Method for isolating embryonic stem cells from avian embryonic cells

Abstract

The invention relates to a method for isolating embryonic stem cells (100) from at least one avian embryonic cell, comprising: isolating at least one embryo (101) around the developmental stage of oviposition; suspending embryonic cells (102) obtained from the embryo by dissociation step a) in an animal serum-free base medium supplemented with a combination of growth factors, at least one inhibitor and an animal serum substitute; seeding the embryonic cell suspension (103) on a feeder cell layer; culturing (104) the embryonic cells at least once passaged.

Description

Invention Field

[0001] This invention belongs to the field of biotechnology. More specifically, this invention relates to a method for isolating embryonic stem cells from avian embryos, as well as kits and culture media for carrying out the method of this invention. Background Technology

[0002] Embryonic stem cells (ESCs) are characterized by their ability to differentiate into any cell type and their capacity for self-renewal. These cells offer some of the most interesting biotechnological tools and are promising candidates in the medical field because of their ability to differentiate into multiple cell types. ESCs are used in tissue engineering, alternative therapies, drug discovery, vaccine production, cultured leather, and more. Furthermore, given the anticipated increase in global meat consumption associated with the booming development of cultured meat technology, the use of ESCs in methods for producing cultured meat is particularly interesting, especially since it is possible to obtain large quantities of cells from a single cell line.

[0003] Several meat alternatives have been developed from insects, plant components, and / or cultured animal, fungal, or plant cells (i.e., cell technology). In particular, cell technology is rapidly evolving to respond to new consumer demands.

[0004] WO 2003 / 076601, WO 2008 / 129058, and WO 2020 / 104650 disclose methods for generating avian cell lines, including the step of isolating embryonic stem cells (ESCs). However, these methods suffer from a lack of reproducibility, which may depend at least in part on the need for animal-derived compounds and culture media, whose composition undergoes uncontrollable variations. Therefore, a reproducible method for isolating ESCs is needed. WO 2023 / 158627 describes the differentiation and proliferation of embryonic stem cells (ESCs) from avian species such as chickens.

[0005] The applicant has been able to develop a reproducible method for isolating avian-derived ESCs that restricts the use of undefined compounds and / or culture media of animal origin, thereby meeting the growing societal concerns for the protection of animal life and welfare and avoiding animal exploitation and slaughter. Summary of the Invention

[0006] This invention relates particularly to a method for isolating avian embryonic stromal cells (ESCs) from at least one avian embryo. The inventors have been able to define specific combinations of growth factors, which, in combination with other specific culture conditions, allow for the reproducible isolation of avian ESCs. Furthermore, the implementation of said method restricts the use of animal-derived compounds and does not use culture media with undefined compositions.

[0007] Therefore, in a first aspect, the present invention relates to a method for isolating embryonic stem cells (100) from at least one avian embryo, comprising the following steps:

[0008] a. Isolate at least one embryo during the developmental stage surrounding oviposition.

[0009] b. Embryonic cells obtained by dissociating the embryos in step a) are suspended in a serum-free basal medium supplemented with:

[0010] - A mixture of growth factors that at least regulate the following pathways: JAK / STAT, PI3K / AKT, SHP2 / MAPK, PLC-γ, MAPK, PI3K / AKT / MTOR, RAS / RAF, RHOA / ROCK

[0011] - At least one inhibitor that causes inhibition of MEK signaling,

[0012] - at least one inhibitor that causes inhibition of Wnt signaling, and

[0013] -Animal serum alternatives,

[0014] c. Inoculate the embryonic cell suspension obtained in step b) onto the feeder cell layer.

[0015] d. The embryonic cells are cultured and passaged at least once (104).

[0016] The regulation of these pathways is sufficient to allow the maintenance of the pluripotency of cells and the culture conditions set by the inventors, and allows for a low loss rate.

[0017] Other optional features of the method according to the invention may optionally include, individually or in combination, one or more of the following features:

[0018] - The mixture of growth factors contains at least one ortholog of the following growth factors: interleukin-6 (IL6), leukemia inhibitory factor (LIF), insulin-like growth factor-1 (IGF-1), and stem cell factor (SCF).

[0019] -IL6 is selected from avian IL6 or mammalian IL6 or a mixture thereof; LIF is selected from avian LIF or mammalian LIF or a mixture thereof; IGF-1 is selected from avian IGF-1 or mammalian IGF-1 or a mixture thereof; or SCF is selected from avian SCF or mammalian SCF or a mixture thereof.

[0020] -The method further comprises at least one inhibitor that causes inhibition of PKC signaling.

[0021] Inhibitors of MEK signaling are selected from small molecules or MEK-1 siRNA; inhibitors of Wnt signaling are selected from small molecules or Wnt-1 siRNA; or inhibitors of PKC signaling (if present) are selected from small molecules or PKC-α siRNA.

[0022] - Inhibitors that inhibit MEK signaling are selected from: binimetinib, cobimetinib, selumetinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901, PD98059, or U0126, or mixtures thereof. Inhibitors that inhibit Wnt signaling are selected from adavivint, capmatinib, CCT251545, FH535, ginsenoside Rh4, ICG-001, iCRT14, IQ-1, isoquercitrin, IWP-2, IWP-4, IWR-1-endo, JW55, KY02111, KY-05009, KYA1797K, lanatoside C. C) LF3, M2912, M435-1279, MSAB, NCB-0846, PNU-74654, PRI-724, styraxin, RCM-1, resibufogenin, salinomycin, triptonide, WIKI4, XAV-939, RXC004, or, when an inhibitor causing PKC signaling inhibition is present, selected from Gö6983, Gö6976, enzastaurin, ruboxistaurin, staurosporine, GF 109203X (bisindolylmaleimide I), ZIP, Ro 31-8220, Ro 32-0432, sotrastaurin, rodttlerin, K252a, bisindolylmaleimide II, protein kinase C, celandine, L-threodihydrosphingosine, melittin or midostaurin, or a mixture thereof.

[0023] -Animal serum substitutes are selected from: KnockOut TM Serum Replacement Basic; Serum Replacement 3; BIT 9500 serum replacement from StemCell.

[0024] -Avian embryonic cells are chicken embryonic cells or duck embryonic cells.

[0025] In a second aspect, the present invention relates to a method for obtaining a continuous diploid cell line derived from avian embryonic stem cells (ESCs), the method comprising:

[0026] a. Providing at least one avian ESC obtained from the method according to the invention for isolating embryonic stem cells from avian embryos,

[0027] b. Gradually remove each growth factor from the culture medium, gradually remove at least one inhibitor from the culture medium, and gradually reduce the concentration of feeder cells in the culture so that the feeder layer can be completely removed after several passages.

[0028] This allows for the acquisition of adherent or non-adherent continuous diploid avian cell lines derived from avian ESCs, which can proliferate in basal medium in the absence of growth factors.

[0029] The highly reproducible and reliable method for isolating ESCs allows for the easy acquisition of their derived cell lines, which can be used in a wide range of fields, from biotechnology and research, medicine (e.g., vaccine production) to cultured meat.

[0030] In a third aspect, the present invention relates to a kit of parts for reconstructing cell culture media, comprising:

[0031] - A direct homolog of interleukin-6 (IL-6)

[0032] - A direct homolog of leukemia inhibitory factor (LIF),

[0033] - An ortholog of insulin-like growth factor 1 (IGF-1)

[0034] - A direct homolog of stem cell factor (SCF)

[0035] - At least one inhibitor of MEK signaling, selected from i) a small molecule selected from bimetinib, cobimetinib, selemetinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901 or a mixture thereof, or ii) MEK-1 siRNA.

[0036] - At least one inhibitor of Wnt signaling, selected from i) a small molecule selected from adavivint, carmatinib, CCT251545, FH535, ginsenoside Rh4, ICG-001, iCRT14, IQ-1, isoquercitrin, IWP-2, IWP-4, IWR-1-endo, JW55, KY02111, KY-05009, KYA1797K, lanolin C, LF3, M2912, M435-1279, MSAB, NCB-0846, PNU-74654, PRI-724, strychnine, RCM-1, bufotoxin, salinomycin, tripterygium wilfordii lactone, WIKI4, XAV-939, RXC004 or mixtures thereof, or ii) Wnt-1 siRNA.

[0037] Depending on optional features, the kit may further comprise at least one inhibitor of PKC signaling, selected from i) small molecules selected from Gö6983, Gö6976, enzatolin, rubestazone, astrococcus, GF109203X (bisindolylmaleimide I), ZIP, Ro 31-8220, Ro 32-0432, sotrastolin, fentanyl, K252a, bisindolylmaleimide II, protein kinase C, chelidonine, L-threodihydrosphingosine, melittin, midostaurin or combinations thereof, or ii) PKC-α siRNA.

[0038] According to another optional feature, the kit further comprises an animal serum substitute. Attached Figure Description

[0039] Figure 1 The steps of the method 100 for isolating embryonic stem cells according to the present invention.

[0040] Figure 2 : A schematic diagram of the steps in the method for obtaining a continuous diploid cell line 200.

[0041] Figure 3 : Determination of pluripotency of duck ESCs isolated by the method of this invention. A: Results of RT-qPCR assay of pluripotency marker gene expression after 5 passages of cells; expression levels (arbitrary units) in ESCs (black) were compared with expression levels in X-stage embryos (gray) and duck fibroblasts; the tested genes were not detectably expressed in fibroblasts. B: Immunofluorescence of SSEA1 and EMA1 after 20 passages of ESCs isolated by the method of this invention.

[0042] Detailed description of the invention and other embodiments

[0043] definition

[0044] As used herein, “embryonic stem cells” (ESCs) refer to undifferentiated cells and their derivatives that possess the capacity for self-renewal through division and development into three primary germ cell layers (i.e., endoderm, ectoderm, and mesoderm). In vertebrates, almost all cell types that constitute the animal at birth originate from a subset of transient pluripotent cells in the preimplantation embryo of mammals during the blastocyst stage, called the inner cell mass (ICM), or in oviparous vertebrates (birds, reptiles, amphibians, and fish), called the blastodisc (BDM). BDM cells can also be readily isolated from early oviparous vertebrate embryos to generate pluripotent ESC lineages (WO 03 / 076601, WO 2008 / 129058, WO 2020 / 104650). Avian BDM cells suitable for generating ESCs can be obtained from any avian species.

[0045] As used herein, “birds” refers to any bird species. “Birds” means any species, subspecies, or variety of organisms belonging to the class Aves. Specifically, birds can belong to the following orders:

[0046] - Order Anseriformes. This order primarily consists of waterfowl. It comprises three families (Anatidae, Anatidae, and Anatidae), with approximately 170 species and 51 genera, covering ducks, geese, squamers, magpie geese, and swans. Most species are highly adapted to aquatic environments;

[0047] - Galliformes (e.g., chickens, quails, turkeys, pheasants, etc.). Galliformes includes 5 families: Phasianidae (including chickens, quails, partridges, pheasants, turkeys, peacocks, and grouse), Quailidae (New World quails), Guinea fowl (guinea fowl), Crested Pheasants (including chachalacas and crested pheasants), and Tomb Pheasants (hatching birds, such as malleefowl and brush-turkeys), comprising approximately 301 species;

[0048] - "Columbiformes" (i.e., pigeons and their close relatives). This order includes only one family, which includes doves and pigeons, comprising 344 species;

[0049] In another specific implementation, birds refer to poultry selected from, but not limited to, the following: chickens, turkeys, ducks, geese, guinea fowl, pigeons, quails, squabs or even pheasants, emus, swans, ostriches, parrots, sparrows, eagles, crows and cassowaries.

[0050] As used herein, “chicken” refers to the species *Gallus domesticus*; “duck” refers to any duck belonging to the order Anseriformes. In a specific implementation, “duck” refers to the species *Malus mallard* (Peking duck) or a hybrid of *Cairinamoschata domestica* and *Anas platyrhynchos domesticus* (mule duck).

[0051] As used herein, when referring to the field of cell culture, "passage" refers to the transfer of cultured cells from one culture vessel to another, with or without intended dilution. The passage number is the number of times adherent cells are passaged in a new vessel. Cells can be passaged or subcultured at any time. Typically, cells are passaged when they reach confluence (for adherent cells) and / or a given density in the culture medium. Passage can occur virtually at any time, depending on cell doubling time, stability of the culture medium components, stability of the feeder layer, predetermined time intervals, etc., provided that the cell density is sufficient to ensure that cell culture can be properly restarted. The passage time can be selected based on colony size, typically when colonies reach a diameter of 100-150 μm. Subsequently, ESCs isolated according to the method of this invention can be subjected to specific culture steps to obtain cell lines that can be cultured for extended periods and / or multiple passages, even continuously, while maintaining their pluripotency, undifferentiated state, and viability.

[0052] As used herein, "small molecule" refers to a small molecule (organic compound) with a molecular weight equal to or less than 1,000 Daltons, which regulates biological processes and leads to biological effects. For example, polymeric molecules such as nucleic acids, proteins, or polysaccharides are not small molecules in the sense of this invention, while ribose or deoxyribonucleotides, amino acids, or monosaccharides are considered small molecules.

[0053] In the context of this invention, references to a specific drug or compound include not only specifically named drugs or compounds, but also any pharmaceutically acceptable salt, hydrate, derivative, isomer, racemate, enantiomer pure composition, conjugate, or corresponding prodrug of the active molecule of the drug or compound. Preferably, references to a compound include specifically named compounds, and any pharmaceutically acceptable salt, hydrate, isomer, racemate, enantiomer, or enantiomer pure composition of said compound. More preferably, the designation of a compound is intended to specify the compound as specifically specified herein, and any pharmaceutically acceptable salt thereof.

[0054] As used herein, when referring to growth factors used in the methods according to the invention, "ortholog" means any protein of any organism that is considered to perform a function equivalent to that of the growth factors specifically named herein. Orthologs can be readily retrieved from genomic databases, such as Ensembl (<http: / / www.ensembl.org / index.html> (or other specialized databases known in the art.) It is noteworthy that a high degree of conservation of genetic pathways and morphogenesis mechanisms controlling embryonic development is observed in vertebrates from fish to humans. In other words, the growth factor used in the method according to the invention can correspond in its sequence to a growth factor from any mammalian (e.g., human or mouse) or avian organism, provided that it performs a function equivalent to that of the growth factor specifically named herein. Thus, in a particular embodiment, an ortholog of the growth factor that can be used in the method of the invention encodes a protein whose sequence in its amino acid sequence corresponds to that of chicken (Gallus domesticus) or duck (Anas esculenta). The corresponding proteins in platyrhynchos share at least 30% homology, preferably greater than 30%, more preferably greater than 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, or even 39%, preferably at least 40%, preferably greater than 40%, preferably 41%, more preferably 42%, 43%, 44%, 45%, 46%, 47%, 48%, even more preferably 49%, preferably at least 50%, preferably greater than 50%, preferably 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, even more preferably 59%, preferably at least 60%, preferably greater than 60%, preferably 61%. The sequence sharing is preferably at least 70%, preferably greater than 70%, preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, and even more preferably 79%. Preferably, the sequence sharing is at least 80%, preferably greater than 80%, preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, and even more preferably 89%. Preferably, the sequence sharing is at least 90%, preferably greater than 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and even more preferably greater than 99% homology.

[0055] The first objective of this invention is to develop a method by which the inventors can isolate ESCs from avian embryos. This method is reproducible, has a high success rate, and does not involve animal-derived compounds and / or culture media, nor does it involve undefined compositions.

[0056] Methods for isolating avian embryonic stem cells.

[0057] like Figure 1 As shown and as described above, the object of the present invention relates to a method for isolating embryonic stem cells 100 from avian embryos, comprising the following steps:

[0058] a. Isolate at least one embryo during the developmental stage surrounding oviposition.

[0059] b. Embryonic cells 102 obtained from the embryo obtained through dissection of step a) are suspended in a serum-free basal medium supplemented with:

[0060] -A mixture of growth factors that regulate at least the following pathways:

[0061] JAK / STAT, PI3K / AKT, SHP2 / MAPK, PLC-γ, MAPK, PI3K / AKT / MTOR, RAS / RAF, RHOA / ROCK,

[0062] - At least one inhibitor that causes inhibition of MEK signaling,

[0063] - at least one inhibitor that causes inhibition of Wnt signaling, and

[0064] -Animal serum substitutes,

[0065] c. Inoculate the embryonic cell suspension obtained in step b) onto the feeder cell layer 103.

[0066] d. Culture embryonic cells and passage them at least once for 10⁴ cells.

[0067] Step a) Isolate at least one embryo 101 at the developmental stage surrounding oviposition.

[0068] Preferably, for the purposes of the method 100 according to the invention, the egg is less than 2 days old, in fact, the embryonic cells can gradually differentiate from the time of spawning, and preferably have the lowest possible content in the differentiated cells.

[0069] The eggs can come from any bird as defined above. In a preferred embodiment, at least one egg is a duck or chicken egg.

[0070] Obviously, the method 100 of the present invention may include a preliminary step of providing at least one fertilized egg, and preferably, first washing it carefully with a detergent solution. A further preliminary step may include washing the egg with a 70% ethanol solution before breaking the at least one egg.

[0071] In a particular implementation, in order to block embryonic development, the eggs may be stored for 0 to 3 days at 18-21°C, 4 to 7 days at 15-17°C, or 8 to 10 days at 10-12°C prior to the embryo separation step.

[0072] As specified, in method 100 of the present invention, avian ESCs are collected from the fertilized eggs laid, from the avian embryos surrounding the egg-laying site. Depending on the species from which the egg originates, the egg-laying corresponds to different developmental stages. These stages are well known to those skilled in the art and are described in Eyal-Giladi and Kochan (1976) or Sellier et al. (2006). Essentially, depending on the species, it ranges from stage VII to stage XI.

[0073] For example, and in a specific implementation, the appropriate stage is stage VII for Muscovy ducks, stage VII-VIII for guinea fowl, stage VII-VIII for turkeys, stage VIII for pekin ducks, stage X for chickens, stage XI for Japanese quail, and stage XI for geese.

[0074] Any method for separating an embryo from an egg can be applied to achieve embryo separation. Such methods are well known in the art. In a particular embodiment, separation step 101 may include the following sub-steps:

[0075] i. Carefully break the eggs to keep the yolks intact.

[0076] ii. Separate the egg white from the yolk, and place the yolk, along with the embryo, on top of a container suitable for preserving the yolk to allow for further dissection. For example, 5 yolks can be preserved in, for example, a 14 cm petri dish.

[0077] iii. Use sterile Whatman® paper to remove excess protein from the surface of the embryo.

[0078] iv. Place a new sterile Whatman® paper with pre-cut discs ranging from 0.4 cm to 1.6 cm in diameter so that the embryo is centered on the disc and the capillaries adhere to it.

[0079] v. To quickly remove the paper carrying the embryo from the yolk.

[0080] vi. Remove as much yolk as possible from the paper and place the disc (embryo side up) in a new 14 cm culture dish, for example, pre-filled with room temperature PBS.

[0081] vii. Separate the epiblast region from the zona pellucida of the egg and collect the zona pellucida of the egg, which has a high content of stem cells.

[0082] Optionally, in step iv, four small incisions are made in the pre-cut opening on the outside of the paper disc using a sterile scalpel, and the embryo is retrieved using sterile forceps.

[0083] In step iv, the diameter of the pre-cut disc should be larger than the embryo to facilitate collection, but not too large to facilitate maintenance and avoid excessive yolk contamination of the embryo. In other words, the diameter is between 0.4 cm and 1.6 cm, preferably between 0.6 cm and 1.2 cm. In a particular embodiment, the diameter is approximately 0.8 cm. Those skilled in the art will know how to adjust the diameter of the disc according to the size of the embryo, which can vary depending on the bird species.

[0084] In step vi, any method can be used to remove as much yolk as possible from the embryo and clean it, for example,

[0085] - Use a 1 mL syringe and a 30G needle to aspirate 0.5 mL of cold PBS, then use the needle to separate and clean the embryos as much as possible without losing any embryos, or

[0086] - Use an inoculation loop to separate and clean the embryos.

[0087] An alternative to step IV-V could be the use of a micropipette with a 1000 μL tip cut to allow for proper aspiration of the embryo directly from the yolk.

[0088] Step b) The embryonic cells obtained by dissecting the embryos in step a) are suspended in a serum-free basal culture. The medium contains 102, and is supplemented with growth factors and inhibitors.

[0089] The inventors have discovered specific culture conditions that allow for the proper separation and maintenance of undifferentiated ESCs. These culture conditions involve the use of a combination of the following substances in the culture medium:

[0090] A mixture of growth factors that activate the JAK / STAT, PI3K / AKT, SHP2 / MAPK, PLC-γ, MAPK, PI3K / AKT / MTOR, RAS / RAF, and RHOA / ROCK pathways.

[0091] -A mixture of inhibitors used to block MEK- and Wnt- signaling.

[0092] - Serum-free culture medium.

[0093] It is noteworthy that, in the method according to the invention, the zona pellucida cells collected in step vii do not require chemical (e.g., using divalent ion chelating agents such as EDTA) and / or enzymatic (e.g., trypsin or any other convenient enzyme) dissociation, which is a further advantage of the method in the art.

[0094] Examples of suitable growth factors for use in growth factor mixtures are listed in Table 1 below.

[0095] Table 1

[0096]

[0097]

[0098] In method 100 of the present invention, any mixture of growth factors that primarily allow upregulation of the JAK / STAT, PI3K / AKT, SHP2 / MAPK, PLC-γ, MAPK, PI3K / AKT / MTOR, RAS / RAF, and RHOA / ROCK pathways can be used. Upregulation of these pathways has been found to be useful for maintaining cells in an undifferentiated state during method 100 of isolating ESCs of the present invention. As described above, the growth factors used in the methods according to the present invention may, in their sequence, correspond to growth factors from any source of mammalian (e.g., human or mouse) or avian organisms, provided that they perform functions equivalent to those of the growth factors specifically named herein.

[0099] In a particular implementation, any mixture of growth factors selected from duck LIF, duck IL6, duck FGF2, duck SCF, duck IGF-1, chicken LIF, chicken IL6, chicken FGF2, chicken SCF, chicken IGF-1, human LIF, human IL6, human IL6-R, human SCF, human IGF-1, human IL11, human FGF2, or their orthologs may be used.

[0100] A useful mixture of growth factors that can regulate these pathways is a mixture containing at least one interleukin-6 (IL6), at least one leukemia inhibitory factor (LIF), at least one insulin-like growth factor-1 (IGF-1), and at least one stem cell factor (SCF), or an ortholog thereof as defined above.

[0101] Preferably, the growth factor is an avian growth factor. Even more preferably, the growth factor is derived from the same avian species as the dissected embryo.

[0102] In a particular embodiment, the embryo is a chicken embryo, and the growth factor mixture is any mixture of growth factors selected from duck LIF, duck IL6, duck FGF2, duck SCF, duck IGF-1, chicken LIF, chicken IL6, chicken FGF2, chicken SCF, chicken IGF-1, human LIF, human IL6, human IL6-R, human SCF, human IGF-1, human IL11, human FGF2, and their orthologs as defined above. In a more specific embodiment, the embryo is a chicken embryo, and the growth factor mixture is a mixture of at least one chicken LIF, at least one chicken IL6, at least one chicken SCF, and at least one chicken IGF-1. In another specific embodiment, the embryo is a chicken embryo, and the growth factor mixture is a mixture of at least one human LIF, at least one human IL6, at least one human SCF, and at least one human IGF-1. In a further specific embodiment, the embryo is a chicken embryo, and the growth factor mixture is a mixture of at least one duck LIF, at least one duck IL6, at least one duck SCF, and at least one duck IGF-1.

[0103] In a particular embodiment, the embryo is a duck embryo, and the growth factor mixture is any mixture of growth factors selected from duck LIF, duck IL6, duck FGF2, duck SCF, duck IGF-1, chicken LIF, chicken IL6, chicken FGF2, chicken SCF, chicken IGF-1, human LIF, human IL6, human IL6-R, human SCF, human IGF-1, human IL11, human FGF2, and their orthologs as defined above. In a more specific embodiment, the embryo is a duck embryo, and the growth factor mixture is a mixture of at least one chicken LIF, at least one chicken IL6, at least one chicken SCF, and at least one chicken IGF-1. In another specific embodiment, the embryo is a duck embryo, and the growth factor mixture is a mixture of at least one human LIF, at least one human IL6, at least one human SCF, and at least one human IGF-1. In a further specific embodiment, the embryo is a duck embryo, and the growth factor mixture is a mixture of at least one duck LIF, at least one duck IL6, at least one duck SCF, and at least one duck IGF-1.

[0104] In a specific embodiment, the relative concentration ratio of LIF / IL6 / SCF / IGF-1 in the final culture medium is 10 / 10 / 1 / 5. In an embodiment, for the purposes of method 100 according to the invention, the final concentration of LIF is between 5 ng / mL and 15 ng / mL, preferably between 7.5 ng / mL and 12.5 ng / mL, more preferably 10 ng / mL. In an embodiment, for the purposes of method 100 according to the invention, the final concentration of IL6 is between 5 ng / mL and 15 ng / mL, preferably between 7.5 ng / mL and 12.5 ng / mL, more preferably 10 ng / mL. In an embodiment, for the purposes of method 100 according to the invention, the final concentration of SCF is between 0.5 ng / mL and 1.5 ng / mL, preferably between 0.75 ng / mL and 1.25 ng / mL, more preferably 10 ng / mL. In the implementation scheme, for the purposes of the method 100 according to the invention, the final concentration of IGF-1 is between 1 ng / mL and 10 ng / mL, preferably between 2.5 ng / mL and 7.5 ng / mL, and more preferably 10 ng / mL.

[0105] Another element of the culture medium used to implement the method 100 according to the invention is a combination of inhibitors capable of preventing cells from initiating early differentiation steps.

[0106] The inventors have discovered that inhibition of MEK- and Wnt- signaling is necessary, but together with other culture conditions according to the method 100 described herein, it is sufficient to prevent stem cells from entering the early differentiation stage. Optionally, although inhibition of MEK- and Wnt- signaling is perfectly effective in isolating ESCs, it has been surprisingly found that further inhibition of PKC- signaling particularly improves the reproducibility and success rate of the method according to the invention. Any compound or molecule capable of causing inhibition of the MEK- and / or Wnt signaling pathways, and optionally PKC- pathway inhibition, can be used. More specifically, the inhibitory compound or molecule may exert inhibitory activity against positive regulators of the target pathway and / or against elements essential for signal transduction in the pathway, ultimately leading to inhibition of signal transduction through that pathway. Alternatively, the inhibitory compound or molecule may exert activating activity against negative regulators of the target pathway, ultimately leading to inhibition of signal transduction in the target pathway.

[0107] In one embodiment, inhibitors that cause inhibition of the MEK signaling pathway inhibit mitogen-activated protein kinases MEK1 and / or MEK2. In another embodiment, inhibitors that cause inhibition of the Wnt signaling pathway directly or indirectly inhibit the typical Wnt pathway, the atypical planar cell polarity pathway, and the atypical Wnt / calcium pathway. More specifically, tankyrase inhibitors have been shown to stabilize axin and antagonize typical Wnt signaling. Therefore, inhibitors of Wnt signaling encompass tankyrase inhibitors.

[0108] In another embodiment, when present, an optional inhibitor that causes inhibition of the PKC signaling pathway, such as one or more members of the protein kinase C family selected from PKCα, PKCβ, PKCγ, PKCδ, PKCε, PKCη, PKCζ, or PKCμ.

[0109] These signaling pathways can be inhibited using any means suitable for cell culture conditions. In embodiments, siRNA can be used as an inhibitor that results in the inhibition of any of the MEK-, Wnt-, or (optionally) PKC- signaling pathways, while simultaneously inhibiting the expression of proteins responsible for the activation and / or signal transduction of any of the PKC-, MEK-, Wnt-, or PKC- signaling pathways, respectively. siRNA is known in the art as a small RNA molecule that interferes with the expression of a gene having a nucleotide sequence complementary to the siRNA's nucleotide sequence. siRNA functions by posttranscriptionally breaking down mRNA. This results in the prevention of gene translation into protein. Candidate genes can be, for example, MEK-1, Wnt1, or tankyrase, or PKC. Customized siRNAs are readily available for any gene on many vendor platforms (see EUROFINS, e.g., https: / / eurofinsgenomics.eu / en / ecom / tools / sirna-design / ).

[0110] Small molecules also provide suitable inhibitors that lead to inhibition of any of the MEK- or Wnt- or (optionally) PKC- pathways.

[0111] Examples of small molecules suitable for inhibitor combinations are listed in Table 2 below.

[0112] Table 2

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] In the embodiments, the inhibitor of the MEK pathway used in the method 100 according to the present invention is selected from: bimetinib, cobimetinib, selmetinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901, PD98059 or U0126, or a mixture thereof.

[0119] In the embodiments, the inhibitor of the Wnt pathway used in method 100 according to the present invention is selected from adavivint, carmatinib, CCT251545, FH535, ginsenoside Rh4, ICG-001, iCRT14, IQ-1, isoquercitrin, IWP-2, IWP-4, IWR-1-endo, JW55, KY02111, KY-05009, KYA1797K, lanolin C, LF3, M2912, M435-1279, MSAB, NCB-0846, PNU-74654, PRI-724, strychnine, RCM-1, bufotoxin, salinomycin, tripterygium wilfordii lactone, WIKI4, XAV-939, RXC004, or mixtures thereof.

[0120] In a specific embodiment, for carrying out the method 100 of the present invention, the culture medium comprises at least one inhibitor selected from bimetinib, cobimetinib, selemetinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901, PD98059 or U0126, and at least one inhibitor selected from adavivint, carmatinib, CCT251545, FH535, ginsenoside Rh4, ICG-001, iCRT14, IQ -1. Inhibitors of isoquercitrin, IWP-2, IWP-4, IWR-1-endo, JW55, KY02111, KY-05009, KYA1797K, lanolin C, LF3, M2912, M435-1279, MSAB, NCB-0846, PNU-74654, PRI-724, styracin, RCM-1, bufotoxin, salinomycin, tripterygium lactone, WIKI4, XAV-939, and RXC004.

[0121] In a specific embodiment, for carrying out method 100 of the present invention, the culture medium comprises at least one inhibitor selected from bimetinib, cobitinib, seletinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901, PD98059 or U0126, and at least one inhibitor selected from adavivint, carmatinib, CCT251545, FH535, ginsenoside Rh4, ICG-001, iCRT14, IQ-1, isoquercitrin, IWP-2, IWP-4. IWR-1-endo, JW55, KY02111, KY-05009, KYA1797K, lanolin C, LF3, M2912, M435-1279, MSAB, NCB-0846, PNU-74654, PRI-724, styracil, RCM-1, bufotoxin, salinomycin, triptolide, WIKI4, XAV-939, RXC004 inhibitors, and at least one inhibitor selected from Gö6983, Gö6976, enzatolin, rubestar, asteroidin, GF 109203X (bisindolylmaleimide I), ZIP, Ro 31-8220, Ro 32-0432, sotrastolin, styrosine, K252a, bisindolylmaleimide II, protein kinase C, chelidonine, L-threodihydrosphingosine, melittin, or an inhibitor of midostaurin.

[0122] In an embodiment, when used in method 100 according to the invention, the inhibitor of the PKC pathway is selected from Gö6983, Gö6976, enzatolin, rubestazone, astrococcus, GF 109203X (bisindolylmaleimide I), ZIP, Ro31-8220, Ro 32-0432, sotrastolin, fenestrated toxin, K252a, bisindolylmaleimide II, protein kinase C, celandine, L-threodihydrosphingosine, melittin, midostorine, or mixtures thereof.

[0123] In an embodiment, in method 100 of the present invention, the culture medium contains PD-325901 as an inhibitor that causes MEK signaling inhibition. In a more specific embodiment, in method 100 of the present invention, the culture medium comprises PD-325901 as an inhibitor causing MEK signaling inhibition and at least one inhibitor selected from the group causing Wnt signaling inhibition: adavivint, carmatinib, CCT251545, FH535, ginsenoside Rh4, ICG-001, iCRT14, IQ-1, isoquercitrin, IWP-2, IWP-4, IWR-1-endo, JW55, KY02111, KY-05009, KYA1797K, lanolin C, LF3, M2912, M435-1279, MSAB, NCB-0846, PNU-74654, PRI-724, strychnine, RCM-1, bufotoxin, salinomycin, tripterygium wilfordii lactone, WIKI4, XAV-939, RXC004, or combinations thereof. In another specific embodiment, in method 100 of the present invention, the culture medium comprises PD-325901 as an inhibitor causing MEK signaling inhibition and at least one inhibitor selected from the following that causes PKC signaling inhibition: Gö6983, Gö6976, enzatolin, rubestazone, astrococcal, GF109203X (bisindolylmaleimide I), ZIP, Ro 31-8220, Ro 32-0432, sotrastolin, fentanyl, K252a, bisindolylmaleimide II, protein kinase C, celandine, L-threodihydrosphingosine, melittin, midostaurin, or combinations thereof.

[0124] In a more specific embodiment, in method 100 of the present invention, the culture medium comprises PD98059 as an inhibitor causing MEK signaling inhibition and at least one inhibitor selected from the group causing Wnt signaling inhibition: adavivint, carmatinib, CCT251545, FH535, ginsenoside Rh4, ICG-001, iCRT14, IQ-1, isoquercitrin, IWP-2, IWP-4, IWR-1-endo, JW55, KY02111, KY-05009, KYA1797K, lanolin C, LF3, M2912, M435-1279, MSAB, NCB-0846, PNU-74654, PRI-724, strychnine, RCM-1, bufotoxin, salinomycin, tripterygium lactone, WIKI4, XAV-939, RXC004, or combinations thereof. In another specific embodiment, in method 100 of the present invention, the culture medium comprises PD98059 as an inhibitor causing MEK signaling inhibition and at least one inhibitor selected from the following that causes PKC signaling inhibition: Gö6983, Gö6976, enzatolin, rubestazone, astrococcal, GF109203X (bisindolylmaleimide I), ZIP, Ro 31-8220, Ro 32-0432, sotrastolin, fentanyl, K252a, bisindolylmaleimide II, protein kinase C, celandine, L-threodihydrosphingosine, melittin, midostorine, or combinations thereof.

[0125] In a more specific embodiment, in method 100 of the present invention, the culture medium comprises U0126 as an inhibitor causing MEK signaling inhibition and at least one inhibitor selected from the group causing Wnt signaling inhibition: adavivint, carmatinib, CCT251545, FH535, ginsenoside Rh4, ICG-001, iCRT14, IQ-1, isoquercitrin, IWP-2, IWP-4, IWR-1-endo, JW55, KY02111, KY-05009, KYA1797K, lanolin C, LF3, M2912, M435-1279, MSAB, NCB-0846, PNU-74654, PRI-724, strychnine, RCM-1, bufotoxin, salinomycin, tripterygium lactone, WIKI4, XAV-939, RXC004, or combinations thereof. In another specific embodiment, in method 100 of the present invention, the culture medium comprises U0126 as an inhibitor causing MEK signaling inhibition and at least one inhibitor selected from the following that causes PKC signaling inhibition: Gö6983, Gö6976, enzatolin, rubestazone, astrococcal, GF109203X (bisindolylmaleimide I), ZIP, Ro 31-8220, Ro 32-0432, sotrastolin, fentanyl, K252a, bisindolylmaleimide II, protein kinase C, celandine, L-threodihydrosphingosine, melittin, midostaurin, or combinations thereof.

[0126] In a more specific embodiment, in method 100 of the present invention, the culture medium comprises bimetinib as an inhibitor causing MEK signaling inhibition and at least one inhibitor selected from the group causing Wnt signaling inhibition: adavivint, carmatinib, CCT251545, FH535, ginsenoside Rh4, ICG-001, iCRT14, IQ-1, isoquercitrin, IWP-2, IWP-4, IWR-1-endo, JW55, KY02111, KY-05009, KYA1797K, lanolin C, LF3, M2912, M435-1279, MSAB, NCB-0846, PNU-74654, PRI-724, strychnine, RCM-1, bufotoxin, salinomycin, tripterygium wilfordii lactone, WIKI4, XAV-939, RXC004, or combinations thereof. In another specific embodiment, in method 100 of the present invention, the culture medium comprises bimetinib as an inhibitor causing MEK signaling inhibition and at least one inhibitor selected from the following that causes PKC signaling inhibition: Gö6983, Gö6976, enzatolin, rubestazone, astrococcus, GF109203X (bisindolylmaleimide I), ZIP, Ro 31-8220, Ro 32-0432, sotrastolin, fentanyl, K252a, bisindolylmaleimide II, protein kinase C, celandine, L-threodihydrosphingosine, melittin, midostaurin, or combinations thereof.

[0127] In a particular embodiment, in method 100 of the present invention, the culture medium contains a tankyrase inhibitor as an inhibitor causing inhibition of Wnt signaling. In a more specific embodiment, in method 100 of the present invention, the culture medium contains a tankyrase inhibitor as an inhibitor causing inhibition of Wnt and at least one inhibitor selected from the group causing inhibition of MEK signaling: bismuthinib, cobimetinib, selumetinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901, PD98059, or U0126, or combinations thereof. In another specific embodiment, in method 100 of the present invention, the culture medium comprises a tankyrase inhibitor as an inhibitor causing inhibition of Wnt signaling and at least one inhibitor selected from the group causing inhibition of PKC signaling: Gö6983, Gö6976, enzatolin, rubestazone, astrococcal, GF109203X (bisindolylmaleimide I), ZIP, Ro 31-8220, Ro 32-0432, sotrastolin, fentanyl, K252a, bisindolylmaleimide II, protein kinase C, celandine, L-threodihydrosphingosine, melittin, midostaurin, or combinations thereof.

[0128] In a particular embodiment, in method 100 of the present invention, the culture medium contains XAV-939 as an inhibitor causing inhibition of Wnt signaling. In a more particular embodiment, in method 100 of the present invention, the culture medium contains XAV-939 as an inhibitor causing inhibition of Wnt signaling and at least one inhibitor selected from the group causing inhibition of MEK signaling: bimetinib, cobimetinib, selumetinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901, PD98059, or U0126, or combinations thereof. In another specific embodiment, in method 100 of the present invention, the culture medium comprises XAV-939 as an inhibitor causing inhibition of Wnt signaling and at least one inhibitor selected from the group causing inhibition of PKC signaling: Gö6983, Gö6976, enzatolin, rubestazone, astrococcal, GF 109203X (bisindolylmaleimide I), ZIP, Ro 31-8220, Ro 32-0432, sotrastolin, fentanyl, K252a, bisindolylmaleimide II, protein kinase C, celandine, L-threodihydrosphingosine, melittin, midostorine, or combinations thereof.

[0129] In a more specific embodiment, in method 100 of the present invention, the culture medium comprises IWP-2 as an inhibitor causing inhibition of Wnt signaling and at least one inhibitor selected from the group causing inhibition of MEK signaling: bimetinib, cobitinib, selmetinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901, PD98059 or U0126 or a combination thereof. In another specific embodiment, in method 100 of the present invention, the culture medium comprises IWP-2 as an inhibitor causing inhibition of Wnt signaling and at least one inhibitor selected from the following that causes inhibition of PKC signaling: Gö6983, Gö6976, enzatolin, rubestazone, astrococcal, GF 109203X (bisindolylmaleimide I), ZIP, Ro 31-8220, Ro 32-0432, sotrastolin, fentanyl, K252a, bisindolylmaleimide II, protein kinase C, celandine, L-threodihydrosphingosine, melittin, midostaurin, or combinations thereof.

[0130] In a more specific embodiment, in method 100 of the present invention, the culture medium comprises adavivint as an inhibitor causing inhibition of Wnt signaling and at least one inhibitor selected from the group causing inhibition of MEK signaling: bimetinib, cobimetinib, selmetinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901, PD98059 or U0126 or a combination thereof. In another specific embodiment, in method 100 of the present invention, the culture medium comprises adavivint as an inhibitor causing inhibition of Wnt signaling and at least one inhibitor selected from the group causing inhibition of PKC signaling: Gö6983, Gö6976, enzatolin, rubestazone, astrococcal, GF 109203X (bisindolylmaleimide I), ZIP, Ro 31-8220, Ro 32-0432, sotrastolin, fenestrated venom, K252a, bisindolylmaleimide II, protein kinase C, celandine, L-threodihydrosphingosine, melittin, midostaurin, or combinations thereof.

[0131] In a more specific embodiment, in method 100 of the present invention, the culture medium comprises carmatinib as an inhibitor causing inhibition of Wnt signaling and at least one inhibitor selected from the group causing inhibition of MEK signaling: bimetinib, cobitinib, selmetinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901, PD98059 or U0126 or a combination thereof. In another specific embodiment, in method 100 of the present invention, the culture medium comprises carmatinib as an inhibitor causing Wnt signaling inhibition and at least one inhibitor selected from the group causing PKC signaling inhibition: Gö6983, Gö6976, enzatolin, rubestazone, astrococcus, GF 109203X (bisindolylmaleimide I), ZIP, Ro 31-8220, Ro 32-0432, sotrastolin, fentanyl, K252a, bisindolylmaleimide II, protein kinase C, celandine, L-threodihydrosphingosine, melittin, midostaurin, or combinations thereof.

[0132] In a further specific embodiment, in method 100 of the present invention, the culture medium contains Gö6983 as an inhibitor causing inhibition of PKC signaling. In a more specific embodiment, in method 100 of the present invention, the culture medium contains Gö6983 as an inhibitor causing inhibition of PKC signaling and at least one inhibitor selected from the group causing inhibition of MEK signaling: bimetinib, cobimetinib, selmetinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901, PD98059 or U0126, or combinations thereof. In another specific embodiment, in method 100 of the present invention, the culture medium comprises Gö6983 as an inhibitor causing PKC signaling inhibition and at least one inhibitor selected from the Wnt pathway: adavivint, carmatinib, CCT251545, FH535, ginsenoside Rh4, ICG-001, iCRT14, IQ-1, isoquercitrin, IWP-2, IWP-4, IWR-1-endo, JW55, KY02111, KY-05009, KYA1797K, lanolin C, LF3, M2912, M435-1279, MSAB, NCB-0846, PNU-74654, PRI-724, styracin, RCM-1, bufotoxin, salinomycin, tripterygium lactone, WIKI4, XAV-939, RXC004, or combinations thereof. In a more specific embodiment, in method 100 of the present invention, the culture medium comprises Gö6976 as an inhibitor causing PKC signaling inhibition and at least one inhibitor selected from the group causing MEK signaling inhibition: bimetinib, cobitinib, selmetinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901, PD98059 or U0126, or a combination thereof. In another specific embodiment, in method 100 of the present invention, the culture medium comprises Gö6976 as an inhibitor causing PKC signaling inhibition and at least one inhibitor selected from the Wnt pathway: adavivint, carmatinib, CCT251545, FH535, ginsenoside Rh4, ICG-001, iCRT14, IQ-1, isoquercitrin, IWP-2, IWP-4, IWR-1-endo, JW55, KY02111, KY-05009, KYA1797K, lanolin C, LF3, M2912, M435-1279, MSAB, NCB-0846, PNU-74654, PRI-724, strychnine, RCM-1, bufotoxin, salinomycin, tripterygium lactone, WIKI4, XAV-939, RXC004, or combinations thereof.In a more specific embodiment, in method 100 of the present invention, the culture medium comprises enzatolin as an inhibitor causing PKC signaling inhibition and at least one inhibitor selected from the group causing MEK signaling inhibition: bimetinib, cobimetinib, selmetinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901, PD98059 or U0126, or a combination thereof. In another specific embodiment, in method 100 of the present invention, the culture medium comprises enzatolin as an inhibitor causing inhibition of PKC signaling and at least one inhibitor selected from the Wnt pathway: adavivint, carmatinib, CCT251545, FH535, ginsenoside Rh4, ICG-001, iCRT14, IQ-1, isoquercitrin, IWP-2, IWP-4, IWR-1-endo, JW55, KY02111, KY-05009, KYA1797K, lanolin C, LF3, M2912, M435-1279, MSAB, NCB-0846, PNU-74654, PRI-724, styracin, RCM-1, bufotoxin, salinomycin, tripterygium wilfordii lactone, WIKI4, XAV-939, RXC004, or combinations thereof. In a more specific embodiment, in method 100 of the present invention, the culture medium comprises rubesta as an inhibitor causing PKC signaling inhibition and at least one inhibitor selected from the group causing MEK signaling inhibition: bimetinib, cobitinib, selmetinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901, PD98059 or U0126, or a combination thereof. In another specific embodiment, in method 100 of the present invention, the culture medium comprises rubesta as an inhibitor causing PKC signaling inhibition and at least one inhibitor selected from the Wnt pathway: adavivint, carmatinib, CCT251545, FH535, ginsenoside Rh4, ICG-001, iCRT14, IQ-1, isoquercitrin, IWP-2, IWP-4, IWR-1-endo, JW55, KY02111, KY-05009, KYA1797K, lanolin C, LF3, M2912, M435-1279, MSAB, NCB-0846, PNU-74654, PRI-724, styracin, RCM-1, bufotoxin, salinomycin, tripterygium wilfordii lactone, WIKI4, XAV-939, RXC004, or combinations thereof.

[0133] In a particular embodiment, the culture medium used in the method 100 according to the invention comprises a combination of at least one inhibitor selected from the following:

[0134] -adavivint and Gö6976,

[0135] - Bimetinib and Gö6983

[0136] -Camatinib and IWP-2,

[0137] - Enzatolin and PD-325901,

[0138] -Gö6976 and PD-325901,

[0139] -Gö6983 and PD98059,

[0140] -IWP-2 and Rubesta,

[0141] -PD-325901 and U0126,

[0142] -PD98059 and adavivint,

[0143] - Lubersta and Bimetinib

[0144] -U0126 and Camatinib,

[0145] -XAV-939 and Enzatolin,

[0146] -XAV-939 and PD98059,

[0147] -XAV-939 and Gö6983,

[0148] -Gö6976 and carmatinib and IWP-2,

[0149] -Gö6983 and PD98059 and adavivint,

[0150] -Gö6983 and PD-325901 and Xav-939,

[0151] - Lubersta and PD-325901 and Bimetinib, or

[0152] - Enzatolin and XAV-939 and U0126.

[0153] In a particular embodiment, each small molecule that acts as an inhibitor in the inhibitor combination described above is present in cell culture medium at a final concentration of 0.05 µM to 4 µM, preferably 0.1 µM to 2 µM, preferably 0.5 µM to 1 µM.

[0154] In a more specific embodiment, in method 100 of the present invention, the culture medium comprises:

[0155] - At least one MEK signaling inhibitor, having a final concentration in cell culture medium of 0.05 μM to 2 μM, preferably 0.5 μM to 1 μM, and / or

[0156] - At least one Wnt signaling inhibitor, having a final concentration in cell culture medium of 0.5 μM to 4 μM, preferably 0.1 μM to 2 μM.

[0157] In a more specific embodiment, in method 100 of the present invention, the culture medium comprises:

[0158] - At least one MEK signaling inhibitor, with a final concentration in cell culture medium of 0.05 μM to 2 μM, preferably 0.5 μM to 1 μM.

[0159] - At least one Wnt signaling inhibitor, with a final concentration in cell culture medium of 0.5 μM to 4 μM, preferably 0.1 μM to 2 µM.

[0160] - At least one PKC inhibitor, having a final concentration in cell culture medium of 0.5 μM to 4 μM, preferably 0.1 μM to 2 μM.

[0161] Another important feature of implementing the method 100 of the present invention and successfully isolating avian ESCs is the culture medium. The culture conditions determined by the inventors allow the use of serum-free culture media. In other words, it is not necessary to supplement the culture medium with animal-derived serum. More preferably, the animal serum substitutes used in the method 100 of the present invention contain very little (if any) animal-derived components. When they do contain animal-derived components, these are known amounts of purified and well-defined compounds (transferrin, BSA, etc.), ensuring no negative impact on reproducibility. This is particularly interesting because it allows for the avoidance of unpredictability associated with undesirable and uncontrollable changes that can occur when using animal-derived products such as fetal bovine serum (FBS). In other words, this feature, in addition to allowing for good isolation of avian ESCs, contributes to the high reproducibility of the method 100 of the present invention. Furthermore, avoiding the use of animal-derived products addresses growing concerns, such as those regarding animal welfare or safety for human consumption of FBS, and is particularly significant when the ESCs are considered for pharmaceutical purposes or for the preparation of cultured meat or any edible food or even laboratory-grown leather.

[0162] Such animal serum substitutes are commercially available. The culture medium that can be used in method 100 of the present invention is, for example:

[0163] -KNOCKOUT TM SR (Gibco),

[0164] -Basic serum replacement (Genaxxon Bioscience)

[0165] - Serum substitute 3 (Sigma),

[0166] - Serum-free B27 Supplement TM (ThermoFisher), or

[0167] -BIT 9500 serum substitute (StemCell).

[0168] These alternatives do not contain animal serum and contain little or no animal-derived compounds. These compounds are clearly identified and used at prescribed concentrations.

[0169] As used herein, "basal medium" refers to a medium that at least allows cell survival, and even better, cell growth. Examples of suitable basal media are BME (Basic Eagle Medium), MEM (Minimum Eagle Medium), 20 Medium 199, DMEM (Dulbecco's Modified Eagle Medium), GMEM (Glasgow Modified Eagle Medium), DMEM F-12, Ham-F12, Ham-F10, Iscove's Modified Dulbecco Medium, MacCoy's 5A Medium, RPMI 1640, GTM3, or combinations thereof. Basal media contain inorganic salts (e.g., CaCl2, KCl, NaCl, NaHCO3, NaH2PO4, MgSO4, etc.), amino acids, vitamins (thiamine, riboflavin, folic acid, D-Ca pantothenate, etc.), and other components such as glucose, 2-mercaptoethanol, and sodium pyruvate. Preferably, the basal medium is a synthetic medium.

[0170] Compositions of DMEM / F-12 suitable as exemplary culture media for the present invention are provided in Table 3 below.

[0171] Table 3

[0172]

[0173]

[0174]

[0175] The basal culture medium is usually supplemented with non-essential amino acids, sodium pyruvate, L-glutamine, 2-mercaptoethanol, and a mixture of penicillin and streptomycin.

[0176] Typically, the basal culture medium of the present invention may be supplemented with additives selected from the following list:

[0177] -0.1 mM to 5 mM L-glutamine, preferably 2 to 3 mM;

[0178] -0.05 mM to 2 mM sodium pyruvate, preferably 0.5 mM to 1.5 mM sodium pyruvate;

[0179] -0.05 mM to 2 mM 2-mercaptoethanol, preferably 0.1 mM to 1 mM;

[0180] -0.5% to 2% non-essential amino acids.

[0181] Step c) The embryonic cell suspension obtained in step b) is inoculated onto the feeder cell layer 103.

[0182] In method 100 of the present invention, cells from at least one dissected embryo obtained in the foregoing steps are seeded onto a feeder cell layer 103 laid at the bottom of a culture well. Alternatively, but less preferably, the feeder cells may be replaced with an extracellular matrix, which may also contain binding growth factors coated on the surface of the culture dish. In an embodiment, the feeder cells are inactivated by mitosis using standard techniques (irradiation or with a double-stranded DNA alkylating agent).

[0183] Typically, STO fibroblasts are preferred and can be any mammalian species (e.g., rats, ungulates, cattle, pigs); or avian species.

[0184] In a specific embodiment, the feeder cells are proliferating mouse STO cells. In even more specific embodiments, the feeder cells are STO CRL-1503. T (United States Type Culture Collection (ATCC))

[0185] In another implementation, the feeder cells are grown in a monolayer in the culture until they reach about 80%, about 90%, or even about 100% confluence.

[0186] In another embodiment, the feeder cells are replaced by an organic extracellular matrix or a feeder cell conditioned medium. Non-limiting examples of organic matrices include matrix gel, vitrin, or any organic matrix known to those skilled in the art. In yet another aspect, the feeder cells are replaced by an organic matrix supplemented with activin-A (e.g., 10 ng / mL activin-A).

[0187] Step d) The cultured and inoculated embryonic cells are passaged at least once for 104 times.

[0188] The culture medium can be changed daily; especially on the first day of culture, when there may be a large amount of cell debris and lipids.

[0189] ESCs should be passed down at the rendezvous time. The definition of passing down is as above.

[0190] Typically, it can be passed down 1 to 20 times or even more.

[0191] Then, the ESCs isolated by method 100 of the present invention can be stored by cryopreservation or by stepwise removal of the above-mentioned growth factors and / or feeder cells and / or inhibitors in order to obtain a stable cell line of ESCs.

[0192] The cells are cultured in a controlled atmosphere at a temperature of 35°C to 39°C, preferably 37°C, and at a CO2 concentration of more than 5% and less than 10%, preferably 6% to 9%, even more preferably 7.5%.

[0193] In a particular embodiment, method 100 of the present invention may include a further step of controlling the pluripotency of cells 105 obtained in step d). This control step may be performed by measuring the expression levels of a set of pluripotency markers. Expression levels may be measured, for example, by RT-qPCR or an immunological method (FACS, immunofluorescence). These pluripotency markers are selected from one or more of, for example, OCT4, TERT, DNMT3B, NANOG, CXCL5, DNMT3B, HESX1, IDO1, LCK, POU5F1, SOX2, TRIM22, EMA1, and SSEA1. Preferably, pluripotency is assessed by determining the pluripotency expression profile of the cells by measuring the expression of at least four pluripotency markers, such as those listed above. A cell is considered to have maintained its pluripotency when its pluripotency expression profile is at least 60%, at least 70%, at least 80%, at least 90%, and even 100% identical to that of a control pluripotent cell. Several bioinformatics assays are commercially available for assessing cell pluripotency (https: / / www.thermofisher.com / fr / fr / home / life-science / stem-cell-research / induced-pluripotent-stem-cells / pluripotent-stem-cell-detection.html). In an embodiment, a cell is considered to have maintained its pluripotency characteristics when at least three of the four pluripotency markers remain at levels higher than those of differentiated cells. Pluripotency expression profiling can be correlated with telomerase expression assays or any pluripotency assays known to those skilled in the art.

[0194] Therefore, the pluripotency of ESCs can be controlled, for example, at each generation, as described above. In a particular embodiment, a group that meets the above criteria after the fifth generation or even further is considered a valuable ESC lineage. Obviously, the more generations an ESC undergoes while maintaining its pluripotency, the more stable the ESC lineage is considered. Thus, in a preferred embodiment of the method 100 according to the invention, the ESC has undergone at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or even 20 generations while maintaining its pluripotency.

[0195] A second object of the present invention is a method 200 for obtaining continuous diploid cell lines from ESCs isolated as described above.

[0196] Methods for obtaining continuous diploid cell lines 200.

[0197] like Figure 2 As shown, the method 200 for obtaining a continuous diploid cell line according to the second objective of the present invention includes all the steps of the method 100 for isolating avian ESCs as described in any of the above embodiments (step 201 of providing at least one obtained ESC) and a further step of removal method 202, which includes the stepwise removal (e.g., reduction of 25% per 2 to 3 passages) of any one of growth factors 203, inhibitors 204 (e.g., reduced by 25% per 2 to 3 passages) and / or feeder cells 205 (e.g., reduced by 25% per 2 to 3 passages) from the ESC culture medium, specifically operated in the same manner as the method disclosed in WO2008 / 129058, with corresponding adjustments, so that the cells are de-dependent on the growth factors, inhibitors, and feeder cells. The removal can be performed simultaneously, sequentially, or separately.

[0198] The order in which 203, 204, and 205 are removed can be, but is not limited to, for example:

[0199] - Feeding cells / inhibitors / growth factors;

[0200] - Feeding cells / growth factors / inhibitors;

[0201] -Inhibitors / growth factors / feeder cells;

[0202] -Inhibitors / feeder cells / growth factors;

[0203] - Growth factors / inhibitors / feeder cells; or

[0204] - Growth factors / feeder cells / inhibitors.

[0205] The cells obtained at the end of step 202 are pluripotent cells capable of dividing indefinitely without growth factors. The pluripotency of the cells can be determined in a further step 206, as explained previously in further step 105 of method 100.

[0206] The inventors have determined that specific compounds and products can be used to isolate avian ESCs and extract them from continuous pluripotent cell lines. Therefore, a third object of the present invention is a component kit that can be used to carry out the methods for achieving these objectives.

[0207] A component kit for reconstructing cell culture media suitable for implementing the method according to the invention.

[0208] A kit for carrying out methods 100 and 200 of the present invention and for reconstructing cell culture media may include:

[0209] -A group of growth factors, including one or more of IL6, leukemia inhibitory factor LIF, IGF-1 or SCF, or any of their orthologs;

[0210] - At least one inhibitor as defined above that causes inhibition of MEK-, Wnt- and optionally PKC- signaling.

[0211] Also in a more specific embodiment, the present invention relates to a kit that further comprises an animal serum substitute.

[0212] In a particular embodiment, the kit according to the present invention comprises:

[0213] a. At least one inhibitor of MEK signaling, selected from i) a small molecule selected from bimetinib, cobimetinib, selemetinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901 or a mixture thereof, or ii) MEK-1 siRNA.

[0214] b. At least one inhibitor of Wnt signaling is selected from i) a small molecule selected from adavivint, carmatinib, CCT251545, FH535, ginsenoside Rh4, ICG-001, iCRT14, IQ-1, isoquercitrin, IWP-2, IWP-4, IWR-1-endo, JW55, KY02111, KY-05009, KYA1797K, lanolin C, LF3, M2912, M435-1279, MSAB, NCB-0846, PNU-74654, PRI-724, strychnine, RCM-1, bufotoxin, salinomycin, tripterygium wilfordii lactone, WIKI4, XAV-939 or RXC004 or mixtures thereof, ii) Wnt-1 siRNA, and

[0215] c. At least one inhibitor of PKC signaling, selected from i) a small molecule selected from Gö6983, Gö6976, enzatolin, rubestazone, astrococcus, GF 109203X (bisindolylmaleimide I), ZIP, Ro 31-8220, Ro 32-0432, sotrastolin, bufotoxin, K252a, bisindolylmaleimide II, protein kinase C, chelidonine, L-threodihydrosphingosine, melittin, midostaurin or a mixture thereof, or a mixture thereof, or ii) PKC-αsiRNA.

[0216] In a particular embodiment, in the kit according to the invention, at least one inhibitor and growth factor are adjusted to allow their final concentrations to be reached in the reconstituted medium used in the method for isolating ESC 100 according to the invention.

[0217] In a further specific embodiment, the kit according to the invention may contain materials that can be used to determine the pluripotency of cells produced by methods 100 and 200. Therefore, in this embodiment, the kit may contain a set of oligonucleotides that allow for the determination of the pluripotency expression profile as described above, for example, at least one set of oligonucleotides suitable for measuring the expression levels of at least one or more of the pluripotency markers OCT4, TERT, DNMT3B, NANOG, CXCL5, DNMT3B, HESX1, IDO1, LCK, POU5F1, SOX2, or TRIM22.

[0218] The following examples are given for illustrative purposes and are not intended to be limiting. Example

[0219] I. Materials and Methods

[0220] a. Materials

[0221] duck eggs

[0222] Certified Beijing duck eggs (Orvia, France) from ducks less than 3 days old:

[0223] -Avian influenza sensitivity control,

[0224] - Free from veterinary drug, pesticide, and pollutant residues

[0225] - Free of Salmonella

[0226] - No intraembryonic vaccination.

[0227] Eggs are collected at the time of spawning and stored between 18-21°C. If it is necessary to stop embryonic development, they are stored. Eggs are used on the day of collection or the day after spawning. Therefore, retrieved embryos are less than 3 days old.

[0228] Chicken eggs

[0229] Certified chicken eggs less than 3 days old (Orvia, France):

[0230] -Avian influenza sensitivity control,

[0231] - Free from veterinary drug, pesticide, and pollutant residues

[0232] - Free of Salmonella

[0233] - No intraembryonic vaccination

[0234] Eggs are collected at the time of spawning and stored between 18-21°C. If it is necessary to stop embryonic development, they are stored. Eggs are used on the day of collection or the day after spawning. Therefore, retrieved embryos are less than 3 days old.

[0235] Culture medium and culture

[0236] DMEM / F12-KO (Thermofisher, #12660-012)

[0237] BIT9500 (Stemcell technologies, # 09500)

[0238] Non-essential amino acids (Thermofisher, #11140-050)

[0239] Sodium pyruvate (Thermofisher, #11530-396)

[0240] L-Glutamine (Thermofisher, #2503123)

[0241] 2-Mercaptoethanol (Thermofisher, #31350-010)

[0242] Penicillin / Streptomycin (Thermofisher, #11548876)

[0243] Feeder cells: STO CRL-1503 TM

[0244] Antibody

[0245] Primary antibodies: Anti-SSEA1: ThermoFisher (catalog number MA1-022) or Abcam (ref: ab16285) or SantaCruz (ref sc-21702); Anti-EMA1: Abcam (ref: ab109110).

[0246] Secondary antibody: Anti-mouse IgM (Alexa-488).

[0247] growth factors

[0248] Based on sequences retrieved from the Uniprot database and listed in Table 1, recombinant growth factors were generated internally using common biological molecular tools. Human and chicken growth factors are commercially available through other means. Concentrations ranging from 1 ng / mL to 10 ng / mL of growth factors were tested.

[0249] Inhibitors

[0250] The test inhibitors were used at a final concentration ranging from 1 to 2 µM. They are commercially available and can be retrieved using the CAS numbers or their names listed in Table 2.

[0251] b. ESCs were isolated from duck or chicken embryos.

[0252] First, wash the five eggs with tissue soaked in ethanol at room temperature, then carefully break them up to keep the yolks intact, gently separate the albumen from the yolks, and place the yolks in a 14 cm petri dish with the embryos on top.

[0253] Use Whatman® paper to detect excess white on the surface of the embryo. Place a Whatman® paper with holes around each embryo on top of the embryo and make 3-4 small incisions on the outside of the paper disc using scissors (while it is in 70% ethanol). Use forceps (also in 70% ethanol) to separate the paper carrying each embryo from the yolk and place it in a new 14 cm culture dish pre-filled with cold PBS, with the embryo facing up.

[0254] Subsequently, using a 1 mL syringe with a 30G needle, each embryo was washed and separated by spraying and aspirating 0.5 mL of PBS. Each embryo was then aspirated using a 1000 μL pipette tip.

[0255] The epiblast region of the embryo was removed to collect only the center, and the embryo was placed in 15 mL of falcon containing 1 mL of PBS at room temperature. The dissected embryo was then centrifuged at 300 g for 5 minutes at room temperature and resuspended in test medium.

[0256] Remove the STO medium from the culture plate containing the feeder cells, then rinse each well once with PBS, seed the cells, and incubate statically at 37°C, 7.5% CO2. Seed three wells with dissected embryos.

[0257] The day after isolation, replace the medium with 1.5 mL of 37°C medium. If excessive lipids and cell debris are present, wash once or twice with room temperature PBS.

[0258] Once the ESC population emerges, perform daily culture medium changes and the first passage. Afterward, cells must be passaged at confluence time.

[0259] Pluripotency can be tested at each generation.

[0260] RT qPCR

[0261] RNA was extracted from cell pellets collected from three wells of a 12-well plate using the Monarch® Total RNA Miniprep Kit (NEB).

[0262] According to the supplier's instructions, the "High-Capacity cDNA Reverse Transcription Kit" (ThermoFicherscientific) was used to reverse transcribe RNA extracted from cells.

[0263] For qPCR, oligonucleotides for OCT4, TERT, DNMT3B, NANOG, CXCL5, DNMT3B, HESX1, IDO1, LCK, POU5F1, SOX2, and TRIM22 were designed using the EUROFIN primer design tool, which is available at: https: / / eurofinsgenomics.eu / en / dna-rna-oligonucleotides / oligo-tools / primer-design-tools / ?gclid=Cj0KCQiAgqGrBhDtARIsAM5s0_lEU3ONpeEQ72pgv1bwpxYKJztJfFPqrj7jWLksG0vXf8GrV8rGsYMaAgS8EALw_wcB, along with gene sequences obtained from the NCBI database.

[0264] IHC assay of EMA1 and SSEA-1

[0265] The cells were cultured in 12-well plates containing glass coverslips coated with feeder cells until they reached at least 80% confluence.

[0266] The wells were then rinsed with room temperature PBS and the cells were fixed for 15 minutes at room temperature by adding 4% paraformaldehyde solution (PFA) (0.5 mL).

[0267] Then remove the PFA and rinse the wells three times with PBS for 5 minutes each time at room temperature with very gentle shaking.

[0268] dyeing

[0269] Remove the PBS and incubate the cells for 20 minutes in blocking solution (BS)-DPBS, 10% goat serum, and 1% bovine serum albumin (BSA) (0.5 mL) with gentle shaking.

[0270] Remove BS and add primary antibody (anti-SSEA1 or anti-EMA1) solution at the supplier-specified dilution for overnight incubation at 4°C with gentle shaking (50-60 rpm).

[0271] Remove the primary antibody solution, rinse the wells three times with DPBS-Tween 20 (0.05%) for 6-10 minutes each time with gentle shaking (50-60 rpm), and then incubate at room temperature with a suitable antibody solution and DAPI for 1 hour, if possible, with gentle shaking (50-60 rpm).

[0272] Rinse the wells three times with DPBS-Tween 20 (0.05%) at room temperature with gentle shaking (50-60 rpm) for 6-10 minutes each time.

[0273] Covering and Imaging

[0274] Place the slide containing cells face down on a slide containing 13-15 μL of mounting solution (ProLong Gold anti-quenching agent – ​​Invitrogen P36930).

[0275] The slides are prepared for analysis under a fluorescence microscope.

[0276] II. result

[0277] Table 4 below lists some of the measurements currently being conducted.

[0278] Table 4

[0279]

[0280] * Evaluation by RT-qPCR

[0281] ** IHC assessment

[0282] As shown in Table 4, the presence of a combination of chicken or human growth factors in the culture medium targeting pathways identified by the inventors allows for the isolation of ESCs from embryos (determined 3-7).

[0283] In addition, the presence of at least a MEK signaling inhibitor and at least a Wnt signaling inhibitor is required for the isolation and maintenance of ESCs (measurements 1 and 2 relative to measurements 3-7).

[0284] For example, measuring 3 ( Figure 3 As shown in assays (3-7), ESCs maintain their pluripotency phenotype during passage. Under these conditions, the use of combinations of growth factors disclosed in the prior art is ineffective (assay 8).

[0285] It is worth noting that, based on the conditions of determination 3-7, a 100% ESC separation process successfully yielded a multifunctional ESC system (n=4). Figure 3 A provides illustrative data on the pluripotency determination of an ESC line obtained using the method of this invention. Pluripotency was confirmed, and results from RT-qPCR confirmed that pluripotency was maintained for up to 20 generations.

[0286] ESC dissociation was successful for any combination of tested MEK-, Wnt-, and optional PKC signaling pathway inhibitors, particularly the following combinations:

[0287] -adavivint and Gö6976,

[0288] - Bimetinib and Gö6983

[0289] -Camatinib and IWP-2,

[0290] - Enzatolin and PD-325901,

[0291] -Gö6976 and PD-325901,

[0292] -Gö6983 and PD98059,

[0293] -IWP-2 and Rubesta,

[0294] -PD-325901 and U0126,

[0295] -PD98059 and adavivint,

[0296] - Lubersta and Bimetinib

[0297] -U0126 and Camatinib,

[0298] -XAV-939 and Enzatolin,

[0299] -XAV-939 and PD98059, or

[0300] -XAV-939 and Gö6983,

[0301] -Gö6976 and carmatinib and IWP-2,

[0302] -Gö6983 and PD98059 and adavivint,

[0303] -Gö6983 and PD-325901 and Xav-939,

[0304] - Lubersta and PD-325901 and Bimetinib, and

[0305] - Enzatolin and XAV-939 and U0126.

[0306] bibliography

[0307] Eyal-Giladi H, Kochav S. From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of the development of the chick. I. General morphology. Dev Biol. 1976 Apr;49(2):321-37.

[0308] Sellier N., Brillard J.-P., Dupuy V., Bakst MR. Comparative Staging of Embryo Development in Chicken, Turkey, Duck, Goose, Guinea Fowl, and Japanese Quail Assessed from Five Hours After Fertilization Through Seventy-Two Hours of Incubation, Journal of Applied Poultry Research, Volume 15,Issue 2, 2006, pages 219-228.

[0309] UniProt Consortium. UniProt: the Universal Protein Knowledgebase in2023. Nucleic Acids Res. 2023 Jan 6;51(D1):D523-D531

Claims

1. A method for isolating embryonic stem cells (100) from at least one avian embryo, the method comprising the steps of: a. Isolate at least one embryo (101) at the developmental stage surrounding oviposition. b. Embryonic cells obtained by dissociating the embryos in step a) are suspended in a serum-free basal medium (102), the medium being supplemented with: - A mixture of growth factors that regulate at least the following pathways: JAK / STAT, PI3K / AKT, SHP2 / MAPK, PLC-γ, MAPK, PI3K / AKT / MTOR, RAS / RAF, RHOA / ROCK - At least one inhibitor that causes inhibition of MEK signaling, - at least one inhibitor that causes inhibition of Wnt signaling, and -Animal serum alternatives c. The embryonic cell suspension (103) obtained in step b) is inoculated onto the feeder cell layer. d. The embryonic cells are cultured and passaged at least once (104).

2. The method for isolating embryonic stem cells (100) according to claim 1, wherein the mixture of growth factors comprises at least one ortholog of the following growth factors: interleukin-6 (IL6), leukemia inhibitory factor (LIF), insulin-like growth factor 1 (IGF-1), and stem cell factor (SCF).

3. The method for isolating embryonic stem cells (100) according to claim 2, wherein: -IL6 is selected from avian IL6 or mammalian IL6 or mixtures thereof. -LIF is selected from avian LIF or mammalian LIF or a mixture thereof. -IGF-1 is selected from avian IGF-1 or mammalian IGF-1 or a mixture thereof, or -SCF is selected from avian SCF or mammalian SCF or a mixture thereof.

4. The method for isolating embryonic stem cells (100) according to any one of claims 1 to 3, further comprising at least one inhibitor that causes inhibition of PKC signaling.

5. A method for isolating embryonic stem cells (100) according to any one of the preceding claims, wherein: Inhibitors of MEK signaling are selected from small molecules or MEK-1 siRNA. Inhibitors of Wnt signaling are selected from small molecules or Wnt-1 siRNA, or - When an inhibitor of PKC signaling is available, the inhibitor of PKC signaling is selected from small molecules or PKC-α siRNA.

6. The method for isolating embryonic stem cells (100) according to any one of the preceding claims, wherein: - Inhibitors that cause MEK signaling inhibition are selected from: bismuthinib, cobimetinib, selemetinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901, PD98059, or U0126, or mixtures thereof. - Inhibitors that cause inhibition of Wnt signaling are selected from adavivint, carmatinib, CCT251545, FH535, ginsenoside Rh4, ICG-001, iCRT14, IQ-1, isoquercitrin, IWP-2, IWP-4, IWR-1-endo, JW55, KY02111, KY-05009, KYA1797K, lanolin C, LF3, M2912, M435-1279, MSAB, NCB-0846, PNU-74654, PRI-724, styracin, RCM-1, bufotalin, salinomycin, tripterygium wilfordii lactone, WIKI4, XAV-939, or RXC004 or mixtures thereof, or - When an inhibitor that causes inhibition of PKC signaling is present, it is selected from Gö6983, Gö6976, enzatolin, rubestazone, astrococcus, GF 109203X (bisindolylmaleimide I), ZIP, Ro 31-8220, Ro 32-0432, sotrastolin, fenestrated toxin, K252a, bisindolylmaleimide II, protein kinase C, celandine, L-threodihydrosphingosine, melittin, or midostaurin or a mixture thereof.

7. The method for isolating embryonic stem cells (100) according to any one of the preceding claims, wherein the animal serum substitute is selected from: KnockOut TM Serum Replacement Basic; Serum Replacement 3; BIT 9500 serum replacement from StemCell.

8. The method for isolating embryonic stem cells (100) according to any one of the preceding claims, wherein the avian embryonic cells are chicken embryonic stem cells or duck embryonic stem cells.

9. A method for obtaining a continuous diploid cell line (200) derived from avian embryonic stem cells (ESCs), the method comprising: a. Providing at least one ESC (201) obtained from the method (100) for isolating embryonic stem cells from at least one avian embryo according to any one of claims 1-8, b. Each growth factor was gradually removed from the culture medium (203), at least one inhibitor was gradually removed from the culture medium (204), and the concentration of feeder cells in the culture was gradually reduced (205) so that all feeder cells could be removed after several passages. This allows for the acquisition of adherent or non-adherent continuous diploid avian cell lines derived from non-human ESCs, which can proliferate in basal medium in the absence of growth factors.

10. A component kit for reconstructing cell culture media, comprising: - A direct homolog of interleukin-6 (IL-6) - A direct homolog of leukemia inhibitory factor (LIF), - An ortholog of insulin-like growth factor 1 (IGF-1) - A direct homolog of stem cell factor (SCF) - At least one inhibitor of MEK signaling, selected from i) a small molecule selected from bimetinib, cobimetinib, seletinib, trametinib, CI-1040, PD035901, TAK-733, PD-325901, PD98059 or U0126, or a mixture thereof, or ii) MEK-1 siRNA. - At least one inhibitor of Wnt signaling, selected from i) small molecules selected from adavivint, carmatinib, CCT251545, FH535, ginsenoside Rh4, ICG-001, iCRT14, IQ-1, isoquercitrin, IWP-2, IWP-4, IWR-1-endo, JW55, KY02111, KY-05009, KYA1797K, lanolin C, LF3, M2912, M435-1279, MSAB, NCB-0846, PNU-74654, PRI-724, strychnine, RCM-1, bufotoxin, salinomycin, tripterygium wilfordii lactone, WIKI4, XAV-939 or RXC004 or mixtures thereof, or ii) Wnt-1 siRNA.

11. The kit according to claim 10, further comprising at least one inhibitor of PKC signaling, said inhibitor being selected from i) small molecules selected from Gö6983, Gö6976, enzatolin, rubestazone, astrococcus, GF 109203X (bisindolylmaleimide I), ZIP, Ro 31-8220, Ro 32-0432, sotrastolin, fenestrated toxin, K252a, bisindolylmaleimide II, protein kinase C, chelidonine, L-threodihydrosphingosine, melittin, or midostaurin or combinations thereof, or ii) PKC-α siRNA.

12. The kit according to any one of claims 10 or 11, further comprising an animal serum substitute.

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