WO2023018995A1 - Animal cell line and process development of cultivated meat products - Google Patents

Animal cell line and process development of cultivated meat products Download PDF

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Publication number
WO2023018995A1
WO2023018995A1 PCT/US2022/040263 US2022040263W WO2023018995A1 WO 2023018995 A1 WO2023018995 A1 WO 2023018995A1 US 2022040263 W US2022040263 W US 2022040263W WO 2023018995 A1 WO2023018995 A1 WO 2023018995A1
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cell
acid
cells
gene
pten
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PCT/US2022/040263
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French (fr)
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Jessica KRIEGER
Narendra BHARATHY ELANGOVAN
Syed Mukhtar AHMED
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Ohayo Valley, Inc
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Publication of WO2023018995A1 publication Critical patent/WO2023018995A1/en

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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
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Definitions

  • the object of the invention is to provide new materials, methods, techniques, cell lines, and strategies to produce and to improve flavor and pigmentation of cell-based meat.
  • DESCRIPTION OF RELATED ART [00002]
  • Cell-based meat is meat grown from isolated animal cells, instead of using the entire animal to produce meat products. The isolated cells are grown in cell culture and are used to develop cell lines that can be grown in bioreactors to produce animal free meat products. Cell- based meat has been called many things, such as cultured meat, cultivated meat, lab-grown meat, and in vitro meat.
  • the present invention provides materials, methods, techniques, cell lines, and strategies to produce cell-based meat, and to improve flavor and pigmentation of cell-based meat, via myogenesis, adipogenesis, increasing the mitotic capacity of cell lines, cell line immortalization, developing anoikis resistance of cell lines, and free fatty acid supplementation.
  • the cells obtained from an animal possess inherent limitations that make them unsuitable for a large-scale and indefinite manufacturing process. Cell lines developed with increased mitotic capacity, however, may be scaled up making the cultivation of these cell lines more amenable to the cultivated meat manufacturing process.
  • the present invention provides a transgenic expression construct comprising a gene or genes selected from the group consisting of ILK, GLUT4, PDK-1, TBX2, Pax3, and/or telomerase, and a constitutively active promoter overexpressing said gene, when compared to the expression the wild-type promoter of said gene, wherein the transgene is inserted into the native genome of the cell.
  • the present invention also provides a method comprising editing the nucleic acid base pair sequences of native promoters of ILK, GLUT4, PDK-1, TBX2, Pax3, and/or telomerase genes in order to increase gene transcription over gene transcription levels found in cells with unmodified promoters.
  • the present invention provides a method for increasing mitotic capacity of cell lines by overexpressing ILK, GLUT4, PDK-1, TBX2, Pax3, telomerase and/or knocking out PTEN expression.
  • Cell culture media often includes the use of expensive growth factors to support cell survival, proliferation, and differentiation, making large scale cell-line cultivation cost- prohibitive. Constitutive activation of the ERK1/2 pathway and insulin-independent glucose transport through activation of the PI3K/Akt pathway can enhance cell proliferation and reduce the amount of growth factor required, thereby lowering the cost of cultivating edible cell lines.
  • the present invention provides a composition comprising a genetically modified cell, wherein a genetic modification comprises mutations to the B-Raf gene, and wherein the modification results in enhanced ERK signaling in comparison to the wild type B- Raf.
  • the genetic modification may be a substitution, deletion, insertion, duplication, inversion, or frameshift mutation.
  • reducing or eliminating insulin from cell culture media may significantly reduce cost of production.
  • the present invention provides a composition comprising a genetically modified cell, wherein a genetic modification comprises modifications to the insulin receptor (INSR) gene.
  • the genetic modification may be a substitution, deletion, insertion, duplication, inversion, or frameshift mutation.
  • Genetically modified cells may contain artificial plasmids (“Synthetic Plasmids”) to amplify or produce many copies of gene of interest (GOI).
  • Cell lines must be able to undergo myogenesis and adipogenesis. The capacity of a cell line to undergo myogenesis and adipogenesis affects meat quality.
  • the present invention provides a method of improving cell differentiation comprising supplementing adipocyte cultures with fatty acids in any combination of monounsaturated fatty acids (MUFA); polyunsaturated fatty acids (PUFA); and saturated fatty acids (SFA).
  • MUFA monounsaturated fatty acids
  • PUFA polyunsaturated fatty acids
  • SFA saturated fatty acids
  • a PUFA, MUFA, or SFA may be added alone, or any combination of PUFAs, MUFAs and SFAs may be added.
  • the MUFA may be oleic acid.
  • the method may further comprise supplementing adipocyte cell cultures with bovine preadipocytes sourced from the subcutaneous stromal vascular cells treated with Oleic acid.
  • the PUFA may include ⁇ -Linolenic acid, Stearidonic acid, Eicosatetraenoic acid, Eicosapentaenoic acid, Docosapentaenoic acid, Docosahexaenoic acid, Linoleic acid, Gamma-linolenic acid, Calendic acid, Dihomo-gamma-linolenic acid, Arachidonic acid, Adrenic acid, and Mead acid.
  • the MUFA may include Oleic acid, Palmitoleic acid, Paullinic acid, Omega-9 FAs, elaidic acid, Gondoic acid, Erucic acid, and Nervonic acid.
  • the SFA may include Myristic acid, Palmitic acid, Stearic acid, Arachidic acid, Behenic acid, Lignoceric acid, and Cerotic acid.
  • the present invention provides a method of improving flavor of cell-based fat comprising supplementing adipocyte cultures with a fatty acid, wherein the fatty acid is polyunsaturated fatty acids (PUFA), monounsaturated fatty acids (MUFA), and/or saturated fatty acids (SFA).
  • PUFA polyunsaturated fatty acids
  • MUFA monounsaturated fatty acids
  • SFA saturated fatty acids
  • a PUFA, MUFA, or SFA may be added alone, or any combination of PUFAs, MUFAs and SFAs may be added.
  • the MUFA may include Palmitoleic acid, Paullinic acid, Omega-9 FAs, elaidic acid, Gondoic acid, Erucic acid, and Nervonic acid.
  • the SFA may include Myristic acid, Palmitic acid, Stearic acid, Arachidic acid, Behenic acid, Lignoceric acid, and Cerotic acid.
  • the present invention provides a method of transdifferentiating myogenic cells into adipogenic cells comprising supplementing the cell culture media with SFAs, MUFAs, and/or PUFAs.
  • the present invention provides a composition comprising a genetically modified bovine or porcine myoblast and a genetically modified bovine or porcine adipoblast.
  • the present invention provides a composition comprising myoblasts, mesenchymal stem cells (MSCs), intramuscular fibroblasts, iPSCs, adipoblasts, adipocytes, preadipocytes, and/or fibro-adipoblasts.
  • MSCs mesenchymal stem cells
  • iPSCs intramuscular fibroblasts
  • adipoblasts adipocytes
  • preadipocytes preadipocytes
  • fibro-adipoblasts fibro-adipoblasts.
  • the present invention provides a cell-based meat product comprising genetically modified fat cells and genetically modified muscle cells.
  • the present invention provides a method of preventing anoikis comprising generation of a cell line with constitutively overexpressed ILK, PDK-1, TBX2, and/or PAX3.
  • the invention also provides a method of preventing anoikis comprising generation of a cell line with a mutated form of B-Raf that has higher activity levels than wild type B-Raf.
  • the invention also provides a method of preventing anoikis comprising generation of a cell line with PTEN gene knocked out.
  • the present invention provides a method of reducing or eliminating insulin from cell culture media comprising truncating the alpha subunits in an INSR gene to produce an exon-free insulin receptor.
  • the present invention also provides a method of reducing or eliminating insulin from cell culture media comprising overexpressing PDK-1, GLUT4, TBX2, and/or PAX3.
  • the present invention provides a method of reducing or eliminating insulin from cell culture media comprising knocking out PTEN.
  • the present invention provides a method for bypassing the hayflick limit comprising overexpressing telomerase.
  • Immortalized cell lines with anoikis resistance, insulin-independent insulin signaling, and mitogen-independent cell cycle progression enhances flavor and sensory properties of edible cell lines and lowers the cost of production.
  • the present invention provides a method for mitogen-independent cell cycle progression comprising generation of a cell line with a mutated form of B-Raf that has higher activity levels than wild type B-Raf.
  • Flavor, pigment, and product quality can be enhanced by constitutive activation of the insulin/GLUT4 pathway, which drives myogenic and adipogenic differentiation. Whereas inactivation of PTEN results in activation of the PI3K/AKT pathway and subsequent increase in cell cycle progression, migration and survival. And overexpressing telomerase helps the cells bypass the hayflick limit by maintaining telomere length through repeated cell divisions, which is necessary to develop scalable cell lines.
  • the present invention provides a cell line wherein the cell line bypasses the hayflick limit via telomerase overexpression; is resistant to anoikis via activation of integrin binding signal transduction via PTEN knockout with increased PIP3, PIP3/PDK-1/pAkt mediated anti-apoptotic signaling, ILK/pAkt mediated anti-apoptotic signaling, ERK1/2 anti-apoptotic signaling; provides insulin-independent glucose transport via a method selected from the group consisting of PIP3- mediated phosphorylation of aPKCs, PDK-1-mediated phosphorylation of aPKCs, pAkt- mediated AS160 inhibition, constitutive insulin receptor activation and GLUT4 overexpression; provides mitogen-independent cell cycle progression via ERK1/2-mediated cell cycle progression; and which has enhanced sensory properties via a method selected from the group consisting of PIP3-mediated phosphorylation of aPKCs, PDK-1-mediated phosphorylation of aPKC
  • Generating a constitutively active insulin receptor pathway provides sensory benefits to myogenic and adipogenic cell lines that can improve flavor, pigment, and overall product quality.
  • the present invention provides methods for improving cell resistance to anoikis and enhancing mitogen independent enhanced proliferation and/or mitotic potential comprising overexpressing of wild type or mutant FAK and/or SRC.
  • the present invention provides methods for insulin- independent glucose transport into the cell comprising overexpressing wild type or mutant GLUT1, GLUT2, and GLUT3.
  • the present invention also includes methods for enhancing the sensory profile of cells comprising overexpressing wild type or mutant GLUT1, GLUT2, and GLUT3.
  • the present invention also includes methods for overexpressing various cell targets via viral based methods comprising transduction and/or inducible gene expression approaches.
  • Inactivation of PTEN results in activation of the PI3K/AKT pathway and subsequently leads to an increase in cell cycle progression, migration and survival.
  • the present invention provides methods for reducing PTEN activity comprising siRNA and/or shRNA mediated knockdown of PTEN protein translation, miRNA-mediated transcriptional interference of PTEN expression, and overexpression of dominant negative form of PTEN, which may contain one or more inactivating mutations.
  • FIG.1 shows Japanese Meat Grading Association [Japan Meat Grading Association (JMGA) Beef Carcass Trading Standards. (2014)] and USDA meat quality standards [Beef Grading Shields. USDA.gov https://www.ams.usda.gov/grades-standards/beef/shields-and- marbling-pictures.].
  • FIG.2 shows cell viability (mitotic capacity) of primary bovine myoblasts as indicated by RLU (Relative Luminescence Units).
  • FIG.2A shows viability for cells transiently transfected to over-express ILK or PDK-1(p ⁇ 0.01), alone or in combination with TERT (p ⁇ 0.02) .
  • FIG.2B shows viability of cells that have undergone lentiviral mediated over-expression of TERT, PAX3, or TBX2, alone or in combination with TERT as compared to untransduced or EGFP control cells (p ⁇ 0.05).
  • FIGs.2C and 2D show proliferation of cells genetically modified to over-express TERT, PAX3, or TBX2 as compared to GFP control cells (p ⁇ 0.05). Error bars represent means with SD; [00023]
  • FIG. 3 shows viability of cells having a combination of siRNA specific for PTEN or TERT alone as compared to the siScrambled control. Error bars represent means with SD; [00024] FIG.
  • FIG. 4A depicts the Sanger sequence for inducible-BRAF-V600E knock-in.
  • FIG. 4B depicts the Sanger sequence for INSR mutant knock-in;
  • FIG. 5 shows cell line anoikis resistance in primary bovine myoblasts or C2C12 cells as indicated by absorbance.
  • FIG 5A shows anoikis resistance for cells transiently transfected to over-express ILK or PDK-1, alone or in combination with TERT.
  • FIG.5B shows anoikis resistance of cells that have undergone lentiviral mediated over-expression of TERT, PAX3, or TBX2, alone or in combination with TERT as compared to untransduced or EGFP control cells.
  • FIG.5C shows anoikis resistance in C2C12 cells genetically modified via CRISPR to over-express B-RAF or SRC compared to wild type.
  • FIG.5D shows anoikis resistance in cells having a combination of siRNA specific for PTEN or TERT alone as compared to the siScrambled control. Error bars represent means with SD; [00026]
  • FIG. 6 shows viability of C2C12 cells with INSR KI alone or in combination with GLUT1-4 in varying serum free environment. Error bars represent means with SD; [00027]
  • FIG. 7 depicts a serum curve and shows viability of C2C12 cells with INSR KI alone or in combination with GLUT1-4 in varying insulin conditions.
  • FIG. 8 shows cell viability of primary bovine myoblasts genetically modified to over-express ILK or PDK-1, alone or in combination with TERT as indicated by RLU (Relative Luminescence Units).
  • FIG 8A shows viability of cells in the absence of FBS (fetal bovine serum)
  • FIG.8B shows cell viability for cells cultured in 5% FBS (fetal bovine serum)
  • FIG.8C shows cell viability for cells cultured in 10% FBS
  • FIG.8D shows cell viability for cells cultured in 20% FBS.
  • Error bars represent means with SD
  • FIG. 9 shows cell viability/mitotic capacity for cells genetically modified to over- express SRC or BRAF.
  • FIG. 10 is an example of GFP tagged cells
  • FIG. 11 shows cell viability for cells genetically modified to over-express C-JUN or FOS in combination with TERT.
  • FIG.11A shows cell viability in the presence of Laminin
  • FIG.11B shows cell viability in the absence of Laminin;
  • FIG. 12 shows bovine fibroblasts cultured in regular PromoCell media alone or in combination with FFAs and bovine myoblasts transdifferentiation when cultured in PromoCell with siRNA targeting MyoD (Horizon Discovery, Waterbeach, UK) or in combination with FFAs and FBS.
  • the present invention provides methods of creating cell-based meat and increasing palatability of cell-based meat, as well as cell-based meat products.
  • the present invention provides strategies for producing immortalized cells lines and anoikis resistance for cultivated meat products.
  • the present invention also provides strategies for reducing the use of insulin in cell culture media.
  • the present invention also provides methods for improving sensory properties of cultivated meat, including use of free fatty acids, and constitutive activation of the insulin/GLUT4 pathway.
  • the present invention provides cell-based meat products and methods with one or more of the following properties: [00035] Property 1 – bypassing the hayflick limit of cell lines through telomerase overexpression. [00036] Property 2 – anoikis-resistance by activating intracellular integrin binding signal transduction pathways. This can be accomplished via PTEN knockout with increased PIP3 signaling, PIP3/PDK-1/pAkt mediated anti-apoptotic signaling, ILK/pAkt mediated anti- apoptotic signaling, and/or ERK1/2 anti-apoptotic signaling. [00037] Property 3 — insulin-independent glucose transport into the cell.
  • Property 6 bypassing the hayflick limit of cell lines through telomerase overexpression and knockdown/knockout of PTEN expression or over expression of dominant negative form/miRNA targeting PTEN for degradation.
  • Property 7 anoikis-resistance by activating intracellular integrin binding signal transduction pathways. This can be accomplished via PTEN knockdown/knockdown or over expression of dominant negative form/miRNA.with increased PIP3 signaling, FAK mediated anti- apoptotic signaling, SRC mediated anti- apoptotic signaling.
  • Property 8 insulin-independent glucose transport into the cell. This can be accomplished through GLUT1, GLUT2, GLU3, and GLUT4 overexpression.
  • Beef color standard assesses meat color and brightness.
  • Beef fat standard measures color, luster and quality of fat.
  • Animal meat is a staple of Western diets. Beef and pork are two of the most popular types of meat in western diets, and other animals such as goat, deer, rabbit, and sheep are consumed as well. Animal meat has specific sensory properties associated with its appearance and taste. The sensory properties of the meat arise from the muscle and fat cells that comprise the meat. Animal meat contains skeletal muscle tissue with intramuscular fat that are created from myogenesis and adipogenesis. Natural myogenic and adipogenic mechanisms in the animal’s body produce optimized sensory experiences upon consumption of animal meat, but cell-based meat is produced in an artificial environment.
  • Cells used for cell cultured meat can be derived from various kinds of stem or precursor cells found in animal embryos, bone marrow, or muscle tissue. Induced pluripotent stem cells (iPSCs) can also be used, which, along with embryonic stem cells, can be differentiated into any cell type in the body, such as skeletal muscle cells. Regardless of the initial cell population, the manufacturing process must result in the production of cells (e.g., muscle and fat) found in animal meat.
  • iPSCs Induced pluripotent stem cells
  • the three dominant cell types that influence meat flavor, juiciness, and texture are skeletal muscle cells, intramuscular fat cells, and connective tissue cells called fibroblasts.
  • Cell lines must be able to undergo myogenesis (the formation of multinucleated, contractile skeletal muscle cells) and adipogenesis (the development of fat cells containing intracellular lipid droplets). The capacity of a cell line to undergo myogenesis and adipogenesis affects meat quality.
  • myogenesis the formation of multinucleated, contractile skeletal muscle cells
  • adipogenesis the development of fat cells containing intracellular lipid droplets.
  • the capacity of a cell line to undergo myogenesis and adipogenesis affects meat quality.
  • Myogenesis Myogenesis
  • Skeletal muscle cells are the main source of the umami flavor in meat that contains muscle-specific proteins. These cells contribute to meat flavor and pigment.
  • Myogenesis begins during embryogenesis and is characterized by mononuclear muscle progenitor cells fusing into multinucleated muscle fibers. This results in the production of high-density skeletal muscle cytoskeletal proteins, myosin heavy chain, and myoglobin, which serves as an oxygen reservoir for cells. Myoglobin is red in hue and its pigmentation arises from various redox forms and concentration within skeletal muscle cells. Myogenesis is controlled by sequential expression of myogenic transcription factors and begins with the activation of quiescent Pax7 + expressing satellite cells, a muscle stem cell. In their activated form, Pax7 + MyoD + Myf5 + satellite cells undergo asymmetric division to produce self-renewing satellite cells and myoblasts committed to undergo myogenesis.
  • Pax7 + MyoD + Myf5 + myoblasts undergo symmetric division to expand in numbers, then commit to terminal differentiation as Pax7-MyoD-Myf5- MyoD + Myogenin + mononuclear myocytes committed to exit the cell cycle and terminally differentiate.
  • These mononuclear cells begin to fuse into multinucleated muscle fibers, or myotubes, and develop sarcomeres, which are the contractile unit of the cell.
  • Sarcomeres are composed of myosin heavy chain and actin filaments that consume ATP (adenine triphosphate) to slide against one another to shorten the length of the sarcomere and consequently the length of the muscle fiber.
  • Adipogenesis regulates energy metabolism in the body by collecting free fatty acids (FFAs) bound to albumin from the blood into adipose cells for future energy use and releasing them back into the blood when glucose levels are low.
  • FFAs free fatty acids
  • Preadipocytes undergo differentiation into adipocytes during adipogenesis.
  • Adipocytes accumulate intracellular lipid droplets that provide juiciness and additional flavor to meat through lipogenesis.
  • Adipogenesis initiates in intramuscular preadipocytes with growth arrest and morphological changes that shifts the cells from a fibroblastic spindle shape to a rounded morphology.
  • Preadipocytes start producing CCAATT enhancer binding proteins ⁇ (C/EBP ⁇ ) and CCAATT enhancer binding proteins ⁇ (C/EBP ⁇ ) transcription factors, which activate gene transcription of peroxisome proliferator-activated receptor ⁇ (PPAR ⁇ ) and CCAATT enhancer binding proteins ⁇ (C/EBP ⁇ ) transcription factors.
  • PPAR ⁇ is a master regulator of adipogenesis and a ligand-activated nuclear membrane receptor protein that can translocate into the nucleus to activate gene expression.
  • PPAR ⁇ and C/EBP ⁇ begin transcribing genes that promote insulin sensitization, glucose utilization, and adipocyte maturation, such as insulin receptor; fatty acid synthase; adipocyte protein 2 (aP2), also known as fatty acid binding protein 4 (FABP4); and glucose transporter type 4 (GLUT 4). These proteins facilitate transportation of FFAs and glucose into the preadipocyte cytosol and promote intracellular lipogenesis.
  • Adipocyte protein 2 also known as fatty acid binding protein 4 (FABP4)
  • FFABP4 fatty acid binding protein 4
  • GLUT 4 glucose transporter type 4
  • Lipogenesis encompasses de novo fatty acid synthesis, the production of FFAs, and triglyceride synthesis, which esterifies FFAs to glycerol to produce triglycerides that incorporate into lipid droplets. Lipolysis occurs when energy levels enter a fasting state.
  • FFAs are enzymatically cleaved from intracellular triglycerides and transported to the blood.
  • Cells in need of energy uptake the FFAs for ⁇ oxidation in the mitochondria to produce ATP.
  • Fasting activates transcription of peroxisome proliferator-activated receptor alpha (PPAR ⁇ ) in the liver, a transcription factor that activates a set of genes involved in fatty acid oxidation.
  • PPAR- ⁇ / ⁇ enhances fatty acid oxidation in skeletal muscle and adipose tissue.
  • PPAR- ⁇ / ⁇ ligands include several 14- to 18-carbon saturated fatty acids as well as 16- to 20-carbon polyunsaturated fatty acids.
  • Adipogenesis can be initiated in vitro by media components that produce metabolic changes in preadipocytes.
  • FBS high glucose
  • insulin dexamethasone
  • IBMX 3- isobutyl-1-methylxanthine
  • thiazolidinediones thiazolidinediones
  • FFAs may be used to induce adipogenesis in preadipocytes.
  • Dexamethasone is an anti-inflammatory corticosteroid used in many medical applications.
  • IBMX is a small chemical compound which together with dexamethasone activates expression of C/EBP ⁇ and C/EBP ⁇ .
  • TZDs are ligands that activate PPAR ⁇ to initiate downstream adipogenic gene transcription.
  • Insulin, glucose, and free fatty acids contribute to intracellular lipogenesis by mediating cellular metabolism.
  • Insulin binds to insulin receptors that activate intracellular signaling pathways to recruit the GLUT 4 transport protein to the plasma membrane, allowing glucose to enter into the cell.
  • Glucose converts to Acetyl-CoA via the glycolytic pathway, which is used to produce fatty acids during de novo lipogenesis.
  • FFAs such as erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid can be transported through the cell membrane and solubilized into the cytosol via aP2.
  • PPAR ⁇ is also activated via FFA binding.
  • telomere shortening begins with extracting individual cells from a tissue biopsy of an animal.
  • the cells obtained from an animal possess inherent limitations that make them unsuitable for a large-scale and indefinite manufacturing process. Aging occurs at the cellular level, and the harvested cells isolated from an animal will also age during culture. The aging process depletes the proliferative capacity of the cells by shortening telomeres through each cell division. Once the telomeres have shortened to their final length, they are no longer able to divide, and the cells undergo senescence. The limited number of cell divisions due to telomere shortening is called the Hayflick limit.
  • the Hayflick limit is a threshold that limits the biomass that can be obtained from primary animal cells. Culturing meat necessitates extending the ability of edible cells to undergo cycles of mitosis by extending or removing a limit to the total number of cell divisions. Engineered cell lines can extend the Hayflick limit and allow more cell divisions while still being subject to the limit. Immortalized cell lines completely bypass cellular aging to allow infinite expansion, like HeLa cells and C2C12s. [00060] For a given cell population, the mitotic capacity is the potential for the cell to undergo mitosis: the ratio of the change in mitotic index over change in time.
  • IMC Innate mitotic capacity
  • EMC Enhanced Mitotic Capacity
  • UMC Unlimited mitotic capacity
  • Immortal cell lines can be created through genetically modifying cells; by selecting a cell type for expansion with naturally enhanced or indefinite proliferation potential, such as stem cells; or depending on spontaneous immortalization of cells through natural genetic mutations that occur during serial cell culture.
  • Cell lines may be stored in a master cell bank, where they are cryopreserved in a state of suspended activity until they are needed. Cryopreserved cells may be thawed and re-animated and expanded in bioreactors.
  • taste and appearance factors [00062] Presentation and sensory characteristics of cell-based meat products may be optimized by identifying which characteristics define an animal meat product and enhancing those characteristics in the cell-based meat product. For example, expression of a muscle-specific protein, myoglobin, that is known to improve meat color and perceived quality, may be increased. Cultured cells are characterized by their color to demonstrate the need for focusing on myoglobin, where myogenic cells in culture are white in color.
  • Myoglobin [00064] Visual perception of red meat quality is determined from myoglobin pigmentation. Myoglobin is a ⁇ 17 kDa cytoplasmic hemoprotein encoded by the MB (myoglobin) gene. It possesses a single heme group, where hemoglobin contains four heme groups. Myoglobin reversibly binds to O2 via the heme group and serves as an oxygen storage system for oxidative phosphorylation.
  • the heme group in myoglobin provides a red pigment to meat, depending on the oxidation state of the Fe + ion, which affiliates with O251.
  • OMb oxymyoglobin
  • DMb deoxymyoglobin
  • MMb metmyoglobin
  • O2 is bound to Fe +2 and OMb is generated, which produces a bright red color.
  • DMb is formed and the heme ring is purplish in color.
  • the present invention provides methods of supplementing with free fatty acids to drive flavor and pigmentation of cultivated meat.
  • the fatty acid (FA) composition of intramuscular fat impacts flavor, juiciness, and tenderness.
  • This composition includes saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs).
  • SFAs like myristic acid (14:0), palmitic acid (16:0), and stearic acid (18:0) are composed of carbon chains that lack double bonds;
  • MUFAs like oleic acid (18:1) and palmitoleic acid (16:1) contain one carbon double bond; and PUFAs have two or more carbon double bonds. Double bonds in fatty acids hinder the formation of the crystal structure of solidified fat. Consequently, every double bond lowers the melting point of the fatty acid. The melting point of lipids greatly influences the juiciness of beef.
  • SFAs have melting points at ⁇ 70 oC, MUFAs melt at ⁇ 20 oC, and PUFAs a liquid at room temperature and melt at -20 oC.
  • High SFA levels can be produced from high calorie feed and is associated with meat toughness and flavor liking.16:0 and 18:0 are two FAs that determine meat hardness due to their high melting point. Lowering the total percentages of these SFAs will soften meat products.
  • Oleic acid is associated with beef palatability due to its lower melting point, which improves tenderness, juiciness, and flavor.
  • High oleic acid levels are found in animals with intense marbling, such as Wagyu and Hanwoo, which can be increased through high calorie feed.
  • PUFAs have the lowest melting point of the FAs. PUFAs contribute minimally to IMF due to their slower deposition rate than SFAs and MUFAs, but have nutritional importance.
  • Linoleic acid (18:2 n-6), also called omega-6, and linolenic acid (18:3 n-3), or omega-3, are essential PUFAs present in animal meat.18:2 is produced in higher concentration with high calorie feeding and is positively correlated with flavor liking, while 18:3 is increased in animals with grass diets and is negatively correlated with liking. A high C18:2/C18:3 ratio is positively correlated with flavor liking and overall liking. [00070] Cell-based meat can be supplemented with fatty acids in order to mimic the fatty acid composition of a naturally meat from an animal.
  • Fatty acids that may be used include, but are not limited to saturated fatty acids (SFAs), such as myristic acid (14:0), palmitic acid (16:0), and stearic acid (18:0), monounsaturated fatty acids (MUFAs), such as oleic acid (18:1) and palmitoleic acid (16:1), and polyunsaturated fatty acids (PUFAs).
  • SFAs saturated fatty acids
  • MUFAs monounsaturated fatty acids
  • PUFAs polyunsaturated fatty acids
  • Fatty acids are added to cell culture media during the adipogenic differentiation to generated cultivated fat cells.
  • FFAs should be supplemented in the culture media at concentrations that result in a fatty acid composition of the IMF of the respective type of meat.
  • any FFA can be used in any combination with other FFAs at concentrations between 1-1000nM, 1-1000 ⁇ M, or 1-1000mM to promote adipogenic differentiation of cultivated cells.
  • SFAs include Myristic acid (14:0), Palmitic acid (16:0), Stearic acid (18:0), Arachidic acid (20:0), Behenic acid (22:0), Lignoceric acid (24:0), and Cerotic acid (26:0).
  • PUFAs include omega-3, -6, and -9 fatty acids.
  • Omega-3 FAs include ⁇ -Linolenic acid (ALA, (18:3(n-3)), Stearidonic acid (SDA, (18:4 (n ⁇ 3)), Eicosatetraenoic acid (ETA, (20:4 (n ⁇ 3)), Eicosapentaenoic acid (EPA, (20:5(n-3)), Docosapentaenoic acid (DPA, (22:5 (n ⁇ 3)), and Docosahexaenoic acid (DHA, (22:6 (n ⁇ 3)).
  • ALA (18:3(n-3)
  • SDA Stearidonic acid
  • ETA Eicosatetraenoic acid
  • EPA Eicosapentaenoic acid
  • DPA Docosapentaenoic acid
  • DHA Docosahexaenoic acid
  • Omega-6 FAs include: Linoleic acid (LA, (18:2 (n ⁇ 6)), Gamma- linolenic acid (GLA, (18:3 (n ⁇ 6)), Calendic acid (18:3 (n ⁇ 6)), Dihomo-gamma-linolenic acid (DGLA, (20:3 (n ⁇ 6)), Arachidonic acid (AA, (20:4 (n ⁇ 6)), and adrenic acid (AdA, (22:4 (n ⁇ 6)).
  • Mead acid (20:3 (n ⁇ 9)) is an omega-9 PUFA.
  • Other omega-9s and omega-7 FAs are MUFAs.
  • MUFAs that are omega-7s include Palmitoleic acid (16:1(n-7)) and Paullinic acid (20:1(n ⁇ 7)).
  • MUFAs that are omega-9 FAs include Oleic acid (18:1, (n ⁇ 9)), elaidic acid (18:1 (n ⁇ 9)), gondoic acid (20:1 (n ⁇ 9)), Erucic acid (22:1(n-9)), and Nervonic acid (24:1 (n ⁇ 9)).
  • the fat cells developed from cell lines can be differentiated to mimic the free fatty acid profile meat from an animal. Supplementation of free fatty acids into the cell culture media will generate a free fatty acid profile of the cultured cells. The molar concentration of each fatty acid will be determined by assessing the efficacy of fatty acid uptake and increasing or lowering the concentration to generate a fat additive that simulates the free fatty acid profile of a specific cut of meat.
  • palmitic acid is the second most common free fatty acid (FFA) in the longissimus muscle at 24.3% of the total FFA content. Palmitic acid should be added to the cell culture media at a concentration that will result in ⁇ 24.3% of the total fatty acid composition being palmitic acid.
  • Cultivated meat products may be designed by modelling the free fatty acid profile of the template meat. For example, pork belly is composed of multiple muscle groups: the cutaneous trunci, latissimus dorsi, pectoralis profundus, rectus abdominis, and internal and external abdominal oblique) and the fat content between these muscles. Bacon is the cured meat from the belly of the pork.
  • Bacon has different names depending on the part of the animal it was derived, and ‘streaky bacon’ is the cut from pork belly. While bacon has its fat content, it isn’t as fatty as pork belly because it is a particular cut of the underside of the animal, which has less fat in it. In order to turn cuts of pork belly into bacon, the meat is drained of all moisture with the help of salt. Pork loin is cut from the animals back and includes the longissimus muscle. [00074] Pigs differ from cattle because they have limited intramuscular fat. A lean cut of the longissimus muscle has 2.9% IMF, but the entire pork loin has 22.6% intramuscular, intermuscular, and subcutaneous fat content.
  • the fat content of pork belly is predominantly intermuscular and at 25.2%.
  • the predominant free fatty acids in pork are palmitic acid (16:0), steric acid (18:0), oleic acid (18:1), palmitoleic acid (16:1), linoleic acid (18:2, omega 6), and linolenic acid (18:3, omega 3).
  • Pork meat tends to contain less palmitic and oleic acid than beef and more PUFAs, such as linoleic acid and linolenic acids. Due to the low levels of marbling in pork, fat cells can be mixed with skeletal muscle cells in the bulk of the product without any marbling structure strategy. Additionally, there is a high amount of intermuscular fat in pork between muscles.
  • the present invention provides methods of transdifferentiating myogenic cells to adipogenic cells, eliminating the need to develop two separate cells lines in the development of a cell-based meat product.
  • mesenchymal stem cells undergo myogenesis, adipogenesis, and fibrogenesis to produce myogenic, adipogenic, and fibroblastic cells.
  • Wnt and ⁇ -catenin regulates MSC differentiation, where Wnt and ⁇ -catenin signaling will promote myogenesis, while suppression of Wnt and ⁇ - catenin signaling induces MSC adipogenesis.
  • rat satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation, Differentiation, 2001 Oct; 68(4-5): 245-53) showed that rat satellite cells can differentiate into adipocytes without treatment of IBMX, dexamethasone, or insulin.
  • Living muscle fibers were isolated and grown on Matrigel, which lacked adipogenic inducers.
  • the satellite cells on the muscle fibers maintained a more stem-like state than they otherwise would on cell culture plates since they were still affiliated with signals from their tissue niche. After 10 days in culture, the satellite cells formed lipid droplets that was visualized with Oil Red O (ORO) and expressed PPAR ⁇ gene, providing supporting evidence to the multipotency of satellite cells.
  • ORO Oil Red O
  • ZTD thiazolidinedione
  • bovine myoblast cells are multipoint and can transdifferentiate into adipocytes.
  • bovine skeletal muscle cells underwent myogenic differentiation through serum reduction, but were cultured in adipogenic media containing insulin, oleic acid, ciglitizone (a drug that serves as a PPAR ⁇ ligand and adipogenesis stimulator for mesenchymal stem cells) and melengestrol acetate (MGA, a feed additive for cattle to improve feedlot performance).
  • adipogenic media containing insulin, oleic acid, ciglitizone (a drug that serves as a PPAR ⁇ ligand and adipogenesis stimulator for mesenchymal stem cells) and melengestrol acetate (MGA, a feed additive for cattle to improve feedlot performance).
  • ORO staining showed multiocular lipid droplets formed inside mononuclear myoblasts and multinucleated myotubes.
  • PPAR ⁇ and C/EBP ⁇ gene expression increased and myogenin gene expression decreased (K.Y. Chung and B.J. Johnson, Melengestrol acetate enhances adipogenic gene expression in cultured muscle-derived cells, Journal of Animal Science, 87(12), December 2009: 3897–3904).
  • oleic acid was tested without a PPAR ⁇ agonist to determine its efficacy to stimulate adipogenesis in bovine satellite cells.
  • oleic acid increased the % of lipid droplets in the cells; increased PPAR ⁇ , C/EBP ⁇ , C/EBP ⁇ , and FABP4 gene expression; and adiponectin protein expression.
  • the satellite cells were still able to undergo myogenesis to form multinucleated muscle fibers and expressed myogenin gene expression, but showed decreased gene expression of Pax3 and MYOD1 (Li et al. Oleic acid in the absence of a PPAR ⁇ agonist increases adipogenic gene expression in bovine muscle satellite cells, Journal of Animal Science, 97(10), October 2019: 4114–4123). These data indicate an incomplete conversion of bovine satellite cells to adipocytes, with both myogenic and adipogenic characteristics maintained in the cells.
  • the present invention provides methods for transdifferentiating myoblasts into adipocytes. Producing myogenic and adipogenic cell lines from a single progenitor cell line is desirable due to a reduction in the complexity of the bioprocess.
  • the time and cost of developing multiple cell culture methods for proliferating muscle and fat cells, multiple media formulations, multiple cell line engineering strategies, and multiple bioprocesses may be condensed into one progenitor cell line by using a transdifferentiation method to obtain adipocytes from myoblasts.
  • PPAR ⁇ agonists such as long-chain fatty acids (LCFA) can be used to activate transdifferentiation of myogenic cells to adipogenic cells. Transdifferentiation can be accomplished through supplementation of cell culture media of myogenic cells with a combination of SFAs, MUFAs, and/or PUFAs.
  • a culture myoblast cells may be expanded to increase the biomass for a meat product. Upon reaching a certain biomass, the culture may be split. The first vessel will continue to grow myogenic cell population in myogenic media, while the cells in the second vessel will undergo transdifferentiation into adipogenic cells.
  • the cell culture media may be supplemented with any combination of the following free fatty acids at any concentration: SFAs include Myristic acid (14:0), Palmitic acid (16:0), Stearic acid (18:0), Arachidic acid (20:0), Behenic acid (22:0), Lignoceric acid (24:0), and Cerotic acid (26:0).
  • SFAs include Myristic acid (14:0), Palmitic acid (16:0), Stearic acid (18:0), Arachidic acid (20:0), Behenic acid (22:0), Lignoceric acid (24:0), and Cerotic acid (26:0).
  • PUFAs include omega-3, -6, and -9 fatty acids.
  • Omega-3 FAs include ⁇ -Linolenic acid (ALA, (18:3(n-3)), Stearidonic acid (SDA, (18:4 (n ⁇ 3)), Eicosatetraenoic acid (ETA, (20:4 (n ⁇ 3)), Eicosapentaenoic acid (EPA, (20:5(n-3)), Docosapentaenoic acid (DPA, (22:5 (n ⁇ 3)), and Docosahexaenoic acid (DHA, (22:6 (n ⁇ 3)).
  • ALA (18:3(n-3)
  • SDA Stearidonic acid
  • ETA Eicosatetraenoic acid
  • EPA Eicosapentaenoic acid
  • DPA Docosapentaenoic acid
  • DHA Docosahexaenoic acid
  • Omega-6 FAs include: Linoleic acid (LA, (18:2 (n ⁇ 6)), Gamma-linolenic acid (GLA, (18:3 (n ⁇ 6)), Calendic acid (18:3 (n ⁇ 6)), Dihomo-gamma-linolenic acid (DGLA, (20:3 (n ⁇ 6)), Arachidonic acid (AA, (20:4 (n ⁇ 6)), and adrenic acid (AdA, (22:4 (n ⁇ 6)).
  • Mead acid (20:3 (n ⁇ 9)) is an omega-9 PUFA.
  • Other omega-9s and omega-7 FAs are MUFAs.
  • Omega-7s include Palmitoleic acid (16:1(n-7)) and Paullinic acid (20:1(n ⁇ 7)).
  • Omega-9 FAs include Oleic acid (18:1, (n ⁇ 9)), elaidic acid (18:1 (n ⁇ 9)), gondoic acid (20:1 (n ⁇ 9)), Erucic acid (22:1(n-9)), and Nervonic acid (24:1 (n ⁇ 9)).
  • Cell culture [00082] The methods for developing cell lines contained herein can be applied to myoblasts, fibroblasts, fibroblast-adipocyte precursor cells, and mesenchymal stem cells. Adipocytes can be differentiated from fibroblasts, fibroblast adipocyte precursor cells, mesenchymal stem cells, and transdifferentiated from myoblasts.
  • Cell Isolation [00084] Any species of mammal may be used as cell donor.
  • Preferred mammals include, but are not limited to bovine (including cattle), caprae (including goats), Cervidae (including deer and elk), lagormorph (including rabbits), oves (including sheep, rodent and suidae (including pork).
  • bovine including cattle
  • caprae including goats
  • Cervidae including deer and elk
  • lagormorph including rabbits
  • oves including sheep, rodent and suidae (including pork).
  • Different species have different intramuscular fat deposits.
  • Skeletal muscle in cattle has intramuscular fat deposits that can be visibly observed as marbling, yet most pig breeds (except the duroc) lack marbling.
  • the IMF content of biceps femoris in pig meat is 1.4 +/- 0.4% at slaughter age, while it is 2.55% in the Dexter cattle breed.
  • a different cell isolation technique can be used to improve the porcine pre-adipoblast isolation yield, due to difference in extracellular matrix composition and localization of pre-adipoblasts.
  • Strategies for producing immortalized animal cell lines for cultivated meat products Cell culture media often includes the use of expensive growth factors to support cell survival, proliferation, and differentiation, making large scale cell-line cultivation cost-prohibitive. Constitutive activation of the ERK1/2 pathway and insulin-independent glucose transport through activation of the PI3K/Akt pathway can enhance cell proliferation and reduce the amount of growth factor required, thereby lowering the cost of cultivating edible cell lines.
  • the present inventions provide methods for enhancing cell proliferation with reduced growth factor concentration through constitutive activation of the ERK1/2 pathway and insulin-independent glucose transport through activation of the PI3K/Akt pathway.
  • Edible cell lines used in cultivated meat products must have low cost production methods to be affordable.
  • Cell lines used in academia or the regenerative medicine industry are not designed to minimize growth factor and insulin requirements in cell culture media required for cell survival, proliferation, and differentiation. These types of cell lines have different cost drivers than cells lines used to produce affordable food for consumers.
  • Cell lines used in research are not designed for scalable manufacturing, and cell lines used in the cell therapy or biopharma industries are expensive products with high profit margins. Consequently, engineering cell lines for cultivated meat products have to have reduced mitogen and insulin requirements desirable.
  • PI3K/Akt signaling pathway PI3Ks (phosphoinositide 3-kinases) are a family of lipid and protein kinases linked to a diverse group of cellular functions, including cell growth, proliferation, differentiation, motility, survival, intracellular trafficking, and insulin receptor signaling. They are separated into classes I, II, and III based on sequence homology and function. PI3K is recruited to the cell membrane via phosphorylated receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs), and can also interact with cytosolic Ras GTPases to initiate signal transduction pathways.
  • RTKs phosphorylated receptor tyrosine kinases
  • GPCRs G protein-coupled receptors
  • Class I PI3Ks are heterodimers composed of IA and IB subsets that contain a catalytic and regulatory subunit.
  • Class IA PI3Ks have p110 catalytic and p85 regulatory subunits, with three variants to the p110 catalytic subunit: p110 ⁇ , p110 ⁇ , or p110 ⁇ (expressed by the genes Pik3ca, Pik3cb, and Pik3cd, respectively).
  • the p85 ⁇ , p55 ⁇ , p50 ⁇ subunits are splice variants of the Pik3r1 gene; p85 ⁇ is expressed from Pik3r2, and p55 ⁇ expressed from Pik3r3. p110 ⁇ and p110 ⁇ isoforms are expressed in all cells. Regulatory subunits stabilize and suppress the catalytic subunit in the absence of upstream signals and mediate the interaction of the catalytic subunit to RTKs and GPCRs during signal transduction.
  • the catalytic subunit of class I PI3Ks contain an amino-terminal adaptor-binding domain (ABD) which interacts with the regulatory unit; a Ras-binding domain (RBD) that facilitates interaction between Ras-GTP and the catalytic subunit, allowing PI3K activation in a Ras-dependent manner; a C2 (protein-kinase-C homology-2) domain that has an affinity for lipid membranes; a helical domain that operates as scaffolding for other p110 domains; and a carboxyl-terminal kinase domain.
  • ABS amino-terminal adaptor-binding domain
  • RBD Ras-binding domain
  • C2 protein-kinase-C homology-2 domain
  • the p85 regulatory unit is composed of several protein- protein interaction domains: there are two SH2 domains, C-SH2 and N-SH2, where N-SH2 interacts with the helical domain of the catalytic subunit; one SH3 domain that binds to phosphorylated tyrosine residues; a breakpoint clustered homology (BH) domain; and an inter- SH2 (iSH2) domain that interacts with the catalytic subunit at ABD and C2.
  • SH2 domains there are two SH2 domains, C-SH2 and N-SH2, where N-SH2 interacts with the helical domain of the catalytic subunit
  • one SH3 domain that binds to phosphorylated tyrosine residues
  • BH breakpoint clustered homology
  • iSH2 domain inter- SH2 domain that interacts with the catalytic subunit at ABD and C2.
  • PIP3 recruits proteins to the cell membrane that contain a pleckstrin homology domains, which includes a serine/threonine specific protein kinase called protein kinase B (PKB, otherwise known as Akt), and PDK1 (phosphoinositide-dependent kinase-1). Binding of Akt to PIP3. Changes it’s conformation and allows interaction with PDK-1 and PDK-1 phosphorylates Akt on threonine 308, which partially activates the enzyme. Akt becomes fully activated upon phosphorylation of serine 473 by the TORC2 complex of the mTOR protein kinase through a positive feedback loop.
  • PPB protein kinase B
  • Akt plays a role in glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration.
  • Akt has three isoforms: Akt1/PKB ⁇ , Akt2/PKB ⁇ , and Akt3/PKB ⁇ .
  • Each isoform contains a N-terminal pleckstrin homology domain (PH), kinase catalytic domain containing threonine 308, and C-terminal regulatory domain containing serine 473 (in Akt1) or serine 474 (in Akt2).
  • the PH domain regulates lipid-protein interactions and is the mechanism for recruitment to the cell membrane.
  • Akt1 is involved in cellular survival pathways by promoting cell cycle progression and inhibiting apoptotic processes.
  • Akt1 is also able to induce protein synthesis pathways, and is therefore a key signaling protein in the cellular pathways that lead to skeletal muscle hypertrophy, and general tissue growth. Since Akt1 can block apoptosis, it has been implicated in numerous cell survival signaling mechanisms. Akt1 can be activated by growth factors. Akt2 is involved in the insulin signaling pathway and is required for glucose transport through the cell membrane. Akt2 is more specific for insulin receptor pathway than Akt1. Akt2 drives differentiation whereas Akt1 appears critical to myoblasts proliferation. Akt1 phosphorylates the cyclin kinase inhibitor p21, triggering its dissociation from CDK2 and leading to cell cycle progression.
  • PDK-1 has a kinase domain and pleckstrin homology domain, and the PH domain localizes the kinase to the cell membrane.
  • PDK-1 is considered to be constitutively active but is regulated by PI3K conversion of PIP2 to PIP3 in the cell membrane along with localization of Akt.
  • the kinase domain has three ligand binding sites: the substrate binding site, the ATP binding site, and the docking site (also known as PIF pocket).
  • PLC protein kinase C
  • SGK serum glucocorticoid-dependent kinase
  • S6K p70 ribosomal protein S6 kinases
  • RSK ribosomal protein S6 kinase
  • the phosphatase and tensin homologue (PTEN) tumor suppressor gene is an antagonist of the PI3K/Akt pathway by dephosphorylating phosphatidylinositol-3,4,5- triphosphate PIP3 to PIP2 - preventing the partial phosphorylation of Akt, and therefore switches off the PI3K- activated pathway.
  • the lipid phosphatase activity is critical for PTEN tumor suppressor function. Inactivation of PTEN results in activation of the PI3K/AKT pathway and subsequent increase in cell cycle progression, migration and survival.
  • PTEN regulates cell cycle progression by downregulating transcriptional expression and protein stability of cyclin D1, as well as by inhibiting its nuclear localization. In addition to cyclin D1, PTEN is also shown to potentially repress cyclin D2 and cyclin D3 to arrest the cell cycle at G1. PTEN is also been shown to modulate the cell cycle by upregulating the cyclin dependent kinase (CDK) inhibitor p27. [00098] PTEN can also be regulated by TBX2 and Pax3. TBX2, a T-box family member, bypasses cell growth control through the repression of the cell cycle regulators p14 and p21.
  • CDK cyclin dependent kinase
  • TBX2 directly represses the tumor suppressor PTEN in both rhabdomyosarcoma (RMS) and normal muscle.
  • RMS rhabdomyosarcoma
  • Exogenous expression of TBX2 in normal muscle cells down regulates PTEN, and depletion of TBX2 in RMS cells upregulates PTEN, resulting in a reduction of phospho- AKT.
  • TBX2 represses PTEN by directly binding to the promoter and recruiting the histone deacetylase, HDAC1.
  • TBX2 is a central component of the PTEN/PI3K/AKT signaling pathway deregulation in rhabdomyosarcoma.
  • the fusion protein PAX3- FOXO1 has been shown to contribute to repression of PTEN.
  • PAX3-FOXO1 Depletion of PAX3-FOXO1 in RMS cells up regulated PTEN and exogenous expression of PAX3 in C2C12 cells downregulated PTEN.
  • the level of PTEN has been shown to be inversely correlated with AKT serine 473 phosphorylation, which is mediated by the rapamycin-insensitive mTOR complex (mTORC2) and required for full activation of AKT.
  • mTORC2 rapamycin-insensitive mTOR complex
  • PAX3 was shown to bind to the PTEN promoter.
  • PAX3 has also been shown to activate TBX2 in melanoma cells.
  • the negative regulation of PTEN by PAX3 may be mediated through TBX2.
  • the MAPK/ERK signaling pathway [00100] As previously mentioned, eliminating or reducing the amount of insulin or growth factors required for cell culture media lowers the cost associated with cultivating edible cell lines.
  • the present invention provides methods of enhancing cell proliferation with reduced growth factor concentration through constitutive activation of the ERK1/2 pathway and insulin-independent glucose transport through activation of the PI3K/Akt pathway MAPKs (mitogen activated protein kinase, originally termed ERKs – extracellular signal regulated kinases) are serine/threonine-selective protein kinases that phosphorylate downstream targets that regulate the cell.
  • MAPKs There are three main families of MAPKs: 1) the ERKs, 2) the c-jun NH 3-terminal kinases/stress activated protein kinase (JNKs), and 3) the p38 MAPKs (p38 ⁇ / ⁇ ).
  • ERKs are activated by growth factor ligands such as NGF, FGF, EGF, PDGF, and BDNF that bind to their tyrosine kinase receptors in the cell membrane to initiate pro-mitotic intracellular signaling cascades.
  • JNKs and p38s are activated by cellular stress and cytokines to cause apoptosis, inflammation, cell cycle arrest, and cell differentiation.
  • MAPKs share structural similarity and have 2 phosphorylation sites (phosphotyrosine and the phosphothreonine residues) for activation in their activation loop domains, substrate recognition sites, and a three-tiered activation cascade.
  • the activation loop contains a characteristic TxY (threonine-x-tyrosine) motif, where both the threonine and tyrosine must be phosphorylated to activate the kinase. It is TEY in mammalian ERK1/2, TYP in JNKs, and TGY in p38 kinases.
  • MAPKs are phosphorylated by MAP2Ks (MKKs, MAP kinase kinases), which in turn are phosphorylated by MAP3Ks, which are localized to the cell membrane where they are activated by various stimuli.
  • MAPKs are the only MAP kinases that can enter the cell nucleus to activate gene transcription.
  • MAPKs and MAP2Ks are regulated via their phosphotyrosine and the phosphothreonine residues, but MAP3Ks have more complex regulation. Some MAP3Ks include Rafs, MEKK4, or MLK3, which require multiple activation steps.
  • a ligand associated with the cell membrane changes the conformation of the MAP3K (Ras for Rafs, GADD45 for MEKK4, or Cdc42 for MLK3) to expose the kinase domains.
  • the accessible kinase domains then allow homo- or heterodimerization which generates a partially activate dimer conformation.
  • the dimers then transphosphorylate each other on their activation loops.
  • the fully activated MAP3Ks can then phosphorylate MAP2Ks.
  • the MAP3Ks are A-, B-, and c-Raf that are activated by growth factors such as EGF, FGF, and PDGF.
  • Rafs then phosphorylate MKK1/2 (aka MEK1/2), which in turn phosphorylate ERK1/2.
  • Mammalian p38 and JKN kinases share some MAP3Ks, such as MEKK1/2 and ASK1/2, and share a MAP2K MKK4.
  • JNK is separately activated by MAP3Ks MLK1/2/3 and MAP2K MKK7, while p38 is activated by MAP2Ks MKK3/6.
  • the Ras-Raf-MEK-ERK signal transduction pathway regulates cell growth and proliferation in response to growth factors, cytokines, and hormones.
  • Rafs are serine/threonine kinases that are stimulated upon binding of GTP-bound Ras to the Ras binding domain (RBD).
  • A-Raf and C-Raf share similar regulatory mechanisms and require additional serine and tyrosine phosphorylation within the N region of the kinase domain for full activity compared to B-Raf, which has higher basal kinase activity than A-Raf and C-Raf.
  • the Ras/MEK/ERK pathway promotes cell cycle progression through phosphorylation of cyclin D1 and c-Myc transcription factor. Phosphorylated cyclin D1 complexes with Cdk4 and Cdk5 (Cdk4/6).
  • Cyclin D-Cdk4/6 regulates the progression of the G1 phase of the cell cycle to S phase. Cyclin D-Cdk4/6 activity increases in late G1 due to the signaling from extracellular mitogens which leads to the hyperphosphorylation of retinoblastoma protein (Rb). Hypophosphorylated Rb is usually bound to E2F transcription factor in early G1 and inhibits its activity. Hyperphosphorylation of Rb dissociates E2F, which can then enter the nucleus and activate gene transcription of cyclins that progress the cell through the S phase of the cell cycle (J. Knight and R. Kothary, The myogenic kinome: protein kinases critical to mammalian skeletal myogenesis, Skeletal Muscle, 1, Art. No.29 (2011)).
  • Rb retinoblastoma protein
  • PI3K/Akt and MEK/ERK signaling pathways converge to jointly regulate c-Myc activation.
  • ERK mediates c-Myc stabilization through phosphorylation at serine 62, and subsequent phosphorylation at Thr58 by GSK-3 is required for c-Myc degradation.
  • Akt phosphorylation causes inhibition of GSK-3, and hyperactivation of the PI3K and MEK-ERK pathways promote c-Myc signaling and cell cycle progression.
  • Activated c-Myc then regulates gene expression by suppressing expression of the cell cycle inhibitor p21 and increasing expression of cyclins A and B and CDK2.
  • c-Myc overexpression enhances cell proliferation rates.
  • c-Myc phosphorylation is majorly attenuated by ERK2 and to a lesser extend ERK1, but c-Myc phosphorylation is highest when both ERK1/2 are active (Marampon et al. Down- regulation of c-Myc following MEK/ERK inhibition halts the expression of malignant phenotype in rhabdomyosarcoma and in non-muscle-derived human tumors, Molecular Cancer, 5, Art. No.31 (2006)).
  • Telomerase is an enzyme that maintains the length of telomeres at the 3’ ends of chromosomes, which prevents cellular senescence based on telomere shortening.
  • Telomeres are present in eukaryotic organisms and are repeating strands of nucleotide sequences. With each round of mitosis, telomeres shorten until the they reach a critical length that prevents DNA replication machinery to initiate another round of cell division. This limits the number of cell divisions and help preserve genomic stability, a phenomenon known as the Hayflick limit. Telomerase is a reverse transcriptase enzyme that carriers an RNA molecule that serves as a template for lengthening telomeres. Telomerase expression is found in embryonic stem cells and iPSCs, and is typically absent or expressed at very low levels in somatic cells.
  • Anchorage-independent proliferation in single cell suspension through anoikis resistance [00110] Primary cell lines such as skeletal muscle cells typically survive and proliferate in an anchorage-dependent manner. Once anchorage dependent cells detach into suspension, a form of cell death called anoikis occurs. This limits the scale up of cell production to microcarrier- or aggregate based growth system, which yield lower cell concentrations per mL than cell populations that can be grown in single cell suspension. To increase the production yield of cell lines, anchorage independent survival and growth can be conferred by manipulating cell signaling pathways that generate anoikis resistance. Failure to initiate anoikis can result in cell survival in suspension.
  • Anoikis pathways terminally converge in activation of caspases, which activate endonucleases, DNA fragmentation, and cell death.
  • the intrinsic pathway that involves perturbation of mitochondria
  • the extrinsic pathway triggered by cell surface death receptors [00112]
  • pro-apoptotic BH3-only proteins Bid and Bim are activated following detachment of cells from ECM, and rapidly promote the oligomerization of Bax- Bak.
  • Bax-Bak translocate to the outer mitochondrial membrane, create a channel that permeabilizes the mitochondrial membrane, and causes cytochrome c release.
  • ECM binds to integrins that transduce signaling cascades which promote survival of attached cells. Activation for four types of integrins, a5B1, avB3, a1B1, and a6B1, plays a specific role in cell survival by protecting cells from apopotosis and anoikis.
  • FAK focal adhesion kinase
  • Src Src kinase
  • ILK integrin-linked kinase
  • MAPK MAPK
  • Activation or overexpression of these signaling molecules confers protection against anoikis.
  • FAK activation causes autophosphorylation, which recruits Src, which further phosphorylates FAK and leads to a fully activated FAK enzyme.
  • FAK then activates the Akt and MAPK pathways.
  • FAK activates PI3K, which then recruits and activates Akt.
  • Akt activation promotes cell survival by several mechanisms.
  • ILK encoded by the ILK gene, is involved with cell proliferation, migration, and adhesion that interacts with focal adhesions. Its main role is to organize the actin cytoskeleton during development and homeostasis.
  • ILK is a scaffolding protein that associates with the cytoplasmic domains of ⁇ -1 and ⁇ -3 integrins to transduce integrin-mediated extracellular signals. [00116] ILK has three different domains: five ankyrin repeats at the N- terminus, followed by a pleckstrin homology (PH)-like domain and a kinase- like domain at the C-terminus.
  • PH pleckstrin homology
  • ILK Before recruitment to the focal adhesion site, ILK forms a complex with two adaptor proteins called Pinch and parvin (termed the IPP complex). This stabilizes the complex and allows targeting to the focal adhesion site. There are different forms of parvin that affiliate directly with the F- actin cytoskeleton or recruit actin binding proteins. ILK can also connect microtubule caps to cortical actin networks through FA sites. [00117] Cells must also release from focal contacts during cell migration. Mesenchymal motility occurs when migrating cells take on an elongated cell morphology dependent on ECM binding to integrins and focal contacts. Another motility style is amoeboid migration which allows cells to overcome ECM barriers by weakening focal contacts.
  • RhoG can suppress anoikis.
  • Mechanisms for cell immortalization and anoikis resistance Immortalized cell lines with anoikis resistance, insulin-independent insulin signaling, and mitogen-independent cell cycle progression enhances flavor and sensory properties of edible cell lines and lowers the cost of production.
  • cell lines used for cultivated meat are designed to grow in single cell suspension without undergoing anoikis. In scaled systems, cells can be grown in single cell suspension, on microcarriers, or as aggregates.
  • Persistent PI3K or Akt activity can lead to cellular immortalization by driving continuous cell cycle progression and promotes anoikis resistance by preventing normal apoptosis mechanisms. Akt blocks apoptosis by inhibiting the activity of caspase 9 and Bad, which prevents apoptosome assembly and permeabilization of the mitochondrial outer lipid membrane, respectively.
  • Akt enhances cell survival and cell progression by increasing activity of mitogens Jun, Myc, and cyclinD, and deceasing activity of p53, p27 and p21, which promote cell cycle arrest.
  • Akt is phosphorylated by PDK-1 and increased proliferation of cells can be achieved with PDK-1 overexpression.
  • Anchorage independent cell growth has been shown with PDK-1 overexpression and membrane localization with activation of SGK3.
  • PDK-1 positively regulates ROCK1 in a kinase-independent manner that promotes amoeboid motility.
  • PDK-1 knockdown in standard tissue culture conditions showed no growth enhancement of cell lines, but show inhibited growth in anchorage independent growth systems.
  • PDK-1 also has Akt-independent downstream signaling mechanisms that have been associated with cell survival and proliferation, such as activation of S6K1, SGK, PKC, and RSK isoforms.
  • S6K regulates protein synthesis and the progression of the cell cycle from G1 to S phase.
  • SGK is activated in response to insulin stimulation and promotes synthesis of glycogen and other proteins.
  • PKC isoforms contribute to cell cycle progression.
  • PKC ⁇ in particular has an antiapoptotic effect since it phosphorylates and activates Bcl-2 (Xie et al.
  • ILK is overexpressed in many types of cells with anchorage independent cell growth. Although it was initially reported to have kinase activity, subsequent studies have shown that the reported catalytic domain is enzymatically inactive. The anoikis resistance effect of ILK may be due to multiple mechanisms.
  • ILK may control the localization of proteins such as Akt to the plasma membrane where it can be phosphorylated. Additionally, Pinch-1 can bind to and inhibit protein phosphatase 1 ⁇ , which results in sustained Akt phosphorylation. Overexpression of ILK blocks anoikis in suspended cells.
  • Constitutive Ras signaling is transduced through MEK/ERK to promote cellular proliferation and anoikis resistance.
  • RMS rhabdomyosarcoma
  • U0126 decreased c-Myc gene expression and phosphorylation levels and induced growth inhibition.
  • U0126 reduced levels of cyclins A, B, D1, E1, and E2 and CDK2 (which forms complexes with cyclin A, B, and E), while increasing levels of cyclin kinase inhibitor (CKI), p21, and p27.
  • CKI cyclin kinase inhibitor
  • Direct inactivation of c-myc protein reduced expression of cyclins A/B/D1 and CDK2, and increased p21 expression.
  • Expression of cyclins E1/2 and p27 were not altered, however, and may be attenuated by ERK depletion, which highlights that both ERK and c-Myc signaling uniquely contribute to cell cycle progression.
  • the RMS cell line shows anoikis resistance and can grow in anchorage-independent colonies on agar plates.
  • a constitutively active mutant form of B- Raf induces persistent MEK/ERK signaling in a Ras-independent mechanism.
  • BRAF-V600E which results from an exchange of a single amino acid: valine 600 into glutamate.
  • thymine is substituted with adenine at nucleotide 1799 to convert valine to glutamate. This mutation causes a conformational change that mimics phosphorylation of the activation loop and renders the kinase domain fully active, yet does not require dimerization to phosphorylate downstream targets.
  • BRAF-V600E has 500 times the signal activity of wild type B-Raf and makes up 90% of the profile of B-Raf mutants (Liu et al. BRAF mutation and its inhibitors in sarcoma treatment, Cancer Medicine, 9(14): 4881- 4896). Most B-Raf mutations enhance kinase activity to increase ERK signaling, but some show reduced kinase activity. However, these mutations with reduced kinase activity activate wild-type C-Raf to initiate downstream ERK signaling (Wan et al., Mechanism of Activation of the RAF-ERK Signaling Pathway by Oncogenic Mutations of B-RAF, Cell, 116(6), 19 March 2004, pages 855-867).
  • KIAA1549-BRAF BRAF fusion mutant found in embryonic rhabadomyosarcoma
  • KIAA1549-BRAF BRAF fusion mutant found in embryonic rhabadomyosarcoma
  • KIAA1549-BRAF BRAF fusion mutant found in embryonic rhabadomyosarcoma
  • KIAA1549-BRAF BRAF fusion mutant found in embryonic rhabadomyosarcoma
  • a gene may be overexpressed by a promoter, if as a result of said expression an altered spatial distribution and/or an increased quantity of the gene product is found, compared to the expression of the wild-type (regular) promoter for this gene.
  • overexpression leads to a more than 2-fold, more than 5-fold, more than 0-fold or even more than 20-fold higher expression of the gene, when compared to the expression of the wild- type (regular) promoter for this gene.
  • Overexpression can be measured using well-known methods in the art, such as mRNA detection (e.g. using rtPCR), promoter activity assays, immunofluorescence and western blotting (immunoblot) analysis.
  • Overexpression of wild type ILK may be accomplished through enhancement of promoter elements of the [ILK] gene.
  • transgenes can be inserted into the genome of the cell under control of constitutively active promoter.
  • Overexpression of PDK-1 may be accomplished through enhancement of promoter elements of the [PDK1] gene.
  • transgenes can be inserted into the genome of the cell under control of constitutively active promoter.
  • Overexpression of telomerase may be accomplished through enhancement of promoter elements of the [TERT] gene.
  • transgenes can be inserted into the genome of the cell under control of constitutively active promoter.
  • Knockout of PTEN Suppression of the PTEN tumor suppressor [PTEN] gene will promote activation of the PI3K/Akt signaling pathway by preventing PTEN from dephosphorylating PIP3.
  • Silencing gene expression may be achieved through genetic knockout/knockdown or over expression of dominant negative form/miRNA or mutation of the PTEN gene or its promoter.
  • Constitutively active B-Raf – The B-Raf gene [BRAF] sequence may be mutated to create a constitutively active form of B-Raf kinase that results in enhanced ERK signaling in comparison to the wild type B-Raf, including but not limited to generating the BRAF-V600E mutant. These may include any substitution, deletion, insertion, duplication, inversion, and frameshift mutations to the BRAF gene.
  • Overexpression of TBX2 may be accomplished through enhancement of promoter elements of the [TBX2] gene.
  • transgenes can be inserted into the genome of the cell under control of constitutively active promoter.
  • Overexpression of Pax3 may be accomplished through enhancement of promoter elements of the [PAX3] gene.
  • transgenes can be inserted into the genome of the cell under control of constitutively active promoter.
  • Media for cell culture [00138] The development of animal and serum free medias is required for commercialization. Finding a chemically defined serum alternative is imperative to scaling up meat biomanufacturing. Fetal bovine serum is used because it contains high concentrations of growth factors that promote cell proliferation and low concentrations of factors that inhibit cell growth.
  • Basic serum free media formulations include growth factors such as EGF, FGF, PDGF, NGF, IGF; cytokines such as interferons and interleukins; insulin and other hormones; proteins like transferrin and albumin; trace elements; and fatty acids.
  • Finding the right media composition can enhance flavor and performance of cell lines.
  • Strategies for reducing the use of insulin in cell culture media [00140] Similarly, reducing or eliminating insulin from cell culture media may significantly reduce cost of production.
  • the present invention provides strategies to reduce to requirement for growth factors or other proteins in cell culture media.
  • the present invention provides methods of targeting the insulin/GLUT4 and PI3K-Akt signaling pathways.
  • the Insulin Receptor-PI3K-AKT pathway [00142] In both skeletal muscle and adipose cells, insulin stimulates PI3K/Akt signaling to promote glucose transport into the cell. In skeletal muscle, insulin stimulates protein synthesis and accelerates mRNA translation by regulating the initiation steps of protein translation. Insulin signaling regulates the metabolism of adipose tissues by promoting glucose utilization, protein synthesis, and lipogenesis. The PI3K/AKT signaling pathway promotes lipid biosynthesis and inhibits lipolysis.
  • Insulin is a hormone that regulates glucose metabolism in the cell and is an essential cell culture media component.
  • insulin activates the insulin receptors (IR, encoded by the INSR gene) present in cell membranes, which transduce an intracellular signaling cascade leading to the translocation of insulin-responsive glucose transporter 4 (GLUT4) from intracellular membranes in the cytoplasm to the cell membrane, and GLUT4 transportation of glucose into the cytoplasm of the cell.
  • the insulin receptor is a dimeric protein with two extracellular ligand-binding alpha domains (the ectodomain), and beta domains consisting of two transmembrane helices and two intracellular tyrosine kinase domains.
  • Binding of insulin ligand to alpha-chains in the dimeric ectodomain causes a conformational change that is propagated structurally through the beta domains.
  • the intracellular tyrosine kinase domains begin to autophosphorylate tyrosine residues that generate binding sites and phosphorylate insulin receptor substrates.
  • the insulin receptor autophorsphorylation leads to activation of PI3K, to activate the PI3K/AKT pathway.
  • PDK-1 and Akt are thus translocated to the cell membrane and activate substrates that lead to GLUT4 translocation to the cell membrane.
  • Akt1 and Akt2 can promote insulin-dependent glucose metabolism, but Akt2 has a more significant impact on glucose metabolism.
  • AKT drives GLUT4 gene expression and directly phosphorylates AS160, inducing GLUT4 translocation to the plasma membrane from storage vesicles.
  • AS160 encoded by the TBC1D4 gene
  • AS160 is a highly insulin-responsive Akt target which negatively regulates insulin receptor signaling.
  • AS160 is a Rab GTPase-activating protein with a GTPase-activating protein (GAP) domain. The GTPase-activating domain accelerates the hydrolysis of GTP (guanine triphosphate) to GDP (guanine diphosphate) on G proteins.
  • Rab proteins are a family of small G proteins with hydrolase enzymatic activity that are part of the larger Ras protein superfamily. Rab proteins regulate membrane trafficking, vesicle formation, vesicle fusion, and membrane fusion by affiliating with microtubules and microtubule associated motor protein kinesin, which plays a role in shuttling GLUT4 vesicles to the cell membrane.
  • RAB2A, RAB8A, RAB10, and RAB14 are stimulated to maintain the GDP-bound form when the AS160 GAP domain is active, but switch from GDP-bound to GTP- binding forms upon inhibition of AS160 to regulate the trafficking and translocation of GLUT4 to the cell membrane.
  • PDK-1 and PIP3 also play a role in insulin-stimulated GLUT4 translocation in an Akt-independent manner. The phosphorylation of residues on atypical PKCs such as PKC- ⁇ (and an interchangeable form, PKC- ⁇ ) is required to stimulate GLUT4 translocation to the cell membrane.
  • PKC- ⁇ is phosphorylated in the kinase activation loop by PDK-1 at Thr-410 and facilitates GLUT4 trafficking to the plasma membrane.
  • Overexpression of PDK-1 increases GLUT4 translocation in the absence of insulin via PKC- ⁇ phosphorylation (G. Bandyopadhyay et al., Dependence of Insulin-Stimulated Glucose Transporter 4 Translocation on 3- Phosphoinositide-Dependent Protein Kinase-1 and its Target Threonine-410 in the Activation Loop of Protein Kinase C- ⁇ , Molecular Endocrinology, 13(1), 1 Oct.1999, pages 1766-1772).
  • PKC- ⁇ also has an autophosphorylation site at T560, and autophosphorylation levels increase with PIP3 activity and insulin treatment.
  • the physical association of PKC- ⁇ with PIP3 provides allosteric interactions that induce a conformational change that increases autophosphorylation activity.
  • Association of PKC- ⁇ with PIP3 also colocalizes it with PDK-1, facilitating phosphorylation at T410. If either phosphorylation site is inactive, the kinase activity decreases and lowers translocation rates of GLUT4 to the cell membrane.
  • Insulin stimulates a reorganization of the actin cytoskeleton and brings PI3K, PKC- ⁇ , and GLUT4 into association in the plasma membrane.
  • PI3K-mediated insulin stimulation caused phosphorylation of PKC- ⁇ , which associated with and remodeled actin, where it colocalizes with GLUT4.
  • Overexpression of PKC- ⁇ lead to increased GLUT4 translocation to the cell membrane and glucose uptake (Liu et al. Protein Kinase C ⁇ Mediates Insulin-induced Glucose Transport through Actin Remodeling in L6 Muscle Cells, Molecular Biology of the Cell, 17(5): 2322-2330 (May 2006)).
  • PI3K-mediated PKC- ⁇ activation stimulation also activates Rab4, which affiliates with kinesin to mediate interaction with microtubules and stimulate transport of GLUT4 to the cell membrane.
  • This truncated receptor spans amino acid residues -27 to 12 fused to residues 915 to 1343, and specifically includes the signal sequence (27 amino acids), the first 12 amino acids of the alpha subunit, and the terminal portion of beta subunit starting three amino acids before the transmembrane domain (23 amino acids), and the tyrosine kinase domain (403 amino acids, 468 amino a ccids total).
  • Another version of the constitutively active, truncated insulin receptor includes the signal sequence, the first 12 amino acids of the alpha subunit, and residues 600- 1355 ( ⁇ 20% of the alpha subunit at the carboxy terminal and all of the beta subunit).
  • a constitutively active insulin receptor will be designed by truncating the alpha subunits in the [INSR] gene. Truncated forms will include the signal sequence, the first 12 amino acids of the alpha subunit, and between 0-40% of the carboxy terminal of the alpha subunit, and all of the beta subunit, and will result in autophosphorylation of the tyrosine residues in the beta subunit.
  • This constitutively active IR will translocate and insert into the cell membrane and lead to downstream phosphorylation of Akt and translocation of GLUT4 into the cell membrane in the reduction or absence of insulin in cell culture media.
  • the truncated IR will be inserted as a transgene under the control of a constitutively active promoter into the native genome of the cell.
  • a mutation can be performed on the native INSR gene that results in the deletion of nucleotide bases which truncates the alpha subunit of the insulin receptor, generating a constitutively active receptor.
  • These methods include all substitution, deletion, insertion, duplication, inversion, and frameshift mutations.
  • PDK-1 overexpression may increase GLUT4 translocation to the nucleus through phosphorylation of Akt and PKCs.
  • Overexpression of GLUT4 gene itself has also been shown to increase GLUT4 protein translation and translocation to the plasma membrane in an insulin-independent manner.
  • GLUT4 overexpression may be achieved in cell lines by modifying the promoter region of the native [GLUT4] gene to activate constitutive expression, or a transgene under the control of constitutively active promoter can be inserted into the native genome of the cell.
  • Generating a constitutively active insulin receptor pathway has additional sensory benefits to myogenic and adipogenic cell lines that can improve flavor, pigment, and overall product quality.
  • glucose can be transported into the cell and used for various cellular metabolism processes. This can include the glycolysis pathway which generates ATP for cells and converts glucose to pyruvate.
  • Pyruvate is then used as the precursor substrate for the citric acid cycle that produces NADH and GTP. NADH then undergoes oxidative phosphorylation in mitochondria to generate high yields of ATP.
  • the protein content of skeletal muscle cells is generated through the consumption of energy supplied by ATP and GTP. Increasing the protein content and the translation of muscle specific proteins enhances the umami meat-like flavor of cell lines.
  • ATP is used by myogenic cells to enhance myogenic differentiation by supplying energy to the contractile protein machinery of sarcomeres. Binding of ATP to myosin filaments releases energy that facilitates the interaction of myosin protein with actin filaments that cause contraction. The development and maturation of sarcomeres increases the protein content of the muscle cell.
  • GTP produced by the citric acid cycle is also used by skeletal muscle cells for protein translation via ribosomes. GTP supplies energy that is consumed during the formation of peptide bonds between amino acids in the ribosome.
  • Glycolysis also plays a direct role in the conversion of glucose to free fatty acids that undergo esterification into triglycerides, which are the basic substrate of lipid droplets inside differentiating adipocytes. Metabolism of glucose produces pyruvate and glycerol, and the pyruvate is converted to fatty acids during fatty acid synthesis pathway. Glycerol is then bound to three fatty acids via esterification to produce the triglyceride.
  • Target Combinations provide a method to enhance cell proliferation with reduced growth factor concentration through constitutive activation of the ERK1/2 pathway and insulin-independent glucose transport through activation of the PI3K/Akt pathway. Edible cell lines used in cultivated meat products must have low cost production methods to be affordable.
  • Cell lines used in academia or the regenerative medicine industry are not designed to minimize growth factor and insulin requirements in cell culture media required for cell survival, proliferation, and differentiation. These types of cell lines have different cost drivers than cells lines used to produce affordable food for consumers.
  • Cell lines used in research are not designed for scalable manufacturing, and cell lines used in the cell therapy or biopharma industries are expensive products with high profit margins. Consequently, engineering cell lines for cultivated meat products to have reduced mitogen and insulin requirements is a novel method to reduce production costs.
  • [00161] The following example combinations can generate immortal cell lines with anoikis resistance, insulin-independent insulin signaling, mitogen-independent cell cycle progression, and improved flavor and sensory properties of cell lines: [00162] ILK+, PDK-1+, Telomerase+, PTEN-, BRAF-V600E [00163] One potential combination of cell line engineering targets is to overexpress ILK, PDK-1, telomerase, knock out PTEN, and replace valine with glutamate at amino acid 600 of B-Raf to confer constitutive activity.
  • Knocking out PTEN prevents the dephosphorylation of PIP3 to PIP2, which allows constant translocation of Akt and PDK-1 to the plasma membrane, where PDK-1 can phosphorylate Akt to initiate downstream signaling pathways related to cell cycle progression and anoikis resistance.
  • Overexpressing PDK-1 increases the rate of Akt phosphorylation while also activating S6K1, SGK, PKC, and RSK, which can all promote cell cycle progression.
  • ILK overexpression and BRAF-V600E activity can simulate integrin- mediated survival signals, block anoikis, and induce anchorage independent cell growth through ILK mediated phosphorylation of Akt and BRAF-V600E activation of ERK1/2.
  • ILK and PDK-1 overexpression and PTEN knockout have the downstream effect of enhancing Akt- mediated cell signaling by increasing phospho-Akt activity and promoting cell cycle progression, anoikis resistance, and insulin-independent insulin signaling through recruitment of GLUT4 to the cell membrane.
  • Flavor, pigment, and product quality can be enhanced by constitutive activation of the insulin / GLUT4 pathway, which drives myogenic and adipogenic differentiation.
  • BRAF-V600E also promotes cell cycle progression through ERK1/2 phosphorylation in a mitogen-independent mechanism. Additionally, overexpressing telomerase helps the cells bypass the hayflick limit by maintain telomere length through repeated cell divisions, which is necessary to develop scalable cell lines.
  • ILK+, PDK-1+, Telomerase+, TBX2+ or PAX3+ Another target combination could be overexpressing ILK, PDK-1, telomerase, TBX2 or Pax3. This follows the same logic above with ILK, PDK-1, and telomerase overexpression. However, instead of knocking out PTEN, TBX2 and PAX3 overexpression (either in combination or individually) suppress PTEN activity which inhibits the dephosphorylation of PIP3 to PIP2.
  • PDK-1+, Telomerase+, PTEN-, BRAF-V600E This target combination does not utilize ILK overexpression.
  • P-Akt confers anoikis resistance by suppressing Bad and procaspase-9 assembly of the apoptosome and increases GLUT4 translocation to the cell membrane.
  • BRAF-V600E activates ERK1/2 signaling, which promotes mitogen-independent transcriptional activation of cyclins and CDKs and phosphorylates c-myc. This increases cell proliferation and provides an ERK- dependent mechanism for anoikis resistance.
  • ILK+, PDK-1+, Telomerase+, PTEN-, BRAF-V600E, GLUT4+ This strategy follows the ILK+, PDK-1+, Telomerase+, PTEN-, BRAF-V600E cell line strategy, but also includes GLUT4 overexpression. GLUT4 overexpression will enhance glucose transport through the cell membrane in an insulin-independent manner, which reduces reliance on PDK-1/pAkt signaling to stimulate GLUT4 translocation.
  • CRISPR-based cell engineering refers to a genetic modification method using a Cas9 enzyme and small guide RNAs (gRNAs).
  • the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system was first discovered in bacteria, which use it to defend against viral infection.
  • the Cas9 enzyme is an endonuclease that uses CRISPR sequences as a guide to cut matching viral DNA sequences.
  • Cas9 is complexed with 20 nucleotide guide RNA sequences attached with a RNA scaffold that match the CRISPR sequences and viral DNA. Cas9 unwinds double-stranded DNA, and once it finds a non-target sequence match to the sgRNA it binds to the protospacer adjacent motif (PAM) region downstream of the target sequence and initiates a double-stranded cut to the DNA.
  • PAM protospacer adjacent motif
  • Cas9 cleaves the DNA 3 base pairs upstream of the PAM region within the target sequence. This can lead to gene inactivation through non- homologous end joining or the insertion of new genes through homologous recombination.
  • a Cas9 mutant called Cas9n is a nickase that creates a single stranded break instead of a double stranded break. Cas9n cleaves only the target strand instead of the both the target and non-target strands. This creates a preference for homology directed DNA repair pathway, which decreases the likelihood of off-target insertion or deletion mutations and increases the efficacy of HDR.
  • Nonhomologous end joining is the DNA repair pathway that repairs breaks in double stranded DNA by joining/ligating two broken ends together without utilizing a homologous template.
  • This repair method is error prone and can lead to insertion or deletion (indel) gene mutations. Indels that occur within a coding exon can inactivate the gene through frameshift mutations or premature stop codons. Commonly used for knockout studies. This repair mechanism occurs when there is no DNA repair (genetic insert) template. This can be performed in mitotically inactive cells.
  • the homology-directed pathway (HDR) repairs single or double stranded breaks in DNA using a homologous template.
  • the repair template can either be double-stranded DNA or single-stranded DNA oligonucleotides (ssODNs).
  • ssODNs can be used to make small edits in the genome, such as adding a single base pairs to a gene sequence to create mutations. This must be performed in mitotically inactive cells.
  • Combination of cells with plant-based ingredients [00177] Manufacturing process for plant-based meat structuring [00178] Plant based products can be used to supplement cell-based meat products.
  • Plant-based meat process engineering can utilize manufacturing processes including but not limited to stretching, kneading, shear-cell processing, phase separation of biopolymer blends, spinning process, press forming, folding, layering, 3D printing, high moisture extrusion and low moisture extrusion, or a combination of the above outlined technologies, which allows cells to mixed into plant protein with aligned fibers and interspersed fatty marbling.
  • the cellular content of the product can be between 1-5%, 5-10%, 10-15%, 15-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60- 70%, or 70-80% of the mass of the product per 100g.
  • Plant-based ingredients that can be used in combination with cell-based meat products may include but are not limited to proteins, including soy protein, legume protein (such as pea, chickpea, lentils, and other types of beans), oilseed proteins (such as rapeseed and canola), wheat gluten, potato protein, hemp protein, mung beans, flavorants, including koji, miso, seaweed, and recombinant proteins, mycoprotein, including mycelium, mushrooms, shiitake mushrooms, fats and oils, including sunflower oil, rapeseed oil, canola oil, corn oil, palm oil, coconut oil, and soya oil, binders, including methyl cellulose, wheat gluten, xanthan gum, albumin, soy protein concentrate, soy flour, pectin, guar gum, carrageenan, transglutaminase, and coloring agents, including beet juice, betanin, and heme proteins.
  • proteins including soy protein, legume protein (such as pea, chickpea, lentils
  • a cell-based meat product may be produced that mimics the taste and sensory properties of a chosen cut of animal meat. For example, one may choose a preferred cut of meat from cattle, pork, or other mammal and create a cell-based meat product that mimics it.
  • Any mammal meat may be used as the inspiration for the cell-based meat product, including but not limited to bovine, caprae, cervidae, lagormorph, oves, rodent and suidae meat.
  • Bovine and porcine cells have different properties that have implications for the bioprocess and cell line requirements for each respective cell type.
  • Bovine meat [00185] The most savory beef cuts originate from the ribs of the cow. Rib-eye steak (derived from the longissimus) is the most flavorful cut of beef due to the high intramuscular fat and marbling content, while the round (derived from the biceps femoris) contains less marbling and fat content than rib eye.
  • the intramuscular fat (IMF) % of the biceps femoris in animals fed a low-fat diet is 2.5% in Dexter, 4.9% in Angus, and 6.8% in Wagyu x Angus crossbreeds (Table 1).
  • the fat content is 12.6% in Angus, 11.6% in Wagyu x Angus, and between 14.7- 26.4% in the proximal and distal cranial head of Japanese Black Wagyu cattle.
  • the IMF% of the longissimus thoracis is 2.3% in Dexter, 6.2% in Angus, 15.3% in Korean Hanwoo, and 31.5% in Japanese Black Wagyu animals.
  • the total IMF% from 21 muscles of Wagyu steers is 32%.
  • Table 1 Average intramuscular fat % and muscle fiber type in the biceps femoris and longissimus thoracis
  • Meat quality grading systems Cell-based meat products m be assessed according to meat quality standards established by their countries of sale. Many countries view intramuscular fat to be important content in meat, such as Korea, Japan, Australia, and the US. European countries, such as France and Germany, prefers leaner cuts of meat. The USDA establishes meat quality with the US Beef Grading System and the Japanese Meat Grading Association (JMGA) establishes meat quality in Japan. Both systems assess quality via intramuscular fat percentage, color, and maturity, and the yield of usable meat on the carcass. These systems differ in how they use quality metrics, however. Understanding the differences between these quality evaluation systems is critical for US competition with Japanese exports.
  • the marbling score of the longissimus muscle is evaluated at the 6th-7th rib interface, in comparison to the USDA marbling score, which is evaluated at the 12th- 13th rib interface.
  • the JMGA uses an overall meat quality scale 1-5 based on four meat quality assessments: beef marbling score (BMS); beef color standard (BCS); firmness and texture of meat; and beef fat standard (BFS).
  • BMS score is an estimate of minimum IMF% and ranks 1-12, with 1 being the least amount of marbling and 12 being the highest degree of marbling (Table 2).
  • the meat must be graded at BMS 3 or above to reach the acceptable minimum IMF of wagyu: 21.4%, and a BMS score of 12 contains at least 56.3% IMF (Table 3).
  • BCS assesses meat color and brightness using a 1-7 scale, where number 1 is a pale red and number 7 is a dark red. The best BCS is 3 or 4, where color and brightness are most strongly correlated (Table 2).
  • the firmness of meat is determined by the amount of exudate on the surface of the cut, and the degree of depression of the indented surface. Meat with high BMS should be firm and have less exudate, since it has higher water holding capacity.
  • the texture reflects a smooth or coarse surface of the cut (Table 2).
  • Meat is graded best when it is very firm with a smooth texture.
  • the BFS measures color, luster and quality of fat using a score between 1-7, where 1 is pale white and 7 is dark cream (Table 2).
  • the BFS is best between 1-4. After each of the quality scores are evaluated according to the 1-5 scale, the lowest of the scores becomes the overall meat quality score for the beef. [00190] Table 2.
  • Japanese and US beef quality scoring metrics [00191] Table 3. Japanese and US beef quality grades determined by intramuscular fat %
  • the USDA beef standard assesses meat quality through marbling score, meat color, and texture of cuts (which is associated with age) and grades good quality beef into three categories: select, choice, and prime (Table 2). Above 21.4% IMF%, marbling is ‘very abundant’ and graded prime+. Typically, prime US beef has 9.9-12.3% IMF, the marbling is ‘moderately abundant’ to ‘slightly abundant’, it’s bright red in color, and moderately firm in texture (Table 3).
  • Choice beef has a ‘moderate’ to ‘small’ amount of marbling (4-7.7% IMF), is light cherry red to slightly dark red in color, and is slightly soft in firmness.
  • Pork meat [00194] Two of the most in-demand pork products are pork belly, bacon, and pork loin. Pork belly is composed of multiple muscle groups: the cutaneous trunci, latissimus dorsi, pectoralis profundus, rectus abdominis, and internal and external abdominal oblique) and the fat content between these muscles. Bacon is the cured meat from the belly of the pork. Bacon has different names depending on the part of the animal it was derived, and ‘streaky bacon’ is the cut from pork belly.
  • the present invention provides methods and compositions of a personally designed cell- based cut of meat.
  • the following example combinations can generate immortal cell lines with anoikis resistance, insulin-independent insulin signaling, mitogen-independent cell cycle progression, and improved flavor and sensory properties of cell lines via overexpression of FAK and/or SRC or by reducing PTEN expression.
  • anchorage independent survival and growth can be conferred by manipulating cell signaling pathways that generate anoikis resistance. Failure to initiate anoikis can result in cell survival in suspension.
  • the present inventions provide methods for enhancing resistance to anoikis and mitogen independent enhanced proliferation and/or mitotic potential via overexpression of FAK and/or SRC.
  • Focal adhesion kinase is a non-receptor tyrosine kinase that provides signaling and scaffolding functions at sites of integrin adhesion.
  • FAK is involved in protein– protein-interaction adaptor functions at sites of cell attachment to the extracellular matrix (ECM), thereby contributing to focal-adhesion 'scaffolding'.
  • ECM extracellular matrix
  • FAK also transmits adhesion- dependent and growth-factor-dependent signals into the cell interior.
  • FAKs with its anti- apoptotic activity helps the cells with anoikis resistance (resistance to suspension induced death).
  • SRC is another family of non-receptor tyrosine kinases which transmit integrin- dependent signals pivotal for cell movement and proliferation.
  • c-Src is shown to bind constitutively and selectively to beta3 integrins through an interaction involving the c-Src SH3 domain and the carboxyl-terminal region of the beta3 cytoplasmic tail.
  • FAK activation causes autophosphorylation, which recruits Src, which further phosphorylates FAK and leads to a fully activated FAK enzyme.
  • FAK then activates the Akt and MAPK pathways.
  • FAK activates PI3K, which then recruits and activates Akt.
  • Akt activation promotes cell survival by several mechanisms.
  • the present inventions provide methods for insulin-independent glucose transport into the cell and enhancing the sensory profile of cells through overexpression of GLUT1, GLUT2, and GLUT3.
  • GLUT1, GLUT2, and GLUT3 may be used in place of or in addition to GLUT4 to achieve these cell properties.
  • knockdown refers to downregulating the expression of a gene or gene product(s). As a result of knockdown, the protein expression and activity will be reduced or ablated. In some embodiments, knockdown is achieved using antisense molecules which are introduced to downregulate the expression of the target gene. In some cases, antisense molecules downregulates the expression of target genes via RNA interference (RNAi). This could comprise a combination of one or more antisense molecules such as short interfering RNA (siRNA), a microRNA (miRNA) or short hairpin RNA (shRNA).
  • siRNA short interfering RNA
  • miRNA microRNA
  • shRNA short hairpin RNA
  • siRNA small interfering RNA
  • siRNA refers to a class of double stranded RNA.
  • siRNA is about 20-23 base pairs in length, similar to miRNA and operates within the RNA interference (RNAi) pathway.
  • RNAi RNA interference
  • siRNAs are highly specific and have the ability to reduce the translation of specific mRNAs and the protein expression of the target gene.
  • siRNA can be chemically synthesized as “siRNA oligonucleotides”. siRNAs can be delivered to cells using lipid-based methods, electroporation or other methods.
  • shRNA or “small hairpin RNA” or “short hairpin RNA” (used interchangeably) refers to another form of RNA interference (RNAi).
  • shRNA is synthesized within the cell by DNA-vector mediated production.
  • shRNA consists of 2 complementary 19- 22 bp RNA sequences linked together by a short nucleotide loop of 4-11 nt.
  • siRNA delivers the siRNA duplex directly to the cytosol, shRNAs are capable of DNA integration.
  • shRNA can be delivered to mammalian cells via infection of the cells with virally produced vectors such as adenoviral, retroviral, or lentiviral-based delivery, called “transduction”.
  • shRNA integrated stable cell lines can be created by selection with antibiotics (drug resistance) in combination with fluorescence markers such as green fluorescent protein (GFP) or red fluorescent protein (RFP).
  • shRNA vector can be constructed with oligonucleotide-based cloning and PCR-based cloning.
  • miRNA or “microRNA” (used interchangeably) refers to endogenous small single-stranded non-coding RNA molecules present in the genome that function in RNA silencing and post-transcriptional regulation of gene expression.
  • miRNA refers to a class of noncoding RNA that plays an important role in regulating gene expression. They are natively present in the genome which could be modulated in their expression using miRNA inhibitors.
  • miRNA refers to the synthetic or artificial miRNA which are chemically modified double stranded RNA molecules designed to mimic endogenous miRNA, resulting in down regulation of target mRNA translation due to mRNA degradation or sequestration.
  • miRNA is an artificially synthesized oligonucleotide as “miRNA mimetics” which is capable of assuming the regulatory role of natural miRNAs. Like siRNA and shRNA, double stranded RNA oligonucleotides with minimal chemical modifications are suitable miRNA replacements in vitro.
  • miRNA or antagomiR could be delivered by lipid-based delivery similar to siRNA delivery or by viral based delivery systems using adeno or retrovirus.
  • siRNA/shRNA/miRNA/antagomiR can be delivered to the cells using cationic polymer polyethylene imine (PEI).
  • PEI cationic polymer polyethylene imine
  • the term “dominant negative” refers to the mutation with the resulting mutant gene product and protein expression resulting in adversely affecting the normal, wild type gene product within the same cell by competitive inhibition by either competing for ligands, substrates and interacting partners. This dominant negative mutant protein overexpression could enable us to achieve the phenotype expected from the knockdown or knockout of the endogenous wildtype gene/protein.
  • the most studied dominant negative PTEN mutants are Cys-124 to Ser (C124S) and the Gly-129 to Glu (G129E).
  • C124S mutation generates a catalytically dead mutant with complete loss of PTEN phosphatase activity whereas the G129E mutation abrogates the phosphoinositide phosphatase function but retains activity towards phospho-peptides.
  • These dead mutants can dimerize with wildtype PTEN protein in a dominant negative manner by disrupting the function.
  • PTENR130G missense mutation generates a stable protein but is catalytically inactive (lipoid phosphatase function is lost).
  • PTENR130X and R233X nonsense mutations generate very unstable PTEN proteins that are almost undetectable mimicking heterozygous conditions.
  • the following describes methods by which various cell targets, for example, FAK and/or SRC, may be overexpressed and therefore result in cell lines having improved flavor and sensory properties.
  • the present invention also includes additional methods for overexpressing various cell targets, such as viral based methods for transduction and inducible gene expression approaches.
  • Viral methods include stable integration of transgenes with lentivirus or retrovirus transduction. Inducible expression systems can be used to achieve overexpression of genes upon the introduction of a signaling molecule.
  • Inducible methodology includes, but are not limited to, transcription factor/promoter activity regulation by ligand inducible transcription factor machinery e.g., cumate-on, cumate-off, tet-on, tet-off,), site-directed recombination technology (e.g., Cre-LoxP, flp-FRT), ligand binding receptor fusion technology (e.g., tamoxifen metabolite, steroid hormone receptor).
  • ligand inducible transcription factor machinery e.g., cumate-on, cumate-off, tet-on, tet-off,
  • site-directed recombination technology e.g., Cre-LoxP, flp-FRT
  • ligand binding receptor fusion technology e.g., tamoxifen metabolite, steroid hormone receptor.
  • Inducible expression system is a regulated expression of transgene. Inducible systems can be classified based
  • inducers include heavy metal ions, heat shock, antibiotics, steroid hormones, IPTG and dimerizer.
  • Inducible gene regulation by small molecules offers time- and dose-dependent control.
  • Inducible gene expression systems are favored over stable expression systems as they are mostly reversible and thus more flexible to use.
  • the protein expression may be titrated as per needs and may completely terminate the expression when not needed.
  • Muscle tissue of 3-4 cm2 (4-6 g) in size was harvested from the thigh muscle of a pig (1- month-old) or a 2-month-old cow’s hind leg (biceps femoris) from a local farm.
  • muscle tissue is cut into small pieces after removing blood vessels and fascia/connective tissue, suspended in a tissue digestion buffer containing DMEM, 1% penicillin/streptomycin and 0.5% collagenase IV (Worthington Bio), and incubated at 37°C for 60-90 minutes. Every 15 minutes the cell and tissue mixture was pipetted to improve tissue disintegration.
  • the cell mixture was further filtered using a 40 ⁇ m cell strainer, neutralized by adding fetal bovine serum, and the suspended cells were collected by a 5-minute centrifuge at 1,200 rpm at room temperature. Following at least one washing steps with PBS and centrifugation, the heterogeneous cell mixture was resuspended in Advanced DMEM/F12, 20% FBS, and 1x P/S (penicillin/streptomycin) and underwent a pre-plating technique. The cells were seeded in standard tissue culture plates to allow fibroblasts attachment overnight at 37°C in 5% CO 2 .
  • Cells between passage 1-4 may be used to generate cell lines.
  • Improving cell differentiation Cell differentiation may be improved by supplementing fibroblasts cultures with fatty acids, for example, oleic acid, linoleic acid & palmitoleic acid. As shown in FIG. 12, when compared to the promo cell media alone, cultures supplemented with fatty acid showed improved differentiation. In-addition, bovine myoblasts were partially transdifferentiated with a combination of MyoD knockdown (to block myogenic differentiation) and supplemented with FFAs to transdifferentiate to adipocytes. Bovine myoblasts grown in regular growth media (lacking FFAs) and myogenic differentiation media were used as negative controls. [00222] 3.
  • fatty acids for example, oleic acid, linoleic acid & palmitoleic acid.
  • FIG. 12 when compared to the promo cell media alone, cultures supplemented with fatty acid showed improved differentiation.
  • bovine myoblasts were partially transdifferentiated with a
  • a gRNA may be designed and assembled to direct the Cas9 nuclease to a first exon of the PTEN gene.
  • a gRNA may be designed to target the PTEN promoter, preventing transcription of the PTEN gene.
  • the gRNA and Cas9 enzyme may be delivered by electroporation nucleofection or lipid transfection into the cells.
  • the gRNA may target Cas9 to a DNA target sequence, where Cas9 may cut the DNA, creating a double stranded break (DSB) and an indel mutation.
  • DSB double stranded break
  • the inactivated form of PTEN may be unable to dephosphorylate PIP3 to PIP2, or the gene may be silenced.
  • HDR Homology Directed Repair
  • ILK, PDK-1, and Telomerase overexpression Three gRNAs may be designed and assembled to direct Cas9n nickase to upstream untranslated regions of the ILK, PDK1, and/or TERT genes. DNA insertions that may add a constitutively active promoter upstream of an AUG sequence start site of the ILK, PDK1, and/or TERT genes using a double stranded DNA template with upstream and downstream homology arms may be performed.
  • the promoter may be native to the cell, such as the native bos taurus beta-actin promoter for bovine cells, that activates transcription for constitutively expressed genes. This technique may generate overexpression of wild type ILK, PDK1, and TERT genes. A double stranded repair template containing the constitutively active promoter may be needed for each gene, since the homology arms must match upstream and downstream sequences of the target sequence for each gene.
  • the three gRNAs, Cas9n enzyme, and three repair templates may be delivered by electroporation nucleofection or lipid transfection into the cells.
  • the gRNA may target Cas9 to the DNA target sequences, where Cas9n cuts the DNA, creating a single stranded break (SSB).
  • HDR may insert the constitutively active promoters upstream of the ILK, PDK1, and TERT genes.
  • three gRNA templates may be designed to target ILK, PDK1, and/or TERT transgenes under the control of constitutively active promoters to the Rosa26 locus of the genome.
  • the gRNAs, Cas9n enzyme, and three transgene DNA constructs may be delivered by electroporation nucleofection or lipid transfection into the cells.
  • B-Raf mutation using single-base substitution [00231] To generate a constitutively active form of B-Raf, BRAF-V600E, from the BRAF gene, thymine may be substituted with adenine at nucleotide 1799 to convert valine to glutamate using CRISPR-mediated single-base substitution and ssODNs.
  • a gRNA may target a Cas9n enzyme to the 1799 nucleotide of the B-Raf gene, and the ssODN sequence may comprise glutamate at 1799 instead of valine and wild type sequences upstream and downstream flanking homology arms.
  • C2C12 mouse myoblasts were genetically modified using the CRISPR knock-in technique outlined above. Genetic modification of C2C12 mouse myoblasts included mutations to the B-Raf gene, resulting in a B-Raf mutant knock-in (B-Raf V600E). A point mutation substituting Valine (V) for Glutamic acid (E) at amino acid position 600 of BRAF was inserted by CRISPR mediated knock-in.
  • V Valine
  • E Glutamic acid
  • the modified B-Raf gene was integrated into ROSA26, the modified sequence was validated and inducible-BRAF-V600E knock-in was verified via Sanger sequencing (FIG.4A). As compared with the wild type B-Raf gene, B-Raf V600E mutants showed enhanced ERK signaling as reflected by an increase in proliferation as measured by CellTiter-Glo® assay (FIG.9).
  • C2C12 myoblasts were genetically modified to include an INSR mutant gene having truncated alpha subunits to produce exon free insulin receptors. The mutant INSR sequence was then inserted by CRISPR mediated knock-in as described above.
  • FIG.4B shows the sequence insert confirmed by Sanger sequencing (CRO company GenScript performed CRISPR Knock-in (KI) projects).
  • KI CRISPR Knock-in
  • FIG.4B shows the sequence insert confirmed by Sanger sequencing (CRO company GenScript performed CRISPR Knock-in (KI) projects).
  • FGF2 Fibroblast growth factor 2
  • EGF epidermal growth factor
  • LIF leukemia inhibitory factor
  • Cell cultures comprising or absent insulin, Fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), leukemia inhibitory factor (LIF), and/or other growth factors may be tested on the cell lines.
  • Cell populations that maintain proliferation and transmembrane glucose transport in media formulations with low concentrations or absent of insulin and growth factors may be continuously passaged and selected for further cell line development.
  • Cells comprising necessary CRISPR edits may be selected via a positive selection process.
  • C2C12 cells having truncated alpha subunits to produce exon free insulin receptor were prepared as described above.
  • C2C12 cells with INSR KI were transfected with plasmids of GLUT1-4 (OriGene, Rockville, MD) and CellTiter-Glo® assay was performed. Cells were tested for ability to proliferate in reduced or insulin free conditions either alone or in combination with transient transfection of GLUT 1-4.
  • FIGs.6 and 7 show C2C12 cells with INSR KI alone or in combination with GLUT1-4 over-expression as having higher ATP activity compared to wild type C2C12 cells. Further, FIG. 7 shows that C2C12 cells with INSR KI and GLUT-4 over-expression showed higher ATP activity (cell proliferation) in reduced serum conditions as measure by the CellTiter-Glo® assay.
  • FIGs. 8A-D show that primary bovine myoblasts over expressing PDK1 or ILK alone or in combination with TERT, exhibit mitogen independent cell cycle progression as evidenced by higher proliferation compared to wild type control cells in serum gradient culture conditions as assessed by CellTiter-Glo® assay. [00240] 5.
  • Anchorage-independent growth Primary bovine myoblasts were genetically modified to over express ILK or PDK-1, alone or in combination with TERT; PAX3 or TBX-2, alone or in combination with TERT; PTEN knockdown; and C2C12 WT or C2C12 with BRAF and SRC were seeded in regular (untreated) 96-well plates and also seeded in anchorage resistant plates.
  • Anoikis resistance properties of genetically modified cells were analyzed using an anoikis assay based on MTT (3-(4,5-Dimethylthiazol-2-yl) dye.
  • 5A-D show measurement of absorbance indicating anoikis resistance by genetically modified cells with all the gene targets tested compared to wild type control cells following a 72-hour incubation.
  • PTEN knockdown in TERT over-expressing cells show significant anchorage independent growth compared to cells seeded and adhered in regular plates.
  • the results show that genetic modification allows the cells to better adapt to anchorage independent conditions as the readings from the absorbance are equivalent to their growth in regular plates.
  • siPTEN knockdown cells show statistically significant increased growth in anchorage independent condition compared to the growth in regular plates.
  • a sample of the cell lines showing reduced growth factor and insulin requirements may be plated onto agarose coated cell culture plates.
  • the cell lines that proliferate in suspension may be continuously passaged, which will positively select for the cells with anoikis resistance.
  • the cells that do not possess anoikis resistance will undergo anoikis and be removed from the cell culture during sub-culturing.
  • Mitotic capacity and senescence assay [00244] Cells that are able to grow in suspension may be continuous sub-cultured over long term periods to determine mitotic capacity. Cell lines that do not undergo senescence may be selected and used for commercial production.
  • Myogenesis protocol [00246] Bovine and porcine myoblasts were cultured in MGM until reaching 80% confluency.
  • fibroblast differentiation media MDM, Advanced DMEM/F12, 2% horse serum, 1% glutamate, 1% P/S
  • FGM fibroblast growth medium
  • fibroblasts were subsequently switched to Promocell medias.
  • the fibroblasts were adapted to pre-adipocyte growth medium (Cat. No. C-27410) for 1 week, then switched to pre-adipocyte differentiation medium (Cat. No. C-27436) for 72 hours. After differentiation, cells were switched to adipocyte nutrition medium (Cat. No. C-27438) for two weeks.
  • Oil Red O staining Lipid droplets inside mature adipocytes were visualized by Oil Red O (ORO) staining.
  • ORO Oil Red O
  • ORO was prepared by dissolving Oil Red O (Cat. No.00625-25G, Sigma Aldrich, St. Louis, MO) in 20 ml of 100% isopropanol, after mixing well, ORO was incubated at room temperature for 20 min.
  • Oil Red O Working Solution was prepared by adding 3 parts of Oil Red O Stock Solution to 2 parts of dH2O, mixing well, and incubated at room temp for 10 min. The solution was filtered with 0.2 ⁇ m syringe filter. The adipocyte cultures were then washed with PBS and fixed with formalin for 30 minutes.
  • a culture of bovine myoblast cells may be expanded to increase the biomass for a beef product. Upon reaching a certain biomass, the culture may be split into two vessels.
  • a first vessel may continue to grow myogenic cell population in myogenic media, while the cells in a second vessel may undergo transdifferentiation into adipogenic cells.
  • Cell culture media may be supplemented with any combination of the following free fatty acids at any concentration: SFAs include Myristic acid (14:0), Palmitic acid (16:0), Stearic acid (18:0), Arachidic acid (20:0), Behenic acid (22:0), Lignoceric acid (24:0), and Cerotic acid (26:0).
  • SFAs include Myristic acid (14:0), Palmitic acid (16:0), Stearic acid (18:0), Arachidic acid (20:0), Behenic acid (22:0), Lignoceric acid (24:0), and Cerotic acid (26:0).
  • PUFAs include omega-3, -6, and -9 fatty acids.
  • Omega-3 FAs include ⁇ -Linolenic acid (ALA, (18:3(n-3)), Stearidonic acid (SDA, (18:4 (n ⁇ 3)), Eicosatetraenoic acid (ETA, (20:4 (n ⁇ 3)), Eicosapentaenoic acid (EPA, (20:5(n-3)), Docosapentaenoic acid (DPA, (22:5 (n ⁇ 3)), and Docosahexaenoic acid (DHA, (22:6 (n ⁇ 3)).
  • ALA (18:3(n-3)
  • SDA Stearidonic acid
  • ETA Eicosatetraenoic acid
  • EPA Eicosapentaenoic acid
  • DPA Docosapentaenoic acid
  • DHA Docosahexaenoic acid
  • Omega-6 FAs include: Linoleic acid (LA, (18:2 (n ⁇ 6)), Gamma-linolenic acid (GLA, (18:3 (n ⁇ 6)), Calendic acid (18:3 (n ⁇ 6)), Dihomo-gamma-linolenic acid (DGLA, (20:3 (n ⁇ 6)), Arachidonic acid (AA, (20:4 (n ⁇ 6)), and adrenic acid (AdA, (22:4 (n ⁇ 6)).
  • Mead acid (20:3 (n ⁇ 9)) is an omega-9 PUFA.
  • Other omega-9s and omega-7 FAs are MUFAs.
  • Omega-7s include Palmitoleic acid (16:1(n-7)) and Paullinic acid (20:1(n ⁇ 7)).
  • Omega-9 FAs include Oleic acid (18:1, (n ⁇ 9)), elaidic acid (18:1 (n ⁇ 9)), gondoic acid (20:1 (n ⁇ 9)), Erucic acid (22:1(n-9)), and Nervonic acid (24:1 (n ⁇ 9)).
  • Adipogenic cultures may differentiate with the fatty acid mixture in the media until a threshold of FFAs are transported into intracellular lipid droplets, which may be an incubation period of 1-3, 3-7, 7-10, or more than 10 days in culture.
  • Wagyu beef product development strategy US beef has much lower IMF% than Japanese beef. The average highest prime grade contains ⁇ 12% IMF, and the average Wagyu BMS is a minimum of 21.4% IMF. Cell-based meat may give US beef a way to compete with Japanese Wagyu beef global market. Cells for cell-based Wagyu may be sourced from an American or Japanese Wagyu calf and grown ex vivo.
  • the intramuscular fat composition can be simulated by adding 21% or greater adipocytes to the meat product.
  • Determine target product characteristics [00258] Designing a cell-based Wagyu rib eye steak first requires assessing meat characteristics that influence sensory properties, such as muscle fiber type composition, fatty acid composition, IMF%, and chemical composition of the longissimus thoracis.
  • the average IMF of the thoracic muscle is 31.5%.
  • the fatty acid composition of the top 5 FFAs are 50.0% 18:1, 26.1% 16:0, 10.4% 18:0, 4.0% 16:1, and 2.8% of 14:0.
  • the chemical composition consists of 22% crude protein, 31% crude fat, and 47% moisture.
  • Cell isolation, cell line development strategy, culture process, and product formulation may be determined by the meat characteristics that influence sensory properties mentioned above.
  • skeletal muscle and pre-adipocytes may be isolated from the longissimus thoracis muscle of a Wagyu calf.
  • the skeletal muscle and pre-adipocyte cell lines may undergo development to increase mitotic capacity for scale up.
  • the skeletal muscle cells may be grown in single cell suspension for scale up, then undergo a differentiation step.
  • the cells may first undergo cell aggregation into micro tissues with low agitation rates inside the vessel. After sufficient tissue synthesis (below ⁇ 300um in thickness, the O2 diffusion limitation), differentiation media may be supplemented to the culture.
  • a cell population that can undergo adipogenesis may be grown in single cell suspension and differentiated with an adipogenic differentiation media to activate PPAR ⁇ expression, which would mature the cells into adipocytes.
  • 18:1, 16:0, 18:0, 16:1, and 14:0 FFAs may be supplemented in the culture media at rates that reflect the fatty acid composition of the muscle. If the adipocytes uptake each FFA at the same rate then a MUSA:SFA ratio may be obtained.
  • the FFAs may be used in combination at respective concentrations between 1nM-1000mM.
  • Production formulation may require recreating the chemical composition of the longissimus thoracis. Adipocytes and skeletal muscle cells may be grown to volumes that may yield a 2:3 ratio of skeletal muscles to fat cells. Cultured cells may not need to compose the entire 22% of crude protein and 31% of crude fat, since plant-based ingredients may add bulk and texture. However, since IMF is the hallmark of Wagyu beef, cell-based products may be held up to the JMGA standard of BMS, and as a result, require a higher percentage (%) of crude fat.
  • the cells and plant- based ingredient may then be blended and hydrated to optimize moisture content closer to around about 47%.
  • a food printer may then be used to print the skeletal muscle blend with a network of intramuscular fat.
  • siRNA and shRNA mediated knockdown of PTEN [00266] Transient transfection of siRNA [00267] Transient transfection of siRNA may be accomplished by seeding 0.1 million cells one day prior to transfection in a 6-well plate. Bovine, porcine, and/or C2C12 myoblasts may be transfected with 100 nM control scrambled siRNA or siRNA specific for PTEN by using Lipofectamine RNAimax. Cells to be harvested for mRNA and/or protein expression may be analyzed for efficiency of PTEN knockdown by RT-PCR and western blot analysis respectively at 48 or 72-hour post transfection.
  • siRNA mediated knockdown of PTEN was performed in TERT over-expressing primary bovine myoblasts as described above using commercially available siRNA targeting PTEN sequence (Horizon Discovery, Waterbeach, UK).
  • Lipofectamine RNAimax® was used to transfect primary bovine myoblasts with 100 nM control scrambled siRNA, siRNA specific for PTEN in TERT over-expressing cells, and/or TERT alone.
  • CellTiter-Glo® assay was used to determine cell viability. Cells were plated in 96-well plates at a starting cell density of 5000 and RLU (relative luminescence units) was measured according to the manufacturer’s instructions.
  • TERT over- expressing cells comprising the PTEN knockdown displayed increased cell viability as compared to the siScrambled control and cells over-expressing TERT alone.
  • cell proliferation may also be analyzed by BrDU proliferation assays, flow cytometry, analysis of cell cycle genes such as p21, pRb, cyclin D1 by RT-PCR and western blot analysis.
  • shRNA mediated knockdown of PTEN [00271] Myoblasts may be seeded at approximately 60-70% confluency in a 24-well plate and incubated overnight prior to infection the next day. Media may be removed and replaced with 500uL of fresh growth media per well.
  • polybrene may be added to each well in such a way to yield a final concentration of 8 ⁇ g/mL.
  • 1mL of viral supernatant may then be added to the wells and plates may be spun at 1-3 hours using a swing- bucket rotor. Supernatant may then be removed, and cells may be replenished with 1mL of growth media. The cells may then be selected 48-72 hours later using antibiotics as the lentiviral and/or retroviral vector may carry antibiotic resistant genes. As a result of the antibiotic resistance, those cells that did not receive the shRNA may be eliminated.
  • the cells may also carry marker genes, for example, GFP or RFP, to assess transduction efficiency via FACS analysis.
  • cell proliferation may be analyzed by BrDU proliferation assays, flow cytometry and analysis of cell cycle genes such as p21, pRb, cyclin D1 by RT-PCR and western blot analysis.
  • BrdU proliferation assays The proliferation capacity of myoblasts with PTEN knockdown may be assessed by BrdU incorporation assays. Cells may be assessed with 10 uM BrdU for 30 minutes. Cells may then be fixed and stained with anti-BrdU antibody. BrdU stained cells may then be analyzed for the percentage of BrdU positive cells using fluorescence microscopy.
  • Cells may be trypsinized and collected by centrifugation. A resulting cell pellet may then be washed at least once using PBS and centrifuged again at 1,200 rpm for 3 minutes. Cells may then be fixed with 70% ethanol while vortexing to avoid clumping of cells. [00277] Cells with ethanol may then be stored at about around -20 0 C for at least 24 hours. Later, cells may be washed with 1X PBS and centrifuged at 1,200 rpm for 5 minutes, 10 ⁇ g/ml propidium iodide solution may be mixed with RNAase A for at least 30 minutes at about room temperature.
  • cell proliferation will be analyzed by BrDU proliferation assays and analysis of cell cycle genes such as p21, pRb, cyclin D1 by RT-PCR and western blot analysis.
  • cell cycle genes such as p21, pRb, cyclin D1 by RT-PCR and western blot analysis.
  • protein lysates will be analyzed for phos-Akt and total AKT.
  • Myoblasts may be transfected with empty vector and plasmids encoding, for example, PAX3, TBX2, ILK1, PDK1, FAK, SRC, GLUT1-4, and/or TERT, by electroporation or using a lipid-based transfection reagent such as Lipofectamine 3000/Lipofectamine plus reagent. Cells may be harvested for mRNA/protein expression and analyzed for over expression 48 or 72-hour post transfection by RT-PCR and/or western blot analysis respectively.
  • a lipid-based transfection reagent such as Lipofectamine 3000/Lipofectamine plus reagent.
  • Transient transfection of commercially available plasmids from OriGene for example, GLUT1-4, PDK-1, ILK, C-JUN and FOS, was done in C2C12 and primary bovine myoblasts using Lipofectamine. Briefly, as described above, lipid-based transfection reagent Lipofectamine 3000/Lipofectamine plus reagent was used to create transgenic expression constructs comprising ILK or PDK-1, either alone or in TERT over-expressing primary bovine myoblasts.
  • FIGs.2A-D show cell viability/cell proliferation (mitotic capacity) for primary bovine myoblasts over-expressing ILK, PDK-1, TBX2, PAX3 and/or TERT. As shown in FIGs. 2A and 2B, metabolic activity of the cells was monitored using measurement of ATP synthesis by CellTiter Glo® assay (Promega, Madison, WI) with ATP being an indicator of number of viable cells.
  • CellTiter Glo® assay may also measure cell proliferation.
  • Primary bovine myoblasts were transiently transfected to over-express ILK or PDK-1, either alone or in combination with TERT and cell viability was assessed using the CellTiter-Glo® assay.
  • Primary bovine cells were seeded into 96-well plates at a starting cell density of 5000 cells, Relative Luminescence Units (RLU) was observed 48-hours post- transfection.
  • RLU Relative Luminescence Units
  • Cell viability was analyzed via CellTiter-Glo® assay, which measures cell proliferation and viability based on ATP synthesis of metabolically active cells. Accordingly, ATP synthesis results in increased luminescence signal and indicates cell viability and/or cell proliferation.
  • FIG 2A shows that over-expression of ILK and PDK-1, either alone or in combination with TERT, increased the proliferation of cells (mitotic capacity of cells) as reflected by increased luminescence signal obtained from the ATP synthesis.
  • FIG.2B shows that cells over-expressing TBX2 or PAX3, either alone or in combination with TERT displayed enhanced mitotic capacity when compared with untreated or GFP control cells.
  • cell proliferation was quantified by cell count at different time points using trypan blue followed by manual counting using hemocytometer or automated cell counter. Viable cells vs. non-viable cells were determined based on the trypan blue staining.
  • FIG. 2C-D shows that C2C12 myoblasts with CRISPR mediated stable knock in of wild type SRC displayed enhanced proliferation as measured by CellTiter-Glo® assay.
  • Cell proliferation may also be analyzed by BrdU incorporation assays.
  • cell proliferation may be measured by analyzing the percentage of cells in different sub-populations using a flow cytometer. Briefly, cells may be stained with propidium iodide mix (10 g/ml propidium iodide solution with RNase 200 g/ml) for 30 min at room temperature and strained using 40 m filters. Cells may be synchronized at the G1/S boundary using 1mM hydroxyurea and released in normal growth media. Cells will be synchronized at mitosis using 300 ng/mL of nocodozole.
  • Cell line resistance to apoptosis (anoikis)
  • Genetically modified cell line resistance to apoptosis will be analyzed using biomarkers such as cleaved caspase 3/7, cleaved PARP-1 and levels may be measured by RT-PCR or Western blot analysis.
  • Caspase 3/7 activity may also be measured by Caspase- Glo 3/7 assay system and/or CellTiter-Glo® assays.
  • Cell cycle may be analyzed by RT-PCR and western blot analysis of cell cycle genes such as p21, pRb, cyclin D1.
  • C2C12 myoblasts cell lines with stable CRISPR mediated knock-in of B-Raf mutation were prepared as described above.
  • FIG. 5C shows B-Raf mutants having increased anoikis resistance compared to wild type C2C12 cells.
  • FIG. 5D shows that anoikis resistance in cells having an siRNA mediated knockdown of PTEN or cells over- expressing TERT is enhanced compared to siScrambled control cells.
  • FIG. 5C shows enhanced anoikis resistance in SRC KI compared to wild type as evidenced by the results Anoikis assay performed with anoikis assay kit from ABCAM (Anoikis Detection Assay Kit; Cat no: ab211153).
  • Transfection & selection Primary bovine myoblasts may be transfected with a 1:10 ratio of an expression vector for GOI (Table 5) and a puromycin/neomycin resistant plasmid. Cells may then be selected 48 hours post transfection, in medium containing appropriate concentration of Geneticin (G-418)/puromycin. After 2-3 weeks, neomycin/puromycin resistant clones may be isolated, expanded and screened by RT-PCR and Western blot analyses. [00301] Table 5. List of Gene Targets
  • cells may be transfected with control vector (lenti/retroviral vector) and lenti and/or retroviral vector containing GOI using Calcium Phosphate transfection kit (Invitrogen)/Lipofectamine Plus reagent (Invitrogen) according to the manufacturer’s instructions.
  • control vector lenti/retroviral vector
  • lenti and/or retroviral vector containing GOI using Calcium Phosphate transfection kit (Invitrogen)/Lipofectamine Plus reagent (Invitrogen) according to the manufacturer’s instructions.
  • 40 ul of 2M CaCl2 may be mixed with 30 ug of DNA in an Eppendorf tube. The volume may be adjusted to 300 ul with sterile water. While vortexing, 300 ul of 2X HBS may be added slowly, and air may be bubbled through the DNA mixture. The resulting DNA complex may be incubated for about 15 minutes at about room temperature.
  • the resulting precipitate may be added to the Phoenix cells with 10 ml of media and incubated for 24 hours. Later, culture medium may be changed to bovine culture growth medium for virus collection from the transfected Phoenix cells. [00308] Every 24 hours, media containing virus may be collected and filtered using 0.45 ⁇ m syringe filter and stored at about -80 0 C. On the 1 st and 2 nd day (the 4th and 5th day, respectively) viral supernatant containing the GOI, may be collected and filtered through 0.45 ⁇ m filters for transduction of target primary bovine/C2C12 myoblasts.
  • GOI over-expressing cells may be generated by transducing cells with virus generated from lenti/retroviral vector alone or lenti/retroviral vector containing GOI.
  • Infection may be carried out using bovine and/or C2C12 myoblasts growth medium comprising virus, for which appropriate concentration of polybrene (Sigma-Aldrich®, St. Louis, MO) may be added and incubated for about 8 hours at about 37 0 C. The media may be changed and replaced with normal growth media 8 hours post-infection. Cells may then be allowed to grow for another 24hrs before selecting with appropriate concentration of puromycin (Sigma-Aldrich®) for 48 hours.
  • polybrene Sigma-Aldrich®, St. Louis, MO
  • EEV CuO-MCS Enhanced Episomal Vector
  • SBI System Biosciences
  • Palo Alto, CA CuO-MCS Enhanced Episomal Vector
  • EEVs offers an advantage for non-integrating, non-viral gene expression and replicate in synchrony with the host cell. As such, gene expression may be stably inherited and may be used for long lasting expression up to several months both in vitro and in vivo without modifying the host genome.
  • CuO-MCS EEV vector may comprise a cumate-inducible promoter upstream of an MCS (cloning gene of interest), CymR and a puromycin selection cassette expression under the control of constitutive EF1 ⁇ promoter.
  • the cumate inducible reporter remains silent until the addition of cumate.
  • the transfected C2C12/primary bovine myoblasts may show a robust gene expression. The expression may be detected even after 72 days.
  • FLP-In System Three different vectors, commercially available from Thermo Fisher (Thermo Fisher Scientific, Waltham, MA), may be used to generate isogenic stable mammalian cell lines expressing gene(s) of interest.
  • the FLP-In system may include, (i). Flp-In target site vector, and pFRT/lacZeo to generate Flp-InTM host cell line(s).
  • the vector may comprise a lacZ- ZeocinTM fusion gene whose expression is controlled by the SV40 early promoter. (ii). Gene of interest may be then cloned into the pcDNA5/FRT expression vector.
  • the vector may also comprise an antibiotic resistance gene with a FRT site embedded in the 5' coding region.
  • a hygromycin resistance gene may lack a promoter and the ATG initiation codon; and (iii).
  • pOG44 plasmid which constitutively expresses the Flp recombinase under the control of the human CMV promoter.
  • the pOG44 plasmid and the pcDNA5/FRT vector comprising gene of interest may be co-transfected into the Flp-InTM host cell line.
  • Flp recombinase expressed from pOG44 may mediate a homologous recombination event between the FRT sites (integrated into the genome and on pcDNA5/FRT) such that the pcDNA5/FRT construct is inserted into the genome at the integrated FRT site. Insertion of pcDNA5/FRT into the genome at the FRT site brings the SV40 promoter and the ATG initiation codon (from pFRT/lacZeo) into proximity and frame with the hygromycin resistance gene. Thus, stable Flp-InTM expression cell lines can be selected for antibiotic resistance and expression of the recombinant protein of interest.
  • Doxycycline (1000 ng/mL) mediated inducible approaches were established in C2C12 myoblasts with CRISPR mediated knock-in of wild type SRC and BRAF mutant.
  • the genetic modification involving lentivirus mediated over expression of PAX3, TERT, & TBX2 were performed and over-expression was confirmed with GFP reporter marker.
  • FIG.10 shows GFP positive cells.
  • the genetic modification involving Doxycycline (Tet system) mediated inducible approaches were also established in C2C12 myoblasts with CRISPR-mediated knock-in of wild type SRC and BRAF mutant.

Abstract

The present disclosure provides materials, methods, techniques, cell lines, and strategies to produce and to improve flavor and pigmentation of cell-based meat.

Description

ANIMAL CELL LINE AND PROCESS DEVELOPMENT OF CULTIVATED MEAT PRODUCTS OBJECT OF THE INVENTION [00001] The object of the invention is to provide new materials, methods, techniques, cell lines, and strategies to produce and to improve flavor and pigmentation of cell-based meat. DESCRIPTION OF RELATED ART [00002] Cell-based meat is meat grown from isolated animal cells, instead of using the entire animal to produce meat products. The isolated cells are grown in cell culture and are used to develop cell lines that can be grown in bioreactors to produce animal free meat products. Cell- based meat has been called many things, such as cultured meat, cultivated meat, lab-grown meat, and in vitro meat. [00003] Palatability and perception of cultivated meat products will determine their success or failure in the market. Products that do not look like meat, such as products that are off-color compared to the animal meat product they are simulating, can be rejected by consumers. Additionally, cultivated meat products have to possess improved flavor over plant-based competitors and have to be likable enough to replace meat from animals in the diets of consumers. Presentation and sensory characteristics of cell-based meat products should be optimized. Cultivated meat products should also reach price parity with animal meat to be commercially viable. Consequently, new materials, methods, techniques and cell lines need to be developed to achieve the goal of growing cultured meat products. SUMMARY OF THE INVENTION [00004] The present invention provides materials, methods, techniques, cell lines, and strategies to produce cell-based meat, and to improve flavor and pigmentation of cell-based meat, via myogenesis, adipogenesis, increasing the mitotic capacity of cell lines, cell line immortalization, developing anoikis resistance of cell lines, and free fatty acid supplementation. [00005] The cells obtained from an animal possess inherent limitations that make them unsuitable for a large-scale and indefinite manufacturing process. Cell lines developed with increased mitotic capacity, however, may be scaled up making the cultivation of these cell lines more amenable to the cultivated meat manufacturing process. In an embodiment, the present invention provides a transgenic expression construct comprising a gene or genes selected from the group consisting of ILK, GLUT4, PDK-1, TBX2, Pax3, and/or telomerase, and a constitutively active promoter overexpressing said gene, when compared to the expression the wild-type promoter of said gene, wherein the transgene is inserted into the native genome of the cell. The present invention also provides a method comprising editing the nucleic acid base pair sequences of native promoters of ILK, GLUT4, PDK-1, TBX2, Pax3, and/or telomerase genes in order to increase gene transcription over gene transcription levels found in cells with unmodified promoters. In another embodiment the present invention provides a method for increasing mitotic capacity of cell lines by overexpressing ILK, GLUT4, PDK-1, TBX2, Pax3, telomerase and/or knocking out PTEN expression. [00006] Cell culture media often includes the use of expensive growth factors to support cell survival, proliferation, and differentiation, making large scale cell-line cultivation cost- prohibitive. Constitutive activation of the ERK1/2 pathway and insulin-independent glucose transport through activation of the PI3K/Akt pathway can enhance cell proliferation and reduce the amount of growth factor required, thereby lowering the cost of cultivating edible cell lines. In another embodiment, the present invention provides a composition comprising a genetically modified cell, wherein a genetic modification comprises mutations to the B-Raf gene, and wherein the modification results in enhanced ERK signaling in comparison to the wild type B- Raf. The genetic modification may be a substitution, deletion, insertion, duplication, inversion, or frameshift mutation. [00007] Similarly, reducing or eliminating insulin from cell culture media may significantly reduce cost of production. In another embodiment the present invention provides a composition comprising a genetically modified cell, wherein a genetic modification comprises modifications to the insulin receptor (INSR) gene. The genetic modification may be a substitution, deletion, insertion, duplication, inversion, or frameshift mutation. Genetically modified cells may contain artificial plasmids (“Synthetic Plasmids”) to amplify or produce many copies of gene of interest (GOI). [00008] Cell lines must be able to undergo myogenesis and adipogenesis. The capacity of a cell line to undergo myogenesis and adipogenesis affects meat quality. In another embodiment, the present invention provides a method of improving cell differentiation comprising supplementing adipocyte cultures with fatty acids in any combination of monounsaturated fatty acids (MUFA); polyunsaturated fatty acids (PUFA); and saturated fatty acids (SFA). A PUFA, MUFA, or SFA may be added alone, or any combination of PUFAs, MUFAs and SFAs may be added. The MUFA may be oleic acid. The method may further comprise supplementing adipocyte cell cultures with bovine preadipocytes sourced from the subcutaneous stromal vascular cells treated with Oleic acid. The PUFA may include α-Linolenic acid, Stearidonic acid, Eicosatetraenoic acid, Eicosapentaenoic acid, Docosapentaenoic acid, Docosahexaenoic acid, Linoleic acid, Gamma-linolenic acid, Calendic acid, Dihomo-gamma-linolenic acid, Arachidonic acid, Adrenic acid, and Mead acid. The MUFA may include Oleic acid, Palmitoleic acid, Paullinic acid, Omega-9 FAs, elaidic acid, Gondoic acid, Erucic acid, and Nervonic acid. The SFA may include Myristic acid, Palmitic acid, Stearic acid, Arachidic acid, Behenic acid, Lignoceric acid, and Cerotic acid. [00009] In another embodiment, the present invention provides a method of improving flavor of cell-based fat comprising supplementing adipocyte cultures with a fatty acid, wherein the fatty acid is polyunsaturated fatty acids (PUFA), monounsaturated fatty acids (MUFA), and/or saturated fatty acids (SFA). A PUFA, MUFA, or SFA may be added alone, or any combination of PUFAs, MUFAs and SFAs may be added. The MUFA may include Palmitoleic acid, Paullinic acid, Omega-9 FAs, elaidic acid, Gondoic acid, Erucic acid, and Nervonic acid. The SFA may include Myristic acid, Palmitic acid, Stearic acid, Arachidic acid, Behenic acid, Lignoceric acid, and Cerotic acid. In another embodiment, the present invention provides a method of transdifferentiating myogenic cells into adipogenic cells comprising supplementing the cell culture media with SFAs, MUFAs, and/or PUFAs. [00010] Genetically modified cell lines may be cultivated to mimic the taste and texture of animal meat products. In another embodiment, the present invention provides a composition comprising a genetically modified bovine or porcine myoblast and a genetically modified bovine or porcine adipoblast. In another embodiment, the present invention provides a composition comprising myoblasts, mesenchymal stem cells (MSCs), intramuscular fibroblasts, iPSCs, adipoblasts, adipocytes, preadipocytes, and/or fibro-adipoblasts. [00011] In another embodiment, the present invention provides a cell-based meat product comprising genetically modified fat cells and genetically modified muscle cells. [00012] To increase production yields while simultaneously lowering production costs, cell lines used for cultivated meat are designed to grow in single cell suspension without undergoing anoikis. In another embodiment, the present invention provides a method of preventing anoikis comprising generation of a cell line with constitutively overexpressed ILK, PDK-1, TBX2, and/or PAX3. The invention also provides a method of preventing anoikis comprising generation of a cell line with a mutated form of B-Raf that has higher activity levels than wild type B-Raf. The invention also provides a method of preventing anoikis comprising generation of a cell line with PTEN gene knocked out. [00013] As previously mentioned, eliminating or reducing the amount of insulin or growth factors required for cell culture media lowers the cost associated with cultivating edible cell lines. In another embodiment, the present invention provides a method of reducing or eliminating insulin from cell culture media comprising truncating the alpha subunits in an INSR gene to produce an exon-free insulin receptor. The present invention also provides a method of reducing or eliminating insulin from cell culture media comprising overexpressing PDK-1, GLUT4, TBX2, and/or PAX3. The present invention provides a method of reducing or eliminating insulin from cell culture media comprising knocking out PTEN. [00014] The cells obtained from an animal possess limitations that make them unsuitable for a large-scale and indefinite manufacturing process. The aging process depletes the proliferative capacity of cells by shortening telomeres through each cell division. The limited number of cell divisions due to telomere shortening is called the Hayflick limit. In another embodiment, the present invention provides a method for bypassing the hayflick limit comprising overexpressing telomerase. [00015] Immortalized cell lines with anoikis resistance, insulin-independent insulin signaling, and mitogen-independent cell cycle progression enhances flavor and sensory properties of edible cell lines and lowers the cost of production. In another embodiment, the present invention provides a method for mitogen-independent cell cycle progression comprising generation of a cell line with a mutated form of B-Raf that has higher activity levels than wild type B-Raf. [00016] Flavor, pigment, and product quality can be enhanced by constitutive activation of the insulin/GLUT4 pathway, which drives myogenic and adipogenic differentiation. Whereas inactivation of PTEN results in activation of the PI3K/AKT pathway and subsequent increase in cell cycle progression, migration and survival. And overexpressing telomerase helps the cells bypass the hayflick limit by maintaining telomere length through repeated cell divisions, which is necessary to develop scalable cell lines. In another embodiment, the present invention provides a cell line wherein the cell line bypasses the hayflick limit via telomerase overexpression; is resistant to anoikis via activation of integrin binding signal transduction via PTEN knockout with increased PIP3, PIP3/PDK-1/pAkt mediated anti-apoptotic signaling, ILK/pAkt mediated anti-apoptotic signaling, ERK1/2 anti-apoptotic signaling; provides insulin-independent glucose transport via a method selected from the group consisting of PIP3- mediated phosphorylation of aPKCs, PDK-1-mediated phosphorylation of aPKCs, pAkt- mediated AS160 inhibition, constitutive insulin receptor activation and GLUT4 overexpression; provides mitogen-independent cell cycle progression via ERK1/2-mediated cell cycle progression; and which has enhanced sensory properties via a method selected from the group consisting of PIP3-mediated phosphorylation of aPKCs, PDK-1-mediated phosphorylation of aPKCs, pAkt-mediated AS160 inhibition, constitutive insulin receptor activation, GLUT4 overexpression. [00017] Generating a constitutively active insulin receptor pathway provides sensory benefits to myogenic and adipogenic cell lines that can improve flavor, pigment, and overall product quality. In another embodiment the present invention provides methods for improving cell resistance to anoikis and enhancing mitogen independent enhanced proliferation and/or mitotic potential comprising overexpressing of wild type or mutant FAK and/or SRC. [00018] In another embodiment the present invention provides methods for insulin- independent glucose transport into the cell comprising overexpressing wild type or mutant GLUT1, GLUT2, and GLUT3. The present invention also includes methods for enhancing the sensory profile of cells comprising overexpressing wild type or mutant GLUT1, GLUT2, and GLUT3. [00019] In another embodiment the present invention also includes methods for overexpressing various cell targets via viral based methods comprising transduction and/or inducible gene expression approaches. [00020] Inactivation of PTEN results in activation of the PI3K/AKT pathway and subsequently leads to an increase in cell cycle progression, migration and survival. In another embodiment the present invention provides methods for reducing PTEN activity comprising siRNA and/or shRNA mediated knockdown of PTEN protein translation, miRNA-mediated transcriptional interference of PTEN expression, and overexpression of dominant negative form of PTEN, which may contain one or more inactivating mutations. BRIEF DESCRIPTION OF THE DRAWINGS The invention will be more fully understood and further advantages will become apparent when reference is made to the following detailed description of the embodiments and the accompanying drawing in which: [00021] FIG.1 shows Japanese Meat Grading Association [Japan Meat Grading Association (JMGA) Beef Carcass Trading Standards. (2014)] and USDA meat quality standards [Beef Grading Shields. USDA.gov https://www.ams.usda.gov/grades-standards/beef/shields-and- marbling-pictures.]. It provides the (A) official picture standard of BMS (Beef Marbling Standard), (B) BCS (Beef Color Standard), and (C) BFS (Beef Fat Standard) by the Japan Meat Grading Association. (D) Official picture standards of the USDA beef grade shield; [00022] FIG.2 shows cell viability (mitotic capacity) of primary bovine myoblasts as indicated by RLU (Relative Luminescence Units). FIG.2A shows viability for cells transiently transfected to over-express ILK or PDK-1(p ≤ 0.01), alone or in combination with TERT (p ≤ 0.02) . FIG.2B shows viability of cells that have undergone lentiviral mediated over-expression of TERT, PAX3, or TBX2, alone or in combination with TERT as compared to untransduced or EGFP control cells (p ≤ 0.05). FIGs.2C and 2D show proliferation of cells genetically modified to over-express TERT, PAX3, or TBX2 as compared to GFP control cells (p ≤ 0.05). Error bars represent means with SD; [00023] FIG. 3 shows viability of cells having a combination of siRNA specific for PTEN or TERT alone as compared to the siScrambled control. Error bars represent means with SD; [00024] FIG. 4A depicts the Sanger sequence for inducible-BRAF-V600E knock-in. FIG. 4B depicts the Sanger sequence for INSR mutant knock-in; [00025] FIG. 5 shows cell line anoikis resistance in primary bovine myoblasts or C2C12 cells as indicated by absorbance. FIG 5A shows anoikis resistance for cells transiently transfected to over-express ILK or PDK-1, alone or in combination with TERT. FIG.5B shows anoikis resistance of cells that have undergone lentiviral mediated over-expression of TERT, PAX3, or TBX2, alone or in combination with TERT as compared to untransduced or EGFP control cells. FIG.5C shows anoikis resistance in C2C12 cells genetically modified via CRISPR to over-express B-RAF or SRC compared to wild type. FIG.5D shows anoikis resistance in cells having a combination of siRNA specific for PTEN or TERT alone as compared to the siScrambled control. Error bars represent means with SD; [00026] FIG. 6 shows viability of C2C12 cells with INSR KI alone or in combination with GLUT1-4 in varying serum free environment. Error bars represent means with SD; [00027] FIG. 7 depicts a serum curve and shows viability of C2C12 cells with INSR KI alone or in combination with GLUT1-4 in varying insulin conditions. Error bars represent means with SD; [00028] FIG. 8 shows cell viability of primary bovine myoblasts genetically modified to over-express ILK or PDK-1, alone or in combination with TERT as indicated by RLU (Relative Luminescence Units). FIG 8A shows viability of cells in the absence of FBS (fetal bovine serum), FIG.8B shows cell viability for cells cultured in 5% FBS (fetal bovine serum), FIG.8C shows cell viability for cells cultured in 10% FBS, and FIG.8D shows cell viability for cells cultured in 20% FBS. Error bars represent means with SD; [00029] FIG. 9 shows cell viability/mitotic capacity for cells genetically modified to over- express SRC or BRAF. Error bars represent means with SD; [00030] FIG. 10 is an example of GFP tagged cells; [00031] FIG. 11 shows cell viability for cells genetically modified to over-express C-JUN or FOS in combination with TERT. FIG.11A shows cell viability in the presence of Laminin, and FIG.11B shows cell viability in the absence of Laminin; and [00032] FIG. 12 shows bovine fibroblasts cultured in regular PromoCell media alone or in combination with FFAs and bovine myoblasts transdifferentiation when cultured in PromoCell with siRNA targeting MyoD (Horizon Discovery, Waterbeach, UK) or in combination with FFAs and FBS. Bovine myoblasts grown in the regular growth media and myogenic differentiation media used as negative control. DETAILED DESCRIPTION OF THE EMBODIMENTS [00033] The present invention provides methods of creating cell-based meat and increasing palatability of cell-based meat, as well as cell-based meat products. The present invention provides strategies for producing immortalized cells lines and anoikis resistance for cultivated meat products. The present invention also provides strategies for reducing the use of insulin in cell culture media. The present invention also provides methods for improving sensory properties of cultivated meat, including use of free fatty acids, and constitutive activation of the insulin/GLUT4 pathway. [00034] Further, the present invention provides cell-based meat products and methods with one or more of the following properties: [00035] Property 1 – bypassing the hayflick limit of cell lines through telomerase overexpression. [00036] Property 2 – anoikis-resistance by activating intracellular integrin binding signal transduction pathways. This can be accomplished via PTEN knockout with increased PIP3 signaling, PIP3/PDK-1/pAkt mediated anti-apoptotic signaling, ILK/pAkt mediated anti- apoptotic signaling, and/or ERK1/2 anti-apoptotic signaling. [00037] Property 3 – insulin-independent glucose transport into the cell. This can be accomplished through PIP3-mediated phosphorylation of aPKCs, PDK-1-mediated phosphorylation of aPKCs, pAkt-mediated AS160 inhibition, constitutive insulin receptor activation, and/or GLUT4 overexpression. [00038] Property 4 – mitogen-independent cell cycle progression through activation of the ERK1/2 signaling pathway. [00039] Property 5 – enhancement of sensory profile of cells. This can be accomplished through PIP3-mediated phosphorylation of aPKCs, PDK-1-mediated phosphorylation of aPKCs, pAkt- mediated AS160 inhibition, constitutive insulin receptor activation, and GLUT4 overexpression. [00040] Property 6 – bypassing the hayflick limit of cell lines through telomerase overexpression and knockdown/knockout of PTEN expression or over expression of dominant negative form/miRNA targeting PTEN for degradation. [00041] Property 7 – anoikis-resistance by activating intracellular integrin binding signal transduction pathways. This can be accomplished via PTEN knockdown/knockdown or over expression of dominant negative form/miRNA.with increased PIP3 signaling, FAK mediated anti- apoptotic signaling, SRC mediated anti- apoptotic signaling. [00042] Property 8 – insulin-independent glucose transport into the cell. This can be accomplished through GLUT1, GLUT2, GLU3, and GLUT4 overexpression. [00043] Property 9 – mitogen-independent cell cycle progression through activation of the ERK1/2 signaling pathway via overexpression of FAK and SRC. [00044] Property 10 – enhancement of sensory profile of cells. This can be accomplished through GLUT1, GLUT2, GLU3, and GLUT4 overexpression. DEFINITIONS [00045] The Japanese Meat Grading Association (JMGA) establishes meat quality in Japan. The JMGA uses an overall meat quality scale 1-5 based on four meat quality assessments: beef marbling score (BMS); beef color standard (BCS); firmness and texture of meat; and beef fat standard (BFS). [00046] Beef marbling score (BMS) is an estimate of minimum intramuscular fat (IMF)%. [00047] Beef color standard (BCS) assesses meat color and brightness. [00048] Beef fat standard (BFS) measures color, luster and quality of fat. [00049] Animal meat is a staple of Western diets. Beef and pork are two of the most popular types of meat in western diets, and other animals such as goat, deer, rabbit, and sheep are consumed as well. Animal meat has specific sensory properties associated with its appearance and taste. The sensory properties of the meat arise from the muscle and fat cells that comprise the meat. Animal meat contains skeletal muscle tissue with intramuscular fat that are created from myogenesis and adipogenesis. Natural myogenic and adipogenic mechanisms in the animal’s body produce optimized sensory experiences upon consumption of animal meat, but cell-based meat is produced in an artificial environment. Consequently, materials and methods are needed that can recreate the experience of eating animal meat using in vitro cell-based meat products. Moreover, cell-based meat products are needed that recreate the taste and texture, and therefore the experience of eating particular animal meat (for example, pork or beef). [00050] Cells used for cell cultured meat can be derived from various kinds of stem or precursor cells found in animal embryos, bone marrow, or muscle tissue. Induced pluripotent stem cells (iPSCs) can also be used, which, along with embryonic stem cells, can be differentiated into any cell type in the body, such as skeletal muscle cells. Regardless of the initial cell population, the manufacturing process must result in the production of cells (e.g., muscle and fat) found in animal meat. The three dominant cell types that influence meat flavor, juiciness, and texture are skeletal muscle cells, intramuscular fat cells, and connective tissue cells called fibroblasts. [00051] Cell lines must be able to undergo myogenesis (the formation of multinucleated, contractile skeletal muscle cells) and adipogenesis (the development of fat cells containing intracellular lipid droplets). The capacity of a cell line to undergo myogenesis and adipogenesis affects meat quality. [00052] Myogenesis [00053] Skeletal muscle cells are the main source of the umami flavor in meat that contains muscle-specific proteins. These cells contribute to meat flavor and pigment. Myogenesis begins during embryogenesis and is characterized by mononuclear muscle progenitor cells fusing into multinucleated muscle fibers. This results in the production of high-density skeletal muscle cytoskeletal proteins, myosin heavy chain, and myoglobin, which serves as an oxygen reservoir for cells. Myoglobin is red in hue and its pigmentation arises from various redox forms and concentration within skeletal muscle cells. Myogenesis is controlled by sequential expression of myogenic transcription factors and begins with the activation of quiescent Pax7+ expressing satellite cells, a muscle stem cell. In their activated form, Pax7+MyoD+Myf5+ satellite cells undergo asymmetric division to produce self-renewing satellite cells and myoblasts committed to undergo myogenesis. Pax7+MyoD+Myf5+ myoblasts undergo symmetric division to expand in numbers, then commit to terminal differentiation as Pax7-MyoD-Myf5- MyoD+Myogenin+ mononuclear myocytes committed to exit the cell cycle and terminally differentiate. These mononuclear cells begin to fuse into multinucleated muscle fibers, or myotubes, and develop sarcomeres, which are the contractile unit of the cell. Sarcomeres are composed of myosin heavy chain and actin filaments that consume ATP (adenine triphosphate) to slide against one another to shorten the length of the sarcomere and consequently the length of the muscle fiber. The unity of sarcomeres and muscle fibers contracting together creates the contractile force of muscle organs. [00054] Adipogenesis [00055] Adipogenesis regulates energy metabolism in the body by collecting free fatty acids (FFAs) bound to albumin from the blood into adipose cells for future energy use and releasing them back into the blood when glucose levels are low. Preadipocytes undergo differentiation into adipocytes during adipogenesis. Adipocytes accumulate intracellular lipid droplets that provide juiciness and additional flavor to meat through lipogenesis. Adipogenesis initiates in intramuscular preadipocytes with growth arrest and morphological changes that shifts the cells from a fibroblastic spindle shape to a rounded morphology. Preadipocytes start producing CCAATT enhancer binding proteins β (C/EBPβ) and CCAATT enhancer binding proteins δ (C/EBPδ) transcription factors, which activate gene transcription of peroxisome proliferator-activated receptor γ (PPARγ) and CCAATT enhancer binding proteins α (C/EBPα) transcription factors. PPARγ is a master regulator of adipogenesis and a ligand-activated nuclear membrane receptor protein that can translocate into the nucleus to activate gene expression. PPARγ and C/EBPα begin transcribing genes that promote insulin sensitization, glucose utilization, and adipocyte maturation, such as insulin receptor; fatty acid synthase; adipocyte protein 2 (aP2), also known as fatty acid binding protein 4 (FABP4); and glucose transporter type 4 (GLUT 4). These proteins facilitate transportation of FFAs and glucose into the preadipocyte cytosol and promote intracellular lipogenesis. [00056] Lipogenesis encompasses de novo fatty acid synthesis, the production of FFAs, and triglyceride synthesis, which esterifies FFAs to glycerol to produce triglycerides that incorporate into lipid droplets. Lipolysis occurs when energy levels enter a fasting state. FFAs are enzymatically cleaved from intracellular triglycerides and transported to the blood. Cells in need of energy uptake the FFAs for β oxidation in the mitochondria to produce ATP. Fasting activates transcription of peroxisome proliferator-activated receptor alpha (PPARα) in the liver, a transcription factor that activates a set of genes involved in fatty acid oxidation. The activation of PPAR-β/δ enhances fatty acid oxidation in skeletal muscle and adipose tissue. PPAR-β/δ ligands include several 14- to 18-carbon saturated fatty acids as well as 16- to 20-carbon polyunsaturated fatty acids. [00057] Adipogenesis can be initiated in vitro by media components that produce metabolic changes in preadipocytes. For example, FBS, high glucose, insulin, dexamethasone, IBMX (3- isobutyl-1-methylxanthine), thiazolidinediones (TZDs), and FFAs may be used to induce adipogenesis in preadipocytes. Dexamethasone is an anti-inflammatory corticosteroid used in many medical applications. IBMX is a small chemical compound which together with dexamethasone activates expression of C/EBPβ and C/EBPδ. TZDs are ligands that activate PPARγ to initiate downstream adipogenic gene transcription. Insulin, glucose, and free fatty acids contribute to intracellular lipogenesis by mediating cellular metabolism. Insulin binds to insulin receptors that activate intracellular signaling pathways to recruit the GLUT 4 transport protein to the plasma membrane, allowing glucose to enter into the cell. Glucose converts to Acetyl-CoA via the glycolytic pathway, which is used to produce fatty acids during de novo lipogenesis. Alternatively, FFAs such as erucic acid, elaidic acid, oleic acid, palmitoleic acid, myristoleic acid, phytanic acid, and pristanic acid can be transported through the cell membrane and solubilized into the cytosol via aP2. PPARγ is also activated via FFA binding. [00058] Increasing mitotic capacity of cell lines [00059] Cell line development, or cell line engineering, begins with extracting individual cells from a tissue biopsy of an animal. The cells obtained from an animal possess inherent limitations that make them unsuitable for a large-scale and indefinite manufacturing process. Aging occurs at the cellular level, and the harvested cells isolated from an animal will also age during culture. The aging process depletes the proliferative capacity of the cells by shortening telomeres through each cell division. Once the telomeres have shortened to their final length, they are no longer able to divide, and the cells undergo senescence. The limited number of cell divisions due to telomere shortening is called the Hayflick limit. The Hayflick limit is a threshold that limits the biomass that can be obtained from primary animal cells. Culturing meat necessitates extending the ability of edible cells to undergo cycles of mitosis by extending or removing a limit to the total number of cell divisions. Engineered cell lines can extend the Hayflick limit and allow more cell divisions while still being subject to the limit. Immortalized cell lines completely bypass cellular aging to allow infinite expansion, like HeLa cells and C2C12s. [00060] For a given cell population, the mitotic capacity is the potential for the cell to undergo mitosis: the ratio of the change in mitotic index over change in time. Innate mitotic capacity (IMC) is the natural capacity of primary cells to undergo mitosis until they reach the Hayflick limit. Enhanced Mitotic Capacity (EMC) extends the Hayflick limit past its innate number of cell divisions, although the cells are still eventually subject to limited rounds of mitosis. Unlimited mitotic capacity (UMC) is an immortal cell line that can undergo cell replication indefinitely without being subject to the Hayflick limit. Immortal cell lines can be created through genetically modifying cells; by selecting a cell type for expansion with naturally enhanced or indefinite proliferation potential, such as stem cells; or depending on spontaneous immortalization of cells through natural genetic mutations that occur during serial cell culture. Cell lines may be stored in a master cell bank, where they are cryopreserved in a state of suspended activity until they are needed. Cryopreserved cells may be thawed and re-animated and expanded in bioreactors. [00061] Taste and appearance factors [00062] Presentation and sensory characteristics of cell-based meat products may be optimized by identifying which characteristics define an animal meat product and enhancing those characteristics in the cell-based meat product. For example, expression of a muscle-specific protein, myoglobin, that is known to improve meat color and perceived quality, may be increased. Cultured cells are characterized by their color to demonstrate the need for focusing on myoglobin, where myogenic cells in culture are white in color. Differentiated cells are preferred over undifferentiated cells, as undifferentiated cells exhibit lower myoglobin levels and muscle-specific protein density, which may change flavor as well as pigment. [00063] Myoglobin [00064] Visual perception of red meat quality is determined from myoglobin pigmentation. Myoglobin is a ~17 kDa cytoplasmic hemoprotein encoded by the MB (myoglobin) gene. It possesses a single heme group, where hemoglobin contains four heme groups. Myoglobin reversibly binds to O2 via the heme group and serves as an oxygen storage system for oxidative phosphorylation. The heme group in myoglobin provides a red pigment to meat, depending on the oxidation state of the Fe+ ion, which affiliates with O251. There are three basic myoglobin redox states that can be detected by reflectance spectra: oxymyoglobin (OMb), deoxymyoglobin (DMb), and metmyoglobin (MMb). In the presence of oxygen, O2 is bound to Fe+2 and OMb is generated, which produces a bright red color. When O2 is absent from Fe+2, DMb is formed and the heme ring is purplish in color. When meat is overexposed to oxygen for a prolonged period, Mb is oxidized to Fe+3, forming MMb and a dark brown color to the meat surface. The electron transport chain in mitochondria can reduce MMb back to Fe+2 and is involved with color stability. Visual meat color acceptance follows the red > purple > brownness sequence. [00065] Driving Flavor and Pigment Profiles via Supplementation with Fatty Acids [00066] The present invention provides methods of supplementing with free fatty acids to drive flavor and pigmentation of cultivated meat. [00067] The fatty acid (FA) composition of intramuscular fat impacts flavor, juiciness, and tenderness. This composition includes saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs). SFAs like myristic acid (14:0), palmitic acid (16:0), and stearic acid (18:0) are composed of carbon chains that lack double bonds; MUFAs like oleic acid (18:1) and palmitoleic acid (16:1) contain one carbon double bond; and PUFAs have two or more carbon double bonds. Double bonds in fatty acids hinder the formation of the crystal structure of solidified fat. Consequently, every double bond lowers the melting point of the fatty acid. The melting point of lipids greatly influences the juiciness of beef. SFAs have melting points at ~70 ºC, MUFAs melt at ~20 ºC, and PUFAs a liquid at room temperature and melt at -20 ºC. [00068] High SFA levels can be produced from high calorie feed and is associated with meat toughness and flavor liking.16:0 and 18:0 are two FAs that determine meat hardness due to their high melting point. Lowering the total percentages of these SFAs will soften meat products. Oleic acid is associated with beef palatability due to its lower melting point, which improves tenderness, juiciness, and flavor. High oleic acid levels are found in animals with intense marbling, such as Wagyu and Hanwoo, which can be increased through high calorie feed. SFA and MUFA deposition increases total IMF% in muscle. The MUFA:SFA ratio increases over the life of the animal due to an increasing oleic acid and MUFA composition, which improves the palatability of beef. The MUFA:SFA ratio also determines the melting point of the fat. [00069] PUFAs have the lowest melting point of the FAs. PUFAs contribute minimally to IMF due to their slower deposition rate than SFAs and MUFAs, but have nutritional importance. Linoleic acid (18:2 n-6), also called omega-6, and linolenic acid (18:3 n-3), or omega-3, are essential PUFAs present in animal meat.18:2 is produced in higher concentration with high calorie feeding and is positively correlated with flavor liking, while 18:3 is increased in animals with grass diets and is negatively correlated with liking. A high C18:2/C18:3 ratio is positively correlated with flavor liking and overall liking. [00070] Cell-based meat can be supplemented with fatty acids in order to mimic the fatty acid composition of a naturally meat from an animal. Fatty acids that may be used include, but are not limited to saturated fatty acids (SFAs), such as myristic acid (14:0), palmitic acid (16:0), and stearic acid (18:0), monounsaturated fatty acids (MUFAs), such as oleic acid (18:1) and palmitoleic acid (16:1), and polyunsaturated fatty acids (PUFAs). Fatty acids are added to cell culture media during the adipogenic differentiation to generated cultivated fat cells. FFAs should be supplemented in the culture media at concentrations that result in a fatty acid composition of the IMF of the respective type of meat. To achieve the desired taste and appearance, any FFA can be used in any combination with other FFAs at concentrations between 1-1000nM, 1-1000 µM, or 1-1000mM to promote adipogenic differentiation of cultivated cells. [00071] SFAs include Myristic acid (14:0), Palmitic acid (16:0), Stearic acid (18:0), Arachidic acid (20:0), Behenic acid (22:0), Lignoceric acid (24:0), and Cerotic acid (26:0). PUFAs include omega-3, -6, and -9 fatty acids. Omega-3 FAs include α-Linolenic acid (ALA, (18:3(n-3)), Stearidonic acid (SDA, (18:4 (n−3)), Eicosatetraenoic acid (ETA, (20:4 (n−3)), Eicosapentaenoic acid (EPA, (20:5(n-3)), Docosapentaenoic acid (DPA, (22:5 (n−3)), and Docosahexaenoic acid (DHA, (22:6 (n−3)). Omega-6 FAs include: Linoleic acid (LA, (18:2 (n−6)), Gamma- linolenic acid (GLA, (18:3 (n−6)), Calendic acid (18:3 (n−6)), Dihomo-gamma-linolenic acid (DGLA, (20:3 (n−6)), Arachidonic acid (AA, (20:4 (n−6)), and adrenic acid (AdA, (22:4 (n−6)). Mead acid (20:3 (n−9)) is an omega-9 PUFA. Other omega-9s and omega-7 FAs are MUFAs. MUFAs that are omega-7s include Palmitoleic acid (16:1(n-7)) and Paullinic acid (20:1(n−7)). MUFAs that are omega-9 FAs include Oleic acid (18:1, (n−9)), elaidic acid (18:1 (n−9)), gondoic acid (20:1 (n−9)), Erucic acid (22:1(n-9)), and Nervonic acid (24:1 (n−9)). [00072] The fat cells developed from cell lines can be differentiated to mimic the free fatty acid profile meat from an animal. Supplementation of free fatty acids into the cell culture media will generate a free fatty acid profile of the cultured cells. The molar concentration of each fatty acid will be determined by assessing the efficacy of fatty acid uptake and increasing or lowering the concentration to generate a fat additive that simulates the free fatty acid profile of a specific cut of meat. By way of example only, palmitic acid is the second most common free fatty acid (FFA) in the longissimus muscle at 24.3% of the total FFA content. Palmitic acid should be added to the cell culture media at a concentration that will result in ~24.3% of the total fatty acid composition being palmitic acid. [00073] Cultivated meat products may be designed by modelling the free fatty acid profile of the template meat. For example, pork belly is composed of multiple muscle groups: the cutaneous trunci, latissimus dorsi, pectoralis profundus, rectus abdominis, and internal and external abdominal oblique) and the fat content between these muscles. Bacon is the cured meat from the belly of the pork. Bacon has different names depending on the part of the animal it was derived, and ‘streaky bacon’ is the cut from pork belly. While bacon has its fat content, it isn’t as fatty as pork belly because it is a particular cut of the underside of the animal, which has less fat in it. In order to turn cuts of pork belly into bacon, the meat is drained of all moisture with the help of salt. Pork loin is cut from the animals back and includes the longissimus muscle. [00074] Pigs differ from cattle because they have limited intramuscular fat. A lean cut of the longissimus muscle has 2.9% IMF, but the entire pork loin has 22.6% intramuscular, intermuscular, and subcutaneous fat content. Similarly, the fat content of pork belly is predominantly intermuscular and at 25.2%. The predominant free fatty acids in pork are palmitic acid (16:0), steric acid (18:0), oleic acid (18:1), palmitoleic acid (16:1), linoleic acid (18:2, omega 6), and linolenic acid (18:3, omega 3). Pork meat tends to contain less palmitic and oleic acid than beef and more PUFAs, such as linoleic acid and linolenic acids. Due to the low levels of marbling in pork, fat cells can be mixed with skeletal muscle cells in the bulk of the product without any marbling structure strategy. Additionally, there is a high amount of intermuscular fat in pork between muscles. This would require separate blends of skeletal muscle cells and fat cells mixed with their respective plant proteins and other food ingredients to be layered into a heterogeneous structure. [00075] Generating cultivated meat products with omega 3, 6 health benefits During adipoogenic differentiation, supplementing adipogenic cell culture media with additional omega-3 fatty acids and omega-6 fatty acids can increase the concentration of omega 3 and omega 6 fatty acids. Increasing omega 3 and 6 in cultivated meat can have health benefits, such as improve brain health, heart health, and reduce inflammation. [00076] Transdifferentiation of myogenic cells to adipogenic cells [00077] Cell lines must be able to undergo myogenesis and adipogenesis. The capacity of a cell line to undergo myogenesis and adipogenesis affects meat quality. The present invention provides methods of transdifferentiating myogenic cells to adipogenic cells, eliminating the need to develop two separate cells lines in the development of a cell-based meat product. During fetal development, mesenchymal stem cells undergo myogenesis, adipogenesis, and fibrogenesis to produce myogenic, adipogenic, and fibroblastic cells. Wnt and β-catenin regulates MSC differentiation, where Wnt and β-catenin signaling will promote myogenesis, while suppression of Wnt and β- catenin signaling induces MSC adipogenesis. In 2001, Rudnicki et al. (Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation, Differentiation, 2001 Oct; 68(4-5): 245-53) showed that rat satellite cells can differentiate into adipocytes without treatment of IBMX, dexamethasone, or insulin. Living muscle fibers were isolated and grown on Matrigel, which lacked adipogenic inducers. The satellite cells on the muscle fibers maintained a more stem-like state than they otherwise would on cell culture plates since they were still affiliated with signals from their tissue niche. After 10 days in culture, the satellite cells formed lipid droplets that was visualized with Oil Red O (ORO) and expressed PPARγ gene, providing supporting evidence to the multipotency of satellite cells. A study focusing on C2C12s showed that myoblasts also have transdifferentiation potential into adipocytes (N.K. Singh, Conversion of C2C12 Myoblast into Adipoblast with Thiazolidinediones - A Possible Basis for Intramuscular Fat Generation in Meat Animals, Anim Biosci 2007; 20(3): 432-439). C2C12s differentiated in adipogenic induction medium with thiazolidinedione (TZD) for 10 days showed a decrease in myotube formation, a simultaneous increase in adipoblast formation (indicated by transition to rounded morphology and lipid droplet formation in the cells), and expressed PPARγ protein. There was not complete conversion into adipocytes, however, and myotubes were still present in the culture. [00078] There is additional evidence that bovine myoblast cells are multipoint and can transdifferentiate into adipocytes. In one study, bovine skeletal muscle cells underwent myogenic differentiation through serum reduction, but were cultured in adipogenic media containing insulin, oleic acid, ciglitizone (a drug that serves as a PPARγ ligand and adipogenesis stimulator for mesenchymal stem cells) and melengestrol acetate (MGA, a feed additive for cattle to improve feedlot performance). ORO staining showed multiocular lipid droplets formed inside mononuclear myoblasts and multinucleated myotubes. PPARγ and C/EBPβ gene expression increased and myogenin gene expression decreased (K.Y. Chung and B.J. Johnson, Melengestrol acetate enhances adipogenic gene expression in cultured muscle-derived cells, Journal of Animal Science, 87(12), December 2009: 3897–3904). In a follow up study, oleic acid was tested without a PPARγ agonist to determine its efficacy to stimulate adipogenesis in bovine satellite cells. The authors found that oleic acid increased the % of lipid droplets in the cells; increased PPARγ, C/EBPβ, C/EBPα, and FABP4 gene expression; and adiponectin protein expression. The satellite cells were still able to undergo myogenesis to form multinucleated muscle fibers and expressed myogenin gene expression, but showed decreased gene expression of Pax3 and MYOD1 (Li et al. Oleic acid in the absence of a PPARγ agonist increases adipogenic gene expression in bovine muscle satellite cells, Journal of Animal Science, 97(10), October 2019: 4114–4123). These data indicate an incomplete conversion of bovine satellite cells to adipocytes, with both myogenic and adipogenic characteristics maintained in the cells. The authors also hypothesized that since Wagyu beef is so high in oleic acid, that perhaps oleic acid can act as a paracrine factor to transdifferentiate satellite cells located between the muscle fiber cell membrane and basal lamina into adipocytes. During fasting states and adipogenic differentiation, FFAs such as oleic acid are released by the adipocytes that regulate skeletal muscle energy metabolism (Kovalik et al. Metabolic Remodeling of Human Skeletal Myocytes by Cocultured Adipocytes Depends on the Lipolytic State of the System, Diabetes 2011 Jul; 60(7): 1882-1893). These studies highlight the potential to create a myogenic cell line that can create both skeletal muscle and fat cells in the same culture, eliminating the need for developing two separate cell lines. [00079] Genetically modified cell lines may be cultivated to mimic the taste and texture of animal meat products. The present invention provides methods for transdifferentiating myoblasts into adipocytes. Producing myogenic and adipogenic cell lines from a single progenitor cell line is desirable due to a reduction in the complexity of the bioprocess. The time and cost of developing multiple cell culture methods for proliferating muscle and fat cells, multiple media formulations, multiple cell line engineering strategies, and multiple bioprocesses may be condensed into one progenitor cell line by using a transdifferentiation method to obtain adipocytes from myoblasts. Thiazolidinediones, IBMX, dexamethasone, and other synthetic PPARγ agonists cannot be used in food applications. Natural PPARγ agonists, such as long-chain fatty acids (LCFA) can be used to activate transdifferentiation of myogenic cells to adipogenic cells. Transdifferentiation can be accomplished through supplementation of cell culture media of myogenic cells with a combination of SFAs, MUFAs, and/or PUFAs. [00080] A culture myoblast cells may be expanded to increase the biomass for a meat product. Upon reaching a certain biomass, the culture may be split. The first vessel will continue to grow myogenic cell population in myogenic media, while the cells in the second vessel will undergo transdifferentiation into adipogenic cells. The cell culture media may be supplemented with any combination of the following free fatty acids at any concentration: SFAs include Myristic acid (14:0), Palmitic acid (16:0), Stearic acid (18:0), Arachidic acid (20:0), Behenic acid (22:0), Lignoceric acid (24:0), and Cerotic acid (26:0). PUFAs include omega-3, -6, and -9 fatty acids. Omega-3 FAs include α-Linolenic acid (ALA, (18:3(n-3)), Stearidonic acid (SDA, (18:4 (n−3)), Eicosatetraenoic acid (ETA, (20:4 (n−3)), Eicosapentaenoic acid (EPA, (20:5(n-3)), Docosapentaenoic acid (DPA, (22:5 (n−3)), and Docosahexaenoic acid (DHA, (22:6 (n−3)). Omega-6 FAs include: Linoleic acid (LA, (18:2 (n−6)), Gamma-linolenic acid (GLA, (18:3 (n−6)), Calendic acid (18:3 (n−6)), Dihomo-gamma-linolenic acid (DGLA, (20:3 (n−6)), Arachidonic acid (AA, (20:4 (n−6)), and adrenic acid (AdA, (22:4 (n−6)). Mead acid (20:3 (n−9)) is an omega-9 PUFA. Other omega-9s and omega-7 FAs are MUFAs. Omega-7s include Palmitoleic acid (16:1(n-7)) and Paullinic acid (20:1(n−7)). Omega-9 FAs include Oleic acid (18:1, (n−9)), elaidic acid (18:1 (n−9)), gondoic acid (20:1 (n−9)), Erucic acid (22:1(n-9)), and Nervonic acid (24:1 (n−9)). [00081] Cell culture [00082] The methods for developing cell lines contained herein can be applied to myoblasts, fibroblasts, fibroblast-adipocyte precursor cells, and mesenchymal stem cells. Adipocytes can be differentiated from fibroblasts, fibroblast adipocyte precursor cells, mesenchymal stem cells, and transdifferentiated from myoblasts. [00083] Cell Isolation [00084] Any species of mammal may be used as cell donor. Preferred mammals include, but are not limited to bovine (including cattle), caprae (including goats), Cervidae (including deer and elk), lagormorph (including rabbits), oves (including sheep, rodent and suidae (including pork). Different species have different intramuscular fat deposits. A smaller population of differentiating adipocytes is observed in porcine cultures than bovine. Skeletal muscle in cattle has intramuscular fat deposits that can be visibly observed as marbling, yet most pig breeds (except the duroc) lack marbling. Additionally, the IMF content of biceps femoris in pig meat is 1.4 +/- 0.4% at slaughter age, while it is 2.55% in the Dexter cattle breed. A different cell isolation technique can be used to improve the porcine pre-adipoblast isolation yield, due to difference in extracellular matrix composition and localization of pre-adipoblasts. [00085] Strategies for producing immortalized animal cell lines for cultivated meat products [00086] Cell culture media often includes the use of expensive growth factors to support cell survival, proliferation, and differentiation, making large scale cell-line cultivation cost-prohibitive. Constitutive activation of the ERK1/2 pathway and insulin-independent glucose transport through activation of the PI3K/Akt pathway can enhance cell proliferation and reduce the amount of growth factor required, thereby lowering the cost of cultivating edible cell lines. The present inventions provide methods for enhancing cell proliferation with reduced growth factor concentration through constitutive activation of the ERK1/2 pathway and insulin-independent glucose transport through activation of the PI3K/Akt pathway. Edible cell lines used in cultivated meat products must have low cost production methods to be affordable. Cell lines used in academia or the regenerative medicine industry are not designed to minimize growth factor and insulin requirements in cell culture media required for cell survival, proliferation, and differentiation. These types of cell lines have different cost drivers than cells lines used to produce affordable food for consumers. Cell lines used in research are not designed for scalable manufacturing, and cell lines used in the cell therapy or biopharma industries are expensive products with high profit margins. Consequently, engineering cell lines for cultivated meat products have to have reduced mitogen and insulin requirements desirable. [00087] The PI3K/Akt signaling pathway [00088] PI3Ks (phosphoinositide 3-kinases) are a family of lipid and protein kinases linked to a diverse group of cellular functions, including cell growth, proliferation, differentiation, motility, survival, intracellular trafficking, and insulin receptor signaling. They are separated into classes I, II, and III based on sequence homology and function. PI3K is recruited to the cell membrane via phosphorylated receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs), and can also interact with cytosolic Ras GTPases to initiate signal transduction pathways. [00089] Class I PI3Ks are heterodimers composed of IA and IB subsets that contain a catalytic and regulatory subunit. Class IA PI3Ks have p110 catalytic and p85 regulatory subunits, with three variants to the p110 catalytic subunit: p110α, p110β, or p110δ (expressed by the genes Pik3ca, Pik3cb, and Pik3cd, respectively). There are 5 variants of the regulatory subunit, including p85α, p55α, p50α, p85β, and p55γ. The p85α, p55α, p50α subunits are splice variants of the Pik3r1 gene; p85β is expressed from Pik3r2, and p55γ expressed from Pik3r3. p110α and p110β isoforms are expressed in all cells. Regulatory subunits stabilize and suppress the catalytic subunit in the absence of upstream signals and mediate the interaction of the catalytic subunit to RTKs and GPCRs during signal transduction. [00090] The catalytic subunit of class I PI3Ks contain an amino-terminal adaptor-binding domain (ABD) which interacts with the regulatory unit; a Ras-binding domain (RBD) that facilitates interaction between Ras-GTP and the catalytic subunit, allowing PI3K activation in a Ras-dependent manner; a C2 (protein-kinase-C homology-2) domain that has an affinity for lipid membranes; a helical domain that operates as scaffolding for other p110 domains; and a carboxyl-terminal kinase domain. The p85 regulatory unit is composed of several protein- protein interaction domains: there are two SH2 domains, C-SH2 and N-SH2, where N-SH2 interacts with the helical domain of the catalytic subunit; one SH3 domain that binds to phosphorylated tyrosine residues; a breakpoint clustered homology (BH) domain; and an inter- SH2 (iSH2) domain that interacts with the catalytic subunit at ABD and C2. [00091] Class I PI3K phosphorylates cell membrane-bound PIP2 to form PIP3. PIP3 recruits proteins to the cell membrane that contain a pleckstrin homology domains, which includes a serine/threonine specific protein kinase called protein kinase B (PKB, otherwise known as Akt), and PDK1 (phosphoinositide-dependent kinase-1). Binding of Akt to PIP3. Changes it’s conformation and allows interaction with PDK-1 and PDK-1 phosphorylates Akt on threonine 308, which partially activates the enzyme. Akt becomes fully activated upon phosphorylation of serine 473 by the TORC2 complex of the mTOR protein kinase through a positive feedback loop. [00092] Akt plays a role in glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration. Akt has three isoforms: Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ. Each isoform contains a N-terminal pleckstrin homology domain (PH), kinase catalytic domain containing threonine 308, and C-terminal regulatory domain containing serine 473 (in Akt1) or serine 474 (in Akt2). The PH domain regulates lipid-protein interactions and is the mechanism for recruitment to the cell membrane. [00093] Akt1 is involved in cellular survival pathways by promoting cell cycle progression and inhibiting apoptotic processes. Akt1 is also able to induce protein synthesis pathways, and is therefore a key signaling protein in the cellular pathways that lead to skeletal muscle hypertrophy, and general tissue growth. Since Akt1 can block apoptosis, it has been implicated in numerous cell survival signaling mechanisms. Akt1 can be activated by growth factors. Akt2 is involved in the insulin signaling pathway and is required for glucose transport through the cell membrane. Akt2 is more specific for insulin receptor pathway than Akt1. Akt2 drives differentiation whereas Akt1 appears critical to myoblasts proliferation. Akt1 phosphorylates the cyclin kinase inhibitor p21, triggering its dissociation from CDK2 and leading to cell cycle progression. [00094] PDK-1 has a kinase domain and pleckstrin homology domain, and the PH domain localizes the kinase to the cell membrane. PDK-1 is considered to be constitutively active but is regulated by PI3K conversion of PIP2 to PIP3 in the cell membrane along with localization of Akt. The kinase domain has three ligand binding sites: the substrate binding site, the ATP binding site, and the docking site (also known as PIF pocket). Atypical protein kinase C (PKC), serum glucocorticoid-dependent kinase (SGK), p70 ribosomal protein S6 kinases (S6K), and p90 ribosomal protein S6 kinase (RSK) bind to the docking site of PDK-1. [00095] Suppression of PI3K/Akt pathway [00096] The phosphatase and tensin homologue (PTEN) tumor suppressor gene is an antagonist of the PI3K/Akt pathway by dephosphorylating phosphatidylinositol-3,4,5- triphosphate PIP3 to PIP2 - preventing the partial phosphorylation of Akt, and therefore switches off the PI3K- activated pathway. The lipid phosphatase activity is critical for PTEN tumor suppressor function. Inactivation of PTEN results in activation of the PI3K/AKT pathway and subsequent increase in cell cycle progression, migration and survival. [00097] PTEN regulates cell cycle progression by downregulating transcriptional expression and protein stability of cyclin D1, as well as by inhibiting its nuclear localization. In addition to cyclin D1, PTEN is also shown to potentially repress cyclin D2 and cyclin D3 to arrest the cell cycle at G1. PTEN is also been shown to modulate the cell cycle by upregulating the cyclin dependent kinase (CDK) inhibitor p27. [00098] PTEN can also be regulated by TBX2 and Pax3. TBX2, a T-box family member, bypasses cell growth control through the repression of the cell cycle regulators p14 and p21. TBX2 directly represses the tumor suppressor PTEN in both rhabdomyosarcoma (RMS) and normal muscle. Exogenous expression of TBX2 in normal muscle cells down regulates PTEN, and depletion of TBX2 in RMS cells upregulates PTEN, resulting in a reduction of phospho- AKT. TBX2 represses PTEN by directly binding to the promoter and recruiting the histone deacetylase, HDAC1. TBX2 is a central component of the PTEN/PI3K/AKT signaling pathway deregulation in rhabdomyosarcoma. [00099] In RMS cells, the fusion protein PAX3- FOXO1 has been shown to contribute to repression of PTEN. Depletion of PAX3-FOXO1 in RMS cells up regulated PTEN and exogenous expression of PAX3 in C2C12 cells downregulated PTEN. In both C2C12 normal myoblasts and RMS cells, the level of PTEN has been shown to be inversely correlated with AKT serine 473 phosphorylation, which is mediated by the rapamycin-insensitive mTOR complex (mTORC2) and required for full activation of AKT. PAX3 was shown to bind to the PTEN promoter. PAX3 has also been shown to activate TBX2 in melanoma cells. Thus, the negative regulation of PTEN by PAX3 may be mediated through TBX2. [00100] The MAPK/ERK signaling pathway [00101] As previously mentioned, eliminating or reducing the amount of insulin or growth factors required for cell culture media lowers the cost associated with cultivating edible cell lines. The present invention provides methods of enhancing cell proliferation with reduced growth factor concentration through constitutive activation of the ERK1/2 pathway and insulin-independent glucose transport through activation of the PI3K/Akt pathway MAPKs (mitogen activated protein kinase, originally termed ERKs – extracellular signal regulated kinases) are serine/threonine-selective protein kinases that phosphorylate downstream targets that regulate the cell. There are three main families of MAPKs: 1) the ERKs, 2) the c-jun NH 3-terminal kinases/stress activated protein kinase (JNKs), and 3) the p38 MAPKs (p38 ^/ ^). ERKs are activated by growth factor ligands such as NGF, FGF, EGF, PDGF, and BDNF that bind to their tyrosine kinase receptors in the cell membrane to initiate pro-mitotic intracellular signaling cascades. JNKs and p38s are activated by cellular stress and cytokines to cause apoptosis, inflammation, cell cycle arrest, and cell differentiation. [00102] Most MAPKs share structural similarity and have 2 phosphorylation sites (phosphotyrosine and the phosphothreonine residues) for activation in their activation loop domains, substrate recognition sites, and a three-tiered activation cascade. The activation loop contains a characteristic TxY (threonine-x-tyrosine) motif, where both the threonine and tyrosine must be phosphorylated to activate the kinase. It is TEY in mammalian ERK1/2, TYP in JNKs, and TGY in p38 kinases. MAPKs are phosphorylated by MAP2Ks (MKKs, MAP kinase kinases), which in turn are phosphorylated by MAP3Ks, which are localized to the cell membrane where they are activated by various stimuli. MAPKs are the only MAP kinases that can enter the cell nucleus to activate gene transcription. [00103] MAPKs and MAP2Ks are regulated via their phosphotyrosine and the phosphothreonine residues, but MAP3Ks have more complex regulation. Some MAP3Ks include Rafs, MEKK4, or MLK3, which require multiple activation steps. First, a ligand associated with the cell membrane changes the conformation of the MAP3K (Ras for Rafs, GADD45 for MEKK4, or Cdc42 for MLK3) to expose the kinase domains. The accessible kinase domains then allow homo- or heterodimerization which generates a partially activate dimer conformation. The dimers then transphosphorylate each other on their activation loops. The fully activated MAP3Ks can then phosphorylate MAP2Ks. In the ERK1/2 signaling pathway, the MAP3Ks are A-, B-, and c-Raf that are activated by growth factors such as EGF, FGF, and PDGF. Rafs then phosphorylate MKK1/2 (aka MEK1/2), which in turn phosphorylate ERK1/2. Mammalian p38 and JKN kinases share some MAP3Ks, such as MEKK1/2 and ASK1/2, and share a MAP2K MKK4. JNK is separately activated by MAP3Ks MLK1/2/3 and MAP2K MKK7, while p38 is activated by MAP2Ks MKK3/6. [00104] The Ras-Raf-MEK-ERK signal transduction pathway regulates cell growth and proliferation in response to growth factors, cytokines, and hormones. Rafs are serine/threonine kinases that are stimulated upon binding of GTP-bound Ras to the Ras binding domain (RBD). A-Raf and C-Raf share similar regulatory mechanisms and require additional serine and tyrosine phosphorylation within the N region of the kinase domain for full activity compared to B-Raf, which has higher basal kinase activity than A-Raf and C-Raf. [00105] The Ras/MEK/ERK pathway promotes cell cycle progression through phosphorylation of cyclin D1 and c-Myc transcription factor. Phosphorylated cyclin D1 complexes with Cdk4 and Cdk5 (Cdk4/6). Cyclin D-Cdk4/6 regulates the progression of the G1 phase of the cell cycle to S phase. Cyclin D-Cdk4/6 activity increases in late G1 due to the signaling from extracellular mitogens which leads to the hyperphosphorylation of retinoblastoma protein (Rb). Hypophosphorylated Rb is usually bound to E2F transcription factor in early G1 and inhibits its activity. Hyperphosphorylation of Rb dissociates E2F, which can then enter the nucleus and activate gene transcription of cyclins that progress the cell through the S phase of the cell cycle (J. Knight and R. Kothary, The myogenic kinome: protein kinases critical to mammalian skeletal myogenesis, Skeletal Muscle, 1, Art. No.29 (2011)). [00106] The PI3K/Akt and MEK/ERK signaling pathways converge to jointly regulate c-Myc activation. ERK mediates c-Myc stabilization through phosphorylation at serine 62, and subsequent phosphorylation at Thr58 by GSK-3 is required for c-Myc degradation. Akt phosphorylation causes inhibition of GSK-3, and hyperactivation of the PI3K and MEK-ERK pathways promote c-Myc signaling and cell cycle progression. Activated c-Myc then regulates gene expression by suppressing expression of the cell cycle inhibitor p21 and increasing expression of cyclins A and B and CDK2. c-Myc overexpression enhances cell proliferation rates. c-Myc phosphorylation is majorly attenuated by ERK2 and to a lesser extend ERK1, but c-Myc phosphorylation is highest when both ERK1/2 are active (Marampon et al. Down- regulation of c-Myc following MEK/ERK inhibition halts the expression of malignant phenotype in rhabdomyosarcoma and in non-muscle-derived human tumors, Molecular Cancer, 5, Art. No.31 (2006)). [00107] Telomerase [00108] Telomerase is an enzyme that maintains the length of telomeres at the 3’ ends of chromosomes, which prevents cellular senescence based on telomere shortening. Telomeres are present in eukaryotic organisms and are repeating strands of nucleotide sequences. With each round of mitosis, telomeres shorten until the they reach a critical length that prevents DNA replication machinery to initiate another round of cell division. This limits the number of cell divisions and help preserve genomic stability, a phenomenon known as the Hayflick limit. Telomerase is a reverse transcriptase enzyme that carriers an RNA molecule that serves as a template for lengthening telomeres. Telomerase expression is found in embryonic stem cells and iPSCs, and is typically absent or expressed at very low levels in somatic cells. [00109] Anchorage-independent proliferation in single cell suspension through anoikis resistance [00110] Primary cell lines such as skeletal muscle cells typically survive and proliferate in an anchorage-dependent manner. Once anchorage dependent cells detach into suspension, a form of cell death called anoikis occurs. This limits the scale up of cell production to microcarrier- or aggregate based growth system, which yield lower cell concentrations per mL than cell populations that can be grown in single cell suspension. To increase the production yield of cell lines, anchorage independent survival and growth can be conferred by manipulating cell signaling pathways that generate anoikis resistance. Failure to initiate anoikis can result in cell survival in suspension. [00111] Anoikis pathways terminally converge in activation of caspases, which activate endonucleases, DNA fragmentation, and cell death. There are two apopototic pathways: the intrinsic pathway that involves perturbation of mitochondria, and the extrinsic pathway triggered by cell surface death receptors. [00112] In intrinsic pathway, pro-apoptotic BH3-only proteins Bid and Bim are activated following detachment of cells from ECM, and rapidly promote the oligomerization of Bax- Bak. Bax-Bak translocate to the outer mitochondrial membrane, create a channel that permeabilizes the mitochondrial membrane, and causes cytochrome c release. Cytochrome C release leads to the formation of the apopotosome, and activation of caspase-9. However, Bcl- 2 overexpression prevents apoptosis by interacting with Bak/Bax, avoiding their oligomerization, and sequesters Bid and Bim. [00113] ECM binds to integrins that transduce signaling cascades which promote survival of attached cells. Activation for four types of integrins, a5B1, avB3, a1B1, and a6B1, plays a specific role in cell survival by protecting cells from apopotosis and anoikis. Their downstream pathways cause activation of focal adhesion kinase (FAK), Src kinase, integrin-linked kinase (ILK), PI3K/Akt, and MAPK. Activation or overexpression of these signaling molecules confers protection against anoikis. [00114] FAK activation causes autophosphorylation, which recruits Src, which further phosphorylates FAK and leads to a fully activated FAK enzyme. FAK then activates the Akt and MAPK pathways. FAK activates PI3K, which then recruits and activates Akt. Akt activation promotes cell survival by several mechanisms. It can phosphorylate the pro- apoptotic protein Bad, which prevents its inhibition of Bcl-2; phosphorylating and inactivating procaspase-9; and activating NFKB survival pathway. MAPK pathway activation causes phosphorylation of Bad, freeing Bcl-2 to suppress cytochrome c release from the mitochondria, and inhibits Bim from causing Bak/Bax oligomerization. MAPK activation additionally upregulates Bcl-2 gene expression. [00115] ILK, encoded by the ILK gene, is involved with cell proliferation, migration, and adhesion that interacts with focal adhesions. Its main role is to organize the actin cytoskeleton during development and homeostasis. Upon activation of integrins via ECM binding, integrins cluster into focal adhesions. This connects the ECM to the actin cytoskeleton of the cell and fine-tunes growth factor receptor signaling. Integrins lack enzymatic activity and rely on recruiting adaptor and signaling proteins. ILK is a scaffolding protein that associates with the cytoplasmic domains of β-1 and β-3 integrins to transduce integrin-mediated extracellular signals. [00116] ILK has three different domains: five ankyrin repeats at the N- terminus, followed by a pleckstrin homology (PH)-like domain and a kinase- like domain at the C-terminus. Before recruitment to the focal adhesion site, ILK forms a complex with two adaptor proteins called Pinch and parvin (termed the IPP complex). This stabilizes the complex and allows targeting to the focal adhesion site. There are different forms of parvin that affiliate directly with the F- actin cytoskeleton or recruit actin binding proteins. ILK can also connect microtubule caps to cortical actin networks through FA sites. [00117] Cells must also release from focal contacts during cell migration. Mesenchymal motility occurs when migrating cells take on an elongated cell morphology dependent on ECM binding to integrins and focal contacts. Another motility style is amoeboid migration which allows cells to overcome ECM barriers by weakening focal contacts. Focal adhesions are not organized in this form of migration, but cell survival is promoted by activation of the Rho family of GTPases. RhoG can suppress anoikis. [00118] Mechanisms for cell immortalization and anoikis resistance [00119] Immortalized cell lines with anoikis resistance, insulin-independent insulin signaling, and mitogen-independent cell cycle progression enhances flavor and sensory properties of edible cell lines and lowers the cost of production. In addition, to increase production yields while simultaneously lowering production costs, cell lines used for cultivated meat are designed to grow in single cell suspension without undergoing anoikis. In scaled systems, cells can be grown in single cell suspension, on microcarriers, or as aggregates. Growing cells in single cell suspension produces the highest concentration of cells per mL, resulting in the highest product yield per production run for cultivated meat products. Accordingly, cell lines used for cultivated meat should be designed to grow in single cell suspension without undergoing anoikis to increase production yields while simultaneously lowering production costs. The present invention provides anoikis resistant cell lines. [00120] Persistent PI3K or Akt activity can lead to cellular immortalization by driving continuous cell cycle progression and promotes anoikis resistance by preventing normal apoptosis mechanisms. Akt blocks apoptosis by inhibiting the activity of caspase 9 and Bad, which prevents apoptosome assembly and permeabilization of the mitochondrial outer lipid membrane, respectively. Alternatively, Akt enhances cell survival and cell progression by increasing activity of mitogens Jun, Myc, and cyclinD, and deceasing activity of p53, p27 and p21, which promote cell cycle arrest. [00121] Akt is phosphorylated by PDK-1 and increased proliferation of cells can be achieved with PDK-1 overexpression. Anchorage independent cell growth has been shown with PDK-1 overexpression and membrane localization with activation of SGK3. PDK-1 positively regulates ROCK1 in a kinase-independent manner that promotes amoeboid motility. PDK-1 knockdown in standard tissue culture conditions showed no growth enhancement of cell lines, but show inhibited growth in anchorage independent growth systems. [00122] PDK-1 also has Akt-independent downstream signaling mechanisms that have been associated with cell survival and proliferation, such as activation of S6K1, SGK, PKC, and RSK isoforms. S6K regulates protein synthesis and the progression of the cell cycle from G1 to S phase. SGK is activated in response to insulin stimulation and promotes synthesis of glycogen and other proteins. PKC isoforms contribute to cell cycle progression. PKC ^ in particular has an antiapoptotic effect since it phosphorylates and activates Bcl-2 (Xie et al. Transformation of mammary epithelial cells by 3-phosphoinositide- dependent protein kinase- 1 activates beta-catenin and c-Myc, and down-regulates caveolin-1, Cancer Res.2003 Sep.1, 63(17): 5370-5). RSK promotes cell survival, motility, and proliferation. RSK lies downstream of the Ras/MAPK signaling pathway (C. Raimondi and M. Falasca, Targeting PDK1 in cancer, Curr Med Chem. 2011, 18(18): 2763-9). PDK-1 activates PKC ^ which can increase cell survival and proliferation. [00123] PTEN signaling is often silenced when there is overactive PI3K signaling. (L. Zhao and P.K. Vogt, Class I PI3K in oncogenic cellular transformation, Oncogene 27, 5486-5496 (2008)). Silencing of PTEN prevents conversion of PIP3 to PIP2 and deregulates the PI3K signaling pathway. Persistent PIP3 levels in the cell membrane allow PDK-1 and Akt to colocalize and initiate phosphorylation of Akt. [00124] ILK is overexpressed in many types of cells with anchorage independent cell growth. Although it was initially reported to have kinase activity, subsequent studies have shown that the reported catalytic domain is enzymatically inactive. The anoikis resistance effect of ILK may be due to multiple mechanisms. First, ILK may control the localization of proteins such as Akt to the plasma membrane where it can be phosphorylated. Additionally, Pinch-1 can bind to and inhibit protein phosphatase 1 ^, which results in sustained Akt phosphorylation. Overexpression of ILK blocks anoikis in suspended cells. [00125] Constitutive Ras signaling is transduced through MEK/ERK to promote cellular proliferation and anoikis resistance. In rhabdomyosarcoma (RMS) cell lines expressing mutant gain of function Ras, mutant p53, and overexpressed c-Myc, a MEK/ERK inhibitor called U0126 decreased c-Myc gene expression and phosphorylation levels and induced growth inhibition. U0126 reduced levels of cyclins A, B, D1, E1, and E2 and CDK2 (which forms complexes with cyclin A, B, and E), while increasing levels of cyclin kinase inhibitor (CKI), p21, and p27. Direct inactivation of c-myc protein reduced expression of cyclins A/B/D1 and CDK2, and increased p21 expression. Expression of cyclins E1/2 and p27 were not altered, however, and may be attenuated by ERK depletion, which highlights that both ERK and c-Myc signaling uniquely contribute to cell cycle progression. [00126] The RMS cell line shows anoikis resistance and can grow in anchorage-independent colonies on agar plates. Treatment with U0126 and inhibition of c-Myc protein activity suppressed anchorage independent growth, however. Conversely, transient c-Myc overexpression enhanced anchorage independent growth compared to the control condition and improved anchorage independent growth rates in cells co-treated with U0126, although the improvement was marginal. This indicates that both constitutive ERK and c-Myc signal activation are important regulators of anoikis resistance. [00127] ERK1/2 and downstream target c-Myc are all important for increased cell cycle progression and anoikis resistance. Upstream signaling targets such as Rafs can be modified to generate constitutive activity of all three proteins. A constitutively active mutant form of B- Raf (encoded by the BRAF gene) induces persistent MEK/ERK signaling in a Ras-independent mechanism. There are over 50 discovered mutant variations but the most common form is BRAF-V600E, which results from an exchange of a single amino acid: valine 600 into glutamate. In the BRAF gene, thymine is substituted with adenine at nucleotide 1799 to convert valine to glutamate. This mutation causes a conformational change that mimics phosphorylation of the activation loop and renders the kinase domain fully active, yet does not require dimerization to phosphorylate downstream targets. BRAF-V600E has 500 times the signal activity of wild type B-Raf and makes up 90% of the profile of B-Raf mutants (Liu et al. BRAF mutation and its inhibitors in sarcoma treatment, Cancer Medicine, 9(14): 4881- 4896). Most B-Raf mutations enhance kinase activity to increase ERK signaling, but some show reduced kinase activity. However, these mutations with reduced kinase activity activate wild-type C-Raf to initiate downstream ERK signaling (Wan et al., Mechanism of Activation of the RAF-ERK Signaling Pathway by Oncogenic Mutations of B-RAF, Cell, 116(6), 19 March 2004, pages 855-867). Additionally, there is a BRAF fusion mutant found in embryonic rhabadomyosarcoma called KIAA1549-BRAF, where duplication event concerning the BRAF gene and KIAA1549 gene that produce a fusion protein that is constitutively active. [00128] Growing cells in single cell suspension produces the highest concentration of cells per mL, resulting in the highest product yield per production run for cultivated meat products. Accordingly, cell lines used for cultivated meat should be designed to grow in single cell suspension without undergoing anoikis to increase production yields while simultaneously lowering production costs. The invention provides for designing anoikis resistant cell lines for growth in single cell suspension for edible meat products through constitutive activation of the PI3K/Akt, ERK1/2, and integrin receptor binding signaling pathways. [00129] The cells obtained from an animal possess inherent limitations that make them unsuitable for a large-scale and indefinite manufacturing process. Cell lines developed with increased mitotic capacity, however, may be scaled up making the cultivation of these cell lines more amenable to the cultivated meat manufacturing process. Increased mitotic capacity of cell lines may be accomplished through overexpression in cell lines as follows. In the context of the present invention, a gene may be overexpressed by a promoter, if as a result of said expression an altered spatial distribution and/or an increased quantity of the gene product is found, compared to the expression of the wild-type (regular) promoter for this gene. Preferred levels of overexpression lead to a more than 2-fold, more than 5-fold, more than 0-fold or even more than 20-fold higher expression of the gene, when compared to the expression of the wild- type (regular) promoter for this gene. Overexpression can be measured using well-known methods in the art, such as mRNA detection (e.g. using rtPCR), promoter activity assays, immunofluorescence and western blotting (immunoblot) analysis. [00130] Overexpression of wild type ILK may be accomplished through enhancement of promoter elements of the [ILK] gene. Alternatively, transgenes can be inserted into the genome of the cell under control of constitutively active promoter. [00131] Overexpression of PDK-1 may be accomplished through enhancement of promoter elements of the [PDK1] gene. Alternatively, transgenes can be inserted into the genome of the cell under control of constitutively active promoter. [00132] Overexpression of telomerase may be accomplished through enhancement of promoter elements of the [TERT] gene. Alternatively, transgenes can be inserted into the genome of the cell under control of constitutively active promoter. [00133] Knockout of PTEN: Suppression of the PTEN tumor suppressor [PTEN] gene will promote activation of the PI3K/Akt signaling pathway by preventing PTEN from dephosphorylating PIP3. Silencing gene expression may be achieved through genetic knockout/knockdown or over expression of dominant negative form/miRNA or mutation of the PTEN gene or its promoter. [00134] Constitutively active B-Raf – The B-Raf gene [BRAF] sequence may be mutated to create a constitutively active form of B-Raf kinase that results in enhanced ERK signaling in comparison to the wild type B-Raf, including but not limited to generating the BRAF-V600E mutant. These may include any substitution, deletion, insertion, duplication, inversion, and frameshift mutations to the BRAF gene. [00135] Overexpression of TBX2 may be accomplished through enhancement of promoter elements of the [TBX2] gene. Alternatively, transgenes can be inserted into the genome of the cell under control of constitutively active promoter. [00136] Overexpression of Pax3 may be accomplished through enhancement of promoter elements of the [PAX3] gene. Alternatively, transgenes can be inserted into the genome of the cell under control of constitutively active promoter. [00137] Media for cell culture [00138] The development of animal and serum free medias is required for commercialization. Finding a chemically defined serum alternative is imperative to scaling up meat biomanufacturing. Fetal bovine serum is used because it contains high concentrations of growth factors that promote cell proliferation and low concentrations of factors that inhibit cell growth. Basic serum free media formulations include growth factors such as EGF, FGF, PDGF, NGF, IGF; cytokines such as interferons and interleukins; insulin and other hormones; proteins like transferrin and albumin; trace elements; and fatty acids. Finding the right media composition can enhance flavor and performance of cell lines. [00139] Strategies for reducing the use of insulin in cell culture media [00140] Similarly, reducing or eliminating insulin from cell culture media may significantly reduce cost of production. The present invention provides strategies to reduce to requirement for growth factors or other proteins in cell culture media. The present invention provides methods of targeting the insulin/GLUT4 and PI3K-Akt signaling pathways. Editing these signaling pathways to generate constitutive activation in edible cell lines eliminates the need for supplying insulin or growth factors into cell culture media that are normally required for activating the pathways. This can lower the cost of media used for meat cultivation. [00141] The Insulin Receptor-PI3K-AKT pathway [00142] In both skeletal muscle and adipose cells, insulin stimulates PI3K/Akt signaling to promote glucose transport into the cell. In skeletal muscle, insulin stimulates protein synthesis and accelerates mRNA translation by regulating the initiation steps of protein translation. Insulin signaling regulates the metabolism of adipose tissues by promoting glucose utilization, protein synthesis, and lipogenesis. The PI3K/AKT signaling pathway promotes lipid biosynthesis and inhibits lipolysis. [00143] Insulin is a hormone that regulates glucose metabolism in the cell and is an essential cell culture media component. In skeletal muscle and adipose tissue, insulin activates the insulin receptors (IR, encoded by the INSR gene) present in cell membranes, which transduce an intracellular signaling cascade leading to the translocation of insulin-responsive glucose transporter 4 (GLUT4) from intracellular membranes in the cytoplasm to the cell membrane, and GLUT4 transportation of glucose into the cytoplasm of the cell. [00144] The insulin receptor is a dimeric protein with two extracellular ligand-binding alpha domains (the ectodomain), and beta domains consisting of two transmembrane helices and two intracellular tyrosine kinase domains. Binding of insulin ligand to alpha-chains in the dimeric ectodomain causes a conformational change that is propagated structurally through the beta domains. The intracellular tyrosine kinase domains begin to autophosphorylate tyrosine residues that generate binding sites and phosphorylate insulin receptor substrates. [00145] The insulin receptor autophorsphorylation leads to activation of PI3K, to activate the PI3K/AKT pathway. PDK-1 and Akt are thus translocated to the cell membrane and activate substrates that lead to GLUT4 translocation to the cell membrane. Akt1 and Akt2 can promote insulin-dependent glucose metabolism, but Akt2 has a more significant impact on glucose metabolism. [00146] AKT drives GLUT4 gene expression and directly phosphorylates AS160, inducing GLUT4 translocation to the plasma membrane from storage vesicles. AS160 (encoded by the TBC1D4 gene) is a highly insulin-responsive Akt target which negatively regulates insulin receptor signaling. AS160 is a Rab GTPase-activating protein with a GTPase-activating protein (GAP) domain. The GTPase-activating domain accelerates the hydrolysis of GTP (guanine triphosphate) to GDP (guanine diphosphate) on G proteins. Akt phosphorylates AS160 during insulin signaling at Thr-642 and Ser-588, causing a conformational change that inhibits the activity of the GAP domain, allowing Rab G proteins to bind to GTP instead of GDP. [00147] Rab proteins are a family of small G proteins with hydrolase enzymatic activity that are part of the larger Ras protein superfamily. Rab proteins regulate membrane trafficking, vesicle formation, vesicle fusion, and membrane fusion by affiliating with microtubules and microtubule associated motor protein kinesin, which plays a role in shuttling GLUT4 vesicles to the cell membrane. RAB2A, RAB8A, RAB10, and RAB14 are stimulated to maintain the GDP-bound form when the AS160 GAP domain is active, but switch from GDP-bound to GTP- binding forms upon inhibition of AS160 to regulate the trafficking and translocation of GLUT4 to the cell membrane. [00148] PDK-1 and PIP3 also play a role in insulin-stimulated GLUT4 translocation in an Akt-independent manner. The phosphorylation of residues on atypical PKCs such as PKC-ζ (and an interchangeable form, PKC-λ) is required to stimulate GLUT4 translocation to the cell membrane. PKC-ζ is phosphorylated in the kinase activation loop by PDK-1 at Thr-410 and facilitates GLUT4 trafficking to the plasma membrane. Overexpression of PDK-1 increases GLUT4 translocation in the absence of insulin via PKC-ζ phosphorylation (G. Bandyopadhyay et al., Dependence of Insulin-Stimulated Glucose Transporter 4 Translocation on 3- Phosphoinositide-Dependent Protein Kinase-1 and its Target Threonine-410 in the Activation Loop of Protein Kinase C-ζ, Molecular Endocrinology, 13(1), 1 Oct.1999, pages 1766-1772). PKC-ζ also has an autophosphorylation site at T560, and autophosphorylation levels increase with PIP3 activity and insulin treatment. The physical association of PKC-ζ with PIP3 provides allosteric interactions that induce a conformational change that increases autophosphorylation activity. Association of PKC-ζ with PIP3 also colocalizes it with PDK-1, facilitating phosphorylation at T410. If either phosphorylation site is inactive, the kinase activity decreases and lowers translocation rates of GLUT4 to the cell membrane. [00149] Insulin stimulates a reorganization of the actin cytoskeleton and brings PI3K, PKC- ζ, and GLUT4 into association in the plasma membrane. PI3K-mediated insulin stimulation caused phosphorylation of PKC-ζ, which associated with and remodeled actin, where it colocalizes with GLUT4. Overexpression of PKC-ζ lead to increased GLUT4 translocation to the cell membrane and glucose uptake (Liu et al. Protein Kinase Cζ Mediates Insulin-induced Glucose Transport through Actin Remodeling in L6 Muscle Cells, Molecular Biology of the Cell, 17(5): 2322-2330 (May 2006)). PI3K-mediated PKC-λ activation stimulation also activates Rab4, which affiliates with kinesin to mediate interaction with microtubules and stimulate transport of GLUT4 to the cell membrane. [00150] Mechanisms for insulin reduction in cell culture media [00151] Constitutive activation of the insulin receptor or its downstream targets may reduce or remove the requirement of insulin in the cell culture media. [00152] Genetically modifying the insulin receptor gene to truncate the receptor to beta subunits is one method to generate a constitutively active IR. These beta subunits can translocate and insert into the cell membrane and lead to persistent Akt phosphorylation (Frazier et al. Expression of a Constitutively Active Human Insulin Receptor in Hippocampal Neurons Does Not Alter VGCC Currents, Neurochem Res 2019 Jan, 44(1): 269-280). This truncated receptor spans amino acid residues -27 to 12 fused to residues 915 to 1343, and specifically includes the signal sequence (27 amino acids), the first 12 amino acids of the alpha subunit, and the terminal portion of beta subunit starting three amino acids before the transmembrane domain (23 amino acids), and the tyrosine kinase domain (403 amino acids, 468 amino a ccids total). Another version of the constitutively active, truncated insulin receptor includes the signal sequence, the first 12 amino acids of the alpha subunit, and residues 600- 1355 (~20% of the alpha subunit at the carboxy terminal and all of the beta subunit). [00153] A constitutively active insulin receptor will be designed by truncating the alpha subunits in the [INSR] gene. Truncated forms will include the signal sequence, the first 12 amino acids of the alpha subunit, and between 0-40% of the carboxy terminal of the alpha subunit, and all of the beta subunit, and will result in autophosphorylation of the tyrosine residues in the beta subunit. This constitutively active IR will translocate and insert into the cell membrane and lead to downstream phosphorylation of Akt and translocation of GLUT4 into the cell membrane in the reduction or absence of insulin in cell culture media. The truncated IR will be inserted as a transgene under the control of a constitutively active promoter into the native genome of the cell. Alternatively, a mutation can be performed on the native INSR gene that results in the deletion of nucleotide bases which truncates the alpha subunit of the insulin receptor, generating a constitutively active receptor. These methods include all substitution, deletion, insertion, duplication, inversion, and frameshift mutations. [00154] PDK-1 overexpression may increase GLUT4 translocation to the nucleus through phosphorylation of Akt and PKCs. [00155] Overexpression of GLUT4 gene itself has also been shown to increase GLUT4 protein translation and translocation to the plasma membrane in an insulin-independent manner. GLUT4 overexpression may be achieved in cell lines by modifying the promoter region of the native [GLUT4] gene to activate constitutive expression, or a transgene under the control of constitutively active promoter can be inserted into the native genome of the cell. [00156] Generating a constitutively active insulin receptor pathway has additional sensory benefits to myogenic and adipogenic cell lines that can improve flavor, pigment, and overall product quality. Through continuous translocation of GLUT4 into the cell membrane of myogenic and adipogenic cells in an insulin-independent manner, glucose can be transported into the cell and used for various cellular metabolism processes. This can include the glycolysis pathway which generates ATP for cells and converts glucose to pyruvate. Pyruvate is then used as the precursor substrate for the citric acid cycle that produces NADH and GTP. NADH then undergoes oxidative phosphorylation in mitochondria to generate high yields of ATP. [00157] The protein content of skeletal muscle cells is generated through the consumption of energy supplied by ATP and GTP. Increasing the protein content and the translation of muscle specific proteins enhances the umami meat-like flavor of cell lines. ATP is used by myogenic cells to enhance myogenic differentiation by supplying energy to the contractile protein machinery of sarcomeres. Binding of ATP to myosin filaments releases energy that facilitates the interaction of myosin protein with actin filaments that cause contraction. The development and maturation of sarcomeres increases the protein content of the muscle cell. GTP produced by the citric acid cycle is also used by skeletal muscle cells for protein translation via ribosomes. GTP supplies energy that is consumed during the formation of peptide bonds between amino acids in the ribosome. [00158] Glycolysis also plays a direct role in the conversion of glucose to free fatty acids that undergo esterification into triglycerides, which are the basic substrate of lipid droplets inside differentiating adipocytes. Metabolism of glucose produces pyruvate and glycerol, and the pyruvate is converted to fatty acids during fatty acid synthesis pathway. Glycerol is then bound to three fatty acids via esterification to produce the triglyceride. The more glucose that is transported into the cell, the more triglycerides can form and contribute to the production and enlargement of lipid droplets. The formation of lipid droplets increase the flavor and juiciness of the cells. Continuous glucose transport into the cell in an insulin-independent manner will drive the fatty flavor profile of adipogenic cell lines. [00159] Target Combinations [00160] The mechanisms herein describe provide a method to enhance cell proliferation with reduced growth factor concentration through constitutive activation of the ERK1/2 pathway and insulin-independent glucose transport through activation of the PI3K/Akt pathway. Edible cell lines used in cultivated meat products must have low cost production methods to be affordable. Cell lines used in academia or the regenerative medicine industry are not designed to minimize growth factor and insulin requirements in cell culture media required for cell survival, proliferation, and differentiation. These types of cell lines have different cost drivers than cells lines used to produce affordable food for consumers. Cell lines used in research are not designed for scalable manufacturing, and cell lines used in the cell therapy or biopharma industries are expensive products with high profit margins. Consequently, engineering cell lines for cultivated meat products to have reduced mitogen and insulin requirements is a novel method to reduce production costs. Furthermore, there has been no published literature that specifically augments the ERK1/2 pathway and the PI3K/Akt pathway to reduce mitogen and insulin requirements in cell culture media to reduce production costs. [00161] The following example combinations can generate immortal cell lines with anoikis resistance, insulin-independent insulin signaling, mitogen-independent cell cycle progression, and improved flavor and sensory properties of cell lines: [00162] ILK+, PDK-1+, Telomerase+, PTEN-, BRAF-V600E [00163] One potential combination of cell line engineering targets is to overexpress ILK, PDK-1, telomerase, knock out PTEN, and replace valine with glutamate at amino acid 600 of B-Raf to confer constitutive activity. Knocking out PTEN prevents the dephosphorylation of PIP3 to PIP2, which allows constant translocation of Akt and PDK-1 to the plasma membrane, where PDK-1 can phosphorylate Akt to initiate downstream signaling pathways related to cell cycle progression and anoikis resistance. Overexpressing PDK-1 increases the rate of Akt phosphorylation while also activating S6K1, SGK, PKC, and RSK, which can all promote cell cycle progression. ILK overexpression and BRAF-V600E activity can simulate integrin- mediated survival signals, block anoikis, and induce anchorage independent cell growth through ILK mediated phosphorylation of Akt and BRAF-V600E activation of ERK1/2. ILK and PDK-1 overexpression and PTEN knockout have the downstream effect of enhancing Akt- mediated cell signaling by increasing phospho-Akt activity and promoting cell cycle progression, anoikis resistance, and insulin-independent insulin signaling through recruitment of GLUT4 to the cell membrane. Flavor, pigment, and product quality can be enhanced by constitutive activation of the insulin / GLUT4 pathway, which drives myogenic and adipogenic differentiation. BRAF-V600E also promotes cell cycle progression through ERK1/2 phosphorylation in a mitogen-independent mechanism. Additionally, overexpressing telomerase helps the cells bypass the hayflick limit by maintain telomere length through repeated cell divisions, which is necessary to develop scalable cell lines. [00164] ILK+, PDK-1+, Telomerase+, TBX2+ or PAX3+ [00165] Another target combination could be overexpressing ILK, PDK-1, telomerase, TBX2 or Pax3. This follows the same logic above with ILK, PDK-1, and telomerase overexpression. However, instead of knocking out PTEN, TBX2 and PAX3 overexpression (either in combination or individually) suppress PTEN activity which inhibits the dephosphorylation of PIP3 to PIP2. [00166] PDK-1+, Telomerase+, PTEN-, BRAF-V600E [00167] This target combination does not utilize ILK overexpression. Instead, it relies on the ability of PDK-1 to phosphorylate Akt in the presence of excess PIP3 due to PTEN knockout. P-Akt confers anoikis resistance by suppressing Bad and procaspase-9 assembly of the apoptosome and increases GLUT4 translocation to the cell membrane. BRAF-V600E activates ERK1/2 signaling, which promotes mitogen-independent transcriptional activation of cyclins and CDKs and phosphorylates c-myc. This increases cell proliferation and provides an ERK- dependent mechanism for anoikis resistance. [00168] ILK+, PDK-1+, Telomerase+, PTEN-, BRAF-V600E, GLUT4+ [00169] This strategy follows the ILK+, PDK-1+, Telomerase+, PTEN-, BRAF-V600E cell line strategy, but also includes GLUT4 overexpression. GLUT4 overexpression will enhance glucose transport through the cell membrane in an insulin-independent manner, which reduces reliance on PDK-1/pAkt signaling to stimulate GLUT4 translocation. [00170] ILK+, PDK-1+, Telomerase+, PTEN-, BRAF-V600E, insulin receptor+ [00171] This strategy follows the ILK+, PDK-1+, Telomerase+, PTEN-, BRAF-V600E, GLUT4+ cell line strategy, but instead of GLUT4 overexpression, it uses a constitutively active insulin receptor that transmits insulin-independent insulin signaling to the cell, leading to GLUT4 translocation to the cell membrane. [00172] CRISPR-based cell engineering [00173] CRISPR/Cas9 refers to a genetic modification method using a Cas9 enzyme and small guide RNAs (gRNAs). The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system was first discovered in bacteria, which use it to defend against viral infection. The Cas9 enzyme is an endonuclease that uses CRISPR sequences as a guide to cut matching viral DNA sequences. Cas9 is complexed with 20 nucleotide guide RNA sequences attached with a RNA scaffold that match the CRISPR sequences and viral DNA. Cas9 unwinds double-stranded DNA, and once it finds a non-target sequence match to the sgRNA it binds to the protospacer adjacent motif (PAM) region downstream of the target sequence and initiates a double-stranded cut to the DNA. Cas9 cleaves the DNA 3 base pairs upstream of the PAM region within the target sequence. This can lead to gene inactivation through non- homologous end joining or the insertion of new genes through homologous recombination. A Cas9 mutant called Cas9n is a nickase that creates a single stranded break instead of a double stranded break. Cas9n cleaves only the target strand instead of the both the target and non-target strands. This creates a preference for homology directed DNA repair pathway, which decreases the likelihood of off-target insertion or deletion mutations and increases the efficacy of HDR. [00174] Nonhomologous end joining (NHEJ) is the DNA repair pathway that repairs breaks in double stranded DNA by joining/ligating two broken ends together without utilizing a homologous template. This repair method is error prone and can lead to insertion or deletion (indel) gene mutations. Indels that occur within a coding exon can inactivate the gene through frameshift mutations or premature stop codons. Commonly used for knockout studies. This repair mechanism occurs when there is no DNA repair (genetic insert) template. This can be performed in mitotically inactive cells. [00175] The homology-directed pathway (HDR) repairs single or double stranded breaks in DNA using a homologous template. This is a high fidelity repair mechanism that preserves the sequence of genes with the option of introducing new genetic inserts. The repair template can either be double-stranded DNA or single-stranded DNA oligonucleotides (ssODNs). ssODNs can be used to make small edits in the genome, such as adding a single base pairs to a gene sequence to create mutations. This must be performed in mitotically inactive cells. [00176] Combination of cells with plant-based ingredients [00177] Manufacturing process for plant-based meat structuring [00178] Plant based products can be used to supplement cell-based meat products. Plant-based meat process engineering can utilize manufacturing processes including but not limited to stretching, kneading, shear-cell processing, phase separation of biopolymer blends, spinning process, press forming, folding, layering, 3D printing, high moisture extrusion and low moisture extrusion, or a combination of the above outlined technologies, which allows cells to mixed into plant protein with aligned fibers and interspersed fatty marbling. The cellular content of the product can be between 1-5%, 5-10%, 10-15%, 15-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60- 70%, or 70-80% of the mass of the product per 100g. [00179] Plant-based ingredients that can be used in combination with cell-based meat products may include but are not limited to proteins, including soy protein, legume protein (such as pea, chickpea, lentils, and other types of beans), oilseed proteins (such as rapeseed and canola), wheat gluten, potato protein, hemp protein, mung beans, flavorants, including koji, miso, seaweed, and recombinant proteins, mycoprotein, including mycelium, mushrooms, shiitake mushrooms, fats and oils, including sunflower oil, rapeseed oil, canola oil, corn oil, palm oil, coconut oil, and soya oil, binders, including methyl cellulose, wheat gluten, xanthan gum, albumin, soy protein concentrate, soy flour, pectin, guar gum, carrageenan, transglutaminase, and coloring agents, including beet juice, betanin, and heme proteins. [00180] Personalized cell-based meat product [00181] A cell-based meat product may be produced that mimics the taste and sensory properties of a chosen cut of animal meat. For example, one may choose a preferred cut of meat from cattle, pork, or other mammal and create a cell-based meat product that mimics it. [00182] Any mammal meat may be used as the inspiration for the cell-based meat product, including but not limited to bovine, caprae, cervidae, lagormorph, oves, rodent and suidae meat. [00183] Bovine and porcine cells have different properties that have implications for the bioprocess and cell line requirements for each respective cell type. [00184] Bovine meat [00185] The most savory beef cuts originate from the ribs of the cow. Rib-eye steak (derived from the longissimus) is the most flavorful cut of beef due to the high intramuscular fat and marbling content, while the round (derived from the biceps femoris) contains less marbling and fat content than rib eye. The intramuscular fat (IMF) % of the biceps femoris in animals fed a low-fat diet is 2.5% in Dexter, 4.9% in Angus, and 6.8% in Wagyu x Angus crossbreeds (Table 1). In cattle fed a high fat diet, the fat content is 12.6% in Angus, 11.6% in Wagyu x Angus, and between 14.7- 26.4% in the proximal and distal cranial head of Japanese Black Wagyu cattle. The IMF% of the longissimus thoracis is 2.3% in Dexter, 6.2% in Angus, 15.3% in Korean Hanwoo, and 31.5% in Japanese Black Wagyu animals. The total IMF% from 21 muscles of Wagyu steers is 32%. [00186] Table 1. Average intramuscular fat % and muscle fiber type in the biceps femoris and longissimus thoracis
Figure imgf000051_0001
[00187] Meat quality grading systems [00188] Cell-based meat products m be assessed according to meat quality standards established by their countries of sale. Many countries view intramuscular fat to be important content in meat, such as Korea, Japan, Australia, and the US. European countries, such as France and Germany, prefers leaner cuts of meat. The USDA establishes meat quality with the US Beef Grading System and the Japanese Meat Grading Association (JMGA) establishes meat quality in Japan. Both systems assess quality via intramuscular fat percentage, color, and maturity, and the yield of usable meat on the carcass. These systems differ in how they use quality metrics, however. Understanding the differences between these quality evaluation systems is critical for US competition with Japanese exports. [00189] In the JMGA meat scale, the marbling score of the longissimus muscle is evaluated at the 6th-7th rib interface, in comparison to the USDA marbling score, which is evaluated at the 12th- 13th rib interface. The JMGA uses an overall meat quality scale 1-5 based on four meat quality assessments: beef marbling score (BMS); beef color standard (BCS); firmness and texture of meat; and beef fat standard (BFS). The BMS score is an estimate of minimum IMF% and ranks 1-12, with 1 being the least amount of marbling and 12 being the highest degree of marbling (Table 2). The meat must be graded at BMS 3 or above to reach the acceptable minimum IMF of wagyu: 21.4%, and a BMS score of 12 contains at least 56.3% IMF (Table 3). BCS assesses meat color and brightness using a 1-7 scale, where number 1 is a pale red and number 7 is a dark red. The best BCS is 3 or 4, where color and brightness are most strongly correlated (Table 2). The firmness of meat is determined by the amount of exudate on the surface of the cut, and the degree of depression of the indented surface. Meat with high BMS should be firm and have less exudate, since it has higher water holding capacity. The texture reflects a smooth or coarse surface of the cut (Table 2). Meat is graded best when it is very firm with a smooth texture. The BFS measures color, luster and quality of fat using a score between 1-7, where 1 is pale white and 7 is dark cream (Table 2). The BFS is best between 1-4. After each of the quality scores are evaluated according to the 1-5 scale, the lowest of the scores becomes the overall meat quality score for the beef. [00190] Table 2. Japanese and US beef quality scoring metrics
Figure imgf000052_0001
[00191] Table 3. Japanese and US beef quality grades determined by intramuscular fat %
Figure imgf000053_0001
[00192] The USDA beef standard assesses meat quality through marbling score, meat color, and texture of cuts (which is associated with age) and grades good quality beef into three categories: select, choice, and prime (Table 2). Above 21.4% IMF%, marbling is ‘very abundant’ and graded prime+. Typically, prime US beef has 9.9-12.3% IMF, the marbling is ‘moderately abundant’ to ‘slightly abundant’, it’s bright red in color, and moderately firm in texture (Table 3). Choice beef has a ‘moderate’ to ‘small’ amount of marbling (4-7.7% IMF), is light cherry red to slightly dark red in color, and is slightly soft in firmness. Select beef has a ‘slight’ amount of marbling (2.3- 3.9%), moderately light red to moderately dark red color, and a moderately soft texture, since it comes from younger animals on average. [00193] Pork meat [00194] Two of the most in-demand pork products are pork belly, bacon, and pork loin. Pork belly is composed of multiple muscle groups: the cutaneous trunci, latissimus dorsi, pectoralis profundus, rectus abdominis, and internal and external abdominal oblique) and the fat content between these muscles. Bacon is the cured meat from the belly of the pork. Bacon has different names depending on the part of the animal it was derived, and ‘streaky bacon’ is the cut from pork belly. While bacon has its fat content, it isn’t as fatty as pork belly because it is a particular cut of the underside of the animal, which has less fat in it. In order to turn cuts of pork belly into bacon, the meat is drained of all moisture with the help of salt. Pork loin is cut from the animals back and includes the longissimus muscle. [00195] The present invention provides methods and compositions of a personally designed cell- based cut of meat. One could use any combination of the methods set forth herein, fatty acid supplementation, as well as methods of modifying cell lines, and create a “personalized” cell derived cut of meat. [00196] The following example combinations can generate immortal cell lines with anoikis resistance, insulin-independent insulin signaling, mitogen-independent cell cycle progression, and improved flavor and sensory properties of cell lines via overexpression of FAK and/or SRC or by reducing PTEN expression. [00197] To increase the production yield of cell lines, anchorage independent survival and growth can be conferred by manipulating cell signaling pathways that generate anoikis resistance. Failure to initiate anoikis can result in cell survival in suspension. The present inventions provide methods for enhancing resistance to anoikis and mitogen independent enhanced proliferation and/or mitotic potential via overexpression of FAK and/or SRC. [00198] Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that provides signaling and scaffolding functions at sites of integrin adhesion. FAK is involved in protein– protein-interaction adaptor functions at sites of cell attachment to the extracellular matrix (ECM), thereby contributing to focal-adhesion 'scaffolding'. FAK also transmits adhesion- dependent and growth-factor-dependent signals into the cell interior. FAKs with its anti- apoptotic activity helps the cells with anoikis resistance (resistance to suspension induced death). [00199] SRC is another family of non-receptor tyrosine kinases which transmit integrin- dependent signals pivotal for cell movement and proliferation. c-Src is shown to bind constitutively and selectively to beta3 integrins through an interaction involving the c-Src SH3 domain and the carboxyl-terminal region of the beta3 cytoplasmic tail. [00200] FAK activation causes autophosphorylation, which recruits Src, which further phosphorylates FAK and leads to a fully activated FAK enzyme. FAK then activates the Akt and MAPK pathways. FAK activates PI3K, which then recruits and activates Akt. Akt activation promotes cell survival by several mechanisms. It can phosphorylate the proapoptotic protein Bad, which prevents its inhibition of Bcl-2; phosphorylating and inactivating procaspase-9; and activating NFKB survival pathway. PTEN interacts with FAK and suppresses the extracellular matrix-dependent PI3K/Akt cell survival pathway In cancer cells, FAK is overexpressed and correlates with invasive potential of tumors. [00201] The present inventions provide methods for insulin-independent glucose transport into the cell and enhancing the sensory profile of cells through overexpression of GLUT1, GLUT2, and GLUT3. In some examples, GLUT1, GLUT2, and GLUT3 may be used in place of or in addition to GLUT4 to achieve these cell properties. [00202] Additional Methods to Reduce Wild Type PTEN Expression [00203] The term “knockdown” refers to downregulating the expression of a gene or gene product(s). As a result of knockdown, the protein expression and activity will be reduced or ablated. In some embodiments, knockdown is achieved using antisense molecules which are introduced to downregulate the expression of the target gene. In some cases, antisense molecules downregulates the expression of target genes via RNA interference (RNAi). This could comprise a combination of one or more antisense molecules such as short interfering RNA (siRNA), a microRNA (miRNA) or short hairpin RNA (shRNA). [00204] The term “small interfering RNA” or “siRNA” (used interchangeably) refers to a class of double stranded RNA. Typically, siRNA is about 20-23 base pairs in length, similar to miRNA and operates within the RNA interference (RNAi) pathway. siRNAs are highly specific and have the ability to reduce the translation of specific mRNAs and the protein expression of the target gene. siRNA can be chemically synthesized as “siRNA oligonucleotides”. siRNAs can be delivered to cells using lipid-based methods, electroporation or other methods. [00205] The term “shRNA” or “small hairpin RNA” or “short hairpin RNA” (used interchangeably) refers to another form of RNA interference (RNAi). shRNA is synthesized within the cell by DNA-vector mediated production. shRNA consists of 2 complementary 19- 22 bp RNA sequences linked together by a short nucleotide loop of 4-11 nt. [00206] While siRNA delivers the siRNA duplex directly to the cytosol, shRNAs are capable of DNA integration. Unlike “siRNA”, “shRNA” can be delivered to mammalian cells via infection of the cells with virally produced vectors such as adenoviral, retroviral, or lentiviral-based delivery, called “transduction”. This results in stable integration, eliminating the need for multiple rounds of transfection and increases the reproducibility of the results. shRNA integrated stable cell lines can be created by selection with antibiotics (drug resistance) in combination with fluorescence markers such as green fluorescent protein (GFP) or red fluorescent protein (RFP). shRNA vector can be constructed with oligonucleotide-based cloning and PCR-based cloning. [00207] The term “miRNA” or “microRNA” (used interchangeably) refers to endogenous small single-stranded non-coding RNA molecules present in the genome that function in RNA silencing and post-transcriptional regulation of gene expression. The majority of miRNAs are transcribed from DNA sequences into primary miRNAs and processed into precursor miRNAs, and finally mature miRNAs. In most cases, miRNAs interact with the 3′ untranslated region (3′ UTR) of target mRNAs to induce mRNA degradation and translational repression. [00208] The term “microRNA” or miRNA refers to a class of noncoding RNA that plays an important role in regulating gene expression. They are natively present in the genome which could be modulated in their expression using miRNA inhibitors. In some embodiments, miRNA refers to the synthetic or artificial miRNA which are chemically modified double stranded RNA molecules designed to mimic endogenous miRNA, resulting in down regulation of target mRNA translation due to mRNA degradation or sequestration. [00209] In some representation, miRNA is an artificially synthesized oligonucleotide as “miRNA mimetics” which is capable of assuming the regulatory role of natural miRNAs. Like siRNA and shRNA, double stranded RNA oligonucleotides with minimal chemical modifications are suitable miRNA replacements in vitro. In some representation, antagomiR, miRNA sponges or decoys to inhibit miRNA functions by preventing stable binding to their targets. Like siRNA, miRNA or antagomiR could be delivered by lipid-based delivery similar to siRNA delivery or by viral based delivery systems using adeno or retrovirus. [00210] In some cases, siRNA/shRNA/miRNA/antagomiR can be delivered to the cells using cationic polymer polyethylene imine (PEI). [00211] The term “dominant negative” refers to the mutation with the resulting mutant gene product and protein expression resulting in adversely affecting the normal, wild type gene product within the same cell by competitive inhibition by either competing for ligands, substrates and interacting partners. This dominant negative mutant protein overexpression could enable us to achieve the phenotype expected from the knockdown or knockout of the endogenous wildtype gene/protein. [00212] The most studied dominant negative PTEN mutants are Cys-124 to Ser (C124S) and the Gly-129 to Glu (G129E). C124S mutation generates a catalytically dead mutant with complete loss of PTEN phosphatase activity whereas the G129E mutation abrogates the phosphoinositide phosphatase function but retains activity towards phospho-peptides. These dead mutants can dimerize with wildtype PTEN protein in a dominant negative manner by disrupting the function. PTENR130G missense mutation generates a stable protein but is catalytically inactive (lipoid phosphatase function is lost). PTENR130X and R233X nonsense mutations generate very unstable PTEN proteins that are almost undetectable mimicking heterozygous conditions. [00213] The following describes methods by which various cell targets, for example, FAK and/or SRC, may be overexpressed and therefore result in cell lines having improved flavor and sensory properties. The present invention also includes additional methods for overexpressing various cell targets, such as viral based methods for transduction and inducible gene expression approaches. [00214] Viral methods include stable integration of transgenes with lentivirus or retrovirus transduction. Inducible expression systems can be used to achieve overexpression of genes upon the introduction of a signaling molecule. Inducible methodology includes, but are not limited to, transcription factor/promoter activity regulation by ligand inducible transcription factor machinery e.g., cumate-on, cumate-off, tet-on, tet-off,), site-directed recombination technology (e.g., Cre-LoxP, flp-FRT), ligand binding receptor fusion technology (e.g., tamoxifen metabolite, steroid hormone receptor). [00215] Inducible expression system is a regulated expression of transgene. Inducible systems can be classified based on the nature of the inducer that is used to regulate the system. Types of inducers that have been used include heavy metal ions, heat shock, antibiotics, steroid hormones, IPTG and dimerizer. Inducible gene regulation by small molecules offers time- and dose-dependent control. Inducible gene expression systems are favored over stable expression systems as they are mostly reversible and thus more flexible to use. The protein expression may be titrated as per needs and may completely terminate the expression when not needed. EXAMPLES [00216] 1. Cell isolation protocol for bovine and porcine myoblasts and fibroblasts [00217] Muscle tissue of 3-4 cm2 (4-6 g) in size was harvested from the thigh muscle of a pig (1- month-old) or a 2-month-old cow’s hind leg (biceps femoris) from a local farm. To isolate bovine and porcine myoblasts, muscle tissue is cut into small pieces after removing blood vessels and fascia/connective tissue, suspended in a tissue digestion buffer containing DMEM, 1% penicillin/streptomycin and 0.5% collagenase IV (Worthington Bio), and incubated at 37°C for 60-90 minutes. Every 15 minutes the cell and tissue mixture was pipetted to improve tissue disintegration. The cell mixture was further filtered using a 40 µm cell strainer, neutralized by adding fetal bovine serum, and the suspended cells were collected by a 5-minute centrifuge at 1,200 rpm at room temperature. Following at least one washing steps with PBS and centrifugation, the heterogeneous cell mixture was resuspended in Advanced DMEM/F12, 20% FBS, and 1x P/S (penicillin/streptomycin) and underwent a pre-plating technique. The cells were seeded in standard tissue culture plates to allow fibroblasts attachment overnight at 37°C in 5% CO2. The next day, unattached cells were collected, centrifuged, washed with PBS, resuspended in myoblast growth media (MGM, DMEM/F-12 with 20% FBS, 1% Glutamax, 1% Penicillin/Streptomycin and 2ng/mL FGF-2), and seeded in laminin-coated culture plates. After a 2-3 day incubation at 37°C and 5% CO2, suspended (unwanted) cells and tissue debris are removed by aspirating the culture medium followed by at least one wash with PBS, and adherent myoblasts can be obtained. [00218] 2. Cell culture protocols [00219] Primary bovine and porcine myoblasts may be grown in MGM and passaged upon reaching 50-60% confluency. Cells between passage 1-4 may be used to generate cell lines. [00220] Improving cell differentiation [00221] Cell differentiation may be improved by supplementing fibroblasts cultures with fatty acids, for example, oleic acid, linoleic acid & palmitoleic acid. As shown in FIG. 12, when compared to the promo cell media alone, cultures supplemented with fatty acid showed improved differentiation. In-addition, bovine myoblasts were partially transdifferentiated with a combination of MyoD knockdown (to block myogenic differentiation) and supplemented with FFAs to transdifferentiate to adipocytes. Bovine myoblasts grown in regular growth media (lacking FFAs) and myogenic differentiation media were used as negative controls. [00222] 3. CRISPR-editing strategy [00223] Non-homologous end to end joining resulting in PTEN knockout [00224] A gRNA may be designed and assembled to direct the Cas9 nuclease to a first exon of the PTEN gene. Alternatively, a gRNA may be designed to target the PTEN promoter, preventing transcription of the PTEN gene. The gRNA and Cas9 enzyme may be delivered by electroporation nucleofection or lipid transfection into the cells. The gRNA may target Cas9 to a DNA target sequence, where Cas9 may cut the DNA, creating a double stranded break (DSB) and an indel mutation. The inactivated form of PTEN may be unable to dephosphorylate PIP3 to PIP2, or the gene may be silenced. [00225] Homology Directed Repair (HDR) [00226] ILK, PDK-1, and Telomerase overexpression [00227] Three gRNAs may be designed and assembled to direct Cas9n nickase to upstream untranslated regions of the ILK, PDK1, and/or TERT genes. DNA insertions that may add a constitutively active promoter upstream of an AUG sequence start site of the ILK, PDK1, and/or TERT genes using a double stranded DNA template with upstream and downstream homology arms may be performed. The promoter may be native to the cell, such as the native bos taurus beta-actin promoter for bovine cells, that activates transcription for constitutively expressed genes. This technique may generate overexpression of wild type ILK, PDK1, and TERT genes. A double stranded repair template containing the constitutively active promoter may be needed for each gene, since the homology arms must match upstream and downstream sequences of the target sequence for each gene. [00228] The three gRNAs, Cas9n enzyme, and three repair templates may be delivered by electroporation nucleofection or lipid transfection into the cells. The gRNA may target Cas9 to the DNA target sequences, where Cas9n cuts the DNA, creating a single stranded break (SSB). HDR may insert the constitutively active promoters upstream of the ILK, PDK1, and TERT genes. [00229] Alternatively, three gRNA templates may be designed to target ILK, PDK1, and/or TERT transgenes under the control of constitutively active promoters to the Rosa26 locus of the genome. The gRNAs, Cas9n enzyme, and three transgene DNA constructs may be delivered by electroporation nucleofection or lipid transfection into the cells. [00230] B-Raf mutation using single-base substitution [00231] To generate a constitutively active form of B-Raf, BRAF-V600E, from the BRAF gene, thymine may be substituted with adenine at nucleotide 1799 to convert valine to glutamate using CRISPR-mediated single-base substitution and ssODNs. A gRNA may target a Cas9n enzyme to the 1799 nucleotide of the B-Raf gene, and the ssODN sequence may comprise glutamate at 1799 instead of valine and wild type sequences upstream and downstream flanking homology arms. The gRNA, Cas9n, and ssODN may be delivered by electroporation nucleofection or lipid transfection into the cells. [00232] Briefly, C2C12 mouse myoblasts were genetically modified using the CRISPR knock-in technique outlined above. Genetic modification of C2C12 mouse myoblasts included mutations to the B-Raf gene, resulting in a B-Raf mutant knock-in (B-Raf V600E). A point mutation substituting Valine (V) for Glutamic acid (E) at amino acid position 600 of BRAF was inserted by CRISPR mediated knock-in. The modified B-Raf gene was integrated into ROSA26, the modified sequence was validated and inducible-BRAF-V600E knock-in was verified via Sanger sequencing (FIG.4A). As compared with the wild type B-Raf gene, B-Raf V600E mutants showed enhanced ERK signaling as reflected by an increase in proliferation as measured by CellTiter-Glo® assay (FIG.9). [00233] In another example, C2C12 myoblasts were genetically modified to include an INSR mutant gene having truncated alpha subunits to produce exon free insulin receptors. The mutant INSR sequence was then inserted by CRISPR mediated knock-in as described above. FIG.4B shows the sequence insert confirmed by Sanger sequencing (CRO company GenScript performed CRISPR Knock-in (KI) projects). [00234] 4. Media formulation analysis [00235] Cell culture media formulations comprising or absent insulin, Fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), leukemia inhibitory factor (LIF), and/or other growth factors may be tested on the cell lines. Cell populations that maintain proliferation and transmembrane glucose transport in media formulations with low concentrations or absent of insulin and growth factors may be continuously passaged and selected for further cell line development. Cells comprising necessary CRISPR edits may be selected via a positive selection process. For example, cells that do not comprise all CRISPR edits will undergo apoptosis and be unable to proliferate in the absence of insulin and growth factors, creating a positive selection process for cells that did achieve all CRISPR edits. Cell banks may be expanded for the selected cell populations. [00236] Briefly, C2C12 cells having truncated alpha subunits to produce exon free insulin receptor were prepared as described above. C2C12 cells with INSR KI were transfected with plasmids of GLUT1-4 (OriGene, Rockville, MD) and CellTiter-Glo® assay was performed. Cells were tested for ability to proliferate in reduced or insulin free conditions either alone or in combination with transient transfection of GLUT 1-4. Assessment of cell proliferation under such conditions is important for understanding whether continuous translocation of GLUT into the cell membrane of myogenic cells in an insulin-independent manner facilitates glucose transport into the cell to be used in various cellular metabolic processes. Such metabolic processes include a glycolysis pathway which generates ATP for cells and converts glucose to pyruvate. Further, NADH undergoes oxidative phosphorylation in mitochondria to generate high yields of ATP. The CellTiter-Glo® assay was used to assess ATP synthesis by the cell as measured by luminescence (RLU). [00237] Additionally, C2C12 cells with INSR KI were transfected with plasmids of GLUT1-4 and CellTiter-Glo® assay was performed. Protein content of skeletal muscle cells may be generated through the consumption of energy supplied by ATP and GTP. Increasing the protein content and the translation of muscle specific proteins enhances the umami meat-like flavor of cell lines. GLUTs transport glucose for ATP synthesis, thus the CellTiter-Glo® assay was performed to confirm increased ATP synthesis. [00238] FIGs.6 and 7 show C2C12 cells with INSR KI alone or in combination with GLUT1-4 over-expression as having higher ATP activity compared to wild type C2C12 cells. Further, FIG. 7 shows that C2C12 cells with INSR KI and GLUT-4 over-expression showed higher ATP activity (cell proliferation) in reduced serum conditions as measure by the CellTiter-Glo® assay. Thus, C2C12 myoblasts with stable knock-in of constitutive INSR and transient transfection of GLUT1/GLUT2/GLUT3/GLUT4 individually resulted in insulin independent glucose transportation as evidenced by increase in the ATP synthesis across different serum and insulin gradient conditions compared to wild type control C2C12 cells. [00239] Moreover, FIGs. 8A-D show that primary bovine myoblasts over expressing PDK1 or ILK alone or in combination with TERT, exhibit mitogen independent cell cycle progression as evidenced by higher proliferation compared to wild type control cells in serum gradient culture conditions as assessed by CellTiter-Glo® assay. [00240] 5. Anchorage-independent growth [00241] Primary bovine myoblasts were genetically modified to over express ILK or PDK-1, alone or in combination with TERT; PAX3 or TBX-2, alone or in combination with TERT; PTEN knockdown; and C2C12 WT or C2C12 with BRAF and SRC were seeded in regular (untreated) 96-well plates and also seeded in anchorage resistant plates. Anoikis resistance properties of genetically modified cells were analyzed using an anoikis assay based on MTT (3-(4,5-Dimethylthiazol-2-yl) dye. FIGs. 5A-D show measurement of absorbance indicating anoikis resistance by genetically modified cells with all the gene targets tested compared to wild type control cells following a 72-hour incubation. In particular, PTEN knockdown in TERT over-expressing cells show significant anchorage independent growth compared to cells seeded and adhered in regular plates. As seen in FIGs 5A-D, the results show that genetic modification allows the cells to better adapt to anchorage independent conditions as the readings from the absorbance are equivalent to their growth in regular plates. Further, siPTEN knockdown cells show statistically significant increased growth in anchorage independent condition compared to the growth in regular plates. [00242] A sample of the cell lines showing reduced growth factor and insulin requirements may be plated onto agarose coated cell culture plates. The cell lines that proliferate in suspension may be continuously passaged, which will positively select for the cells with anoikis resistance. The cells that do not possess anoikis resistance will undergo anoikis and be removed from the cell culture during sub-culturing. [00243] 6. Mitotic capacity and senescence assay [00244] Cells that are able to grow in suspension may be continuous sub-cultured over long term periods to determine mitotic capacity. Cell lines that do not undergo senescence may be selected and used for commercial production. [00245] Myogenesis protocol [00246] Bovine and porcine myoblasts were cultured in MGM until reaching 80% confluency. Media was then switched to myoblast differentiation media (MDM, Advanced DMEM/F12, 2% horse serum, 1% glutamate, 1% P/S) and cultured for 72 hours. [00247] Adipogenesis protocol [00248] Bovine or porcine intramuscular fibroblasts were cultured in fibroblast growth medium (FGM) until adipogenesis assays. Fibroblasts were subsequently switched to Promocell medias. First, the fibroblasts were adapted to pre-adipocyte growth medium (Cat. No. C-27410) for 1 week, then switched to pre-adipocyte differentiation medium (Cat. No. C-27436) for 72 hours. After differentiation, cells were switched to adipocyte nutrition medium (Cat. No. C-27438) for two weeks. [00249] Oil Red O staining [00250] Lipid droplets inside mature adipocytes were visualized by Oil Red O (ORO) staining. ORO was prepared by dissolving Oil Red O (Cat. No.00625-25G, Sigma Aldrich, St. Louis, MO) in 20 ml of 100% isopropanol, after mixing well, ORO was incubated at room temperature for 20 min. Oil Red O Working Solution was prepared by adding 3 parts of Oil Red O Stock Solution to 2 parts of dH2O, mixing well, and incubated at room temp for 10 min. The solution was filtered with 0.2 µm syringe filter. The adipocyte cultures were then washed with PBS and fixed with formalin for 30 minutes. After fixation, cultures were washed twice with dH2O. Isopropanol (60%) was prepared by adding 3 parts Isopropanol (100%) to 2 parts of water. Isopropanol (60%) was added to each well and incubated for 5 min. Isopropanol was removed and Oil Red O working solution added to the cultures. Samples were incubated for 20 min. Oil Red O solution was removed and samples were washed 2-5x with dH2O. [00251] Transdifferentiation of myogenic cells to adipogenic cells [00252] A culture of bovine myoblast cells may be expanded to increase the biomass for a beef product. Upon reaching a certain biomass, the culture may be split into two vessels. A first vessel may continue to grow myogenic cell population in myogenic media, while the cells in a second vessel may undergo transdifferentiation into adipogenic cells. Cell culture media may be supplemented with any combination of the following free fatty acids at any concentration: SFAs include Myristic acid (14:0), Palmitic acid (16:0), Stearic acid (18:0), Arachidic acid (20:0), Behenic acid (22:0), Lignoceric acid (24:0), and Cerotic acid (26:0). PUFAs include omega-3, -6, and -9 fatty acids. Omega-3 FAs include α-Linolenic acid (ALA, (18:3(n-3)), Stearidonic acid (SDA, (18:4 (n−3)), Eicosatetraenoic acid (ETA, (20:4 (n−3)), Eicosapentaenoic acid (EPA, (20:5(n-3)), Docosapentaenoic acid (DPA, (22:5 (n−3)), and Docosahexaenoic acid (DHA, (22:6 (n−3)). Omega-6 FAs include: Linoleic acid (LA, (18:2 (n−6)), Gamma-linolenic acid (GLA, (18:3 (n−6)), Calendic acid (18:3 (n−6)), Dihomo-gamma-linolenic acid (DGLA, (20:3 (n−6)), Arachidonic acid (AA, (20:4 (n−6)), and adrenic acid (AdA, (22:4 (n−6)). Mead acid (20:3 (n−9)) is an omega-9 PUFA. Other omega-9s and omega-7 FAs are MUFAs. Omega-7s include Palmitoleic acid (16:1(n-7)) and Paullinic acid (20:1(n−7)). Omega-9 FAs include Oleic acid (18:1, (n−9)), elaidic acid (18:1 (n−9)), gondoic acid (20:1 (n−9)), Erucic acid (22:1(n-9)), and Nervonic acid (24:1 (n−9)). [00253] Adipogenic cultures may differentiate with the fatty acid mixture in the media until a threshold of FFAs are transported into intracellular lipid droplets, which may be an incubation period of 1-3, 3-7, 7-10, or more than 10 days in culture. [00254] Genetic modification including lentiviral mediated transduction (over-expression), transient transfection (lipofectamine-mediated), and CRISPR mediated knock-in were used to obtain genetically modified primary bovine myoblast and/or C2C12 mouse myoblasts. [00255] Wagyu beef product development strategy [00256] US beef has much lower IMF% than Japanese beef. The average highest prime grade contains ~12% IMF, and the average Wagyu BMS is a minimum of 21.4% IMF. Cell-based meat may give US beef a way to compete with Japanese Wagyu beef global market. Cells for cell-based Wagyu may be sourced from an American or Japanese Wagyu calf and grown ex vivo. The intramuscular fat composition can be simulated by adding 21% or greater adipocytes to the meat product. [00257] Determine target product characteristics [00258] Designing a cell-based Wagyu rib eye steak first requires assessing meat characteristics that influence sensory properties, such as muscle fiber type composition, fatty acid composition, IMF%, and chemical composition of the longissimus thoracis. The average IMF of the thoracic muscle is 31.5%. The fatty acid composition of the top 5 FFAs are 50.0% 18:1, 26.1% 16:0, 10.4% 18:0, 4.0% 16:1, and 2.8% of 14:0. The chemical composition consists of 22% crude protein, 31% crude fat, and 47% moisture. [00259] Design bioprocess to meet product characteristics [00260] Cell isolation, cell line development strategy, culture process, and product formulation may be determined by the meat characteristics that influence sensory properties mentioned above. First, skeletal muscle and pre-adipocytes may be isolated from the longissimus thoracis muscle of a Wagyu calf. The skeletal muscle and pre-adipocyte cell lines may undergo development to increase mitotic capacity for scale up. The skeletal muscle cells may be grown in single cell suspension for scale up, then undergo a differentiation step. The cells may first undergo cell aggregation into micro tissues with low agitation rates inside the vessel. After sufficient tissue synthesis (below ~300um in thickness, the O2 diffusion limitation), differentiation media may be supplemented to the culture. Differentiation may increase myoglobin, myosin heavy chain, and other myogenic protein expression by muscle fibers. [00261] A cell population that can undergo adipogenesis may be grown in single cell suspension and differentiated with an adipogenic differentiation media to activate PPAR ^ expression, which would mature the cells into adipocytes. 18:1, 16:0, 18:0, 16:1, and 14:0 FFAs may be supplemented in the culture media at rates that reflect the fatty acid composition of the muscle. If the adipocytes uptake each FFA at the same rate then a MUSA:SFA ratio may be obtained. The FFAs may be used in combination at respective concentrations between 1nM-1000mM. This ratio, and the fatty acid composition, may improve tenderness, flavor, palatability, and fat melting in cooked meat. [00262] Production formulation [00263] Product formulation may require recreating the chemical composition of the longissimus thoracis. Adipocytes and skeletal muscle cells may be grown to volumes that may yield a 2:3 ratio of skeletal muscles to fat cells. Cultured cells may not need to compose the entire 22% of crude protein and 31% of crude fat, since plant-based ingredients may add bulk and texture. However, since IMF is the hallmark of Wagyu beef, cell-based products may be held up to the JMGA standard of BMS, and as a result, require a higher percentage (%) of crude fat. The cells and plant- based ingredient may then be blended and hydrated to optimize moisture content closer to around about 47%. A food printer may then be used to print the skeletal muscle blend with a network of intramuscular fat. [00264] Inactivation of PTEN results in activation of the PI3K/AKT pathway and subsequently leads to an increase in cell cycle progression, migration and survival. The present invention provides methods that may be used to reduce PTEN activity including siRNA and/or shRNA mediated knockdown of PTEN protein translation, miRNA-mediated transcriptional interference of PTEN expression, and overexpression of dominant negative form of PTEN. [00265] siRNA and shRNA mediated knockdown of PTEN [00266] Transient transfection of siRNA [00267] Transient transfection of siRNA may be accomplished by seeding 0.1 million cells one day prior to transfection in a 6-well plate. Bovine, porcine, and/or C2C12 myoblasts may be transfected with 100 nM control scrambled siRNA or siRNA specific for PTEN by using Lipofectamine RNAimax. Cells to be harvested for mRNA and/or protein expression may be analyzed for efficiency of PTEN knockdown by RT-PCR and western blot analysis respectively at 48 or 72-hour post transfection. [00268] Accordingly, siRNA mediated knockdown of PTEN was performed in TERT over-expressing primary bovine myoblasts as described above using commercially available siRNA targeting PTEN sequence (Horizon Discovery, Waterbeach, UK). Lipofectamine RNAimax® was used to transfect primary bovine myoblasts with 100 nM control scrambled siRNA, siRNA specific for PTEN in TERT over-expressing cells, and/or TERT alone. At 48- hours post transfection, CellTiter-Glo® assay was used to determine cell viability. Cells were plated in 96-well plates at a starting cell density of 5000 and RLU (relative luminescence units) was measured according to the manufacturer’s instructions. FIG. 3 shows that TERT over- expressing cells comprising the PTEN knockdown displayed increased cell viability as compared to the siScrambled control and cells over-expressing TERT alone. [00269] Once the knockdown efficiency is confirmed, cell proliferation may also be analyzed by BrDU proliferation assays, flow cytometry, analysis of cell cycle genes such as p21, pRb, cyclin D1 by RT-PCR and western blot analysis. [00270] shRNA mediated knockdown of PTEN [00271] Myoblasts may be seeded at approximately 60-70% confluency in a 24-well plate and incubated overnight prior to infection the next day. Media may be removed and replaced with 500uL of fresh growth media per well. In addition, polybrene may be added to each well in such a way to yield a final concentration of 8µg/mL. Further, 1mL of viral supernatant may then be added to the wells and plates may be spun at 1-3 hours using a swing- bucket rotor. Supernatant may then be removed, and cells may be replenished with 1mL of growth media. The cells may then be selected 48-72 hours later using antibiotics as the lentiviral and/or retroviral vector may carry antibiotic resistant genes. As a result of the antibiotic resistance, those cells that did not receive the shRNA may be eliminated. The cells may also carry marker genes, for example, GFP or RFP, to assess transduction efficiency via FACS analysis. [00272] Once the knockdown efficiency is confirmed, cell proliferation may be analyzed by BrDU proliferation assays, flow cytometry and analysis of cell cycle genes such as p21, pRb, cyclin D1 by RT-PCR and western blot analysis. [00273] BrdU proliferation assays [00274] The proliferation capacity of myoblasts with PTEN knockdown may be assessed by BrdU incorporation assays. Cells may be assessed with 10 uM BrdU for 30 minutes. Cells may then be fixed and stained with anti-BrdU antibody. BrdU stained cells may then be analyzed for the percentage of BrdU positive cells using fluorescence microscopy. [00275] Flow cytometric analysis [00276] Cells may be trypsinized and collected by centrifugation. A resulting cell pellet may then be washed at least once using PBS and centrifuged again at 1,200 rpm for 3 minutes. Cells may then be fixed with 70% ethanol while vortexing to avoid clumping of cells. [00277] Cells with ethanol may then be stored at about around -200C for at least 24 hours. Later, cells may be washed with 1X PBS and centrifuged at 1,200 rpm for 5 minutes, 10μg/ml propidium iodide solution may be mixed with RNAase A for at least 30 minutes at about room temperature. Cells may then be stained with 300 μl propidium iodide (PI) and strained using 40 µm filters to avoid clumps and run through BD FACS machine. At least 10,000 cells may be acquired during the run. [00278] Overexpression of miR-26A/miR-486-5P/miR-382-5P mediated knockdown of PTEN [00279] For miR-mediated transcriptional interference of PTEN expression, miR- 26A/miR-486-5P/miR-382-5P/miR-144-3P to be over expressed in myoblasts by transient transfection of miRNA mimetics and negative control using lipofectamine RNAimax or lentiviral mediated transduction. 48 or 72-hour post transfection or transduction, cells to be harvested for mRNA and/or protein expression and analyzed for downregulation of PTEN by RT-PCR and western blot analysis respectively. [00280] Table 4. miR-mediated transcriptional interference of PTEN expression.
Figure imgf000071_0001
[00281] Overexpression of dominant negative form of PTEN to reduce wild type PTEN activity [00282] Myoblasts to be transfected with empty vector and catalytically inactive PTEN mutants (for example, PTENC124S, PTENG129E, PTENR130G, PTENR130X, and PTENR233X) using Lipofectamine 3000 or lentiviral and/or retroviral mediated transduction. Once the overexpression is confirmed, cell proliferation will be analyzed by BrDU proliferation assays and analysis of cell cycle genes such as p21, pRb, cyclin D1 by RT-PCR and western blot analysis. In addition, protein lysates will be analyzed for phos-Akt and total AKT. [00283] Additional Methods for Overexpressing Gene Targets [00284] Transient transfection [00285] Myoblasts may be transfected with empty vector and plasmids encoding, for example, PAX3, TBX2, ILK1, PDK1, FAK, SRC, GLUT1-4, and/or TERT, by electroporation or using a lipid-based transfection reagent such as Lipofectamine 3000/Lipofectamine plus reagent. Cells may be harvested for mRNA/protein expression and analyzed for over expression 48 or 72-hour post transfection by RT-PCR and/or western blot analysis respectively. [00286] Transient transfection of commercially available plasmids from OriGene, for example, GLUT1-4, PDK-1, ILK, C-JUN and FOS, was done in C2C12 and primary bovine myoblasts using Lipofectamine. Briefly, as described above, lipid-based transfection reagent Lipofectamine 3000/Lipofectamine plus reagent was used to create transgenic expression constructs comprising ILK or PDK-1, either alone or in TERT over-expressing primary bovine myoblasts. In addition, GLUT4 was similarly transiently transfected into INSR knock-in C2C12 cell lines (ILK (Cat no: MR207213), PDK-1(Cat no: MR206918 and GLUT4 (Cat no: MG208202)). Plasmids were purchased from OriGene. [00287] FIGs.2A-D show cell viability/cell proliferation (mitotic capacity) for primary bovine myoblasts over-expressing ILK, PDK-1, TBX2, PAX3 and/or TERT. As shown in FIGs. 2A and 2B, metabolic activity of the cells was monitored using measurement of ATP synthesis by CellTiter Glo® assay (Promega, Madison, WI) with ATP being an indicator of number of viable cells. CellTiter Glo® assay may also measure cell proliferation. [00288] Primary bovine myoblasts were transiently transfected to over-express ILK or PDK-1, either alone or in combination with TERT and cell viability was assessed using the CellTiter-Glo® assay. Primary bovine cells were seeded into 96-well plates at a starting cell density of 5000 cells, Relative Luminescence Units (RLU) was observed 48-hours post- transfection. Cell viability was analyzed via CellTiter-Glo® assay, which measures cell proliferation and viability based on ATP synthesis of metabolically active cells. Accordingly, ATP synthesis results in increased luminescence signal and indicates cell viability and/or cell proliferation. [00289] FIG 2A shows that over-expression of ILK and PDK-1, either alone or in combination with TERT, increased the proliferation of cells (mitotic capacity of cells) as reflected by increased luminescence signal obtained from the ATP synthesis. Further, FIG.2B shows that cells over-expressing TBX2 or PAX3, either alone or in combination with TERT displayed enhanced mitotic capacity when compared with untreated or GFP control cells. [00290] Once the overexpression was confirmed, cell proliferation was quantified by cell count at different time points using trypan blue followed by manual counting using hemocytometer or automated cell counter. Viable cells vs. non-viable cells were determined based on the trypan blue staining. Primary bovine myoblasts over-expressing TERT, PAX3, TBX2, or GFP were seeded onto 12-well plates at a starting cell density of 0.025 x106. Following a 48-hour incubation period, cells were stained with tryphan blue and counted using a hemocytometer. As seen in FIG. 2C-D, when compared to GFP control cells, cells over- expressing TERT, PAX3, or TBX2 exhibit increased cell proliferation. [00291] In addition, FIG.9 shows that C2C12 myoblasts with CRISPR mediated stable knock in of wild type SRC displayed enhanced proliferation as measured by CellTiter-Glo® assay. [00292] Cell proliferation may also be analyzed by BrdU incorporation assays. In- addition, cell proliferation may be measured by analyzing the percentage of cells in different sub-populations using a flow cytometer. Briefly, cells may be stained with propidium iodide mix (10 g/ml propidium iodide solution with RNase 200 g/ml) for 30 min at room temperature and strained using 40 m filters. Cells may be synchronized at the G1/S boundary using 1mM hydroxyurea and released in normal growth media. Cells will be synchronized at mitosis using 300 ng/mL of nocodozole. [00293] Cell line resistance to apoptosis (anoikis) [00294] Genetically modified cell line resistance to apoptosis (anoikis) will be analyzed using biomarkers such as cleaved caspase 3/7, cleaved PARP-1 and levels may be measured by RT-PCR or Western blot analysis. Caspase 3/7 activity may also be measured by Caspase- Glo 3/7 assay system and/or CellTiter-Glo® assays. Cell cycle may be analyzed by RT-PCR and western blot analysis of cell cycle genes such as p21, pRb, cyclin D1. [00295] Over-expression of ILK or PDK-1 in primary bovine myoblasts was achieved via Lipofectamine mediated transient transfection. In addition, lentiviral mediated over expression was also performed in primary bovine myoblasts to over-express TBX2 and PAX3, alone or in combination with TERT. Cell line resistance to apoptosis (anoikis) was assessed using the commercially available anoikis assay kit from ABCAM (Anoikis Detection Assay Kit; Cat no: ab211153) according to the manufacturer’s instructions. As shown in FIGs.5A and 5B, all the targets showed statistically significant anoikis resistance as measured by absorbance (RLU). [00296] In addition, C2C12 myoblasts cell lines with stable CRISPR mediated knock-in of B-Raf mutation (B-Raf V600E) were prepared as described above. FIG. 5C shows B-Raf mutants having increased anoikis resistance compared to wild type C2C12 cells. [00297] Primary bovine myoblasts with siRNA mediated knockdown of PTEN or TERT over-expressing cells were prepared as previously described. As such, FIG. 5D shows that anoikis resistance in cells having an siRNA mediated knockdown of PTEN or cells over- expressing TERT is enhanced compared to siScrambled control cells. Anoikis assay performed with anoikis assay kit from ABCAM (Anoikis Detection Assay Kit; Cat no: ab211153). [00298] Finally, C2C12 myoblasts with CRISPR mediated stable knock-in of SRC were prepared as described previously. FIG. 5C shows enhanced anoikis resistance in SRC KI compared to wild type as evidenced by the results Anoikis assay performed with anoikis assay kit from ABCAM (Anoikis Detection Assay Kit; Cat no: ab211153). [00299] Transfection & selection [00300] Primary bovine myoblasts may be transfected with a 1:10 ratio of an expression vector for GOI (Table 5) and a puromycin/neomycin resistant plasmid. Cells may then be selected 48 hours post transfection, in medium containing appropriate concentration of Geneticin (G-418)/puromycin. After 2-3 weeks, neomycin/puromycin resistant clones may be isolated, expanded and screened by RT-PCR and Western blot analyses. [00301] Table 5. List of Gene Targets
Figure imgf000075_0001
Figure imgf000076_0001
[00302] [00303] Viral Methods for Transduction and Overexpression of Gene Targets [00304] Lentiviral and Retroviral production and infection [00305] For stable expression using Phoenix cells, our gene of interest (for example, PAX3, TBX2, ILK1, PDK1, FAK, SRC, GLUT1-4, and/or TERT) may be cloned into a lentiviral and/or retroviral vector which may comprise a packaging signal and an antibiotic resistance marker for selection. Phoenix cells may be plated at a density of 1.5 x 106 cells per 10 cm-diameter culture dish. [00306] On the 2nd day, cells may be transfected with control vector (lenti/retroviral vector) and lenti and/or retroviral vector containing GOI using Calcium Phosphate transfection kit (Invitrogen)/Lipofectamine Plus reagent (Invitrogen) according to the manufacturer’s instructions. [00307] Briefly, 40 ul of 2M CaCl2 may be mixed with 30 ug of DNA in an Eppendorf tube. The volume may be adjusted to 300 ul with sterile water. While vortexing, 300 ul of 2X HBS may be added slowly, and air may be bubbled through the DNA mixture. The resulting DNA complex may be incubated for about 15 minutes at about room temperature. The resulting precipitate may be added to the Phoenix cells with 10 ml of media and incubated for 24 hours. Later, culture medium may be changed to bovine culture growth medium for virus collection from the transfected Phoenix cells. [00308] Every 24 hours, media containing virus may be collected and filtered using 0.45 μm syringe filter and stored at about -800C. On the 1st and 2nd day (the 4th and 5th day, respectively) viral supernatant containing the GOI, may be collected and filtered through 0.45 μm filters for transduction of target primary bovine/C2C12 myoblasts. GOI over-expressing cells may be generated by transducing cells with virus generated from lenti/retroviral vector alone or lenti/retroviral vector containing GOI. Infection may be carried out using bovine and/or C2C12 myoblasts growth medium comprising virus, for which appropriate concentration of polybrene (Sigma-Aldrich®, St. Louis, MO) may be added and incubated for about 8 hours at about 370C. The media may be changed and replaced with normal growth media 8 hours post-infection. Cells may then be allowed to grow for another 24hrs before selecting with appropriate concentration of puromycin (Sigma-Aldrich®) for 48 hours. After selection with puromycin for two days, cells may be analyzed to confirm over-expression of the GOI and for any change in phenotype. [00309] Genetic modifications involving lentivirus mediated over-expression of PAX3, TERT, TBX2 were performed. Briefly, TBX2, PAX3 and/or TERT genes were packaged into lentiviral particles as described above and over-expressed in primary bovine myoblasts either alone or in combination. As shown in FIG.2B, cells over-expressing TBX2 or PAX3, alone or in combination with TERT, displayed enhanced cell viability when compared to controls. In addition, there was an increase in the number of cells over-expressing TBX2, PAX3, or TERT compared to GFP control cells (FIG.2D). [00310] Inducible Gene Expression Approaches [00311] Cumate controlled operator system [00312] CuO-MCS Enhanced Episomal Vector (EEV) (Cat. # EEV610A-1) developed by System Biosciences (SBI, Palo Alto, CA), an improved cumate controlled operator system without limitation on insert size may be used for expression of GOIs. EEVs offers an advantage for non-integrating, non-viral gene expression and replicate in synchrony with the host cell. As such, gene expression may be stably inherited and may be used for long lasting expression up to several months both in vitro and in vivo without modifying the host genome. [00313] Briefly, CuO-MCS EEV vector may comprise a cumate-inducible promoter upstream of an MCS (cloning gene of interest), CymR and a puromycin selection cassette expression under the control of constitutive EF1α promoter. The cumate inducible reporter remains silent until the addition of cumate. Upon addition of cumate to the medium, the transfected C2C12/primary bovine myoblasts may show a robust gene expression. The expression may be detected even after 72 days. [00314] FLP-In System [00315] Three different vectors, commercially available from Thermo Fisher (Thermo Fisher Scientific, Waltham, MA), may be used to generate isogenic stable mammalian cell lines expressing gene(s) of interest. The FLP-In system may include, (i). Flp-In target site vector, and pFRT/lacZeo to generate Flp-In™ host cell line(s). The vector may comprise a lacZ- Zeocin™ fusion gene whose expression is controlled by the SV40 early promoter. (ii). Gene of interest may be then cloned into the pcDNA5/FRT expression vector. The vector may also comprise an antibiotic resistance gene with a FRT site embedded in the 5' coding region. A hygromycin resistance gene may lack a promoter and the ATG initiation codon; and (iii). pOG44 plasmid which constitutively expresses the Flp recombinase under the control of the human CMV promoter. [00316] The pOG44 plasmid and the pcDNA5/FRT vector comprising gene of interest may be co-transfected into the Flp-In™ host cell line. Upon co-transfection, Flp recombinase expressed from pOG44 may mediate a homologous recombination event between the FRT sites (integrated into the genome and on pcDNA5/FRT) such that the pcDNA5/FRT construct is inserted into the genome at the integrated FRT site. Insertion of pcDNA5/FRT into the genome at the FRT site brings the SV40 promoter and the ATG initiation codon (from pFRT/lacZeo) into proximity and frame with the hygromycin resistance gene. Thus, stable Flp-In™ expression cell lines can be selected for antibiotic resistance and expression of the recombinant protein of interest. [00317] Doxycycline (1000 ng/mL) mediated inducible approaches were established in C2C12 myoblasts with CRISPR mediated knock-in of wild type SRC and BRAF mutant. The genetic modification involving lentivirus mediated over expression of PAX3, TERT, & TBX2 were performed and over-expression was confirmed with GFP reporter marker. FIG.10 shows GFP positive cells. The genetic modification involving Doxycycline (Tet system) mediated inducible approaches were also established in C2C12 myoblasts with CRISPR-mediated knock-in of wild type SRC and BRAF mutant. The genetic modification involving transient transfection of commercially available plasmids from OriGene such as GLUT1-4 (GLUT1 Cat no: MR207871, GLUT 2 Cat no: MR208388, GLUT 3 Cat no: 207915), PDK-1, ILK, C-JUN and FOS over-expression was performed in C2C12 and primary bovine myoblasts using Lipofectamine. [00318] The invention is not restricted by the examples given in the specification. The invention comprises any feature or combination of features described in the specification and claims, even if not explicitly depicted in this specific combination. [00319] The foregoing disclosure has been set forth merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof. [00320] Other objects, advantages and novel features of the present invention are apparent from the foregoing detailed description of the one or more preferred embodiments, examples and aspects. It should be recognized that the one or more examples in the disclosure are non-limiting examples and that the present invention is intended to encompass variations and equivalents of these examples.

Claims

CLAIMS: 1. A transgenic expression construct comprising a gene or genes selected from the group consisting of ILK, GLUT4, PDK-1, TBX2, Pax3, and/or telomerase, and a constitutively active promoter overexpressing said gene, when compared to the expression the wild-type promoter of said gene, wherein the transgene is inserted into the native genome of the cell.
2. A method comprising editing the nucleic acid base pair sequences of native promoters of ILK, GLUT4, PDK-1, TBX2, Pax3, and/or telomerase genes to increase gene transcription over gene transcription levels found in cells with unmodified promoters.
3. A method for increasing mitotic capacity of a cell line comprising overexpressing ILK, GLUT4, PDK-1, TBX2, Pax3, telomerase and/or knocking out PTEN expression.
4. A composition comprising a genetically modified cell, wherein a genetic modification comprises mutations to the B-Raf gene, and wherein the modification results in enhanced ERK signaling in comparison to the wild type B-Raf.
5. The composition of claim 4, wherein the genetic modification is a substitution, deletion, insertion, duplication, inversion, or frameshift mutation.
6. A composition comprising a genetically modified cell, wherein a genetic modification comprises modifications to the INSR gene.
7. The composition of claim 6, wherein the genetic modification is a substitution, deletion, insertion, duplication, inversion, or frameshift mutation.
8. A method of improving cell differentiation comprising supplementing adipocyte cultures with fatty acids selected from the group consisting of monounsaturated fatty acids (MUFA); polyunsaturated fatty acids (PUFA); and saturated fatty acids (SFA).
9. The method of claim 8, wherein the MUFA is oleic acid.
10. The method of claim 8, further comprising supplementing adipocyte cell cultures with bovine preadipocytes sourced from the subcutaneous stromal vascular cells treated with oleic acid.
11. The method of claim 8, wherein the PUFA is selected from the group consisting of α- Linolenic acid, Stearidonic acid, Eicosatetraenoic acid, Eicosapentaenoic acid, Docosapentaenoic acid, Docosahexaenoic acid, Linoleic acid, Gamma-linolenic acid, Calendic acid, Dihomo-gamma-linolenic acid, Arachidonic acid, Adrenic acid, and Mead acid.
12. A method of improving flavor of cell-based fat comprising supplementing adipocyte cultures with fatty acids selected from the group consisting of monounsaturated fatty acids (MUFA); polyunsaturated fatty acids (PUFA); and saturated fatty acids (SFA).
13. The method of claim 12, wherein the MUFA is selected from the group consisting of Palmitoleic acid, Paullinic acid, Omega-9 FAs, elaidic acid, Gondoic acid, Erucic acid, and Nervonic acid.
14. The method of claim 12, wherein the SFA is selected from the group consisting of Myristic acid, Palmitic acid, Stearic acid, Arachidic acid, Behenic acid, Lignoceric acid, and Cerotic acid.
15. A composition comprising a genetically modified bovine or porcine myoblast and a genetically modified bovine or porcine adipoblast.
16. A composition comprising myoblasts, MSCs, intramuscular fibroblasts, iPSCs, adipoblasts, adipocytes, preadipocytes, and/or fibro-adipoblasts.
17. A cell-based meat product comprising genetically modified fat cells and genetically modified muscle cells.
18. A method of preventing anoikis comprising generating a cell line, wherein the cell line is constitutively overexpressing ILK, PDK-1, TBX2, and/or PAX3.
19. A method of preventing anoikis comprising generating a cell line having a mutated form of B-Raf that has higher activity levels than wild type B-Raf.
20. A method of preventing anoikis comprising generating a cell line having a PTEN gene knocked out.
21. A method of reducing or eliminating insulin from cell culture media comprising truncating alpha subunits in an INSR gene to produce an exon-free insulin receptor.
22. A method of reducing or eliminating insulin from cell culture media comprising overexpressing PDK-1, GLUT4, TBX2, and/or PAX3.
23. A method of reducing or eliminating insulin from cell culture media comprising knocking out PTEN.
24. A method for bypassing the hayflick limit comprising overexpressing telomerase.
25. A method for mitogen-independent cell cycle progression comprising generating a cell line having a mutated form of B-Raf having higher activity levels than wild type B-Raf.
26. A method of transdifferentiating myogenic cells into adipogenic cells comprising supplementing the cell culture media with SFAs, MUFAs, and/or PUFAs.
27. A cell line wherein the cell line a. bypasses the hayflick limit via telomerase overexpression b. is resistant to anoikis via activation of integrin binding signal transduction via PTEN knockout with increased PIP3, PIP3/PDK-1/pAkt mediated anti-apoptotic signaling, ILK/pAkt mediated anti-apoptotic signaling, ERK1/2 anti-apoptotic signaling c. provides insulin-independent glucose transport via a method selected from the group consisting of PIP3-mediated phosphorylation of aPKCs, PDK-1-mediated phosphorylation of aPKCs, pAkt-mediated AS160 inhibition, constitutive insulin receptor activation and GLUT4 overexpression d. provides mitogen-independent cell cycle progression via ERK1/2-mediated cell cycle progression; and e. which has enhanced sensory properties via a method selected from the group consisting of PIP3-mediated phosphorylation of aPKCs, PDK-1-mediated phosphorylation of aPKCs, pAkt-mediated AS160 inhibition, constitutive insulin receptor activation, GLUT4 overexpression.
28. A composition comprising a genetically modified cell, wherein genetic modification comprises at least one mutated PTEN gene, and wherein the modification results in a reducing wild type PTEN activity through competitive inhibition.
29. The composition of claim 28, wherein the PTEN mutant is selected from a group including PTENC124S, PTENG129E, PTENR130G, PTENR130X, and PTENR233X.
30. A method for knocking down PTEN activity in a cell, the method comprising genetically modifying the cell using siRNAs, shRNAs, and microRNAs.
31. The method of claim 30, wherein the siRNA, shRNA, and/or microRNA includes miR- 26A, miR-486-5P, miR-144-3P, miR-382-5P, miR-142-5p, miR-21, and/or miR-200A.
32. A method for improving cell line resistance to anoikis comprising overexpressing mutant or wild type FAK and/or SRC.
33. A method for enhancing mitogen independent enhanced proliferation and/or mitotic potential comprising overexpressing mutant or wild type FAK and/or SRC.
34. A method for insulin-independent glucose transport into a cell comprising overexpressing GLUT1, GLUT2, and/or GLUT3.
35. A method for enhancing a sensory profile of a cell line comprising overexpressing GLUT1, GLUT2, and/or GLUT3.
36. A composition comprising a genetically modified cell, wherein genetic modification results in the overexpression of at least one gene.
37. The composition of claim 36, wherein the genetic modification includes a lentivirus and/or retrovirus.
38. The composition of claim 36, wherein the genetic modification includes inducible approaches to gene overexpression including a TeT system, FLP-FRT system, and/or Cumate system.
39. The composition of claim 36, wherein the genetic modification includes transient transfection of extrachromosomal expression vectors (plasmids).
40. A method for developing edible cell lines, the method comprising genetically modifying a cell line to overexpress at least one gene, wherein genetic modification includes a method selected from a group consisting of lentivirus and/or retrovirus, a TeT system, FLP-FRT system, and/or Cumate system, and includes transient transfection of extrachromosomal expression vectors (plasmids).
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Citations (1)

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Publication number Priority date Publication date Assignee Title
US20090111157A1 (en) * 1997-11-26 2009-04-30 Geron Corporation Protein and peptide fragments from mouse telomerase reverse transcriptase

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US20090111157A1 (en) * 1997-11-26 2009-04-30 Geron Corporation Protein and peptide fragments from mouse telomerase reverse transcriptase

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