 % of triple positive (Oct4 / Nanog / Sox2) cells (top row) or did not survive past 5 passages (bottom row).

[0190] FGF2 has an effective range of approximately 0-50 ng / mL, resulting in 1) Oct4 / Nanog / Sox2 expression above 75% and 2) survival past passage 5. At 200 ng / mL FGF2, the pESC culture shows signs of differentiation. Activin A has an effective range of approximately 10-100 ng / mL, resulting in 1) Oct4 / Nanog / Sox2 expression above 75% and 2) survival past passage 5. At 0 ng / mL Activin A, the pESC culture shows signs of differentiation and reduced proliferation. IWR-l-endo has an effective range of approximately around 2.5 pM, resulting in 1) Oct4 / Nanog / Sox2 expression above 75% and 2) survival past passage 5. At 1.0 pM and 0.1 pM IWR-l-endo, the pESC culture shows signs of differentiation.

[0191] Conclusion

[0192] The optimal concentration for FGF2 was below 200 ng / mL, preferably in the range of 0-50 ng / mL. For Activin A, the optimal concentration range was above 0 ng / mL, preferably between 10-100 ng / mL, whereas the concentration of IWR-l- endo was 2.5 pM.

[0193] Example 4 - Aggregate formation

[0194] Aim of study

[0195] The aim of this study was to investigate whether the porcine embryonic stem cells could form aggregates when cultured in the FF2 and FF3 medium as aggregate formation (without the use of scaffolding) is a key characteristic of pluripotent stem cells.

[0196] Materials and methods

[0197] Suspension culturing of porcine pluripotent stem cells (pESCs) in feeder-free and defined medium conditions

[0198] The MT003 cell line were cultured in FF2 or FF3 medium in suspension. To culture pESCs in suspension cultures, pESCs from feeder-free cultures were dissociated to single-cells (Accutase) and transferred to low-attachment 6-well plates (Greiner) and agitated on a shaker (75 RPM) in FF2 or FF3 medium.

[0199] Results The pESCs line, MT003, can be successfully cultured in suspension in both FF2 and FF3 medium and form homogenous and proliferative aggregates after only 24 hours (DIV1) (Figure 7) which is a key characteristic of pluripotent stem cells. After 48 hours (DIV2), the pluripotent stem cells have proliferated and generated more dense aggregates.

[0200] Conclusion

[0201] Porcine pluripotent stem cells cultured in the culture medium of the present invention optionally comprising FGF2 (FF3) can form homogeneous and proliferative aggregates.

[0202] Example 5 - Pluripotency quality in FF3 medium after 45 passages

[0203] Aim of study

[0204] The aim of this study was to investigate whether the porcine pluripotent stem cells (MT003) could be cultured for more than 45 passages in FF3 medium whilst maintaining the expression of the pluripotency markers Oct4, Nanog and Sox2.

[0205] Results

[0206] The pESCs line, MT003, can be successfully cultured for more than 45 passages and successfully maintain the high expression of Oct4, Nanog and Sox2 (Figure 8).

[0207] Conclusion

[0208] The culture medium of the present invention can be used over an extended time period to culture pluripotent stem cells which retain their pluripotency during culture.

[0209] Example 6 - Pluripotency quality in FF3 medium with high or low protein content

[0210] Aim of study

[0211] The aim of this study was to investigate whether the porcine pluripotent stem cells (MT003) could be cultured in a low protein medium while maintaining the expression of the pluripotency markers Oct4, Nanog and Sox2. Materials and methods

[0212] The MT003 cell line were cultured in either FF3 High Protein Medium or FF3 Low Protein Medium in adherent culture for five passages. The FF3 High Protein Medium is identical to the FF3 mediums described in the previous examples and consisted of DMEM / Ham's F-12 medium (86%, Gibco), Glutamax™ (1%, Gibco), NEAA (non-essential amino acids, 1%, Gibco), PenStrep (lOU / mL, Gibco), Ascorbic acid (50 pg / mL, Merck), B-27™ supplement (1%) without vitamin A, N-2 supplement (0.5%), Knockout™ Serum Replacement (4%), bovine serum albumin (6.25%), and supplemented with Activin A (25 ng / mL, Miltenyi Biotec), IWR-1- endo (2.5 pM, Tocris), and FGF2 (50 ng / mL, Miltenyi).

[0213] Of these constituents, only the B-27™ supplement without vitamin A, N-2 supplement, Knockout™ Serum Replacement, bovine serum albumin, Activin A, and FGF2 attributed to the total protein content in the culture medium.

[0214] The B-27™ supplement without vitamin A comprises the constituents listed in table 2 and attributes with approximately 1,250 mg / L protein in the FF3 High-

[0215] Protein culture medium.

[0216] Table 2: Overview of the constituents in the B-27™ supplement without vitamin A

[0217] The N-2 supplement comprises the constituents listed in table 3 and attributes with approximately 50 mg / L protein in the FF3 High-Protein culture medium. Table 3: Overview of the constituents in the N-2 supplement

[0218] The Knockout™ Serum Replacement comprises the constituents listed in table 4 and attributed with approximately 3300 mg / L protein in the FF3 High-Protein culture medium.

[0219] Table 4: Overview of the constituents in the Knockout™ Serum Replacement

[0220] * AlbuMAX™ comprises free fatty acids, lysophosphatidylcholine, triacylglycerides, phosphatidylcholine, phosphatidic acid, cholesterol, and sphingomyelin.

[0221] Bovine serum albumin, Activin A, and FGF2 attributed with approximately 4000 mg / L protein, 25 pg / L protein and 50 pg / L protein, respectively, in the FF3 High- Protein medium.

[0222] The FF3 Low Protein Medium consisted of DMEM / Ham's F-12, insulin (19.4 pg / mL), transferrin (10.7 pg / mL), selenium (14 ng / mL), ascorbic acid-2- phosphate (64 pg / mL), NaHCC (543 pg / mL), FGF2 (50 ng / mL), IWR-1 (2.5 pM) and Activin A (25 ng / mL). The FF2 Low Protein Medium consisted of DMEM / Ham's F-12, insulin (19.4 pg / mL), transferrin (10.7 pg / mL), selenium (14 ng / mL), ascorbic acid-2-phosphate (64 pg / mL), NaHCC (543 pg / mL), IWR-1 (2.5 pM) and Activin A (25 ng / mL). Table 5 describes the protein content in FF3 High Protein Medium, FF3 Low Protein Medium, FF2 Low Protein Medium, as well as the protein content of previously published mediums for culturing porcine embryonic stem cells.

[0223] Table 5: Overview of the protein content in selected culture mediums

[0224] Results

[0225] The pESCs line, MT003, can be successfully cultured in a FF3 Low Protein Medium and successfully maintain the high expression of Oct4, Nanog and Sox2 at levels comparable to FF3 High Protein Medium (Figure 9).

[0226] Conclusion

[0227] The FF3 low protein medium of the present invention can be used to culture pluripotent stem cells which retain their pluripotency during culture.

[0228] Example 7 - Bovine ESC pluripotency quality in Low Protein FF3 and FF2 medium

[0229] Aim of study

[0230] The aim of this study was to validate that the culture medium of the present invention can be used to maintain high pluripotency quality of any ungulate species.

[0231] Materials and methods

[0232] Two bovine ESC lines (MTbOOl, MTb002) normally cultured in high-protein medium on MEFs were transferred to Low-Protein Medium (FF2 and FF3) on Laminin-521 or Geltrex (1: 100) and maintained for multiple passages. Example 6 describes the components of the FF2 Low-Protein Medium and FF3 Low-Protein Medium.

[0233] Results

[0234] Both bovine ESC lines (MTbOOl, MTb002) express high levels of pluripotency markers, Oct3 / 4, Nanog and Sox2 (Figures 10A-B, Figure 12B) and display superior proliferation in Low-Protein Medium independent of the presence of FGF2 (Low Protein medium FF3 and FF2) (Figures 11 and 12A).

[0235] Conclusion

[0236] The culture medium of the present invention can successfully maintain high pluripotency quality regardless of the ungulate species.

[0237] Example 8 - Optimal concentration ranges of FGF2, IWR-1 and Activin A Aim of study

[0238] The aim of this study was to validate the optimal concentrations of a Wnt pathway inhibitor (IWR-l-endo), a SMAD2 / 3 activator (Activin A) and an activator of the Fibroblast Growth Factor receptor (FGFR) (FGF2) in FF3 Low-Protein medium.

[0239] Materials and methods pESCs (MT002) were cultured in FF3 Low-Protein medium (medium composition described in Example 6) on cell culture plates (Sarstedt) with suitable coating such as Geltrex (Gibco) for a full passage. Immunofluorescence staining was performed as described in Example 2.

[0240] Results

[0241] FGF2 has an optimal concentration range of approximately 0-100 ng / mL. At 200 ng / mL FGF2, the pESC culture start to differentiate (Figure 13). IWR-l-endo has an optimal concentration range of approximately 1.25-5 pM (Figure 14). At 0 pM, the pESC culture differentiates, i.e., a Wnt pathway inhibitor is required for the ungulate pluripotent stem cell culture medium of the present invention. Activin A has an optional concentration range of approximately 25-100 ng / mL. At 0 ng / mL Activin A, the pESC culture shows signs of differentiation and reduced proliferation, i.e., a SMAD2 / 3 activator is required for the ungulate pluripotent stem cell culture medium of the present invention (Figure 15). Conclusion

[0242] The optimal concentration for FGF2 was below 200 ng / mL, preferably in the range of 0-100 ng / mL, more preferably 0-50 ng / mL. For Activin A, the optimal concentration range was above 0 ng / mL, preferably between 10-100 ng / mL, whereas the optimal concentration range of IWR-l-endo was above 0 pM, such as 1.25-5 pM, preferably 2.5 pM.

[0243] Example 9 - SMAD activation by various activators

[0244] Aim of study

[0245] The aim of this study was to investigate whether any SMAD activator can be used in culture medium of the present invention to maintain high pluripotency quality.

[0246] Materials and methods

[0247] Porcine ESCs were maintained in Low-Protein FF3 medium containing either Activin A, GDF8 (myostatin), GDF11, TGF-pi and the absence of a SMAD2 / 3 activator. Example 6 describes the components of the FF3 Low-Protein Medium; however, this example provides data wherein activin A is replaced with other SMAD2 / 3 activators. The concentrations of TGF-pi tested were 2.5 ng / mL, 10 ng / mL, 25 ng / mL and 50 ng / mL. The concentrations tested for GDF8 and GDF11 were 2.5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL and 100 ng / mL. However, broader ranges were equally effective (data not shown). Immunofluorescence staining was performed as described in Example 2.

[0248] Results

[0249] Activin A, GDF8 and GDF11 all resulted in very high levels of pluripotency while TGF-gl was able to maintain pluripotency at slightly lower levels. Pluripotency was lost already after one passage in the absence of a SMAD2 / 3 activator (Figure 16).

[0250] Conclusion

[0251] The culture medium of the present invention can successfully maintain high pluripotency quality regardless of the SMAD activator used. Example 10 - Wnt inhibition by various inhibitors

[0252] Aim of study

[0253] The aim of this study was to investigate whether any Wnt pathway inhibitor can be used in culture medium of the present invention to maintain high pluripotency quality.

[0254] Materials and methods

[0255] Porcine ESCs were maintained in Low-Protein FF3 medium containing either IWR- 1, XAV-939, IWP-2, Wnt-C59 and the absence of a Wnt pathway inhibitor.

[0256] Example 6 describes the components of the FF3 Low-Protein Medium; however, this example provides data wherein IWR-l-endo is replaced with other Wnt pathway inhibitors. The concentration of the different Wnt pathway inhibitors are 2.5 pM for IWR-1, XAV-939 and IWP-2 and 1 pM for Wnt-C59.

[0257] Immunofluorescence staining was performed as described in Example 2.

[0258] Results

[0259] IWR-1, XAV-939, IWP-2, Wnt-C59 all resulted in very high levels of pluripotency. Pluripotency was lost already after one passage in the absence of a Wnt pathway inhibitor (Figure 17).

[0260] Conclusion

[0261] The culture medium of the present invention can successfully maintain high pluripotency quality regardless of the Wnt pathway inhibitor used.

[0262] Example 11 - Pluripotency maintenance in various base media

[0263] Aim of study

[0264] The aim of this study was to investigate whether any base media can be used in culture medium of the present invention to maintain high pluripotency quality.

[0265] Materials and methods

[0266] Porcine ESCs were maintained in Low-Protein FF3 medium containing either DMEM / Ham's F-12, F-12, MEM or RPMI 1640 as base medium for one full passage. Example 6 describes the components of the FF3 Low-Protein Medium.

[0267] Immunofluorescence staining was performed as described in Example 2. Results

[0268] All base media were able to maintain pluripotency (Figure 18).

[0269] Conclusion

[0270] The culture medium of the present invention can successfully maintain high pluripotency quality regardless of the base media used.

[0271] Example 12 - Transferrin removal

[0272] Aim of study

[0273] Key commercial human PSC media such as Essential 8™ (Gibco) and mTeSR™ (STEMCELL Technologies) rely on transferrin. The aim of this study was to investigate whether removal of transferrin from the medium of the present invention affect the growth and pluripotency of ungulate pluripotent stem cells.

[0274] Materials and methods

[0275] Porcine ESCs were maintained in Low-Protein FF3 medium with or without transferrin for one full passage. Example 6 (Table 5) describes the components of the FF3 Low-Protein Medium with and without transferrin. Immunofluorescence staining was performed as described in Example 2.

[0276] Results

[0277] Removal of transferrin did not appear to affect the growth or pluripotency of porcine ESCs (Figures 19 and 20).

[0278] Conclusion

[0279] The culture medium of the present invention can successfully maintain ungulate PSC cultures in the absence of transferrin.

[0280] Example 13 - Suspension culture

[0281] Aim of study

[0282] The aim of this study was to investigate whether total protein content of the culture medium of the present invention affected the growth and pluripotency of ungulate PSCs in suspension culture. Materials and methods

[0283] Porcine ESCs were maintained in High-Protein or Low-Protein FF3 medium in suspension culture of four days (96 hours, DIV4). Example 6 describes the components of the FF3 Low-Protein Medium and FF3 High-Protein Medium. Immunofluorescence staining was performed as described in Example 2.

[0284] Results

[0285] Low-Protein FF3 medium resulted in considerably improved growth relative to High-Protein FF3 medium. In Low-Protein FF3 the pESCs proliferated extensively and formed aggregates with good morphology and high pluripotency levels while they in High-Protein FF3 formed aggregates of poorer morphology, display loss of Oct4 expression, and did not result in noticeable proliferation (Figures 21, 22 and 23).

[0286] Conclusion

[0287] Low-Protein FF3 medium improved suspension culture conditions extensively and resulted in superior growth, pluripotency and aggregate formation of ungulate pluripotent stem cells compared with high-protein FF3 medium.

[0288] Conclusion on the examples

[0289] Examples 2-5 demonstrate that porcine pluripotent stem cells can be cultured and retain their pluripotency in a medium composition comprising DMEM / Ham's F-12 medium, Glutamax™, NEAA (non-essential amino acids), PenStrep, Ascorbic acid, B-27™ supplement without vitamin A, N-2 supplement, Knockout™ Serum Replacement, bovine serum albumin, Activin A, IWR-l-endo, and optionally FGF2. Examples 2-4 further demonstrate that FGF2 is not an essential constituent of a culture medium for culturing porcine pluripotent stem cells. The inventors further demonstrated that the pluripotent stem cells could be cultured for an extended period of time (up to at least 45 passages) in the complex medium listed above (example 5), and the porcine pluripotent stem cells could also form aggregates which is a key characteristic of pluripotent stem cells (example 4).

[0290] Next, the inventors simplified the culture medium used in the previous examples and therefore generated and tested the FF3 Low protein medium used in example 6. Said Low protein medium comprised DMEM / Ham's F-12, insulin, transferrin, selenium, ascorbic acid, NaHCOs, FGF2, IWR-1 and Activin A. Since the previous examples showed that FGF2 was not required for the pluripotent stem cells to retain their pluripotency, it is reasonable to assume that FGF2 can also be omitted in the low protein culture medium (FF2 Low protein medium). This is demonstrated in Example 7 for bovine ESCs and Example 8 for porcine ESCs. Finally, as previously published bovine ESC culture systems using complex and protein-rich mediums also include IWR-1 and Activin A, in addition to other growth factors and supplements, it is reasonable to assume that the Low protein medium presented in this invention is applicable for other ungulate species, in particular bovine and ovine pluripotent stem cells. Example 7 clearly demonstrates that the ungulate pluripotent stem cell culture medium of the present invention (FF2 Low Protein Medium and FF3 Low Protein Medium) can be used to culture bovine ESCs. The concentrations of FGF2, IWR-1 and Activin A were further optimized in Example 8 and different varieties of SMAD2 / 3 activators and Wnt pathway inhibitors were tested in example 9 and 10, respectively. The present inventors have further demonstrated in Example 11 that the culture medium of the present invention can successfully maintain high pluripotency quality regardless of the base media used. Additionally, Example 12 demonstrates that transferrin can be omitted from the culture medium which is surprising since said compound is typically used in most commercially available culture medium for pluripotent stem cells. Interestingly, Example 13 demonstrates that the low protein culture medium of the present invention improves the growth, pluripotency and aggregate formation of ungulate pluripotent stem cells compared with high-protein culture medium.

[0291] References

[0292] • Habekost, M. et al., Transcriptomic profiling of porcine pluripotency identifies species-specific reprogramming requirements for culturing iPSCs. Stem Cell Research, 41, 101645 (2019)

[0293] • Bog I iotti, Y.S., Wu, J., Vilarino, M. et al., Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts. PNAS, 115-9, 2090-2095 (2018)

[0294] • Kinoshita, M et al., Pluripotent stem cells related to embryonic disc exhibit common self-renewal requirements in diverse livestock species. Development, 148-23 (2021)

[0295] • Zhi, M., Zhang, J., Tang, Q. et al., Generation and characterization of stable pig pregastrulation epiblast stem cell lines. Cell Res 32, 383-400 (2022)

[0296] • Choi, K.H. et a / ., Chemically defined media can maintain pig pluripotency network in vitro, Stem Cell Reports, 13-1, 221-234 (2019)

[0297] • Soto, D.A., Navarro, M., Zheng, C. et a / ., Simplification of culture conditions and feeder-free expansion of bovine embryonic stem cells. Sci Rep 11, 11045 (2021)

[0298] • WO23177181 Al

[0299] • WO2019245278 Al

[0300] • W02019140260 Al

[0301] Items

[0302] 1. An ungulate pluripotent stem cell culture medium comprising a Wnt pathway inhibitor and a SMAD2 / 3 activator, wherein said culture medium comprises less than or equal to 1,000 mg / L protein.

[0303] 2. The ungulate pluripotent stem cell culture medium according to item 1, wherein the culture medium comprises less than or equal to 800 mg / L protein, such as less than or equal to 700 mg / L protein, more preferably less than or equal to 500 mg / L protein, such as less than or equal to 200 mg / L protein, most preferably less than or equal to 150 mg / L protein, such as less than or equal to 100 mg / L protein, such as less than or equal to 60 mg / L protein, such as less than or equal to 40 mg / L protein, such as less than or equal to 30 mg / L protein, such as less than or equal to 20 mg / L protein, such as 0 mg / L protein.

[0304] 3. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the culture medium does not comprise a component of animal origin.

[0305] 4. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein said culture medium comprises less than or equal to 25 mg / L fatty acids, such as less than or equal to 22 mg / L fatty acids, such as less than or equal to 20 mg / L fatty acids, such as less than or equal to 15 mg / L fatty acids, preferably less than or equal to 10 mg / L fatty acids, such as less than or equal to 5 mg / L fatty acids, such as less than or equal to 3 mg / L fatty acids, more preferably less than or equal to 1 mg / L fatty acids, most preferably the culture medium is essentially free of fatty acids.

[0306] 5. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein said culture medium does not comprise Fibroblast Growth Factor 2 (FGF2).

[0307] 6. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein said culture medium does not comprise an activator of the Fibroblast Growth Factor receptor (FGFR), such as Fibroblast Growth Factor 1 (FGF1), FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF22, or DJ-1.

[0308] 7. The ungulate pluripotent stem cell culture medium according to item 6, wherein the Fibroblast Growth Factor receptor (FGFR) is FGFR1, FGFR2, FGFR3, or FGFR4.

[0309] 8. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the Wnt pathway inhibitor inhibits the canonical Wnt pathway.

[0310] 9. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the Wnt pathway inhibitor is selected from the group consisting of IWR-l-endo (CAS No. 1127442-82-3), IWR-1 (CAS No. 1127442- 82-3), IWR-exo-1 (CAS No. 1127442-87-8), IWR-2 (PubMED CID: 52944858), ICG-001 (CAS No. 780757-88-2), Cerberus, Wnt-C59 (CAS No. 1243243-89-1), IWP-1 (CAS No. 2074607-48-8), IWP-2 (CAS No. 686770-61-6), IWP-3 (CAS No. 687561-60-0), IWP-4 (CAS No. 686772-17-8), AZD5055, DKK-1, KY02111 (CAS No. 1118807-13-8), KYA1797K (CAS No. 1956356-56-1), JW67 (CAS No. 442644- 28-2), JW74 (CAS No. 863405-60-1), LGK974 (CAS No. 1243244-14-5), Niclosamide (CAS No. 50-65-7), Notum, NSC668036 (CAS No. 144678-63-7), CGP049090 (CAS No. 35082-49-6), BC2059 (CAS No. 1227637-23-1), ETC-159 (CAS No. 1638250-96-0), ETC-131, sFRPl, sFRP2, Shizokao ID (CAS No. 142279- 42-3), XAV939 (CAS No. 284028-89-3), and WIFI, or a mixture thereof, preferably IWR-l-endo, IWR-1, XAV-939, IWP-2, or Wnt-C59, more preferably IWR-l-endo.

[0311] 10. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein said culture medium comprises 0.1-20 pM of the Wnt pathway inhibitor, such as 0.5-15 pM of the Wnt pathway inhibitor, such as 0.7-10 pM of the Wnt pathway inhibitor, preferably 1-5 pM of the Wnt pathway inhibitor, such as 1-4 pM of the Wnt pathway inhibitor, more preferably 2-3 pM of the Wnt pathway inhibitor, most preferably 2.5 pM of the Wnt pathway inhibitor.

[0312] 11. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the SMAD2 / 3 activator activates SMAD2 and / or SMAD3 using Anaplastic lymphoma kinase 4 (ALK4), Anaplastic lymphoma kinase 5 (ALK5) and / or Anaplastic lymphoma kinase 7 (ALK7).

[0313] 12. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the SMAD2 / 3 activator is selected from the group consisting of activin A, activin B, transforming growth factor beta 1 (TGF-pi), TGF-P2, TGF-P3, Growth Differentiation Factor 1 (GDF1), GDF3, GDF8, GDF9a, GDF10, GDF11, Inducer of Definitive Endoderm 1 (IDE1), IDE2, Nodal, PD0332991 (CAS No. 827022-32-2), JNK inhibitor IX (CAS No. 312917-14-9), LY- 294002 (CAS No. 934389-88-5), JNJ-42041935 (CAS No. 1193383-09-3), and BRD-K42644990, or a mixture thereof, preferably activin A, activin B, GDF1, GDF3, GDF8, GDF11, TGF-fJl, TGF-g2, or TGF-03, more preferably activin A, GDF8, or GDF11, most preferably activin A.

[0314] 13. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein said culture medium comprises 5-100 ng / mL of the SMAD2 / 3 activator, such as 10-100 ng / mL of the SMAD2 / 3 activator, preferably 5-50 ng / mL of the SMAD2 / 3 activator, more preferably 10-30 ng / mL of the SMAD2 / 3 activator, most preferably 25 ng / mL of the SMAD2 / 3 activator.

[0315] 14. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the ratio between the Wnt pathway inhibitor and the SMAD2 / 3 activator (w / w) is selected from the range of 1: 10 to 1: 100, such as

[0316] 1: 15 to 1:80, such as 1:20 to 1: 50, such as 1:20 to 1: 30, preferably 1:25.

[0317] 15. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the culture medium does not comprise a compound of formula (I):

[0318] 16. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the culture medium does not comprise a compound of formula (II):

[0319] 17. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the culture medium does not comprise a retinoic acid receptor inhibitor.

[0320] 18. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the culture medium further comprises a base medium, such as Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Ham's Nutrient Mixture F-12 (F-12), Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F-12), RPMI 1640, Minimum Essential Medium Eagle - Alpha Modification (Alpha MEM), or a mixture thereof, preferably DMEM / Ham's F-12, MEM, F-12, or RPMI 1640, more preferably DMEM / Ham's F-12.

[0321] 19. The ungulate pluripotent stem cell culture medium according to item 18, wherein the base medium is selected from the group consisting of Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Ham's Nutrient Mixture F-12 (F-12), Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F-12), RPMI 1640, and Minimum Essential Medium Eagle - Alpha Modification (Alpha MEM), or a mixture thereof, preferably DMEM / Ham's F-12, MEM, F-12, or RPMI 1640, more preferably DMEM / Ham's F-12.

[0322] 20. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the culture medium further comprises selenium and / or a compound comprising selenium, such as SeOs2-, such as sodium selenite, such as selenium dioxide.

[0323] 21. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the culture medium further comprises insulin, insulinlike Growth Factor-1 (IGF-1), or insulin-like growth factor 2 (IGF-2), or a mixture thereof.

[0324] 22. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the culture medium further comprises transferrin.

[0325] 23. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the culture medium further comprises ascorbic acid.

[0326] 24. The ungulate pluripotent stem cell culture medium according to any one of items 1-4 or 8-23, wherein the culture medium further comprises an activator of the Fibroblast Growth Factor receptor (FGFR), such as Fibroblast Growth Factor 1 (FGF1), FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF22, or DJ-1. 25. The ungulate pluripotent stem cell culture medium according to item 24, wherein the Fibroblast Growth Factor receptor (FGFR) is FGFR1, FGFR2, FGFR3, or FGFR4.

[0327] 26. The ungulate pluripotent stem cell culture medium according to any one of items 24 or 25, wherein the activator of the Fibroblast Growth Factor receptor (FGFR) is Fibroblast Growth Factor 2 (FGF2).

[0328] 27. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the culture medium further comprises albumin, such as bovine serum albumin (BSA).

[0329] 28. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the culture medium further comprises a pH buffer, such as NaHCC .

[0330] 29. The ungulate pluripotent stem cell culture medium according to any one of items 1-4 or 8-28, wherein the culture medium further comprises a base medium, such as Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F-12), insulin, transferrin, ascorbic acid, an activator of the Fibroblast Growth Factor receptor (FGFR), albumin, NaHCOs, and selenium and / or a compound comprising selenium.

[0331] 30. The ungulate pluripotent stem cell culture medium according to any one of items 1-4, 8-26, or 28, wherein the culture medium further comprises a base medium, such as Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F-12), insulin, transferrin, ascorbic acid, an activator of the Fibroblast Growth Factor receptor (FGFR), and selenium and / or a compound comprising selenium.

[0332] 31. The ungulate pluripotent stem cell culture medium according to any one of items 1-23 or 27-28, wherein the culture medium further comprises a base medium, such as Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F-12), insulin, transferrin, ascorbic acid, and selenium and / or a compound comprising selenium. 32. The ungulate pluripotent stem cell culture medium according to items 1-4, 8-

[0333] 18, or 20-25, wherein the culture medium further comprises a base medium, such as Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F-12), insulin, ascorbic acid, an activator of the Fibroblast Growth Factor receptor (FGFR), and selenium and / or a compound comprising selenium.

[0334] 33. The ungulate pluripotent stem cell culture medium according to any one of item 1-21 or 23-28, wherein said culture medium does not comprise transferrin.

[0335] 34. The ungulate pluripotent stem cell culture medium according to any one of items 18, 19 or 29-32, wherein the DMEM / Ham's F-12 comprises: Valine

[0336] 35. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the culture medium does not comprise serum, such as fetal bovine serum (FBS).

[0337] 36. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the culture medium does not comprise albumin, such as bovine serum albumin (BSA).

[0338] 37. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein the culture medium does not comprise fetal bovine serum (FBS), Knockout™ Serum Replacement, N-2 supplement, and / or B-27™ supplement.

[0339] 38. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein said culture medium further comprises an antibiotic, such as penicillin and / or streptomycin.

[0340] 39. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein said culture medium further comprises L-glutamine or a source thereof, such as L-alanyl-L-glutamine dipeptide or GlutaMax™.

[0341] 40. The ungulate pluripotent stem cell culture medium according to any one of item 1-32, 34-36, or 38-39, wherein said culture medium further comprises N-2 supplement.

[0342] 41. The ungulate pluripotent stem cell culture medium according to item 40, wherein said N-2 supplement comprises transferrin, insulin, progesterone, putrescine and SeOs2-.

[0343] 42. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein said culture medium further comprises non-essential amino acids selected from the group consisting of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, serine, arginine, cysteine, glutamine, and tyrosine, or a mixture thereof, preferably glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine, or a mixture thereof, more preferably a mixture of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine.

[0344] 43. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein said culture medium comprises no more than three growth factors, such as no more than two growth factors.

[0345] 44. The ungulate pluripotent stem cell culture medium according to any one of the preceding items, wherein said culture medium is a porcine pluripotent stem cell culture medium.

[0346] 45. Use of the ungulate pluripotent stem cell culture medium according to any one of the preceding items for culturing ungulate pluripotent stem cells.

[0347] 46. Use of the ungulate pluripotent stem cell culture medium according to item

[0348] 45, wherein the ungulate pluripotent stem cells belong to the Artiodactyla order, preferably terrestrial artiodactyl.

[0349] 47. Use of the ungulate pluripotent stem cell culture medium according to item

[0350] 46, wherein the Artiodactyla order has a paraxonic foot structure.

[0351] 48. Use of the ungulate pluripotent stem cell culture medium according to any one of items 45-47, wherein the ungulate pluripotent stem cells belong to the Suina order or Ruminantia order.

[0352] 49. Use of the ungulate pluripotent stem cell culture medium according to any one of items 45-48, wherein the ungulate pluripotent stem cells belong to the Suidae order or Bovidae order. 50. Use of the ungulate pluripotent stem cell culture medium according to item 45, wherein the ungulate pluripotent stem cells are selected from the group consisting of porcine, bovine, bubaline, cameline, giraffa, rhinocerotic, ovine, caprine, equine, and cervine pluripotent stem cells, preferably porcine, bovine, or ovine pluripotent stem cells, more preferably porcine or bovine pluripotent stem cells, most preferably porcine pluripotent stem cells.

[0353] 51. Use of the ungulate pluripotent stem cell culture medium according to any one of items 45-50, wherein the ungulate pluripotent stem cells are porcine pluripotent stem cells or bovine pluripotent stem cells, preferably porcine pluripotent stem cells.

[0354] 52. Use of the ungulate pluripotent stem cell culture medium according to any one of items 45-51, wherein the cell culture does not comprise feeder cells, such as mouse embryonic fibroblasts (MEFs).

[0355] 53. Use of the ungulate pluripotent stem cell culture medium according to any one of items 45-52, wherein the cultured ungulate pluripotent stem cells express POU5F1, NANOG and SOX2.

[0356] 54. Use of the ungulate pluripotent stem cell culture medium according to item 53, wherein at least 75 %, such as 80 %, such as 85 %, preferably at least 90 % of the cultured ungulate pluripotent stem cells express POU5F1, NANOG and SOX2.

[0357] 55. Use of the ungulate pluripotent stem cell culture medium according to any one of items 45-54, wherein the doubling time of the ungulate pluripotent stem cells is less than 30 hours, such as less than 28 hours, preferably less than 25 hours.

[0358] 56. Use of the ungulate pluripotent stem cell culture medium according to any one of items 45-55, wherein the cultured ungulate pluripotent stem cells display absence of Erk translocation to the nucleus, and / or absence of Erk- phosphorylation, and / or absence of ETV4 / 5 and / or absence of SPRY2 / 4 gene activation. 57. Use of the ungulate pluripotent stem cell culture medium according to any one of items 45-56, wherein the cultured ungulate pluripotent stem cells display presence of Beta-catenin phosphorylation, and / or absence of Beta-catenin translocation to the nucleus, and / or absence of SP gene (SP 1-9) activation, and / or absence of AXIN2 gene activation.

[0359] 58. Use of the ungulate pluripotent stem cell culture medium according to any one of items 45-57, wherein the cultured ungulate pluripotent stem cells display presence of SMAD2 / 3 phosphorylation, and / or presence of SMAD2 / 3 translocation to the nucleus.

[0360] 59. Use of the ungulate pluripotent stem cell culture medium according to any one of items 45-58, wherein the cultured ungulate pluripotent stem cells or derivatives thereof are used for the production of cultivated meat products.

[0361] 60. Use of the ungulate pluripotent stem cell culture medium according to any one of items 45-59, wherein the cultured ungulate pluripotent stem cells or derivatives thereof are used in animal breeding programs.

[0362] 61. Use of the ungulate pluripotent stem cell culture medium according to any one of items 45-59, wherein the cultured ungulate pluripotent stem cells or derivatives thereof are used for the production of therapeutic compounds.

[0363] 62. Use of the ungulate pluripotent stem cell culture medium according to any one of items 45-59, wherein the cultured ungulate pluripotent stem cells or derivatives thereof are used for toxicological screening, drug discovery, or disease modelling.

[0364] 63. Use of the ungulate pluripotent stem cell culture medium according to any one of items 45-62, wherein the ungulate pluripotent stem cells are cultured in 2D.

[0365] 64. Use of the ungulate pluripotent stem cell culture medium according to any one of items 45-62, wherein the ungulate pluripotent stem cells are cultured in 3D. 65. Use of the ungulate pluripotent stem cell culture medium according to any one of items 45-62, wherein the ungulate pluripotent stem cells are cultured in suspension.

[0366] 66. A method for culturing ungulate pluripotent stem cells, said method comprising the steps: a) mixing a base medium with a Wnt pathway inhibitor and a SMAD2 / 3 activator to obtain a culture medium, b) adding the culture medium of step a) to ungulate pluripotent stem cells, and c) culturing the ungulate pluripotent stem cells in a culture vessel, wherein said culture medium comprises less than or equal to 1,000 mg / L protein.

[0367] 67. A method for culturing ungulate pluripotent stem cells, said method comprising the steps: a) mixing a Wnt pathway inhibitor and a SMAD2 / 3 activator to obtain a culture medium according to any one of items 1-44, b) adding the culture medium of step a) to ungulate pluripotent stem cells, and c) culturing the ungulate pluripotent stem cells in a culture vessel.

[0368] 68. The method according to item 66 or 67, wherein the temperature in step c) is in the range of 35 to 42°C, such as 37 °C to 40 °C, preferably 38 °C.

[0369] 69. The method according to any one of items 66-68, wherein the CO2 level in step c) is in the range of 3 to 7 %, preferably 5 %.

[0370] 70. The method according to any one of items 66-69, wherein the relative humidity in step c) is in the range of 85 to 95 %.

[0371] 71. The method according to any one of items 66-70, wherein the culture vessel is selected from the group consisting of a petri dish, a culture flask, a multiwell plate, a shaker flask or bioreactor, a spinner flask or bioreactor, and a scaffoldbased culture. 72. The method according to any one of items 66-71, wherein the culture medium further comprises a base medium selected from the group consisting of Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Ham's Nutrient Mixture F-12 (F-12), Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F-12), RPMI 1640, and Minimum Essential Medium Eagle - Alpha Modification (Alpha MEM), or a mixture thereof, preferably DMEM / Ham's F-12, MEM, F-12, or RPMI 1640, more preferably DMEM / Ham's F-12.

[0372] 73. The method according to any one of items 66-72, wherein the ungulate pluripotent stem cells are cultured in 2D.

[0373] 74. The method according to any one of items 66-72, wherein the ungulate pluripotent stem cells are cultured in 3D.

[0374] 75. The method according to any one of items 66-74, wherein the ungulate pluripotent stem cells are defrosted prior to step b).

[0375] 76. The method according to any one of items 66-75, wherein the ungulate pluripotent stem cells are cryopreserved after step c).

[0376] 77. The method according to any one of items 66-76, wherein the ungulate pluripotent stem cells are differentiated after step c).

Claims

1. Claims1. An ungulate pluripotent stem cell culture medium comprising a Wnt pathway inhibitor and a SMAD2 / 3 activator, wherein said culture medium comprises less than or equal to 1,000 mg / L protein.

2. The ungulate pluripotent stem cell culture medium according to claim 1, wherein the culture medium comprises less than or equal to 800 mg / L protein, such as less than or equal to 700 mg / L protein, more preferably less than or equal to 500 mg / L protein, such as less than or equal to 200 mg / L protein, most preferably less than or equal to 150 mg / L protein, such as less than or equal to 100 mg / L protein, such as less than or equal to 60 mg / L protein, such as less than or equal to 40 mg / L protein, such as less than or equal to 30 mg / L protein, such as less than or equal to 20 mg / L protein, such as 0 mg / L protein.

3. The ungulate pluripotent stem cell culture medium according to any one of the preceding claims, wherein said culture medium does not comprise Fibroblast Growth Factor 2 (FGF2).

4. The ungulate pluripotent stem cell culture medium according to any one of the preceding claims, wherein the Wnt pathway inhibitor inhibits the canonical Wnt pathway.

5. The ungulate pluripotent stem cell culture medium according to any one of the preceding claims, wherein the Wnt pathway inhibitor is selected from the group consisting of IWR-l-endo, IWR-1, IWR-exo-1, IWR-2, ICG-001, Cerberus, Wnt- C59, IWP-1, IWP-2, IWP-3, IWP-4, AZD5055, DKK-1, KY02111, KYA1797K, JW67, JW74, LGK974, Niclosamide, Notum, NSC668036, CGP049090, BC2059, ETC-159, ETC-131, sFRPl, sFRP2, Shizokao ID, XAV939, and WIFI, or a mixture thereof, preferably IWR-l-endo, IWR-1, XAV-939, IWP-2, or Wnt-C59, more preferably IWR-l-endo.

6. The ungulate pluripotent stem cell culture medium according to any one of the preceding claims, wherein the SMAD2 / 3 activator is selected from the group consisting of activin A, activin B, transforming growth factor beta 1 (TGF-pi),TGF-P2, TGF-P3, Growth Differentiation Factor 1 (GDF1), GDF3, GDF8, GDF9a, GDF10, GDF11, Inducer of Definitive Endoderm 1 (IDE1), IDE2, Nodal, PD0332991, JNK inhibitor IX, LY-294002, JNJ-42041935, and BRD-K42644990, or a mixture thereof, preferably activin A, activin B, GDF1, GDF3, GDF8, GDF11, TGF-pi, TGF-P2, or TGF-[33, more preferably activin A, GDF8, or GDF11, most preferably activin A.

7. The ungulate pluripotent stem cell culture medium according to any one of the preceding claims, wherein the culture medium further comprises a base medium, such as Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Ham's Nutrient Mixture F-12 (F-12), Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F-12 (DMEM / Ham's F-12), RPMI 1640, Minimum Essential Medium Eagle - Alpha Modification (Alpha MEM), or a mixture thereof, preferably DMEM / Ham's F-12, MEM, F-12, or RPMI 1640, more preferably DMEM / Ham's F-12.

8. The ungulate pluripotent stem cell culture medium according to any one of the preceding claims, wherein said culture medium does not comprise transferrin.

9. The ungulate pluripotent stem cell culture medium according to any one of the preceding claims, wherein said culture medium comprises no more than three growth factors.

10. The ungulate pluripotent stem cell culture medium according to any one of the preceding claims, wherein said culture medium is a porcine pluripotent stem cell culture medium.

11. Use of the ungulate pluripotent stem cell culture medium according to any one of the preceding claims for culturing ungulate pluripotent stem cells.

12. Use of the ungulate pluripotent stem cell culture medium according to claim11, wherein the ungulate pluripotent stem cells belong to the Artiodactyla order, preferably terrestrial artiodactyl.

13. Use of the ungulate pluripotent stem cell culture medium according to claim 11, wherein the ungulate pluripotent stem cells are selected from the group consisting of porcine, bovine, bubaline, cameline, giraffa, rhinocerotic, ovine, caprine, equine, and cervine pluripotent stem cells, preferably porcine, bovine, or ovine pluripotent stem cells, more preferably porcine or bovine pluripotent stem cells, most preferably porcine pluripotent stem cells.

14. Use of the ungulate pluripotent stem cell culture medium according to any one of claims 11-13, wherein the ungulate pluripotent stem cells are porcine pluripotent stem cells.

15. Use of the ungulate pluripotent stem cell culture medium according to any one of claims 11-13, wherein the ungulate pluripotent stem cells are bovine pluripotent stem cells.

16. Use of the ungulate pluripotent stem cell culture medium according to any one of claims 11-15, wherein the cell culture does not comprise feeder cells, such as mouse embryonic fibroblasts (MEFs).

17. Use of the ungulate pluripotent stem cell culture medium according to any one of claims 11-16, wherein the cultured ungulate pluripotent stem cells express POU5F1, NANOG and SOX2.

18. Use of the ungulate pluripotent stem cell culture medium according to claim 17, wherein at least 75 %, such as 80 %, such as 85 %, preferably at least 90 % of the cultured ungulate pluripotent stem cells express POU5F1, NANOG and SOX2.

19. Use of the ungulate pluripotent stem cell culture medium according to any one of claims 11-18, wherein the ungulate pluripotent stem cells are cultured in 3D.

20. A method for culturing ungulate pluripotent stem cells, said method comprising the steps: a) mixing a base medium with a Wnt pathway inhibitor and a SMAD2 / 3 activator to obtain a culture mediumb) adding the culture medium of step a) to ungulate pluripotent stem cells, and c) culturing the ungulate pluripotent stem cells in a culture vessel, wherein said culture medium comprises less than or equal to 1,000 mg / L protein.

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