Pluripotent cell aggregates and their uses

Abstract

The present invention relates to compositions and methods for the mass production of pluripotent stem cells derived from non-human animals, particularly in the form of aggregates suitable for a variety of uses, and in particular the mass production of aggregates of bovine-derived pluripotent stem cells for use in cell-expanded meat cultures and the production of cell-based meat products.

Description

[Technical Field]

[0001] The present invention relates to compositions and methods for the mass production of pluripotent stem cells derived from non-human animals, particularly in the form of aggregates suitable for a variety of uses, and in particular the mass production of aggregates of bovine-derived pluripotent stem cells for use in cell-expanded meat cultures and the production of cell-based meat products. [Background technology]

[0002] Many consumable resources, including water, grain, land, and energy, are utilized to support the growth of livestock, particularly cattle for meat production. The world's rapidly growing population will likely lead to further increases in the use of these valuable assets. Therefore, it is most desirable to produce meat and meat products in a way that reduces the number of cattle needed to feed the entire population. Replacing cattle as a primary meat source is ethically beneficial because it prevents the crowded and sometimes inappropriate living conditions that plague bovine subjects. For this reason, cell-grown meat products may potentially be consumed by people who abstain from meat for humanitarian reasons. Cell-based meat (also known as cultured meat, cultivated meat, cell-grown meat, clean meat, engineered meat, in vitro meat, etc.) is also a way to control the content of consumed food. Today, many cattle are administered growth hormones, which ultimately end up on consumers' plates. Furthermore, producing meat in culture can improve the nutritional value of meat and make it healthier through control of protein content, fat amount and composition, iron, vitamin B12, and zinc levels. Furthermore, consuming meat produced in bioreactors (or other culture systems) under strict regulatory and clean conditions is less likely to transmit microbial contamination and therefore safer for consumption.

[0003] Pluripotent stem cells (PSCs) are cells capable of self-renewal by division while maintaining the ability to differentiate into all cell types in the body. Both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are pluripotent stem cells. ESCs are typically derived from a population of cells in the inner cell mass of preimplantation blastocysts. Induced pluripotent stem cells (iPSCs), a type of PSC, are generated from somatically differentiated cells that have been reprogrammed to restore their pluripotent characteristics. Bogliotti et al. (PNAS. 2018 11(9):2090-2095) described the derivation of pluripotent bovine ESCs (bESCs) with stable morphology, transcriptome, karyotype, population doubling time, pluripotency marker gene expression, and epigenetic characteristics by utilizing a culture system containing fibroblast growth factor 2 (also known as basic fibroblast growth factor, bFGF) and an inhibitor of the canonical Wnt signaling pathway.

[0004] Reprogramming of cells into iPSCs was first described by Takahashi and Yamanaka in 2006 (Takahashi and Yamanaka, Cell 2006, 126:663-76). Since then, numerous publications have described the reprogramming of stem cells into iPSCs from various cell types of multiple species (Yu et al., Science 2007, 318:1917-20; Takahashi et al., Cell 2007, 131:861-72; Ogorerc et al. J Anim Sci Biotechnol 2016, 7:10; Ezashi et al., Annu Rev Anim Biosci. 2016, 4:223-53). In cattle, fibroblasts have been reprogrammed into iPSCs using a combination of reprogramming factors by several methods, including retroviral / lentiviral transduction, polypromoter plasmids, and the piggyBac transposon system (Ogorevc 2016 ibid.; Han et al., Cell Res. 2011, 21:1509-12; Huang et al., Plos One 2011, 6:e24501; Cao et al., Int J Biol Sci. 2012, 8:498-511; Talluri et al., Cell Reprogram. 2015, 17:131-40; Ezashi et al. 2016 ibid.). There are no reports of more modern methods (e.g., non-integrating viral or non-integrating episomal vectors or mRNA-based methods) being used to reprogram cells in livestock species (Ogorevc 2016 ibid.). The use of mRNA, small molecules, and proteins as reprogramming factors has not been demonstrated in bovine cells.

[0005] WO 1999 / 031223 discloses a process for producing meat products, comprising culturing animal cells in vitro on an industrial scale to provide three-dimensional animal muscle tissue suitable for human and / or animal consumption, optionally followed by further processing of the cell culture into a finished food product similar to known processes for meat-containing foods, without the need for deboning, removal of offal and / or tendons and / or sinews and / or fat. Preferably, the meat product comprises solidified cellular tissue, the cells being selected from muscle cells, somite cells, and stem cells. Meat products comprising solidified cellular tissue are also provided.

[0006] International Patent Application Publication No. 2006 / 041429 and U.S. Patent No. 6,835,390 disclose non-human tissue engineered meat products and methods for producing such meat products, which contain muscle cells grown ex vivo and used for food consumption.

[0007] International Patent Application Publication No. 2010 / 017562 provides iPSCs, compositions containing the same, methods for obtaining iPSCs, and methods for using iPSCs. Additionally, methods and materials are provided for using iPSCs to repair tissue (e.g., cardiovascular tissue) in vivo, as well as methods and materials for using such cells to evaluate their therapeutic potential in appropriate animal models.

[0008] WO 2013 / 188679 discloses a method for preparing iPSCs in which a combination of mRNA and miRNA is introduced into the cells.

[0009] WO 2015 / 066377 discloses a method for producing cultured muscle tissue, comprising modifying a self-renewing cell line of an animal species with a myogenic transcription factor to produce a myogenic transcription factor-modified cell line.

[0010] International Patent Application Publication No. 2018 / 011805 discloses a system for growing cells, including a bioreactor chamber for growing the cells. The application further discloses an in vitro method for producing meat, comprising culturing spontaneously immortalized fibroblasts in a serum-free medium under conditions suitable for converting the fibroblasts into adipocytes and / or conditions suitable for converting the fibroblasts into muscle cells, thereby producing meat.

[0011] International Patent Publication No. 2019 / 016795 discloses a method for producing an edible composition, comprising incubating a three-dimensional porous scaffold and a plurality of cell types, including at least one of myoblasts or their precursor cells, extracellular matrix (ECM)-secreting cells, and endothelial cells or their precursor cells, and inducing differentiation of myoblasts into myotubes.

[0012] WO 2019 / 140260, published after the priority date of the present application, discloses a method for obtaining ungulate embryonic stem cells (ESCs) derived from the inner cell mass of pre-implantation blastocysts or embryo-derived pluripotent cells, useful for genomic testing and selection and genetic manipulation of domestic ungulates, and as an experimental tool for studying human disease.

[0013] U.S. Patent No. 9,944,894 discloses a closed system in a rocking platform bioreactor for growing and passaging cell aggregates containing stem cells and / or differentiated cells. A method is also disclosed that allows for closed-system serial propagation of pluripotent stem cells and / or their progeny, along with associated pluripotency markers and differentiation potential.

[0014] U.S. Patent No. 9,834,749 discloses a method for expanding and maintaining human embryonic stem cells (ESCs) in an undifferentiated state by culturing ESCs in suspension culture under culture conditions lacking substrate adhesion. This method is useful for generating lineage-specific cells from ESCs. The patent also discloses a method for deriving ESC lineages in suspension culture.

[0015] There remains an unmet need for compositions and methods to enable the large-scale production of non-genetically modified (GM) cell-grown meat from cells derived from non-human animals, particularly bovine. Summary of the Invention

[0016] The present invention provides compositions and methods for mass production of pluripotent stem cells (PSCs) derived from non-human animals, particularly in the form of homogenous, three-dimensional (3D), genetically unmodified (GM) aggregates. The present invention provides a previously unavailable continuous reservoir of PSCs derived from non-human animals that maintains their pluripotency in large-scale liquid culture conditions. PSCs can be used in the growing industry of cell-expanded meat products, as well as in experimental tools for studying cell proliferation and differentiation and developing new non-human animal-related drugs.

[0017] The present invention is based in part on the unexpected discovery that bovine-derived pluripotent cells, and / or bovine embryo-derived embryonic stem cells (ESCs), obtained by the flushing procedure, can proliferate, maintain pluripotency, and further form 3D homogeneous aggregates under large-scale liquid culture conditions when specific combinations of growth factors and small molecules are added to the culture medium. The cells within the aggregates maintain their pluripotency and division rate, thereby increasing the diameter of the aggregates and achieving cell division rates of up to 10 per liter of liquid culture, which has not previously been reported for cells derived from non-human animals. 9 ~10 12 This results in the mass production of pluripotent stem cells to reach a final concentration of cells.

[0018] Without wishing to be bound by any particular theory or mechanism of action, the mass production and aggregate formation of pluripotent stem cells can be attributed to a system of growth settings that includes a source of non-human animal cells, particularly bovine cells, a unique serum-free medium containing a combination of growth factors and small molecules, and the use of a closed system in which further cell aggregation and disaggregation steps are performed, and an incubation temperature that is compatible with the body temperature of the non-human animal.

[0019] In certain aspects, the 3D non-human animal-derived PSC aggregates of the present invention are a source for cell-expanded meat products. According to certain exemplary embodiments, the PSCs are bovine-derived cells.

[0020] According to one aspect, the present invention provides a method for mass-producing aggregates of non-genetically modified, non-human animal-derived pluripotent stem cells (PSCs), comprising: (a) seeding at least one PSC in a growth medium to form a suspension culture, wherein the growth medium is a serum-free liquid medium comprising a combination of the growth factor bFGF, at least one additional growth factor, and / or an inhibitor of the Wnt-β-catenin signaling pathway, CHIR 99021 (C22H18Cl2N8), PD 0325901 (C16H14F3IN2O4), and at least one small molecule selected from the group consisting of A 83-01 (C25H19N5S); and (b) growing the suspension culture under conditions that permit aggregate formation and aggregate growth, thereby forming homogenous aggregates of the PSCs.

[0021] According to certain embodiments, seeding and growth are performed in a vessel having walls made of a material to which PSCs and / or aggregates containing them do not adhere. It should be clearly understood that PSC aggregates are formed in the vessel without the addition of inactivated feeder cells, organic extracellular matrix, feeder cell-conditioned medium, and / or microcarriers. Thus, the PSCs and / or aggregates containing them are free in suspension culture and do not adhere to a surface. According to certain embodiments, the vessel is a bioreactor.

[0022] According to certain embodiments, the homogenous aggregates comprise at least 70% of cells that express at least one pluripotency marker.

[0023] According to certain embodiments, the at least one pluripotency marker is selected from the group consisting of stage-specific embryonic antigen-4 (SSEA4), octamer-binding transcription factor 4 (Oct4), Sall4 transcription factor, Nanog homeobox transcription factor, translation factor Lin28A, DNA methyltransferase Dnmt3b, and combinations thereof. Each possibility represents a separate embodiment of the present invention. According to certain exemplary embodiments, the pluripotency marker is SSEA4.

[0024] According to certain embodiments, step (b) of growing the suspension culture includes disaggregating the formed homogeneous aggregates into smaller aggregates and / or single cells, and re-aggregating the smaller aggregates and / or single cells to reform the homogeneous aggregates.

[0025] According to certain exemplary embodiments, disaggregating the homogeneous aggregates comprises exposing the aggregates to a dissociation reagent and / or a dissociation force. According to some embodiments, the dissociation reagent comprises at least one protease and, optionally, at least one DNase and / or a chelating agent. According to certain embodiments, the chelating agent is selected from EDTA and EGTA. According to some exemplary embodiments, the protease is trypsin.

[0026] According to some embodiments, the dissociation force is a shear force. According to certain embodiments, the shear force rate is set by an impeller embedded within the vessel.

[0027] According to certain embodiments, the disaggregating step further comprises washing the homogenous aggregates with an aqueous wash medium before exposing the aggregates to a dissociation reagent and / or a dissociation force.

[0028] According to certain embodiments, the reaggregating step comprises seeding smaller aggregates and / or single cells in growth medium.

[0029] According to certain embodiments, the step of seeding and reaggregating further comprises adding an inhibitor of Rho-associated protein kinase (Rock) to the culture medium.

[0030] According to certain embodiments, the entire production of aggregates is carried out in a closed system. The use of a closed system has significant advantages in the mass production of PSC aggregates on a large scale, allowing for the maintenance of a sterile environment and an automated process. According to certain embodiments, the steps of disaggregation and reaggregation are carried out in separate containers connected to the bioreactor in a manner that maintains a closed system. According to certain embodiments, the container is a cell retention device.

[0031] According to certain embodiments, the disaggregation and re-aggregation steps are repeated at least once, at least twice, at least three times, at least four times, at least five times, and more. It should be clearly understood that the number of repetitions may be unlimited and will depend on the number of PSCs required for the intended use. According to certain exemplary embodiments, when the PSC aggregates are for the production of cell-grown meat, aggregate formation and disaggregation may be repeated for about 10 6 cells / ml~about 10 9 The process is repeated until a final cell concentration of about 5×10 cells / ml is reached. According to certain embodiments, the formation and disaggregation of aggregates is repeated until a final cell concentration of about 5×10 cells / ml is reached. 6 ~Approx. 5×10 8 This is repeated until a final cell concentration of 100 cells / ml is reached.

[0032] According to certain embodiments, the at least one additional growth factor is a protein of the transforming growth factor beta (TGF-β, TGFB) superfamily. According to certain embodiments, the TGF-β is selected from the group consisting of TGF-β-1, TGF-β-3, activin-A, and any combination thereof.

[0033] According to certain embodiments, the growth medium comprises a combination comprising bFGF and TGF-β.

[0034] According to certain embodiments, the growth medium comprises a combination of bFGF and at least one additional growth factor. According to certain exemplary embodiments, the additional growth factor is a protein of the TGF-β superfamily. According to certain embodiments, the TGF-β is selected from the group consisting of TGF-β-1, TGF-β-3, activin-A, and any combination thereof.

[0035] According to certain embodiments, the growth medium comprises a combination consisting of bFGF and at least one small molecule.

[0036] According to certain embodiments, the inhibitor of the Wnt-β-catenin signaling pathway is selected from the group consisting of IWR1, JW67, NSC668036, KY02111, niclosamide, DKK1 or si-beta-catenin, a porcupine inhibitor, and IWP-2, each possibility representing a separate embodiment of the present invention.

[0037] According to certain exemplary embodiments, the inhibitor of the Wnt-β-catenin signaling pathway is IWR1.

[0038] According to certain embodiments, the growth medium comprises a combination of growth factors bFGF and a protein of the TGF-β superfamily and a small molecule selected from the group consisting of IWR1, CHIR 99021, PD 0325901, A 83-01, and any combination thereof.

[0039] According to certain exemplary embodiments, the growth medium comprises a combination of at least one of the growth factors bFGF, TGF-β-1, TGF-β-3, and activin-A, and the small molecule IWR1. According to some embodiments, the growth medium comprises a combination of at least one of the growth factors bFGF, TGF-β-1, TGF-β-3, and activin-A, and the small molecule IWR1.

[0040] According to further specific exemplary embodiments, the growth medium comprises a combination consisting of the growth factor bFGF and the small molecule IWR1.

[0041] According to certain embodiments, the growth medium comprises a combination comprising the growth factor bFGF and the small molecules IWR1 and CHIR 99021. According to certain embodiments, the growth medium comprises a combination consisting of the growth factor bFGF and the small molecules IWR1 and CHIR 99021.

[0042] According to a further exemplary embodiment, the growth medium comprises the growth factor bFGF and a combination of small molecules consisting of IWR1, CHIR 99021, PD 0325901 and A 83-01.

[0043] According to certain embodiments, the growth medium is further free of animal-derived components.

[0044] According to certain embodiments, the non-human animal is selected from the group consisting of ungulates, poultry, aquatic animals, invertebrates, and reptiles, each possibility representing a separate embodiment of the present invention.

[0045] According to certain embodiments, the ungulate is selected from the group consisting of a cow, a sheep, a horse, a pig, a giraffe, a camel, a deer, a hippopotamus, or a rhinoceros, each possibility representing a separate embodiment of the present invention.

[0046] According to certain exemplary embodiments, the non-human animal is a bovine. According to some exemplary embodiments, the bovine is of the species Bos Taurus.

[0047] According to certain embodiments, the PSCs are bovine-derived embryonic stem cells (ESCs).

[0048] According to certain exemplary embodiments, the bovine-derived embryonic stem cells are (1) obtaining at least one bovine early embryo; (2) culturing at least one early embryo to reach the blastocyst or enhanced blastocyst stage; (3) obtaining at least one cell from the blastocyst; (4) culturing at least one cell in a culture medium comprising a combination of the growth factor bFGF and (i) at least one additional growth factor and (ii) at least one small molecule selected from the group consisting of an inhibitor of the Wnt-β-catenin signaling pathway, CHIR 99021 (C22H18Cl2N8), PD 0325901 (C16H14F3IN2O4), and A 83-01 (C25H19N5S), to obtain a plurality of bovine-derived embryonic stem cells.

[0049] According to certain embodiments, the at least one early bovine embryo is obtained by an embryo flushing procedure.

[0050] According to certain embodiments, the at least one early bovine embryo is a frozen embryo. According to these embodiments, the frozen embryo is thawed prior to culturing.

[0051] It should be clearly understood that the at least one PSC seeded in the growth medium produced by any method known in the art and as described herein above may be a fresh PSC or may be obtained from a frozen stock.

[0052] According to certain embodiments, the inhibitor of the Wnt-β-catenin signaling pathway is selected from the group consisting of IWR1, JW67, NSC668036, KY02111, niclosamide, DKK1 or si-beta-catenin, porcupine inhibitor, and IWP-2, each possibility representing a separate embodiment of the present invention.

[0053] According to certain exemplary embodiments, the medium comprises bFGF and IWR1.

[0054] According to certain embodiments, the PSCs are induced PSCs (iPSCs) reprogrammed from non-human animal cells. According to certain exemplary embodiments, the non-human animal is a mammal. According to even more specific exemplary embodiments, the mammal is a bovine.

[0055] According to other embodiments, the PSCs are non-embryonic stem cells (ESCs).

[0056] According to certain exemplary embodiments, the ungenetically modified PSCs are reprogrammed non-human animal-derived cells produced by a method comprising introducing a combination of (a) at least one reprogramming mRNA encoding a reprogramming factor, and (b) at least one double-stranded microRNA into at least one non-human animal-derived cell, thereby producing at least one iPSC.

[0057] According to certain embodiments, the method further comprises introducing at least one immune evasion mRNA into at least one cell derived from a non-human animal.

[0058] According to certain embodiments, at least one reprogramming mRNA is a synthetically modified mRNA.

[0059] According to certain further exemplary embodiments, the PSCs are reprogrammed non-human animal-derived cells produced by a method comprising introducing into at least one non-human animal-derived cell a combination of (a) at least one reprogramming mRNA encoding a reprogramming factor, and (b) at least one inhibitor of at least one microRNA endogenous to the non-human animal-derived cell, thereby producing at least one iPSC.

[0060] According to certain embodiments, the method further comprises introducing at least one immune evasion mRNA into at least one cell derived from a non-human animal.

[0061] According to certain embodiments, at least one reprogramming mRNA is a synthetically modified mRNA.

[0062] According to the teachings of the present invention, the at least one reprogramming mRNA does not integrate into the genome of the cell, and the iPSCs produced thereby are not genetically modified.

[0063] According to certain embodiments, introducing at least one reprogramming mRNA, and / or at least one immune evasion mRNA and / or at least one double-stranded microRNA and / or at least one microRNA inhibitor is performed in a serum-free liquid growth medium.

[0064] According to some embodiments, the method further comprises culturing at least one iPSC produced in a serum-free liquid growth medium to form a plurality of iPSCs. The serum-free liquid growth medium is as described hereinabove. According to some embodiments, the serum-free liquid growth medium used is supplemented with a Rock inhibitor.

[0065] According to some embodiments, the reprogramming mRNA introduced into the non-human animal-derived cell or cells encodes a reprogramming factor selected from the group consisting of OCT4, SOX2, KLF4, cMYC, NANOG, LIN28, KLF5, and any combination thereof, with each possibility representing a separate embodiment of the present invention.

[0066] According to yet another embodiment, the double-stranded microRNA is selected from the group consisting of miR-302a, miR-302b, miR-302c, miR-302d, miR-367, miR-218, miR-449b, and any combination thereof, with each possibility representing a separate embodiment of the present invention.

[0067] According to certain embodiments, the at least one microRNA inhibitor is an RNA inhibitor (RNAi) molecule. According to certain exemplary embodiments, the at least one microRNA inhibitor targets miR-145.

[0068] According to certain embodiments, when used, the immune evasion mRNA introduced into the non-human animal-derived cell or cells is selected from the group consisting of E3, K3, B18R[EKB] from vaccinia virus, and any combination thereof, each possibility representing a separate embodiment of the present invention.

[0069] According to certain embodiments, the at least one non-human animal-derived cell and / or the at least one iPSC formed are incubated at a temperature of 37.5 to 39.5°C.

[0070] According to certain embodiments, the non-human animal is of a species selected from the group consisting of ungulates, poultry, aquatic animals, invertebrates, and reptiles, each possibility representing a separate embodiment of the present invention.

[0071] According to certain embodiments, the ungulate is selected from the group consisting of a cow, a sheep, a horse, a pig, a giraffe, a camel, a deer, a hippopotamus, or a rhinoceros.

[0072] According to certain exemplary embodiments, the ungulate is a bovine. According to these embodiments, the at least one bovine-derived cell and / or the at least one formed iPSC is incubated at about 38.6°C, the average body temperature of a bovine.

[0073] According to some embodiments, bovine-derived cells are obtained from bovine umbilical cord, bovine nasopharyngeal mucosa, and bovine milk and blood. According to certain exemplary embodiments, somatic bovine cells are harvested by non-invasive techniques.

[0074] According to certain exemplary embodiments, the bovine-derived cells used to produce the induced and / or reprogrammed PSCs are obtained from bovine umbilical cord, and the cells are selected from the group consisting of endothelial cells, umbilical cord lining cells, Wharton's Jelly cells, or combinations thereof. According to yet additional embodiments, the bovine-derived cells are obtained from the beef cattle breed Belgium Blue, which is characterized by a dual muscle phenotype due to a natural mutation in the myostatin gene.

[0075] According to certain embodiments, the PSCs are encapsulated within the particles. According to certain exemplary embodiments, the particles further comprise essential agents for cell growth and proliferation, as described herein. Encapsulation of PSCs within particles comprising growth factors and / or small molecules according to the teachings of the present invention can provide significant reductions in production costs due to the smaller amounts of these agents that may be required to reach a particular concentration within the particles compared to the amount required to reach the same concentration in the entire culture medium volume.

[0076] Thus, according to certain embodiments, step (a) of seeding at least one PSC further comprises encapsulating at least one of the PSCs within a particle and seeding a plurality of the particles within a growth medium.

[0077] According to certain embodiments, the particles comprise an inner core containing at least one PSC surrounded by at least one outer shell layer. According to certain embodiments, the outer shell layer is of a fluid-permeable food-grade material. According to certain embodiments, the fluid-permeable food-grade material is dissolvable. According to these embodiments, the encapsulation is reversible, resulting in the release of PSC aggregates formed within the particle upon dissolution of the outer shell layer. According to certain embodiments, the outer shell layer comprises at least one food-grade prepolymer and / or polymer and / or copolymer. According to certain embodiments, the outer shell layer is composed of at least one hydrogel. According to certain embodiments, the hydrogel is a thermoreversible hydrogel that forms the outer shell layer at cell growth temperatures. According to certain embodiments, the food-grade polymer is selected from the group consisting of alginate, gellan gum, agar, agarose, chitosan, hyaluronic acid, curdlan, carrageenan, pectin, modified starch, and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0078] According to certain embodiments, the inner core provides a microenvironment that supports the growth of at least one PSC. According to certain embodiments, the microenvironment of the inner core comprises at least one compound that supports the growth of at least one PSC. According to some embodiments, the at least one agent is formulated in a slow-release and / or sustained-release formulation, as known in the art.

[0079] According to certain exemplary embodiments, the inner core comprises the growth factor bFGF in combination with at least one of (i) at least one additional growth factor and (ii) at least one small molecule selected from the group consisting of inhibitors of the Wnt-β-catenin signaling pathway, CHIR 99021 (C22H18Cl2N8), PD 0325901 (C16H14F3IN2O4), and A 83-01 (C25H19N5S). The combination of growth factors and / or small molecules is as described herein above.

[0080] According to certain embodiments, the encapsulated PSCs grow within the inner core to form PSC aggregates within the particle.

[0081] According to certain embodiments, the suspension culture is incubated under dynamic rotation, under static conditions, or a combination thereof.

[0082] According to certain exemplary embodiments, the suspension culture is incubated under dynamic rotation conditions.

[0083] The present invention now demonstrates that maintaining the growth medium at a temperature that is the body temperature of the species from which the cells are derived throughout the process is important to obtain cell growth and aggregates containing a large number of cells.

[0084] According to a particular embodiment, the incubation is carried out at a temperature of 37.5 to 39.5°C.

[0085] According to certain exemplary embodiments in which the non-human animal is a bovine, the incubation temperature is approximately 38.6°C, the average body temperature of a bovine.

[0086] According to certain embodiments, the volume of the bioreactor is adapted to contain a suspension culture in a volume of from about 50 ml to about 15,000 liters.

[0087] According to some embodiments, the volume of the suspension culture is from about 100 ml to about 15,000 liters. According to some embodiments, the volume of the suspension culture is from about 1 liter to about 15,000 liters.

[0088] An advantage of the methods of the present invention is that all steps can be performed in commercial-scale volumes. According to some embodiments, the suspension culture volume is from about 1,000 liters to about 15,000 liters per bioreactor.

[0089] According to certain embodiments, the formed PSC aggregates have an average diameter of about 30 μm to about 500 μm. According to certain embodiments, the formed PSC aggregates have an average diameter of about 30 μm to about 550 μm. According to certain exemplary embodiments, the formed PSC aggregates have an average diameter of about 100 μm to about 350 μm.

[0090] According to certain embodiments, the aggregates contain PSCs that express at least one pluripotency marker. According to certain embodiments, the at least one pluripotency marker is selected from the group consisting of stage-specific embryonic antigen-4 (SSEA4), octamer-binding transcription factor 4 (Oct4), Sall4 transcription factor, Nanog homeobox transcription factor, translation factor Lin28A, DNA methyltransferase Dnmt3b, and combinations thereof. Each possibility represents a separate embodiment of the present invention. According to certain exemplary embodiments, the pluripotency marker is SSEA4.

[0091] According to a further aspect, the present invention provides homogeneous aggregates of non-genetically modified, non-human animal-derived PSCs produced according to the methods of the present invention.

[0092] According to certain embodiments, the homogeneous aggregates have an average diameter of about 30 μm to about 500 μm. According to certain embodiments, the formed PSC aggregates have an average diameter of about 30 μm to about 550 μm. According to certain exemplary embodiments, the formed PSC aggregates have an average diameter of about 100 μm to about 350 μm.

[0093] According to another aspect, the present invention provides a suspension comprising a serum-free liquid medium and non-human animal-derived cell aggregates, wherein the aggregates comprise at least 70% viable, non-genetically modified, non-human animal-derived PSCs.

[0094] According to certain embodiments, the non-genetically modified, non-human animal-derived PSCs divide approximately every 16-32 hours.

[0095] According to certain embodiments, the non-genetically modified, non-human animal-derived PSCs express at least one pluripotency marker.

[0096] According to certain embodiments, the pluripotency marker is selected from the group consisting of stage-specific embryonic antigen-4 (SSEA4), octamer-binding transcription factor 4 (Oct4), Sall4 transcription factor, Nanog homeobox transcription factor, translation factor Lin28A, DNA methyltransferase Dnmt3b, and combinations thereof.

[0097] According to certain exemplary embodiments, the aggregates in the suspension have an average size of about 200 μm to about 500 μm.

[0098] According to certain exemplary embodiments, the aggregates in the suspension have an average size of about 200 μm to about 350 μm.

[0099] According to a particular embodiment, the suspension comprises about 10 6 ~about 10 9 / ml of genetically unmodified, non-human animal-derived PSCs.

[0100] According to certain embodiments, the PSCs of the aggregates express at least one surface protein that contributes to cell-cell adhesion.

[0101] According to certain embodiments, the non-human animal is selected from the group consisting of ungulates, poultry, aquatic animals, invertebrates, and reptiles, each possibility representing a separate embodiment of the present invention.

[0102] According to certain embodiments, the ungulate is selected from the group consisting of cattle, sheep, horses, pigs, giraffes, camels, deer, hippos, and rhinoceroses, each possibility representing a separate embodiment of the present invention.

[0103] According to certain exemplary embodiments, the non-human animal is a bovine. According to some exemplary embodiments, the bovine is of the species Bos Taurus.

[0104] The PSC aggregates of the present invention can be used as such, for example, as experimental and research tools or as starting material for further uses.

[0105] According to certain embodiments, the PSC aggregates of the present invention can be used as a target material in the cell-grown meat industry. According to these embodiments, the PSCs are further differentiated into at least one of adipocyte, myocyte, stromal, and endothelial cell lineages.

[0106] According to yet another aspect, the present invention provides a cell-expanded meat culture comprising homogenous aggregates of non-genetically modified, non-human animal-derived PSCs, or a suspension comprising same, according to the teachings of the present invention.

[0107] According to yet an additional aspect, the present invention provides a cell-expanded meat product comprising progeny of homogenous aggregates of the non-genetically modified, non-human animal-derived PSCs of the present invention. According to certain embodiments, the progeny comprise cells that differentiate from the PSCs to form at least one of myocytes, stromal cells, endothelial cells, and adipocytes.

[0108] According to yet another aspect, the present invention provides a method for differentiating non-human animal cells selected from the group consisting of PSCs, their progeny, somatic cells, and combinations thereof, into adipocyte cells, the method comprising incubating the cells in a serum-free liquid medium containing (i) at least one inhibitor of the Wnt-β-catenin signaling pathway, and (ii) at least one of at least one type of fatty acid, thereby differentiating the cells into adipocyte cells.

[0109] According to certain embodiments, the culture medium comprises a combination of at least one inhibitor of the Wnt-β-catenin signaling pathway and at least one type of fatty acid.

[0110] According to certain embodiments, the medium further comprises the growth factor bFGF. According to certain embodiments, the medium further comprises a Rock inhibitor.

[0111] According to a particular embodiment, the incubation of the cells is for at least 4 days.

[0112] According to certain embodiments, the inhibitor of the Wnt-β-catenin signaling pathway is selected from the group consisting of IWR1, JW67, NSC668036, KY02111, niclosamide, DKK1 or si-beta-catenin, porcupine inhibitor, and IWP-2.

[0113] According to certain exemplary embodiments, the at least one inhibitor of the Wnt-β-catenin signaling pathway is IWR-1.

[0114] According to certain embodiments, the fatty acid is selected from the group consisting of free fatty acids, low molecular weight fatty acids, esters thereof, salts thereof, and any combination thereof.

[0115] According to certain embodiments, the PSCs are selected from the group consisting of induced PSCs (iPSCs) and embryonic stem cells.

[0116] According to certain embodiments, the PSC derivatives are stromal stem cells isolated from adipose tissue.

[0117] According to certain embodiments, the cells comprise a population of cells derived from embryonic muscle tissue. According to certain embodiments, the population of cells derived from embryonic muscle tissue comprises embryonic fibroblasts (EF).

[0118] According to certain embodiments, the cells to be differentiated are derived from a non-human animal selected from the group consisting of ungulates, poultry, aquatic animals, invertebrates and reptiles, each possibility representing a separate embodiment of the present invention.

[0119] According to certain embodiments, the ungulate is selected from the group consisting of a cow, a sheep, a horse, a pig, a giraffe, a camel, a deer, a hippopotamus, or a rhinoceros, each possibility representing a separate embodiment of the present invention.

[0120] According to certain exemplary embodiments, the non-human animal is a bovine.

[0121] It is to be understood that any combination of each of the aspects and embodiments disclosed herein is expressly encompassed within the present disclosure.

[0122] Other objects, features and advantages of the present invention will become apparent from the following description. [Brief explanation of the drawings]

[0123] [Figure 1] Embryonic stem cells (bESCs) derived from flushed bovine embryos are shown. Figure 1A shows the typical morphology of early embryos isolated from the zona pellucida and attached to a feeder layer one day after seeding. Figure 1B shows migration of cells with ESC-like morphology five days after seeding. [Figure 2] 1 shows the growth of bESCs in serum-free medium mTeSR1® supplemented with 2.5 μM IWR1 on inactivated mouse embryonic fibroblasts (iMEFs) as a feeder layer. [Figure 3] Figure 3A shows the pluripotency of expanded bESCs. Figure 3A shows the percentage of positive staining for Alp 0501 (17 experiments) and Alp 0505 (5 experiments) with the SSEA4 antibody by flow cytometry. Figure 3B shows the expression of pluripotency markers compared to expression in BEFs as measured by real-time PCR. [Figure 4] Figure 4 shows the expression of the pluripotency marker SSEA4 by bESCs grown in feeder-free cultures, as measured by flow cytometry. Figure 4A: PSC line Alp 0501. Figure 4B: PSC line Alp 0505. [Figure 5]The differentiation potential of bESC lines Alp 0501 and Alp 0505, as determined by expression of a pluripotency marker (Oct4) versus a mesoderm marker (Brachyury), is shown. [Figure 6] Figure 6 shows the formation and proliferation of bESC aggregates in 6-well plates. Figure 6A: 1 day after seeding in suspension. Figure 6B: 2 days after seeding in suspension. Figure 6C: 7 days after seeding in suspension. Representative images, scale bar = 200 μm. [Figure 7] 1 shows the incidence of bESC aggregation (viable / viable cell %) in a stirred tank (STR) bioreactor. [Figures 8A-8C] Growth of bESC aggregates in a stirred-tank (STR) bioreactor after 1, 3, and 4 days of growth is shown (Figures 8A-C, respectively). Representative images, scale bar = 650 μm. [Figure 8D-8E] Cell concentration and viability (FIG. 8D) and average aggregate diameter (FIG. 8E) are shown. The time course of pluripotency (expressed as SSEA4% expression) is shown in FIG. 8E. [Figure 9] The process of disaggregation and reaggregation is shown. Figure 9A shows 3-day-old aggregates that served as the source for disaggregation. Figure 9B shows aggregates formed from small aggregates / single cells obtained from the aggregates in Figure 9A that were reseeded in medium containing dissociation reagent. Representative images, scale bar = 650 µm. [Figure 10] Representative photographs of various stages in the isolation of umbilical cord cells are shown: Figure 10A: umbilical cord (UC); Figure 10B: Wharton's Jelly; Figure 10C: tissue section; Figure 10D: isolated UC cells. [Figure 11] Representative photographs of adipocytes differentiated from cells in BEF culture are shown. Differentiated cells appear dark due to staining with Oil Red O. Oil Red O stains triglycerides and lipids. [Figure 12]Figure 12 shows the growth of biPSC clones on iMEFs in 12-well plates. Figure 12A: Embryonic stem cell (ES)-like colonies two weeks after transfection. Figure 12B: Brightfield image showing bovine ES-like colony morphology after adaptation to a vitronectin-coated surface. Figure 12C: The episomal CoMiP plasmid did not integrate into the bovine host genome. [Figure 13] Figure 13A shows BEF cells efficiently transfected with modified mRNA, as indicated by expression of mCherry and bovine OCT4. Fluorescence (Figure 13A) and brightfield (Figure 13B) images of BEF cells transfected with modified mRNA encoding bovine OSKM, mCherry, and B18R. Fluorescence images show transfection efficiency 24 hours after initial mRNA transfection. Figure 13C: qPCR analysis of bovine OCT4 transcripts in untreated BEF cells, iBEF cells (BEF cells transfected with modified mRNA encoding bovine OSKM to induce reprogramming), and human iPSC cells. Values ​​are compared to untreated BEF cells (RQ=1) and normalized to bovine beta-actin. DETAILED DESCRIPTION OF THE INVENTION

[0124] The present invention provides a method for forming homogenous 3D aggregates at commercial scale from non-human animal-derived PSCs suspended in a liquid serum-free medium that contains a specific combination of growth factors and small molecules added to the medium and lacks a cell adhesive surface.

[0125] The present invention fulfills a long-felt need to produce non-GM cell-grown meat for food consumption from PSCs derived from non-human animals, particularly bovine, under large-scale conditions.

[0126] definition The terms "comprise," "comprising," "includes," "including," "having," and variations thereof mean "including but not limited to."

[0127] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0128] Throughout this application, various embodiments of the present invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0129] As used herein, the term "about" refers to a variation of the numerical designation of plus or minus 10% of the numerical designation.

[0130] The terms "animal" and "non-human animal" with respect to cells of origin are used interchangeably herein and refer only to cells of non-human animals.

[0131] As used herein, the term "pluripotent stem cells (PSCs)" refers to cells that can proliferate indefinitely and give rise to all other cell types in the body.

[0132] As used herein, the term "induced pluripotent stem cells (iPSCs)" refers to a type of pluripotent stem cell that can be generated directly from somatic cells.

[0133] As used herein, the term "embryonic stem cells (ESCs)" refers to a type of pluripotent stem cell derived from the blastocyst of a non-human animal, particularly a bovine.

[0134] The term "three-dimensional (3D)" refers to a cell culture of cells grown in liquid suspension and not attached to a solid and / or semi-solid surface. 3D cell cultures are artificially created environments in which living cells can grow or interact with their surrounding cells. 3D cell cultures allow for the in vitro growth of cells in any orientation, similar to how cells grow in vivo.

[0135] The term "genetically unmodified (non-GM)" refers to intact cells that have no genomic alterations due to transfection with / integration of exogenous nucleic acid into their genome.

[0136] As used herein, the term "aggregates" refers to the phenomenon whereby dissociated cells mixed in vitro tend to aggregate with themselves and with other cells, also called "floating aggregates."

[0137] As used herein, the term "homogeneous" with respect to aggregates of the present invention refers to a plurality of aggregates comprising at least 70% non-human animal-derived PSCs and having an average diameter of about 30 μm to about 500 μm. According to certain embodiments, the average diameter of the aggregates is about 30 μm to about 450 μm, about 30 μm to about 400 μm, or about 30 μm to about 350 μm. According to some embodiments, the average diameter of the aggregates is greater than 300 μm. According to certain exemplary embodiments, the average diameter of the aggregates is about 30 μm to about 350 μm. According to certain embodiments, the final suspension product comprises aggregates having an average diameter of about 250 μm to about 350 μm.

[0138] As used herein, the term "serum-free" in reference to a medium refers to a medium that does not contain animal serum.

[0139] As used herein, the term "animal-derived component-free" in reference to a medium refers to a medium that does not contain any components of animal origin, particularly a medium that does not contain components of mammalian origin.

[0140] The term "cell-grown meat" is used herein to describe meat grown in vitro from animal cell cultures, as distinguished from meat from slaughtered animals. Additional terms that can be used in the art to describe meat grown in vitro from animal cell cultures include cultured meat, cultivated meat, clean meat, laboratory-grown meat, test tube meat, in vitro meat, tube steak, synthetic meat, cell-cultured meat, cell-grown meat, tissue-engineered meat, engineered meat, artificial meat, and artificial meat.

[0141] As used herein, the term "closed system" with respect to a cell culture system refers to a system that contains all the elements necessary to complete the proliferation cycle of PSC aggregates according to the teachings of the present invention.

[0142] The term "reprogramming" refers to the conversion of one particular cell type into another cell type. According to certain embodiments of the invention, reprogramming is the conversion of a somatic cell type into a pluripotent cell type known as an induced pluripotent stem cell, or iPSC.

[0143] As used herein, the term "reprogramming mRNA" refers to an mRNA encoding any one of the following transcription factors: OCT4, SOX2, KLF4, cMYC, NANOG, LIN28 and KLF5.

[0144] The terms "vessel" or "tissue culture vessel" are used interchangeably herein to refer to any receptacle in which PSCs can grow in suspension without adhering to the receptacle material. Receptacles may vary in size, ranging from the few milliliter range (e.g., non-adherent plates or Erlenmeyer flasks) to the thousands of liter range (e.g., bioreactors).

[0145] According to one aspect, the present invention provides a method for producing aggregates of non-genetically modified, non-human animal-derived PSCs, comprising the steps of: (a) seeding at least one PSC in a growth medium to form a suspension culture, wherein the growth medium contains at least one growth factor, bFGF, at least one additional growth factor, and / or an inhibitor of the Wnt-β-catenin signaling pathway, CHIR 99021 (C 22 H 18 Cl2N8), PD 0325901(C 16 H 14 F3IN2O4), A 83-01(C 25 H 19 and (b) growing the suspension culture under conditions that permit aggregate formation and aggregate growth, thereby forming homogenous aggregates of the PSCs. Each possibility represents a separate embodiment of the present invention.

[0146] According to certain embodiments, seeding and growth is in a container having walls of a material to which the PSCs and / or aggregates comprising them do not adhere.

[0147] According to certain embodiments, the growth medium is further free of animal-derived components.

[0148] bFGF (basic fibroblast growth factor, also known as FGF2) has extensive cell division and cell survival activities and is involved in various biological processes, including embryonic development, cell proliferation, morphogenesis, tissue repair, tumor growth, and invasion. bFGF activates JAK / STAT, PI3K, ERK1 / 2, and other receptor tyrosine kinase (RTK) signaling pathways. It supports the maintenance of undifferentiated human embryonic stem cells.

[0149] According to certain embodiments, the growth medium comprises a combination of bFGF and at least one additional growth factor. According to certain exemplary embodiments, the additional growth factor is a protein of the transforming growth factor beta (TGF-β, also referred to as TGFB) family.

[0150] TGF-β is a multifunctional cytokine belonging to the transforming growth factor superfamily, which includes three distinct mammalian isoforms (TGF-β1-3, or TGFB1, TGFB2, and TGFB3) and many other signaling proteins, including activin-A. Activated TGF-β complexes with other factors to form a serine / threonine kinase complex that binds to the TGF-β receptor. The TGF-β receptor is composed of both type 1 and type 2 receptor subunits. After TGF-β binding, the type 2 receptor kinase phosphorylates and activates the type 1 receptor kinase, which activates a signaling cascade. This induces the activation of different downstream substrates and regulatory proteins, including the transcription of different target genes that function in differentiation, chemotaxis, and proliferation, as well as the activation of many immune cells.

[0151] According to certain embodiments, the growth medium comprises a combination consisting of bFGF and at least one small molecule.

[0152] WNT-β-catenin signaling is involved in maintaining the homeostasis of many developmental processes and adult tissues by regulating cell proliferation, differentiation, migration, genetic stability, and apoptosis, and known inhibitors and future inhibitors developed from this pathway can be used in accordance with the teachings of the present invention. Known inhibitors are listed, for example, in Kahn M. 2014. Nat Rev Drug Discov. 13(7):513-32. doi:10.1038 / nrd4233.

[0153] According to certain embodiments, the inhibitor of the Wnt-β-catenin signaling pathway is selected from the group consisting of IWR1, JW67, NSC668036, KY02111, niclosamide, DKK1 or si-beta-catenin, porcupine inhibitor, and IWP-2, each possibility representing a separate embodiment of the present invention.

[0154] According to certain exemplary embodiments, the inhibitor of the Wnt-β-catenin signaling pathway is IWR1. IWR1 potently inhibits WNT signaling by blocking the cell-based WNT / β-catenin pathway. When used in combination with CHIR 99021, it has been shown to promote self-renewal and maintain the pluripotency of human embryonic stem cells and mouse epithelial stem cells (Kim et al., Nat Commun. 2013, 4:2403). A culture medium containing a combination of IWR1 and bFGF has also been shown to support the proliferation and maintenance of bovine blastocyst-derived pluripotent embryonic stem cells (Bogliotii YS et al., ibid.). According to certain embodiments, the concentration of IWR1 in the medium is about 1 μM to about 10 μM. According to some embodiments, the concentration of IWR1 in the medium is about 2 μM to about 10 μM.

[0155] CHIR 99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile) is an aminopyrimidine derivative that is a selective and potent inhibitor of glycogen synthase kinase 3 (GSK-3).

[0156] PD0325901 (N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide) is a potent and selective MEK1 and MEK2 inhibitor.

[0157] PD 0325901 can be used in conjunction with CHIR 99021 to reprogram somatic cells into iPSCs and promote cellular self-renewal.

[0158] A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide) is a potent inhibitor of the TGF-β type I receptor ALK5 kinase, the type I activin / nodal receptor ALK4, and the type I nodal receptor ALK7 (IC50 values ​​are 12, 45, and 7.5 nM, respectively). A83-01 blocks Smad2 phosphorylation and inhibits TGF-β-induced epithelial-mesenchymal transition. A83-01 is used to inhibit iPSC differentiation and maintain cell self-renewal in vitro.

[0159] According to another embodiment, the growth medium contains two growth factors bFGF and TGF-β, as well as IWR1, CHIR 99021 (C 22 H 18 Cl2N8), PD 0325901(C 16 H 14 F3IN2O4), and A 83-01(C 25 H 19 N5S).

[0160] According to certain embodiments, the growth medium comprises a combination of the growth factor bFGF and at least one small molecule selected from the group consisting of IWR1, CHIR 99021, PD 0325901, A 83-01, and any combination thereof. According to some embodiments, the growth medium comprises a combination of the growth factor bFGF and the small molecule IWR1.

[0161] According to certain exemplary embodiments, the growth medium comprises a combination of the growth factors bFGF, and at least one of TGF-β-1, TGF-β-3, and activin-A, and the small molecule IWR1. According to some embodiments, the growth medium comprises a combination of the growth factors bFGF, at least one of TGF-β-1, TGF-β-3, and activin-A, and the small molecule IWR1.

[0162] According to certain embodiments, the growth medium comprises a combination comprising the growth factor bFGF and the small molecules IWR1 and CHIR 99021. According to certain embodiments, the growth medium comprises a combination consisting of the growth factor bFGF and the small molecules IWR1 and CHIR 99021.

[0163] According to certain exemplary embodiments, the growth medium comprises a combination of the growth factor bFGF and the small molecule A 83-01. According to further exemplary embodiments, the growth medium comprises a combination of at least one of the growth factors bFGF, TGF-β-1, TGF-β-3, and activin-A, and a combination of the small molecules CHIR 99021, PD 0325901, and A 83-01.

[0164] Pluripotent stem cells from any non-human animal, as known in the art, can be used in accordance with the teachings of the present invention. According to certain embodiments, the non-human animal is of a species selected from the group consisting of ungulates, poultry, aquatic animals, invertebrates, and reptiles. Each possibility represents a separate embodiment of the present invention.

[0165] According to certain embodiments, the ungulate is selected from the group consisting of a cow, a sheep, a horse, a pig, a giraffe, a camel, a deer, a hippopotamus, or a rhinoceros. According to some embodiments, the ungulate is a cow. According to certain exemplary embodiments, the ungulate is a dairy cow.

[0166] Commercial preparations of bovine-derived PSCs are also available, including, for example, blastocyst-derived PSCs.

[0167] According to some embodiments, the PSCs are embryonic stem cells (ESCs).

[0168] According to some embodiments, the PSCs are non-embryonic stem cells (ESCs).

[0169] According to certain embodiments, the PSCs are induced PSCs (iPSCs) reprogrammed from somatic cells.

[0170] According to certain embodiments, the PSCs are induced PSCs (iPSCs) reprogrammed from somatic cells that do not include ESCs.

[0171] Reprogramming of cells to produce iPSCs can be carried out by any method known in the art, including, for example, the method described in Peleganove et al. (Poleganov et al., Hum. Gene Ther. 2015;26:751-766).

[0172] According to certain exemplary embodiments, a method for producing aggregates of non-genetically modified, non-human animal-derived PSCs comprises: (a) seeding at least one PSC in a growth medium to form a seed suspension culture; (b) incubating the seeded suspension culture to form PSC aggregates; (c) disaggregating the aggregates into smaller aggregates and / or single cells; (d) repeating steps (a)-(c) at least once to obtain a desired PSC concentration; (e) Proliferating the PSCs by repeating steps (a)-(b) to obtain a desired aggregate concentration.

[0173] According to certain exemplary embodiments, disaggregating the aggregates comprises exposing the aggregates to a dissociation reagent and / or a dissociation force. According to some embodiments, the dissociation reagent comprises at least one protease and, optionally, at least one DNase and / or a chelating agent. According to some embodiments, the dissociation reagent comprises at least one protease, at least one DNase, and a chelating agent. According to certain embodiments, the chelating agent is selected from EDTA and EGTA. According to some exemplary embodiments, the protease is trypsin.

[0174] According to some embodiments, the dissociation force is a shear force. According to certain embodiments, the shear force rate is set by an impeller embedded within the vessel.

[0175] According to certain embodiments, the disaggregating step further comprises washing the homogenous aggregates with an aqueous wash medium before exposing the aggregates to a dissociation reagent and / or a dissociation force.

[0176] According to certain embodiments, steps (a)-(c) are repeated at least two times, at least three times, at least four times, at least five times, and more. It should be clearly understood that the number of repetitions may be unlimited and depends on the amount of PSC required for the intended use. According to certain embodiments, the aggregating and disaggregating steps are repeated for at least 10 6 The steps of aggregating and disaggregating are repeated until a cell concentration of at least 5×10 cells / ml is reached. 6 cells / ml, at least 10 7 cells / ml, at least 5 × 10 7 cells / ml, at least 10 8 cells / ml, at least 5 × 10 8 cells / ml, or at least 10 9 This is repeated until a cell concentration of 100 cells / ml or more is reached.

[0177] According to certain embodiments, steps (a) to (c) are repeated in the same medium. According to some embodiments, the medium comprises a dissociation reagent.

[0178] According to certain embodiments, the seeding is in a non-adherent tissue culture vessel.

[0179] According to certain embodiments, step (a) of seeding at least one PSC further comprises adding an inhibitor of Rho-associated protein kinase (Rock).

[0180] Any ROCK inhibitor currently known in the art or developed in the future can be used according to the teachings of the present invention. According to certain embodiments, the ROCK inhibitor is selected from the group consisting of Thiazovivin, Fasudil, Ripasudil, Netarsudil, RKI-1447, Y-27632, GSK429286A, and Y30141. Each possibility represents a separate embodiment of the present invention. According to certain exemplary embodiments, the ROCK inhibitor is Y-27632 dihydrochloride (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide).

[0181] According to certain embodiments, step (a) of seeding at least one PSC further comprises encapsulating at least one non-genetically modified, non-human animal-derived PSC within a particle and seeding a plurality of the particles in a growth medium.

[0182] According to certain embodiments, the particles comprise an inner core surrounded by at least one outer shell, the inner core further comprising at least one compound that supports the growth of at least one non-genetically modified, non-human animal-derived PSC. According to certain embodiments, the outer shell layer is made of a food-grade, fluid-permeable material. According to certain embodiments, the outer shell layer is designed for reversible encapsulation, such that PSC aggregates formed within the capsule are released upon exposure of the particle to certain conditions. According to certain embodiments, the fluid-permeable, food-grade material of the outer shell layer is dissolvable. It should be clearly understood that the microenvironment of the particle's inner core provides free space that allows initial cell-cell interactions and subsequent cell growth, but is free of cell aggregation and hydrodynamic stress. Therefore, particles encapsulating PSCs can significantly improve culture efficiency and aggregate formation.

[0183] According to certain embodiments, the outer shell layer comprises at least one food-grade polymer or copolymer. According to certain embodiments, the outer shell layer comprises one or more polymers that form a hydrogel. According to certain embodiments, the hydrogel is a thermosensitive and / or thermoreversible hydrogel. Suitable polymers and hydrogel materials are known in the art. For example, alginate is a well-known polymer used for encapsulation. Gellan gum forms a hydrogel at low temperatures (a gel at body temperature) when mixed with monovalent or divalent cations. Chitosan forms a strong ionotropic hydrogel with phosphate ions. Curdlan can form low-set gels (thermoreversible) and high-set gels (thermoreversible), primarily based on different heating temperatures. Agarose, hyaluronic acid, carrageenan, modified starch, and pectin are all known to be suitable for forming the outer shell layer according to the teachings of the present invention.

[0184] According to certain embodiments, at least one growth-supporting compound present within the inner core microenvironment is formulated in a slow and / or sustained release formulation, as known in the art.

[0185] According to certain embodiments, the tissue culture vessel is made of a material to which the PSCs do not adhere, and therefore the PSCs and / or aggregates comprising them are free in suspension and not attached to any surface.

[0186] According to certain embodiments, the suspension culture is expanded to reach a desired pluripotent cell concentration of about 100,000 cells / ml to about 50,000,000 cells / ml. According to certain embodiments, the suspension culture is expanded to reach PSCs at a concentration of about 300,000 cells / ml to about 10,000,000 cells / ml or about 1,000,000 cells / ml.

[0187] According to certain embodiments, the suspension culture is incubated under dynamic rotation, under static conditions, or a combination thereof.

[0188] According to certain exemplary embodiments, the suspension culture is incubated under dynamic rotation conditions.

[0189] According to certain embodiments, the incubation is carried out at the body temperature of the animal from which the cells are derived.

[0190] According to a particular embodiment, the incubation is carried out at a temperature of 37.5 to 39.5°C.

[0191] According to certain exemplary embodiments, when the non-human animal is a bovine, the incubation temperature is approximately 38.6°C, the average body temperature of a bovine.

[0192] According to certain embodiments, the volume of the suspension culture is from about 100 ml to about 15,000 liters per vessel.

[0193] According to some embodiments, the volume of the suspension culture is from about 500 ml to about 15,000 liters per vessel. According to some embodiments, the volume of the suspension culture is from about 1 liter to about 15,000 liters per vessel. According to some embodiments, the volume of the suspension culture is from about 1 liter to about 1,500 liters per vessel. According to some embodiments, the volume of the suspension culture is from about 1 liter to about 150 liters per vessel.

[0194] An advantage of the methods of the present invention is that all steps can be performed in commercial-scale volumes and in a closed system. According to some embodiments, the volume of the suspension culture is about 1,000 liters to about 15,000 liters per vessel.

[0195] According to certain embodiments, the formed PSC aggregates have an average diameter of about 30 μm to about 500 μm. According to some embodiments, the aggregates have an average diameter of about 100 μm to about 500 μm. According to some embodiments, the aggregates have an average diameter of about 150 μm to about 500 μm. According to certain exemplary embodiments, the aggregates have an average diameter of about 300 μm to 550 μm. According to certain exemplary embodiments, the average diameter of the aggregates is about 30 μm to about 350 μm. According to certain exemplary embodiments, the average diameter of the aggregates is about 250 μm to about 350 μm.

[0196] According to certain exemplary embodiments, the PSCs are reprogrammed non-human animal-derived cells produced by a method comprising introducing a combination of (a) at least one reprogramming mRNA encoding a reprogramming factor, and (b) at least one double-stranded microRNA into at least one non-human animal-derived cell, thereby producing at least one iPSC.

[0197] According to certain embodiments, the method further comprises introducing at least one immune evasion mRNA into at least one cell derived from a non-human animal.

[0198] According to certain embodiments, at least one reprogramming mRNA is a synthetically modified mRNA.

[0199] According to certain further exemplary embodiments, the PSCs are reprogrammed cells derived from a non-human animal produced by a method comprising introducing into at least one cell derived from a non-human animal a combination of (a) at least one reprogramming mRNA and (b) at least one inhibitor of at least one microRNA endogenous to the cell derived from the non-human animal, thereby producing at least one iPSC.

[0200] According to certain embodiments, the method further comprises introducing at least one immune evasion mRNA into at least one cell derived from a non-human animal.

[0201] According to certain embodiments, the at least one reprogramming mRNA and / or the at least one double-stranded microRNA encoding a reprogramming factor is of human or non-human animal origin.

[0202] According to certain embodiments, the non-human animal is a bovine. According to these embodiments, the at least one reprogramming mRNA and / or the at least one double-stranded microRNA encoding a reprogramming factor is of human or bovine origin.

[0203] Introduction of the oligo- or polynucleotides of the invention into PSCs can be carried out by any method known in the art.

[0204] According to the teachings of the present invention, the at least one mRNA does not integrate into the genome of the cell, and the iPSCs produced thereby are not genetically modified.

[0205] According to certain embodiments, the introduction of at least one reprogramming mRNA, and / or at least one immune evasion mRNA and / or at least one double-stranded microRNA and / or at least one microRNA inhibitor is performed in a serum-free, animal-derived component-free liquid growth medium. According to some embodiments, the medium is further free of animal-derived components.

[0206] According to some embodiments, the method further comprises culturing at least one iPSC produced in a serum-free liquid growth medium to form a plurality of iPSCs. The serum-free liquid growth medium is as described hereinabove. According to some embodiments, the serum-free liquid growth medium used is supplemented with a Rock inhibitor.

[0207] According to some embodiments, the reprogramming mRNA introduced into the cell or cells from the non-human animal is selected from the group consisting of OCT4, SOX2, KLF4, cMYC, NANOG, LIN28, KLF5, and any combination thereof, with each possibility representing a separate embodiment of the present invention.

[0208] According to further embodiments, the immune evasion mRNA introduced into the cell or cells derived from the non-human animal is selected from the group consisting of E3, K3, B18R[EKB] from vaccinia virus, and any combination thereof, each possibility representing a separate embodiment of the present invention.

[0209] According to yet another embodiment, the double-stranded microRNA is selected from the group consisting of miR-302a, miR-302b, miR-302c, miR-302d, miR-367, miR-218, miR-449b, and any combination thereof, with each possibility representing a separate embodiment of the present invention.

[0210] According to certain embodiments, the at least one microRNA inhibitor is an RNA inhibitor (RNAi) molecule. According to certain exemplary embodiments, the at least one microRNA inhibitor targets miR-145.

[0211] According to certain embodiments, when used, the immune evasion mRNA introduced into the cell or cells derived from a non-human animal is selected from the group consisting of E3, K3, B18R[EKB] from vaccinia virus, and any combination thereof, each possibility representing a separate embodiment of the present invention.

[0212] According to certain embodiments, the at least one non-human animal-derived cell and / or the at least one iPSC formed are incubated at a temperature of 37.5 to 39.5°C.

[0213] According to certain embodiments, the non-human animal is of a species selected from the group consisting of ungulates, poultry, aquatic animals, invertebrates, and reptiles, each possibility representing a separate embodiment of the present invention.

[0214] According to certain embodiments, the ungulate is selected from the group consisting of a cow, a sheep, a horse, a pig, a giraffe, a camel, a deer, a hippopotamus, or a rhinoceros.

[0215] According to certain exemplary embodiments, the ungulate is a bovine. According to these embodiments, the at least one bovine-derived cell and / or the at least one formed iPSC is incubated at 38.6°C, the average body temperature of a bovine.

[0216] According to some embodiments, bovine-derived cells are obtained from bovine umbilical cord, bovine nasopharyngeal mucosa, and bovine blood. According to certain exemplary embodiments, somatic bovine cells are harvested by non-invasive techniques.

[0217] According to certain exemplary embodiments, the bovine-derived cells are obtained from bovine umbilical cord, and the cells are selected from the group consisting of endothelial cells, umbilical cord lining cells, Wharton's Jelly cells, or combinations thereof. According to yet additional embodiments, the bovine-derived cells are obtained from the beef cattle breed Belgium Blue, which is characterized by a dual muscle phenotype due to a natural mutation in the myostatin gene.

[0218] According to a further aspect, the present invention provides homogeneous aggregates of non-genetically modified, non-human animal-derived PSCs produced according to the methods of the present invention. According to certain embodiments, the aggregates comprise at least 70% viable cells expressing at least one pluripotency marker. According to certain embodiments, the pluripotency marker is selected from the group consisting of SSEA4, Oct4, Nanog, Lin28A, Sall4, Dnmt3b, and combinations thereof. According to certain exemplary embodiments, the pluripotency marker is SSEA4.

[0219] In another aspect, the present invention provides a suspension comprising a serum-free liquid medium and cell aggregates derived from a non-human animal, the cell aggregates comprising at least 70% viable PSCs expressing at least one pluripotency marker. According to certain embodiments, the PSCs divide approximately every 16-32 hours. According to certain embodiments, the PSCs divide approximately every 16-24 hours.

[0220] According to certain embodiments, the PSCs of the aggregates express at least one surface protein that contributes to cell-cell adhesion.

[0221] According to yet another aspect, the present invention provides a cell-expanded meat culture comprising homogenous aggregates of non-genetically modified, non-human animal-derived PSCs according to the present invention, or a suspension comprising them.

[0222] According to yet an additional aspect, the present invention provides a cell-grown meat product comprising progeny of homogenous aggregates of the non-genetically modified, non-human animal-derived PSCs of the present invention.

[0223] The non-human animal is as described herein above. According to certain exemplary embodiments, the non-human animal is a bovine.

[0224] According to certain embodiments, the progeny include cells that differentiate from the PSC to form at least one of myocytes, stromal cells, endothelial cells, and adipocytes.

[0225] The present invention now unexpectedly demonstrates that the presence of fatty acids in growth media according to certain embodiments of the invention results in the differentiation of cultures of bovine-derived PSCs or BEFs into adipocytes.

[0226] According to yet another aspect, the present invention provides a method for differentiating non-human animal cells selected from the group consisting of PSCs, their progeny, somatic cells, and combinations thereof, into adipocyte cells, the method comprising incubating the cells in a serum-free liquid medium containing (i) at least one inhibitor of the Wnt-β-catenin signaling pathway, and (ii) at least one of at least one type of fatty acid, thereby differentiating the cells into adipocyte cells.

[0227] According to certain embodiments, the culture medium comprises a combination of at least one inhibitor of the Wnt-β-catenin signaling pathway and at least one type of fatty acid.

[0228] According to a particular embodiment, the medium further comprises the growth factor bFGF.

[0229] According to certain embodiments, the medium further comprises a Rock inhibitor.

[0230] According to certain embodiments, the cells are incubated for at least 4 days. According to other embodiments, the cells are incubated for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days or more. Each possibility represents a separate embodiment of the present invention.

[0231] According to certain embodiments, the inhibitor of the Wnt-β-catenin signaling pathway is selected from the group consisting of IWR1, JW67, NSC668036, KY02111, niclosamide, DKK1 or si-beta-catenin, porcupine inhibitor, and IWP-2.

[0232] According to certain exemplary embodiments, the at least one inhibitor of the Wnt-β-catenin signaling pathway is IWR-1.

[0233] According to certain embodiments, the fatty acid is selected from the group consisting of free fatty acids, low molecular weight fatty acids, esters thereof, salts thereof, and any combination thereof.

[0234] According to certain embodiments, the PSCs comprise stromal stem cells isolated from adipose tissue.

[0235] According to certain embodiments, the cells comprise a population of cells derived from embryonic muscle tissue. According to some embodiments, the cells derived from embryonic muscle tissue comprise embryonic fibroblasts (EF).

[0236] The non-human animal is as defined herein above.

[0237] Food products comprising the cell-grown meat of the present invention are also included within the scope of the present invention.

[0238] In another aspect, the present invention provides a growth medium for expanding PSCs derived from a non-human animal, comprising the growth factor bFGF in combination with (i) at least one additional growth factor and (ii) at least one small molecule selected from the group consisting of an inhibitor of the Wnt-β-catenin signaling pathway, CHIR 99021, PD 0325901, and A 83-01, or any combination thereof, wherein the growth medium is serum-free and lacks inactivated feeder cells. According to certain embodiments, the growth medium further lacks an organic matrix. According to certain embodiments, the growth medium further does not contain animal-derived components.

[0239] According to certain embodiments, the growth medium is capable of maintaining non-human mammalian pluripotent stem cells in a pluripotent state when cultured in suspension culture lacking substrate attachment.

[0240] According to certain embodiments, the growth medium lacks proteinase inhibitors.

[0241] According to certain embodiments, the growth medium comprises a combination of bFGF and at least one additional growth factor. According to certain exemplary embodiments, the additional growth factor is a protein of the TGF-β superfamily. According to certain exemplary embodiments, the TGF-β is selected from the group consisting of TGF-β-1, TGF-β-3, activin-A, and any combination thereof.

[0242] According to certain embodiments, the growth medium comprises a combination consisting of bFGF and at least one small molecule.

[0243] According to certain embodiments, the inhibitor of the Wnt-β-catenin signaling pathway is selected from the group consisting of IWR1, JW67, NSC668036, KY02111, niclosamide, DKK1 or si-beta-catenin, porcupine inhibitor, and IWP-2.

[0244] According to certain exemplary embodiments, the inhibitor of the Wnt-β-catenin signaling pathway is IWR1.

[0245] According to certain exemplary embodiments, the growth medium comprises a combination of at least one of the growth factors bFGF, TGF-β-1, TGF-β-3, and activin-A, and a small molecule selected from the group consisting of IWR1, CHIR 99021, PD 0325901, A 83-01, and any combination thereof.

[0246] According to certain exemplary embodiments, the growth medium comprises a combination of at least one of the growth factors bFGF, TGF-β-1, TGF-β-3, and activin-A, and the small molecule IWR1. According to some embodiments, the growth medium comprises a combination of at least one of the growth factors bFGF, TGF-β-1, TGF-β-1, and activin-A, and the small molecule IWR1.

[0247] According to certain embodiments, the growth medium comprises a combination comprising the growth factor bFGF and the small molecules IWR1 and CHIR 99021. According to certain embodiments, the growth medium comprises a combination consisting of the growth factor bFGF and the small molecules IWR1 and CHIR 99021.

[0248] According to further specific exemplary embodiments, the growth medium comprises a combination consisting of the growth factor bFGF and the small molecule IWR1.

[0249] According to another aspect, the present invention provides a kit for expanding PSC aggregates derived from non-human animals, comprising a serum-free growth medium lacking inactivated feeder layer cells, the growth factor bFGF, at least one additional growth factor, and at least one small molecule selected from the group consisting of inhibitors of the Wnt-β-catenin signaling pathway, CHIR 99021, PD 0325901, and A 83-01. According to certain embodiments, the kit further comprises instructions on growth conditions for expanding the PSC aggregates derived from non-human animals.

[0250] According to certain embodiments, the serum-free medium further lacks an organic matrix. According to certain embodiments, the growth medium further does not contain animal-derived components.

[0251] According to certain embodiments, the inhibitor of the Wnt-β-catenin signaling pathway is selected from the group consisting of IWR1, JW67, NSC668036, KY02111, niclosamide, DKK1 or si-beta-catenin, porcupine inhibitor, and IWP-2.

[0252] According to certain exemplary embodiments, the inhibitor of the Wnt-β-catenin signaling pathway is IWR1.

[0253] According to certain exemplary embodiments, the at least one additional growth factor is a protein of the TGF-β superfamily, wherein the TGF-β is selected from the group consisting of TGF-β-1, TGF-β-3, activin-A, and any combination thereof.

[0254] According to a further aspect, the present invention provides a kit for growing PSC aggregates derived from a non-human animal, comprising a serum-free growth medium lacking inactivated feeder layer cells, the growth factor bFGF, the growth factor TGF-β-3, activin A, the small molecule IWR1, and instructions for growth conditions for growing PSC aggregates derived from a non-human animal.

[0255] According to yet an additional aspect, the present invention provides a method for reprogramming a cell derived from a non-human animal into an iPSC, the method comprising introducing a combination of (a) at least one reprogramming mRNA encoding a reprogramming factor, and (b) at least one double-stranded microRNA into at least one cell derived from the non-human animal, thereby producing at least one iPSC.

[0256] According to certain embodiments, the method further comprises introducing at least one immune evasion mRNA into at least one cell derived from a non-human animal.

[0257] According to certain embodiments, at least one reprogramming mRNA is a synthetically modified mRNA.

[0258] According to yet another aspect, the present invention provides a method of reprogramming a cell derived from a non-human animal into an iPSC, the method comprising introducing into at least one cell derived from the non-human animal a combination of (a) at least one reprogramming mRNA and (b) at least one inhibitor of at least one microRNA endogenous to the cell derived from the non-human animal, thereby producing at least one iPSC.

[0259] According to certain embodiments, the method further comprises introducing at least one immune evasion mRNA into at least one cell derived from a non-human animal.

[0260] According to certain embodiments, the at least one reprogramming mRNA and / or the at least one double-stranded microRNA encoding a reprogramming factor is of human or non-human animal origin.

[0261] According to certain embodiments, the non-human animal is a bovine. According to these embodiments, at least one reprogramming mRNA and / or at least one double-stranded microRNA encoding a reprogramming factor is of human or bovine origin. Each possibility represents a separate embodiment of the present invention.

[0262] Introduction of the oligo- or polynucleotides of the invention into PSCs can be carried out by any method known in the art.

[0263] According to the teachings of the present invention, the at least one mRNA does not integrate into the genome of the cell, and the iPSCs produced thereby are not genetically modified.

[0264] According to certain embodiments, the introduction of at least one reprogramming mRNA, and / or at least one immune evasion mRNA and / or at least one double-stranded microRNA and / or at least one microRNA inhibitor is performed in a serum-free, animal-derived component-free liquid growth medium. According to some embodiments, the medium is further free of animal-derived components.

[0265] According to some embodiments, the method further comprises culturing at least one iPSC produced in a serum-free liquid growth medium to form a plurality of iPSCs. The serum-free liquid growth medium is as described hereinabove. According to some embodiments, the serum-free liquid growth medium used is supplemented with a Rock inhibitor.

[0266] The reprogramming mRNA, the immune evasion mRNA, the double-stranded microRNA, and the at least one microRNA inhibitor are as described herein above.

[0267] According to certain embodiments, the at least one non-human animal-derived cell and / or the at least one iPSC formed are incubated at a temperature of 37.5 to 39.5°C.

[0268] According to certain embodiments, the non-human animal is of a species selected from the group consisting of ungulates, poultry, aquatic animals, invertebrates, and reptiles, each possibility representing a separate embodiment of the present invention.

[0269] According to certain embodiments, the ungulate is selected from the group consisting of a cow, a sheep, a horse, a pig, a giraffe, a camel, a deer, a hippopotamus, or a rhinoceros.

[0270] According to certain exemplary embodiments, the ungulate is a bovine. According to these embodiments, the at least one bovine-derived cell and / or the at least one formed iPSC is incubated at 38.6°C, the average body temperature of a bovine.

[0271] According to some embodiments, bovine-derived cells are obtained from bovine umbilical cord, bovine nasopharyngeal mucosa, and bovine blood. According to certain exemplary embodiments, somatic bovine cells are harvested by non-invasive techniques.

[0272] According to certain exemplary embodiments, the bovine-derived cells are obtained from bovine umbilical cord, and the cells are selected from the group consisting of endothelial cells, umbilical cord lining cells, Wharton's Jelly cells, or combinations thereof. According to yet additional embodiments, the bovine-derived cells are obtained from the beef cattle breed Belgium Blue, which is characterized by a dual muscle phenotype due to a natural mutation in the myostatin gene.

[0273] The present invention also uses principles known from the art of PSC aggregate formation. However, the present invention differs from the known art in several important areas. First, the present invention provides methods for forming aggregates of non-human animal-derived PSCs, particularly bovine-derived PSCs. Second, the present invention provides methods for large-scale production of aggregates. Third, the present invention provides methods for forming aggregates of non-human animal-derived PSCs, particularly bovine-derived PSCs, that are not genetically modified.

[0274] The following examples are presented to further illustrate some embodiments of the present invention. However, they should in no way be construed as limiting the broad scope of the invention. Those skilled in the art can readily devise numerous variations and modifications of the principles disclosed herein without departing from the scope of the invention. [Example]

[0275] Example 1: Derivation of pluripotent bovine embryonic stem cells (bESCs) from flushed embryos Cell induction The non-surgical procedure of flushing preimplantation embryos after insemination is a routine procedure performed in conventional bovine breeding (Castro Neto ASet et al., Theriogenology 63, 2005, pp. 1249-1255). These preimplantation embryos were used to derive embryonic stem cells (bESCs). Embryo flushing was coordinated and performed by Sion, an Israeli artificial insemination and breeding company. Flushed embryos were examined and counted in situ under a light microscope and then rapidly transported to the applicant's laboratory. To derive bESCs, preimplantation embryos must be at the blastocyst stage. However, the flushed embryos were at various embryonic developmental stages (from early morula to mature blastocysts) and different grades (from poor to excellent). To reach the blastocyst or enhanced blastocyst stage, the flushed embryos were transferred to embryo maturation medium under a microscope in a biological hood using a micropipette and incubated for 2–3 hours. The stage and grade of the embryos were assessed (Stringfellow DA, Givens MD, Manual of the International Embryo Transfer Society, 4 th Blastocyst-stage embryos were selected using the International Embryo Transfer Society (IEM) (International Embryo Transfer Society, 2010). Microsurgery was then used to isolate the inner cell mass (ICM) of the embryo (Figure 1A). The isolated ICM was then incubated at 38.6°C and 5% CO2, and the establishment of bESC colonies was monitored and documented (Figure 1B).

[0276] Characterization of bESCs Two exemplary lines of bESCs derived as described herein above, designated herein as Alp 0501 and Alp 0505, were examined for pluripotency properties.

[0277] These bESC lines were grown in serum-free medium essentially as described by Bogliotti et al. (Bogliotti YS et al., 2018. PNAS 115(9):2090-2095). Briefly, cells were grown in serum-free medium mTeSR1® (STEMCELL Technologies Inc. Canada) supplemented with 2.5 μM IWR1, with inactivated mouse embryonic fibroblasts (iMEFs) as a feeder layer. Rho-associated kinase inhibitor (Rock inhibitor) was added, and cells were passaged every 3–4 days. bESC morphology was consistent with that of stem cells, and cells were expanded in defined colonies.

[0278] The bESC population doubling time (PDT) was measured by seeding cells in 12-well plates (in triplicate) and counting their number on days 2, 3, and 4 after seeding (Figure 2). Cell proliferation reached 12–14 fold in one passage (day 4). The population doubling time (PDT) was calculated to be 26 h for Alp 0501 and 24 h for Alp 0505, which is consistent with the stem cell division rate.

[0279] The bESC lines were further examined for expression of the pluripotency markers OCT4, SSEA4, Nanog, Sall4 and Dnmt3b.

[0280] Oct4 immunostaining was performed as follows. 1. Cells were washed with PBS and fixed by incubation with 4% paraformaldehyde (PFA) at room temperature for 20 minutes. 2. Cells were permeabilized by incubation with 0.3% Triton-X for 10 minutes at room temperature. 3. The cells were washed with PBS. Cells were blocked by incubation with 4.5% BSA and 0.1% Triton-X for 30 min at room temperature. 5. Cells were stained overnight at 4°C with anti-Oct4 antibody (from Abcam) diluted 1:100 in 1% BSA and 0.1% Triton-X. 6. The cells were washed three times with PBS and analyzed by fluorescence microscopy.

[0281] Flow cytometry analysis of SSEA4 was performed as follows. 1. Cells were washed twice with PBS. 2. Cells were incubated with anti-SSEA4 antibody (from R&D Systems) at 1:50 in staining buffer (from R&D Systems) for 1 hour at 4°C protected from light. 3. The cells were washed twice with PBS and analyzed using a flow cytometer.

[0282] Quantitative PCR (qPCR) analysis was performed as follows. 1. Total RNA was extracted from cells using a GeneJET RNA purification kit (Thermo Fisher Scientific). 2. Reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). 3. Quantitative PCR (qPCR) was performed to analyze the expression of specific genes using specific primers designed for use in probe-based detection methods. 4. All expression data were normalized to beta-actin. Relative quantification of fold changes was calculated using the comparative Ct (ΔΔCt) method.

[0283] Oct4 immunostaining of cells demonstrated positive staining of cells within bES colonies (data not shown). Flow cytometry demonstrated that 80% of the cells in the cultures were positive for the characteristic pluripotency marker SSEA4 (the majority of unstained cells were iMEFs, which typically comprise 15% of cells in confluent cultures) (Figure 3A). High expression of the pluripotency markers Nanog, Sall4, Dnmt3b, and Oct4 was demonstrated by quantitative PCR (qPCR) analysis in two cell lines at passage 2 for each (Figure 3B).

[0284] Overall, based on cell morphology, proliferation rate, and pluripotency marker expression, bESC lines Alp 0501 and Alp 0505 are shown to be pluripotent.

[0285] Example 2: Development of feeder-free conditions for bESC proliferation and aggregation Feeder-free growth conditions iMEF feeder layers support the proliferation and pluripotency of bESCs by secreting necessary factors into the culture medium and providing a growth matrix for the cells. However, if the cells are to be used in cultured foods, it is crucial to grow bovine cells without additional cells for simplicity, safety concerns, and consumer acceptance. Coating cell culture vessels with the coating matrix vitronectin and adding activin A to the culture medium successfully replaced the use of iMEF feeder layers. bESCs derived from flushed embryos and grown as described above, but using vitronectin and activin A instead of an iMEF feeder layer, were found to be positive for OCT4 using immunostaining (data not shown) and SSEA4 using flow cytometry (Figure 4).

[0286] Example 3: bESC Differentiation to Mesoderm The PSC aggregates produced according to the teachings of the present invention are intended for use in the cultivated meat industry. Cultivated meat preferably contains a variety of cells, including muscle tissue, including myoblasts, adipocytes, stromal and endothelial cells, all of which are derivatives of the mesodermal lineage. Therefore, the ability of PSCs to differentiate into mesoderm was investigated.

[0287] Early differentiation of bESCs into mesoderm: 1. Cells were harvested and seeded into 6-well plates for aggregate formation as detailed in Example 4 below. 2. The initial conditions were as follows: a. bESCs in growth medium mTeSR1 (StemCell technologies) b. bESCs in growth medium Essential 8 (Thermo Fisher Scientific) 3. Four days after seeding, the medium was changed to form the following conditions. a. The medium of bESC aggregates formed in mTeSR1 was left as is. b. The medium of bESC aggregates formed in Essential 8 medium was replaced with Essential 6 medium (Thermo Fisher Scientific). or c. The medium of bESC aggregates formed in Essential 8 medium was replaced with Essential 6 supplemented with 8 μM CHIR 99021. 4. Cell aggregates were grown for 3 days, after which they were harvested and disaggregated using recombinant trypsin. 5. Cell pellets were used to extract RNA and qPCR was performed as detailed in Example 1 for Oct4 and Brachyury gene expression.

[0288] As shown in Figure 5, replacing the mTeSR1 growth medium of PSCs with E6 essential medium reduced the expression of the pluripotency marker Oct4 in both bESC lines examined (Alp 0501 and Alp 0505). Addition of 8 μM CHIR 99021 further reduced Oct4 expression and increased the early mesoderm marker Brachyury, indicating that the cells had the potential to differentiate into mesoderm.

[0289] Example 4: Production of aggregates of non-genetically modified bovine-derived pluripotent stem cells (PSCs) 1. Seeding Bovine-derived PSCs in Growth Medium to Form Seeded Suspension Cultures and PSC Aggregates bESCs obtained as described in Example 1 above are used to prepare 3D bESC aggregate suspension cultures of the invention in Growth Medium, which is a serum-free liquid medium containing a combination of growth factors.

[0290] 1.1 Cell harvesting and seeding: iPSC clones were grown on iMEF feeder layers in serum-free growth medium supplemented with 50 ng / ml bFGF and 2.5 μM IWR1. After 3–4 days of culture, when colonies reached 70–80% confluence, cells were detached using recombinant trypsin to form single cells by following the steps below. a. Aspirate the medium from the initial monolayer culture in a biological hood. b. Rinse cells with PBS (-Ca / -Mg). c. Add pre-heated recombinant trypsin solution. d. Incubate in a 38.6°C incubator for 3-5 minutes. e. Place the container in a biological hood and add soybean antitrypsin at a ratio of 1:50 (v / v). Gently tap the side of the container with the palm of your hand to facilitate detachment. Add basal medium (for washing - unreplenished) and gently pipette up and down 3-4 times to dissociate the cells into single cells. f. Collect the cells from the container and transfer them to an appropriate tube. Centrifuge the cells, remove the supernatant from the cell pellet, and resuspend in fresh medium. g. Count the cells and culture in an appropriate culture vessel at 0.2–0.5 × 10 6 Seed the cells in 3D suspension at a concentration of 1000 cells / ml (see sections 1.2 and 1.3 below). Add 10 μM Rho-associated protein kinase inhibitor (Rock Inhibitor, Y27632) and 1X Poloxamer 188 solution (Pluronic F-68). h. For reseeding into 2D conditions, iMEFs were seeded at 20–50K cells / cm. 2 Sow at a density of .

[0291] 1.2 Aggregate formation in 6-well plates: Plate 0.3 x 10 bESCs in 3 ml of growth medium per well in ultra-low attachment 6-well plates supplemented with 50 ng / ml bFGF, 2.5 μM IWR1, and 10 μM Rho-associated protein kinase inhibitor. 6Cells were seeded at 1000 cells / ml. Cells were incubated at 38.6°C, 5% CO2, and 80% humidity with a shaking speed of 90-95 rpm for 2-7 days. The medium was changed daily from day 2 of seeding by carefully aspirating 80% of the used medium and replacing it with fresh growth medium containing 10 μM of Rho-associated protein kinase inhibitor. Aggregates were monitored on days 1, 2, and 7 after seeding (Figure 6). One day after seeding, the diameter of the aggregates was 38 ± 22 μm (Figure 6A). After two days of culture, the size of the aggregates increased to 58 ± 36 μm (Figure 6B). Although measurements were not taken on day 7 due to technical difficulties, the aggregates continued to grow to approximately 200 μm (Figure 6C).

[0292] Example 5: Formation of bESC aggregates in a stirred tank bioreactor system After examining the feasibility of bESC aggregation, we investigated growth in a stirred-tank bioreactor system. Stirred-tank bioreactor systems allow for monitoring and control of additional factors, such as pH, dissolved oxygen (DO2), and the connections between various pumps and the feed / harvest media, which are important for long-term and automated processes. bESCs were seeded at an initial concentration of 200,000–500,000 cells / ml in 70–100 ml of growth medium per well, supplemented with 50 ng / ml bFGF, 2.5 μM iWR1, and 10 μM Rho-associated protein kinase inhibitor. The pH was set to 7.0, and DO2 was set to 40–70%. The impeller rotation speed was set to 90–190 rpm. Trials were performed with clone Alp0505. As shown in Figure 7, an average of 104% growth was observed across 12 separate trials. Calculate the aggregation incidence as the percentage of viable cell concentration 24 hours after inoculation relative to the viable cell concentration at the time of seeding (viable / viable%). An incidence of over 100% across multiple attempts indicates that not only did most of the bESCs aggregate, but they also began to proliferate and expand in aggregates within the first 24 hours.

[0293] Example 6: Expansion of bESCs in aggregates in a stirred tank bioreactor system After successfully achieving a rate of aggregation greater than 70%, cell growth in the aggregates was examined. To promote cell growth, the medium was freshly supplemented with 25 ng / ml bFGF (relative to the total working volume), considering that all bFGF from the previous day had been used or heat-inactivated. Cell metabolism was also tracked, and glucose was added if the measured concentration was less than 1 gr / L. These steps were performed manually in a fed-batch culture format. However, once the aggregates exceeded a diameter of 45 μm, the medium was automatically exchanged via a perfusion filter system (typically after 2–3 days of growth). As indicated by an increase in cell concentration, the medium reached 0.2 × 10 6 1.08 x 10 viable cells / ml on day 4 after seeding 6 A 5.4-fold expansion to 1.5x10 viable cells / ml (in 100 ml) was observed in the bioreactor (Figure 8D). In parallel, aggregate diameter increased from 31 μm on day 1 to 56 μm on day 4 (Figures 8A-C, 8E). Pluripotency was analyzed on days 0, 3, and 5 and remained above 70% at all time points (Figure 8D, day 5 data not shown, %SSEA4 = 83%). Initial aggregate size, cell number, and pluripotency after 4 days were approximately 2.5x10 7 Theoretical calculations based on observed fold expansion of cells up to 100-fold cell growth to over 100 cells / ml are predicted as shown in Table 1 herein below. [Table 1]

[0294] Example 7: Reaggregation of small aggregates and single cells According to some embodiments of the present invention, some of the PSC aggregates function as a continuous reservoir for cell growth cycles in large-scale liquid culture conditions via disaggregation and reaggregation procedures. Furthermore, it is crucial for large-scale production of PSC aggregates, particularly bovine-derived PSC aggregates for use in the production of cell-grown meat products, that the entire aggregate-forming procedure, including repeated disaggregation and reaggregation steps, be performed in a closed system. A closed system refers to either performing the disaggregation and reaggregation steps in the same vessel without discarding the aggregate dissociation solution, or, for example, using a cell retention / separation device in a closed system connected to the vessel, forming a loop that allows the dissociation solution to be removed and maintain a sterile environment and an automated process. Cell retention / separation devices are common and can be added to any vessel system ranging from 3 to 2000 L.

[0295] In the initial reaggregation test, small aggregates / single cells obtained from disaggregation of 4- and 7-day-old aggregates replated in fresh medium formed very few very large aggregates. Furthermore, the pluripotency of the cells after such reaggregation was not maintained. In a further reaggregation test, small aggregates / single cells obtained from disaggregation of 3-day-old aggregates (Figure 9A) were replated in medium containing dissociation reagents (protease, DNase, and chelator). Unexpectedly, the presence of the dissociation reagents allowed reaggregation, and the numerous small aggregates that formed (Figure 9B) allowed cell proliferation and expansion of the aggregates.

[0296] Example 8: Isolation of stromal mesenchymal cells from bovine umbilical cord. 1. Bovine umbilical cords (bUC) and placentas were collected from the collection site wrapped in saline and transported to the laboratory at 4°C. Tissue processing began 2–4 h prior to collection. 2. The tissue was placed in a 150 mm Petri dish kept on ice in a biosafety cabinet and rinsed multiple times with ice-cold PBS containing antibiotics (composed of Pen-strep solution (Pen-100u / ml, Strep-0.1mg / ml), Gentamicin solution (5mg / ml), Amphotericin B (0.25mg / ml)) using a needle and syringe to remove blood clots. 3. The bUC (Figure 10A) was isolated and cut longitudinally to completely expose the blood vessels and surrounding Wharton's Jelly (WJ) without disturbing the epithelium. 4. WJs (Figure 10B) were scraped from blood vessels and the inner epithelium and transferred to a Petri dish. The blood vessels were removed. 5. The tissues were cut into 1-2 mm pieces by mechanical shearing and enzymatic dissociation (Figure 10C) (WJ and umbilical cord lining (CL)). 6. WJ tissue fragments were washed four times with PBS containing antibiotics (as described above). 30–40 tissue fragments were plated onto a 150 cm tissue culture dish and either complete medium (composed of DMEM (containing glutamine and glucose), 1% Pen-strep solution, 1% gentamicin solution, and 10% fetal bovine serum) or serum-free medium with or without a cell adhesion matrix was added to a total volume of 20 ml. The cultures were then incubated at 38.6°C for 2–3 days to allow the fragments to adhere. 7. CL tissue fragments were washed four times with PBS containing antibiotics (as described above). The fragments were spread across the plate. Cultures were then incubated at 38.6°C for 2-3 days to allow the fragments to adhere. 8. For all treatments, the complete medium was changed after 2–3 days of incubation. 9. Cells from explants were examined daily for the appearance of proliferation. Cell proliferation was evident from attached explants after 7–10 days of incubation with bovine-derived CL. From this point onward, medium was changed every 3 days or as needed, and cells were expanded using trypsin at 80% confluency (Figure 10D). 10. After 1–3 passages, pool the cells and resuspend them in growth medium containing 10% DMSO at 0.2–1.5 × 10 per cryovial. 6 The cells were frozen as a cell bank at a concentration of 1000 kJ / ml. 11. The cells were thawed at 38.6°C for 1-2 minutes and their viability was counted. Viability upon thawing was over 73%.

[0297] Umbilical cord endothelial cells obtained from both cell banks used possess typical characteristics of mesenchymal stromal cells, with a fibroblast-like morphology and a high proliferation rate at lower passages up to P8 / P9, and they become senescent with greater cytoplasm at P10. PCR analysis of umbilical cord endothelial cells showed that they were positive for NCAD, THY1 / CD90, and NCAM / CD146, typical markers of mesenchymal cells.

[0298] Example 9: Isolation of Bovine Embryonic Fibroblasts (BEF) and Myoblasts (BEM) 1. The hind limbs of bovine embryos were removed from the fetus and transferred to the laboratory in a 50 ml tube containing cold PBS (-Ca / -Mg) + 1% Pen-Strep. 2. In a biological hood, blood vessels, adipose tissue, and connective tissue were removed from the muscle tissue using stainless steel scissors and tweezers. 3. The selected muscle tissue pieces were transferred to a 150 mm dish containing fresh PBS, and the solution was changed twice. 4. All fluid was removed and the selected amount of tissue was cut into a paste-like consistency. 5. The paste was transferred to a 50 ml tube (=tube #1) containing 30 mg of collagenase (type 1, Gibco) in 10 ml of DPBS (-Ca / -Mg) and filtered through a 0.2 μm filter. The 6.50 ml tube was sealed with parafilm and placed horizontally on an orbital shaker (90 RPM) at 38.6°C for 1 hour. 7. Trypsin was heated for 15 minutes before the end of step 5. 8. Tube #1 was centrifuged at 130 RCF for 1.5 minutes, then the upper sup (containing cells + fat) was transferred to a new 50 ml tube (= tube #2). 9.20 ml of pre-warmed trypsin was added to tube #1 (containing the remaining tissue) and placed horizontally on an orbital shaker (90 RPM) at 38.6°C for 20 minutes. 10. In parallel, tube #2 was centrifuged at 285 RCF for 5 minutes (pelleted cells + tissue residues), then the sup was discarded and the pellet was resuspended in 10 ml of standard medium. 11. Once trypsinization was complete (step #8), tube #1 was centrifuged at 285 RFC for 5 minutes. 12. Removed most of the sup from tube #1 (up to the 5ml mark). 13. Standard medium was added to tube #1 up to the 10 ml mark. 14. The tissue pieces were mechanically disrupted by pipetting the entire volume of tube #1 approximately 15 times with a 13 ml pipette. 15. Tube #1 was centrifuged at 130 RCF for 1.5 minutes. 16. The interphase (approximately 5 ml) from tube #1 was collected and transferred to tube #2. 17. Steps #14 to 17 were repeated two more times (3 times total). 18. Finally, cell pool tube #2 was centrifuged at 285 RCF for 5 minutes to pellet all collected cells. 19. The sup was discarded and the cells were resuspended in 20 ml of standard medium. 20. The cell suspension was passed through a 40 μm strainer into 2 × 100 mm dishes (approximately 10 ml each). 21. Both dishes were placed in an incubator at 38.6°C and 5% CO2 for 2 hours. 22. One hour before finishing, 2 × 100 mm dishes were precoated with 0.1% gelatin at room temperature. 23. Two hours after plating the cells, the dish was returned to the hood and as much of the top sup as possible was carefully discarded (containing dead, unattached, floating cells). 24. The dish was tilted slightly to maintain a constant tilt angle. The dish was then gently rinsed with pre-warmed 20% FBS medium and transferred to a 15 ml tube (myoblast pool). Rinsing was repeated at least two more times (a total of three times). 25. Finally, all cells were plated onto freshly coated 100 mm dishes. 26. The cells that remain attached to the uncoated TC plate are the desired fibroblasts.

[0299] Example 10: Cell differentiation into adipocytes The conditions for differentiation of bovine cells into adipocytes required serum-free medium (adipocyte differentiation medium) supplemented with fatty acids and a combination of bFGF 20 ng / μl, IWR1 2.5 μM and Rock inhibitor 10 μM.

[0300] Materials and solutions 1. BEF Cultures up to Passage 6 2. Standard medium (DMEM HG, 10% FBS, 4 mM L-glutamine, 1% Pen-Strep) 3. PBS, (-)Ca +2 , (-)Mg +2 , BI 02-023-1A 4. Serum-free growth medium: ES medium (DMEM / F12 medium, 15% KOSR Gibco, 1% NEAA, 4 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 1% Pen-Strep) 5. Adipocyte Differentiation Medium: 5.1 Serum-free growth medium 5.2 bFGF 20ng / μl 5.3 IWR1 2.5μM 5.4 Rock inhibitor 10 μM

[0301] procedure Day 0: 1.15,000 cells / cm 2 The BEF culture is inoculated into standard medium at a concentration of 0.05%. 2. Incubate overnight at 38.6°C and 5% CO2. Day 1: Discard the medium and add freshly made adipocyte differentiation medium. Days 3-9: From now on, change the adipogenic differentiation medium every other day. The medium must be prepared fresh each time. Days 5-10: Cultures are inspected daily and oil vesicles are expected to become visible. Days 11-30: An increase in adipocyte differentiation products is predicted.

[0302] Fixation and staining of adipocytes: 1. To make the Oil Red O working solution, add 3 parts Oil Red O stock solution to 2 parts dH2O. 2. Mix thoroughly and let stand for 10 minutes. 3. Filter through a 0.2 μm syringe filter or Whatman No. 1 filter paper or equivalent. 4. Add isopropanol (60%) to each well and incubate for 5 minutes. 5. Remove the isopropanol and add Oil Red O working solution to completely and evenly cover the cells (1 ml / well in a 6-well plate). 6. Rotate the plate or dish and incubate for 10-20 minutes. 7. Remove the Oil Red O solution and wash 2-5 times with dH2O as needed until excess staining is no longer visible.

[0303] When exposed to the adipocyte differentiation medium described above, bovine embryonic fibroblast (BEF) cultures demonstrated differentiation into adipocytes. The differentiated cells were stained with Oil Red O, which stains triglycerides and lipids (Figure 11). The conditions for cell differentiation into adipocytes require serum-free medium (adipocyte differentiation medium) supplemented with fatty acids and a specific combination of bFGF 20 ng / μl, IWR1 2.5 μM, and Rock inhibitor 10 μM.

[0304] The appearance of adipocytes began on day 5 after culturing the cells in adipocyte differentiation medium, and adipocyte differentiation increased over time.

[0305] Among other roles, bFGF induces the expression of PPARgamma2, a key transcription factor for adipogenesis. IWR-1 is a WNT signaling inhibitor. WNT signaling suppresses adipogenesis by blocking the induction of peroxisome proliferator-activated receptor gamma (PPARgamma) and CEBPA. Addition of IWR-1 inhibits the WNT pathway, and adipogenesis is induced accordingly. Y-26732, a ROCK inhibitor, enhances cell survival and proliferation.

[0306] Bovine stromal stem cells isolated from adipose tissue were also shown to effectively differentiate into adipocytes using the adipocyte differentiation medium described above.

[0307] Example 11: Reprogramming of bovine cells using episomal plasmids: We reprogrammed bovine induced pluripotent stem cells (biPSCs) using a single episomal plasmid (CoMiP plasmid #63727) encoding human sequences for four standard reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) along with a dTomato red marker, all controlled by a single promoter. Using a plasmid DNA-based reprogramming method dramatically reduces the potential risk of random integration of transfected DNA into the host cell genome, and we found that most human iPSCs generated by plasmid transfection were integration-free (Diecke S 2015, Sci. Rep., Article number: 8081).

[0308] BEF cells at passage 4 (P4) were transfected with 12 μg of CoMiP plasmid by electroporation. To increase cell viability, the CoMiP plasmid was purified using an endotoxin-free Qiagen plasmid miniprep kit.

[0309] Transfected cells were immediately plated onto 10 cm plates coated with 0.1% gelatin in DMEM 10% FBS medium. Transfection efficiency was assessed one day later by measuring red fluorescent protein expression of the dTomato color marker in the CoMiP plasmid. Cells were allowed to recover for four days after electroporation. After recovery, cells were trypsinized and plated onto irradiated mouse embryonic feeder layers (iMEFs). The next day, the cell medium was replaced with serum-free growth medium (also known as "reprogramming medium") containing 2.5 μM human IWR1 and 20 ng / ml human bFGF. 10 μM rock inhibitor was added at the time of plating. The cell medium was refreshed every other day. Embryonic stem cell (ES)-like colonies appeared in the culture two weeks after transfection (Figure 12A).

[0310] Single colonies were manually picked and expanded for cell banking and characterization. iPSCs maintained ES-like morphology even after extended passage. After clonal expansion, iPSCs were adapted to feeder-free growth conditions and demonstrated proliferation on vitronectin in growth medium supplemented with 2.5 μM IWR1 and 50 ng / ml human bFGF (Figure 12B).

[0311] iPSCs were screened for possible plasmid integration events by PCR analysis using primers targeting the junction region between OCT4 and KLF4 in the reprogramming vector and found to be absent (Figure 12C).

[0312] Example 12: Reprogramming of bovine cells using modified mRNA BEF cells were induced to reprogram using RNAs encoding mRNAs for the four canonical reprogramming factors Oct4, Sox2, Klf4, and c-Myc according to the bovine genome sequence together with two of the bovine microRNA cluster sequences encoding miR203b and miR302d.

[0313] To reduce cytotoxicity upon RNA transfection, cells were further transfected with mRNA encoding the B18R vaccinia virus sequence.To assess transfection efficiency and mRNA expression, cells were transfected with modified mRNA encoding the mCherry red marker.

[0314] To obtain enhanced translation and higher stability of our designed mRNA molecules, we used mRNA modifications (Ψ5mC at all bases, CleanCAP AG, 120-nt Poly-A).

[0315] RNA transfection protocol: 1. BEFs were harvested and seeded in DMEM + 10% FBS + 1% Pen-Strep medium (10K cells / cm). 2 ), and incubated overnight at 38.6°C. 2. The medium was changed to pen-strep-free DMEM + 10% FBS and the cells were returned to the incubator. 3. Transfection mix was prepared (per 100K cells): mRNA and microRNA were diluted in 250 μl of Opti-MEM serum-free medium. Transfection reagent was added, the reaction was mixed, and incubated at RT for 5 minutes. 4. The transfection mix was added dropwise directly to the cell culture medium. 5. The cells were incubated overnight at 38.6°C. 6. Transfection was carried out four times over four consecutive days (steps 2–5 × 4). 7. After the final transfection, the medium was changed and the cells were incubated at 38.6°C for 2-3 days to recover. 8. The cells were harvested and their viability was counted. 9.10 6 Cells were set aside for RNA isolation and qPCR analysis of OCT4 mRNA expression levels. 10. Plate the cells at 5K-10K cells / cm on inactivated mouse embryonic feeder layers (iMEFs) in DMEM + 10% FBS + 1% Pen-Strep medium. 2 The cells were seeded at 1000 x g for 1 hour. Cell pellets were also collected. 11. Cells were incubated overnight at 38.6°C. 12. The cell culture medium was aspirated and replaced with growth medium supplemented with 2.5 μM human IWR, 20 ng / ml human bFGF and 10 μM rock inhibitor. 13. The cell medium was refreshed every other day for 3 weeks and the cultures were screened for the appearance of ES-like colonies.

[0316] To assess transfection efficiency, cells were analyzed for both mCherry and OCT4 expression. Twenty-four hours after each transfection, mCherry expression was assessed using a fluorescent microscope (Figure 13A, fluorescent filter; 13B, bright field). After the fourth transfection, cells were allowed to recover over the weekend, then harvested using trypsin, and cell pellets were taken for qPCR analysis, which showed high transcription levels of OCT4 in BEFs induced by mRNA and miRNA (iBEF) reprogramming (Figure 13C).

[0317] The foregoing descriptions of specific embodiments fully reveal the general nature of the present invention, so that others, by applying their current knowledge, can easily modify and / or adapt such specific embodiments to various uses without undue experimentation and without departing from the general concept; therefore, such adaptations and modifications should be understood within the meaning and range of equivalents of the embodiments of the present disclosure, and are so intended. It should be understood that the expressions or terminology used herein are for purposes of description and not limitation. The means, materials, and steps for carrying out the various functions of the present disclosure may take a variety of alternative forms without departing from the invention.

Claims

1. A method for producing aggregates of pluripotent stem cells (PSCs) derived from a non-human animal, comprising: a. seeding at least one PSC in a growth medium to form a suspension culture, the growth medium being a serum-free liquid medium comprising a combination of the growth factor bFGF and at least one of (i) a protein of the TGF-β superfamily, which is activin-A, and (ii) an inhibitor of the Wnt-β-catenin signaling pathway, which is IWR1; b. growing the suspension culture under conditions that permit aggregate formation and aggregate growth, thereby forming homogenous aggregates of the PSCs; The non-human animal is a bovine, the genome of the PSCs is not genetically modified, and inactivated mouse embryonic fiber (iMEF) is used at each step. A method characterized in that no feeder layer (blast cell) is used.

2. 2. The method of claim 1, wherein step (b) of growing the suspension culture comprises performing at least one of the following steps: (i) disaggregating the formed homogeneous aggregates into smaller aggregates and / or single cells, and (ii) re-aggregating the smaller aggregates and / or single cells to reform homogeneous aggregates.

3. 3. The method of claim 1 or 2, wherein the method is carried out in a closed system.

4. The method of any one of claims 1 to 3, wherein the growth medium comprises a combination comprising bFGF and an inhibitor of the Wnt-β-catenin signaling pathway.

5. 5. The method of any one of claims 1 to 4, wherein the step (a) of seeding the PSCs further comprises encapsulating the at least one PSC within a particle and seeding a plurality of the particles in the growth medium, the particles having an outer shell layer composed of at least one hydrogel selected from the group consisting of alginate, gellan gum, agar, agarose, chitosan, hyaluronic acid, curdlan, carrageenan, pectin, modified starch, and combinations thereof.

6. 6. The method of any one of claims 1 to 5, wherein the suspension culture is grown at a temperature that is the body temperature of the non-human animal species from which the PSCs are derived.

7. 7. The method of any one of claims 1 to 6, wherein the PSC aggregates formed have an average diameter of about 50 μm to about 500 μm.

8. The non-human animal is a bovine, and the bovine PSCs are i. obtaining at least one early bovine embryo; ii. Culturing the at least one early embryo to reach the blastocyst or enhanced blastocyst stage; iii. Obtaining at least one cell from the blastocyst; iv. The method of any one of claims 1 to 7, wherein the embryonic stem cells are produced by a method comprising the step of culturing the at least one cell in a culture medium comprising a combination of the growth factor bFGF and at least one of: (i) at least one additional growth factor that is a protein of the TGF-β superfamily selected from the group consisting of TGF-β-1, TGF-β-3, and activin-A; and (ii) an inhibitor of the Wnt-β-catenin signaling pathway.

9. The PSC is a. at least one reprogramming mRNA encoding a reprogramming factor; b. at least one double-stranded microRNA, or c. at least one inhibitor of at least one microRNA endogenous to the non-human animal-derived cell into at least one cell derived from a non-human animal, thereby producing at least one artificial PSC (iPSC), wherein the at least one reprogramming mRNA and the at least one double-stranded microRNA are not integrated into the genome of the iPSC.

10. 10. The method of claim 9, wherein the method further comprises introducing at least one immune escape mRNA into the at least one non-human animal-derived cell.

11. 11. The method of claim 9 or 10, wherein introducing the at least one reprogramming mRNA, and / or the at least one immune evasion mRNA and / or the at least one double-stranded microRNA and / or the at least one miRNA inhibitor is performed in a serum-free liquid growth medium.

12. The method of any one of claims 1 to 11, wherein the homogenous aggregates comprise at least 70% of cells that express at least one pluripotency marker.

13. A plurality of homogenous aggregates comprising at least 70% viable PSCs derived from a non-human animal, wherein the genomes of the PSCs are not genetically modified, and wherein the aggregates are produced by the method of any one of claims 1 to 12.

14. 14. A suspension comprising a serum-free liquid medium and a plurality of the homogeneous aggregates of claim 13.

15. the non-genetically modified, non-human animal-derived PSCs divide approximately every 16 to 32 hours; the non-genetically modified, non-human animal-derived PSCs express at least one pluripotency marker; and the non-genetically modified, non-human animal-derived PSCs express at least one surface protein that contributes to cell-cell adhesion; 15. The suspension of claim 14, wherein at least one of:

16. 16. Cell-grown meat comprising the plurality of homogenous aggregates of PSCs or progeny thereof derived from a non-human animal according to claim 13, or a suspension comprising the same according to claim 14 or 15, wherein the genomes of the PSCs have not been genetically modified.

17. 17. The cell-grown meat product of claim 16, wherein the progeny comprise cells that differentiate from the PSCs to form at least one of muscle cells, stromal cells, endothelial cells, and adipocytes.

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