CN117580943A - Method for inducing hypertrophic muscle fibers for industrial meat production - Google Patents

Method for inducing hypertrophic muscle fibers for industrial meat production Download PDF

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Publication number
CN117580943A
CN117580943A CN202280046185.2A CN202280046185A CN117580943A CN 117580943 A CN117580943 A CN 117580943A CN 202280046185 A CN202280046185 A CN 202280046185A CN 117580943 A CN117580943 A CN 117580943A
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cells
receptor
inhibitor
ryr
agonist
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埃尔达德·扎霍尔
奥里·波拉特·阿维诺姆
塔玛·米里亚姆·罗兹·艾格勒·赫什
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Yeda Research and Development Co Ltd
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Yeda Research and Development Co Ltd
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Priority claimed from PCT/IL2022/050474 external-priority patent/WO2022234586A1/en
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Abstract

Methods of inducing multi-core myotube formation are provided. The method comprises contacting myogenic precursor cells from a cultured animal with an extracellular regulated signal kinase (ERK 1/2) inhibitor and/or an up-regulating factor of intracellular ca2+ and/or a RXR/RAR agonist to enhance fusion and myogenic maturation.

Description

Method for inducing hypertrophic muscle fibers for industrial meat production
RELATED APPLICATIONS
The present application claims priority from israel patent application No. 283011 filed 5/6/2021, the entire contents of which are incorporated herein by reference. The present application also claims priority from U.S. provisional patent application Ser. No. 63/283,242 filed on 11/25 of 2021. The entire contents of the above application are incorporated by reference as if fully set forth herein.
Technical field and background of the invention
The present invention, in some embodiments thereof, relates to methods of cell culture, and more particularly, but not exclusively, to culturing meat.
The meat industry is one of the biggest factors responsible for environmental stress, through pollution, through fossil fuel use, methane and other waste production, and water and land consumption. At the same time, the global population, estimated to be up to approximately 97 billion in 2050 and up to 110 billion in 2100, has accompanied increased demand for meat products, which is not sustainable under current environmental conditions. Therefore, replacement of meat sources is indispensable.
Common meats consist mainly of muscle tissue. The concept of cultivating meat or in vitro meat or laboratory cultivated meat is based on a technology that has been used in laboratory environments for many years in the field of research of muscle biology related processes. Briefly, muscle biopsies are harvested and subjected to enzymatic hydrolysis. Muscle precursor (stem cells) cells are then isolated and expanded several orders of magnitude under growth conditions (i.e., proliferation medium). Then, once enough cells are obtained, they are transferred to a low serum medium (differentiation medium), which causes them to eventually exit the cell cycle, initiate the muscle differentiation procedure, and finally myoblasts fuse to form multinucleated myotubes. Myotubes are similar to adult muscle fibers found in the original organism. Thus, the myotubes obtained by this process are considered to be equivalent to meat.
The process of myoblast proliferation, differentiation, fusion is complex, with several molecular signaling pathways involved in regulating the individual components of this process. The culture meat industry utilizes this well characterized process and this differentiation protocol to produce multinuclear myotubes on a large scale from primary derived myoblasts or muscle cell lines. This is typically done by expanding large numbers of precursor cells over time (30-40 days) in a bioreactor, then collecting the cells and seeding them onto the surface while changing them from proliferation medium to differentiation medium and allowing differentiation and fusion to proceed spontaneously until a multinucleated myotube is obtained. Currently, the process of in vitro differentiation and myotube formation is very inefficient and time consuming. The time for myotube formation varies depending on the original species of muscle tissue (i.e., birds, 4-6 days; cows, 10-14 days). The use of molecules that target specific activation differentiation, enhance myoblast fusion and mechanisms of polynuclear myotube formation can increase the efficiency of the meat industry, thereby increasing overall productivity/yield.
Mitogen Activated Protein Kinases (MAPKs), including p38, JNK, ERK1/2, and ERK 5, mediate a variety of signaling pathways, and are involved in muscle development and myoblast differentiation. The role of ERK1/2 in muscle differentiation and fusion is not clear, as both positive and negative effects have been proposed. ERK1/2 promotes myoblast proliferation in response to various growth factors; inhibition of the signaling pathway leading to ERK1/2 activation leads to cell cycle withdrawal and differentiation.
Calcium (ca2+) has long been recognized as a regulator of mammalian muscle fusion; transient ca2+ depletion of the Sarcoplasmic Reticulum (SR) is associated with myoblast differentiation and fusion. Furthermore, ca2+ sensitive transcription factor NFATC2 has been reported to mediate myoblast recruitment and myotube expansion. However, the signaling cascade leading to ca2+ -mediated myoblast fusion remains elusive. CaMKII is a member of the Ca2+/calmodulin (CaM) -dependent serine/threonine kinase family. CaMKII delta and gamma and to some extent beta are the major isoforms expressed in skeletal muscle (primary isoforms). Upon binding of ca2+/CaM to the individual subunits (subnits), cross-phosphorylation of adjacent subunits at T287 leads to an autonomously activated state by increasing the affinity of ca2+/CaM by thousands of times. Previously, caMKII was identified for its role in the regulation of ca2+ dependent gene expression associated with muscle oxidative metabolism and components of the contractile machinery. However, to date, the specific role of CaMKII as a myoblast fusion medium has not been demonstrated.
Additional background art includes: US patent No. US7,270,829, international patent application WO2018/189738A1 (US publication No. 2020/100525 A1), international patent application WO2018/227016A1, international patent application WO2017/124100A1, US patent application publication US2016/0227830A1, US patent application publication US20200165569, US patent application publication US2020/0140821, US patent application publication US2017/0218329, US patent application publication US20200392461, US20200245658, US20200140810, US20200080050, US20160251625, US20190376026, US20210037870 and US20200140821. Related non-patent publications include Bunge, J, wall street Japanese (Wall Street Journal) 3 months 15 (2017-03-15) 2017; hong, tae Kyung et al, animal resources food science (Food Science of Animal Resources), 41:355-372,2021 and Michailovici, I.et al, development (141:2611-2620,2014).
Disclosure of Invention
According to an aspect of some embodiments of the present invention there is provided a method of inducing myotube formation comprising contacting myogenic precursor cells from a cultured animal with an extracellular regulated signal kinase (ERK 1/2) inhibitor and/or an intracellular ca2+ up-regulator (upregulator).
According to an aspect of some embodiments of the present invention there is provided a method of inducing myotube formation comprising contacting myogenic precursor cells from a farmed animal with at least one molecule selected from the group consisting of: an extracellular regulated signal kinase (ERK 1/2) inhibitor, a mitogen activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranialkali receptor (RYR 1, RYR 3) agonist, a ranialkali receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, a calcium ionophore, and a calmodulin-dependent protein kinase II (CaMKII) activator.
According to some embodiments of the invention, the ERK1/2 inhibitor is selected from the group consisting of: MK-8353 (SCH 900353), SCH772984, CC-90003, dehydrogambir (Corynoxyine), ERK1/2 inhibitor 1, magnolin (magnolin), ERK IN-1, ERK IN-2, ERK IN-3, LY3214996, ravoxertiinib, hydrochloric acid Ravoxertinib, VX-11e, FR180204, ulixitinib (Ulixiviatinib), ulixiviatinib hydrochloride, ADZ0364, KO947, FRI-20 (ON-01060), bromoacetoxycalcitol (B3 CD), BVD523, DEL22379, FR180204, GDC0994, KO947, AEZ-131 (AEZS-131), AEZS-136, AZ-13767370, BL-EI-001, LTT, ASTX-029, TCS ERK 11e and CAY10561.
According to some embodiments of the invention, the MEK1 inhibitor is selected from the group consisting of: trametinib, PD98059, U0126 (U0126-EtOH), PD0325901, semetinib (AZD 6244), cobratinib (GDC-0973, RG 7420), bimetinib (MEK 162), CI-1040 (PD 184352), remiratinib (BAY 869766; RDEA119), pimecrtib (Pimassertib) (AS 703026), semetinib (AZD 6244), cobratinib hemi-fumarate (Cobimetinib hemifumarate), GDC-0623 (RG7421), RO 499755, AZD8330, (ARRY-424704), SL327, MEK inhibitors, PD318088, cobratinib racemate (GDC-0973 racemate; XL518 racemate) and EBI-1051.
According to some embodiments of the invention, the FGF inhibitor is selected from the group consisting of delazantinib (Derazantinib), PD 161570, SSR 128129E, CH5183284, PD 166866, and Pemigatinib (Pemigatinib).
According to some embodiments of the invention, TGF- β1 is selected from the group consisting of SD208, LY364947, repox, SB 525334, R268712, and GW 788388.
According to some embodiments of the invention, the RXR/RAR agonist is selected from the group consisting of: CD3254, docosahexaenoic acid, LG100268, SR11237, AC261066, AC55649, adapalene (Adapalene), BMS961, CD1530, CD2314, CD437, BMS453, EC23, all-trans retinoic acid, all-trans-4-hydroxy retinoic acid, all-trans retinoic acid-D5, cyantraniliprole, vitamin A, all-trans retinol, LG100754, beta-carotene, beta-apo-13 carotene, lycopene, all-trans-5, 6-epoxyretinoic acid, all-trans-13, 14-dihydroretinol, retinyl Acetate (Retinyl Acetate), and magnolol, valproic acid, HX630, CD 600, LG101506, 9cUAB30, AGN194204, LG101305, UVI3003, net-4IB, CBt-PMN, XCT0135908, PA024, methoprene (methoprene), methoprene, LG-9, LG-13, 14-dihydroretinol, retinol Acetate (Rzepine) and Floride (TTO) 20, 14-D1067, 14-dihydroretinol, floride (ALOfjone) and Floride (ALOfP) 20-D, floride) are included.
According to some embodiments of the invention, the RYR1, RYR3 agonist is selected from the group consisting of: caffeine, chlorocresol, CHEBI 67113, chlorantraniliprole, S107 hydrochloride, JTV519, trifluoperazine (TFP), xanthine, suramin, sodium suramin, NAADP tetrasodium salt, S100A1, cyclic ADP-ribose (ammonium salt), hexidine, 4-chloro-3-methylphenol (4-chloro-m-cresol), tetrazolium carboxamide, trifluoperazine (TFP), cyclic bromaromide and cyantraniliprole.
According to some embodiments of the invention, the intracellular ca2+ up-regulating factor is selected from the group consisting of NAADP tetrasodium salt, cyclic ADP-ribose, 4-bromo a23187, ionomycin, a23187 and isoprenaline.
According to some embodiments of the invention, the CaMKII agonist is selected from the group consisting of calcium, calmodulin, CALP1 and CALP 3.
According to some embodiments of the invention, the myogenic precursor cells are selected from the group consisting of myoblasts, satellite cells, myosided crowd (mSP) cells, myogenic stem cells (MDSC), mesenchymal Stem Cells (MSC), myogenic pericytes (muscle-derived pericytes), embryonic Stem Cells (ESC), induced muscle progenitor cells (induced muscle progenitor cell, icc), and Induced Pluripotent Stem Cells (iPSC).
According to some embodiments of the invention, the myogenic precursor cells express MyoD, pax3 and Pax7, or their corresponding interspecies homologous genes (orthologs).
According to some embodiments of the invention, the myogenic precursor cell is a myoblast.
According to some embodiments of the invention, the myogenic precursor cells are from a biopsy of the farmed animal.
According to some embodiments of the invention, the biopsy is a muscle biopsy.
According to some embodiments of the invention, myogenic precursor cells are isolated from a biopsy by enzymatic and/or mechanical dissociation.
According to some embodiments of the invention, the myogenic progenitor cells are undifferentiated myogenic precursor cells that are cultured in a proliferation medium prior to inducing multinuclear myotube formation.
According to some embodiments of the invention, the proliferation medium is free of molecules selected from the group consisting of: an extracellular regulated signal kinase (ERK 1/2) inhibitor, a mitogen activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranialkali receptor (RYR 1, RYR 3) agonist, a ranialkali receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, a calcium ionophore, and a calmodulin-dependent protein kinase II (CaMKII) activator.
According to some embodiments of the invention, the myogenic progenitor cells are myogenic precursor cells that are cultured in a differentiation medium prior to inducing multinucleated myotube formation.
According to some embodiments of the invention, the culturing is performed in a single vessel.
According to some embodiments of the invention, the method of the invention is performed by supplementing the culture medium with any of the molecules.
According to some embodiments of the invention, the method is performed in the presence of serum or serum replacement in an amount that allows for cell proliferation and/or under normoxic conditions.
According to some embodiments of the invention, the farmed animal is selected from the group consisting of mammals, birds, fish, invertebrates, reptiles and amphibians.
According to some embodiments of the invention, the multinuclear myotube comprises at least three nuclei.
According to some embodiments of the invention, the multinuclear myotube comprises at least ten nuclei.
According to some embodiments of the invention, the multinucleated myotubes express myogenic differentiation and fusion factors selected from the group consisting of MyoD, myoG, mymk and Mymx.
According to some embodiments of the invention, inducing the multinuclear myotubes results in a partial increase in MYOG positive nuclei compared to nuclei of myogenic progenitor cells cultured in differentiation medium without the at least one molecule.
According to some embodiments of the invention, induction of multinuclear myotube formation results in classical ladder-like streaks of actin and troponin signals and/or phalloidin staining representing actin filaments (phalloidin staining).
According to some embodiments of the invention, the multinucleated myotube formation comprises mononuclear myocell-myotube fusion and/or binuclear and trinuclear myotube expansion into large multinucleated fibers.
According to some embodiments of the invention, contacting myogenic precursor cells is performed for 12-48 hours.
According to some embodiments of the invention, contacting myogenic precursor cells is performed for 16-24 hours.
According to an aspect of some embodiments of the present invention there is provided a cultured meat composition comprising multinuclear myotubes produced by the method of the present invention.
According to an aspect of some embodiments of the present invention, there is provided a food (composition) comprising the cultured meat composition of the present invention.
According to some embodiments of the invention, the food is processed to impart a organoleptic sensation and texture to the meat.
According to some embodiments of the invention, the food further comprises a food product (foodstuff) of vegetable and/or animal origin.
According to some embodiments of the invention, the food further comprises adipocytes, muscle cells, blood cells, chondrocytes, bone cells, connective tissue cells, fibroblasts, and/or cardiomyocytes.
According to some embodiments of the invention, the food of the invention further comprises a plant-based protein.
According to an aspect of some embodiments of the present invention there is provided a method of producing a food product comprising combining a cultured meat composition or food product of the present invention with an edible composition for human or animal consumption.
According to an aspect of some embodiments of the present invention there is provided a method of treating muscle damage in a farm animal, the method comprising contacting damaged muscle tissue with at least one molecule selected from the group consisting of: an extracellular regulated signal kinase (ERK 1/2) inhibitor, a mitogen activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranialkali receptor (RYR 1, RYR 3) agonist, a ranialkali receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, and a calmodulin-dependent protein kinase II (CaMKII) activator, thereby inducing myotube regeneration and treating the muscle injury.
According to an aspect of some embodiments of the present invention there is provided at least one molecule for use in inducing myotube regeneration and treating muscle injury in a farmed animal, the molecule being selected from the group consisting of: an extracellular regulatory signal kinase (ERK 1/2) inhibitor, a mitogen-activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranitidine receptor (RYR 1, RYR 3) agonist, a ranitidine receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, a calmodulin-dependent protein kinase II (CaMKII) activator.
According to an aspect of some embodiments of the present invention there is provided a cell culture medium for preparing a multinuclear myotube from myogenic precursor cells, the medium comprising a basal medium and an extracellular regulated signal kinase (ERK 1/2) inhibitor.
According to some embodiments of the invention, the cell culture medium further comprises at least one of: mitogen-activated protein kinase 1 (MEK 1) inhibitors, fibroblast Growth Factor (FGF) inhibitors, transforming growth factor-beta (TGF-beta) inhibitors, retinoid-X receptor (RXR) agonists, retinoid-X receptor (RXR) activators, retinoic Acid Receptor (RAR) agonists, retinoic Acid Receptor (RAR) activators, ranitidine receptor (RYR 1, RYR 3) agonists, ranitidine receptor (RYR 1, RYR 3) activators, intracellular ca2+ upregulation factors, calmodulin-dependent protein kinase II (CaMKII) agonists, calcium ionophores, and calmodulin-dependent protein kinase II (CaMKII) activators.
According to some embodiments of the invention, the cell culture medium is composed of components that are certified as a generally recognized safety (GRAS).
According to some embodiments of the invention, the cell culture medium is a serum-free medium.
According to some embodiments of the invention, the cell culture medium comprises a serum replacement component.
According to some embodiments of the invention, the cell culture medium consists of ingredients that are certified to be heterogeneous.
According to an aspect of some embodiments of the present invention there is provided a method of inducing multinuclear myotube formation, the method comprising contacting a myogenic precursor cell from a cultured animal with an extracellular regulated signal kinase (ERK 1/2) inhibitor and/or an intracellular ca2+ up-regulating factor, wherein when the myogenic precursor cell is a chicken myogenic precursor cell, the contacting is performed in the presence of an extracellular regulated signal kinase (ERK 1/2) inhibitor and an intracellular ca2+ up-regulating factor.
According to an aspect of some embodiments of the present invention there is provided a method of inducing myotube formation comprising contacting myogenic precursor cells from a farmed animal with at least one molecule selected from the group consisting of: an extracellular regulated signal kinase (ERK 1/2) inhibitor, a mitogen activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor- β (TGF- β) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a ranitidine receptor (RYR 1, RYR 3) agonist, a ranine receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, and a calmodulin-dependent protein kinase II (CaMKII) activator, wherein the contacting occurs in the presence of the extracellular regulated signal kinase (ERK 1/2) inhibitor and the up-regulator of intracellular ca2+ when the myogenic precursor cell is a chicken myogenic precursor cell.
According to some embodiments of the invention, the ERK1/2 inhibitor is selected from the group consisting of: MK-8353 (SCH 900353), CC-90003, dehydrorhynchophylline, ERK1/2 inhibitor 1, magnolin, ERK IN-1, ERK IN-2, ERK IN-3, LY3214996, ravoxertinib, hydrochloric acid Ravoxertinib, VX-11e, FR 180204, ulitinib hydrochloride, ADZ0364, KO947, FRI-20 (ON-01060), bromoacetoxycalcitonin (B3 CD), AEZ-131 (AEZS-131), AEZS-136, AZ-13767370, BL-EI-001, LTT, peptide inhibitor EPE, ERK activation inhibitor peptide I (ERK inhibitor IV), ERK activation inhibitor peptide II (ERK inhibitor V).
According to some embodiments of the invention, the MEK1 inhibitor is selected from the group consisting of: trametinib, PD98059, U0126 (U0126-EtOH), PD0325901, semetinib (AZD 6244), cobratinib (GDC-0973, RG 7420), bimetanib (MEK 162), CI-1040 (PD 184352), refatinib (BAY 869766; RDEA119), pimatinib (AS 703026), semetinib (AZD 6244), cobratinib hemi-fumarate (Cobimetinib hemifumarate), GDC-0623 (RG 7421), RO 4987555, AZD8330 (ARRY-424704), SL327, MEK inhibitors, PD318088, cobratinib racemate (GDC-0973 racemate; XL518 racemate) and EBI-1051.
According to some embodiments of the invention, the FGF inhibitor is selected from the group consisting of delazantinib (Derazantinib), PD 161570, SSR 128129E, CH5183284, PD 166866, and Pemigatinib (Pemigatinib).
According to some embodiments of the invention, the TGF- β1 inhibitor is selected from the group consisting of SD208, LY364947, repox, SB 525334, R268712 and GW 788388.
According to some embodiments of the invention, the RXR agonist is selected from the group consisting of CD3254, LG100268, LG-100064, SR11237 (BMS-649), flubensalrotene (compound 20), bexarotene (LGD 1069), NBD-125 (B-12), bexarotene D4 and 9-cis-retinoic acid (ALRT 1057).
According to some embodiments of the invention, the RYR1, RYR3 agonist is selected from the group consisting of: chlorocresol, CHEBI 67113-chlorantraniliprole, S107 hydrochloride, JTV519, trifluoperazine (TFP), xanthine, suramin, NAADP tetrasodium salt, S100A1, cyclic ADP-ribose (ammonium salt) and cyantraniliprole.
According to some embodiments of the invention, the intracellular ca2+ up-regulating factor is selected from the group consisting of NAADP tetrasodium salt, cyclic ADP-ribose, 4-bromo a23187, ionomycin, a23187 and isoprenaline.
According to some embodiments of the invention, the CaMKII agonist is selected from the group consisting of calcium, calmodulin, CALP1 and CALP 3.
According to some embodiments of the invention, the myogenic precursor cells are selected from the group consisting of: myoblasts, satellite cells, myoside group (mSP) cells, myogenic stem cells (MDSCs), mesenchymal Stem Cells (MSCs), myogenic pericytes, embryonic Stem Cells (ESCs), and induced pluripotent stem cells (ipscs).
According to some embodiments of the invention, the myogenic precursor cell is a myoblast.
According to some embodiments of the invention, the myogenic precursor cells are from a biopsy of the farmed animal.
According to some embodiments of the invention, the biopsy is a muscle biopsy.
According to some embodiments of the invention, myogenic precursor cells are isolated from a biopsy by enzymatic and/or mechanical dissociation.
According to some embodiments of the invention, the myogenic progenitor cells are undifferentiated myogenic precursor cells that are cultured in a proliferation medium prior to inducing multinuclear myotube formation.
According to some embodiments of the invention, the proliferation medium is free of molecules selected from the group consisting of: an extracellular regulatory signal kinase (ERK 1/2) inhibitor, a mitogen-activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a RYR1, RYR3 agonist, a RYR1, RYR3 activator, an intracellular ca2+ up-regulator, a calmodulin-dependent protein kinase II (CaMKII) agonist, and a calmodulin-dependent protein kinase II (CaMKII) activator.
According to some embodiments of the invention, the method is performed in the presence of serum or serum replacement in an amount that allows for cell proliferation and/or under normoxic conditions.
According to some embodiments of the invention, the farmed animal is selected from the group consisting of mammals, birds, fish, invertebrates, reptiles and amphibians.
According to some embodiments of the invention, the multinuclear myotube comprises at least three nuclei.
According to some embodiments of the invention, the multinuclear myotube comprises at least 10 nuclei.
According to some embodiments of the invention, the multinucleated myotubes express myogenic differentiation and fusion factors selected from the group consisting of MyoD, myoG, mymk and.
According to some embodiments of the invention, inducing the multinuclear myotubes results in a partial increase in MYOG positive nuclei compared to nuclei of myogenic progenitor cells cultured in differentiation medium without the at least one molecule.
According to some embodiments of the invention, multinuclear myotube formation is evident by classical ladder-like streaks of actin and troponin signals and/or phalloidin staining representing actin filaments.
According to some embodiments of the invention, the yield of myotubes is higher than that obtained with myogenic precursor cells incubated with DMEM 2% Horse Serum (HS) containing 1% penicillin/streptomycin (DM), as evident by any fiber surface coverage, cell weight and protein content as can be determined by Bradford.
According to some embodiments of the invention, the multinucleated myotube formation comprises mononuclear myocell-myotube fusion and/or binuclear and trinuclear myotube expansion into large multinucleated fibers.
According to some embodiments of the invention, the contacting myogenic precursor cells is performed for 12-48 hours.
According to some embodiments of the invention, the contacting myogenic precursor cells is performed for 16-24 hours.
According to an aspect of some embodiments of the present invention there is provided a cultured meat composition comprising multinuclear myotubes produced by the method of the present invention.
According to an aspect of some embodiments of the present invention there is provided a food comprising the cultured meat composition of the present invention.
According to some embodiments of the invention, the food of the invention is processed to impart a organoleptic sensation and texture to the meat.
According to some embodiments of the invention, the food of the invention further comprises food products of vegetable and/or animal origin.
According to some embodiments of the invention, the food of the invention further comprises adipocytes, muscle cells, blood cells, chondrocytes, bone cells, connective tissue cells, fibroblasts and/or cardiomyocytes.
According to some embodiments of the invention, the food of the invention further comprises a plant-based protein.
According to an aspect of some embodiments of the present invention there is provided a method of producing a food product comprising combining a cultured meat composition of the present invention or a food product of the present invention with an edible composition for human or animal consumption.
According to an aspect of some embodiments of the present invention there is provided a method of treating muscle damage in a farm animal, the method comprising contacting damaged muscle tissue with at least one molecule selected from the group consisting of: an extracellular regulated signal kinase (ERK 1/2) inhibitor, a mitogen activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor- β (TGF- β) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a RYR1, RYR3 agonist, a RYR1, RYR3 activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, and a calmodulin-dependent protein kinase II (CaMKII) activator, thereby inducing myotube regeneration and treating the muscle injury, wherein the contacting is performed in the presence of the extracellular regulated signal kinase (ERK 1/2) inhibitor and the up-regulator of intracellular ca2+.
Unless defined otherwise, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be necessarily limiting.
Brief description of several views of the drawings
Some embodiments of the invention are described herein, by way of example, with reference to the accompanying drawings. Referring now in specific detail to the drawings, it is emphasized that the details shown are exemplary and are for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how the embodiments of the present invention may be embodied.
In the drawings:
FIGS. 1A-1I are a series of images and graphs showing the induction of myoblast differentiation and hyperfusion by ERK1/2 inhibition.
(1A) Representative images of myoblasts at various time points after treatment with DMSO control (Ctrl) or 1 μmsch772984 (ERKi) in growth medium or Differentiation Medium (DM). Cells were fixed and stained 8, 24 and 48 hours after treatment with the differentiation markers myosin heavy chain (MyHC, red) and nuclear Hoechst (blue). Scale bar = 200 μm. (1B) Fusion index indicates differentiation in 1A (MyHC + ) The proportion of nuclei found in the cells. Total number of cores detected n= 88,518. (1C) Representative qRT-PCR results show transient gene expression profiles of Myod, myog, mymk and Mymx during myogenesis. Gene expression values were normalized to Gapdh and expressed as fold change from control at 0 hours. (1D, 1F, 1H) treatment with DMSO control or 1. Mu.M ERKi, or DM in growth medium for 24 hours and staining with MyHC (red) and MYOG (green) (1D), myHC (red) and Ki-67 (green) (1F) and MyHC (red) and pH3 (green) into representative images of myocytes. Nuclei were stained with DAPI (blue). Scale bar = 100 μm. (1D, 1E, 1G). The percentages of MYOG, ki-67 and PH3, respectively. All data represent at least 3 biological replicates. Error bars represent SEM;
FIGS. 2A-2J are a series of images and graphs showing ERK1/2 inhibition initiates RXR/RYR dependent fusion reactions.
(2A) Immunoprecipitation of ERK1/2 and RXR. (2B) Representative images of cells stained with control, 1 μM ERKi, 20 μM HX531 (RXRi), ERKi and RXRi, 50 μm Dantrolene (RYRi), ERKi and RYRi, 10uM BAPTA-AM, or ERKi and BAPTA-AM for 24 hours and with differentiation markers MyHC (red), MYOG (green) and nuclei (blue). The white box represents the enlarged view portion on the right side. (2C) fusion index of ERKi and RXRI co-processing experiments. (2D) Quantification of MYOG positive nuclei per field of the ERKi and RXRi co-treatment experiments. Total number of cores detected by 2C and 2D, n=106,116. (2E) qRT-PCR analysis of fold change in expression of calcium channels and sensors in vector (control) compared to cells treated with ERKi for 24 hours; gene expression was normalized to Hprt. Values represent fold changes relative to controls. (2F) qRT-PCR analysis of RYR1/3 gene expression demonstrated modulation of ERK1/2 and RXR. (2G) fusion index of ERKi and RYRi co-processing experiments. (2H) Quantification of MYOG positive nuclei per field of view for ERKi and RYRi co-treatment experiments. Total number of nuclei detected by 2G and 2H, n= 113,448. (2I) fusion index of ERKi and BAPTA-AM co-processing experiments. (2J) Quantification of MYOG positive nuclei per field of ERKI and BAPTA-AM co-treatment experiments. Total number of nuclei detected by 2I and 2J, n= 109,360. All data represent at least 3 biological replicates. Scale = 100 μm;
Figures 3A-3M are a series of images and graphs showing that asymmetric myoblast fusion requires calcium dependent CaMKII activation.
(3A) Representative western blots of CaMKII activation after 24 hours of treatment with ERKi or DM. (3B, 3C and 3D) shows representative Western blots of CaMKII activation in myoblasts after 24 hours of treatment. Treatment (3B) was DMSO (control), 1 μm ERKi, 20 μm HX531 (RXRi), or co-treatment with ERKi and RXRi, respectively. (3C) Treatments were control, 1. Mu.M ERKi, 50. Mu.M Dandelion (RYRi), or co-treatment with ERKi and RYRi. (3D) The treatments were control, 1. Mu.M ERKi, 10. Mu.M BAPTA-AM, or co-treatment with ERKi and BAPTA-AM. (3E) Representative image immunofluorescence images of cells treated for 24 hours with control, 1 μM (ERKi), 5 μM KN93 (CaMKIIi), or with ERKi and CaMKIIi. Cells were stained for differentiation markers MyHC (red), MYOG (green) and DAPI (blue). The indication area is enlarged on the right side. (3F) fusion index of 3E; the values are stratified by the number of cores per myhc+ fiber. Total number of cores detected n= 61,510. (3G) quantification of MYOG positive nuclei per field of 3E. Total number of cores detected n= 112,901. (3H) qRT-PCR gene expression analysis of the experiment shown in 3D; gene expression was normalized to Hprt. Values represent fold changes relative to controls. (3I) With wild CaMKII (Ad-CaMKII) WT ) Or a phosphate null mutant (Ad-CaMKII) T287V ) Representative western blots of CaMKII activation in function acquisition/loss studies performed 72 hours after transfer to DM. Bands for endogenous and exogenous CaMKII are shown. (3J) For each MyHC when processed in DM for 72 hours + The number of nuclei of cells was quantified for CaMKII function acquisition/deletion studies, expressed as fold change relative to control virus. Total number of cores detected n= 18,758. (3K) Representative western blot time course after treatment with 1 μm ERKi. (3L) shows that the Raney receptor (RYR) is found in the control andrepresentative images of localization in ERKi-treated muscle fibers. The indicated area in the ERKi image is enlarged on the right, displaying the individual fluorescent channels and overlays. The arrow indicates the lack of differentiation of the Raney receptor (MyHC + ) A muscle cell. (3M) representative immunofluorescent staining, showing that p-CaMKII was predominantly localized to myotubes (green) 24 hours after ERKi treatment. The indicated area in the ERKi image is enlarged on the right, displaying the individual fluorescent channels and the cover. The arrow indicates p-CaMKII negative mononuclear MyHC+ cells, while the asterisks indicate double-nucleated MyHC+ cells that are p-CaMKII+. Arrows indicate myhc+ cells that have fused with myotubes and are p-camkii+. All data represent at least 3 biological replicates. Error bars represent SEM. Scale = 100 μm;
Figures 4A-4E are a series of images showing asymmetric myotube growth by recruiting mononuclear myoblasts at the fusion synapse.
(4A) The hour fusion index shows the distribution of mononuclear, binuclear, trinuclear and polynuclear (n.gtoreq.4) cells. A significant increase in the number of polynuclear fibers, with a concomitant decrease in the number of monocytes, was observed about 16 hours after treatment with ERKi. The average number of binuclear and trinuclear cells remained relatively constant over about 12 hours. Total number of cores detected n= 13,044. (4B) Data-driven simulations show that if fusion occurs with equal probability (inverted triangle) or weighted probability (regular triangle) the proportion of nuclei in multi-nuclei (n.gtoreq.4) cells will not be reiterated (recapitulates) considering that larger cells have higher fusion probability. Simulations were performed by estimating the number of fusion events per hour in the experiment. The estimated fusion number was used to model two simple cases: in the stochastic simulation, cells have a uniform fusion probability, and the weighted simulation adjusts the probability according to the number of nuclei in the cells. Statistical significance is determined by a bootstrap method, see methods for complete detailed information. (4C) Frames obtained from time series microscopy (time lapse microscopy) of individual myotubes undergoing asymmetric fusion. At time 0, binuclear early myotubes were observed to be labeled with cytoplasmic DsRed (purple) and approached mononuclear myoblasts (yellow squares) expressing membrane-targeted GFP (farnesylated-GFP; white). When the cells fuse, the mixing of the cytoplasm and the membrane becomes evident (t=00:28). Time: hh: mm. Scale bar: 50 μm. (4D) two examples of synapses are fused (time: hh: mm). The scale bar is 10 μm. "front view" represents the fusion event represented in 4C (yellow square). Upper graph: the Z-projection of the membrane marker highlights the 3D structure of the protrusion extending from the myoblasts to the myotubes, eventually fusing, which can be seen by the simultaneous diffusion of the cytoplasmic markers into the myoblasts and the disappearance of the membrane markers from the protrusion between the two fused cells. Middle diagram: indicating a specific Z-plane of the membrane marker, it is seen that the fusion holes are expanding. The following figures: from the Z-plane of the same time lapse shot, different fusion events are seen in the side view. The cyan and yellow arrows of the middle and lower panels point to the pre-and post-fusion synapses, respectively. (4E) Frames of GCaMP6S calcium reporter fluorescence acquired in growing myotubes undergoing asymmetric fusion. The fluorescent signal is depicted as a heat map. The solid arrows indicate that myotubes are about to recruit some myoblasts to fuse with them. The dashed arrow represents one of these myoblasts before and during the first asymmetric fusion event. At (00:10) it is indicated that calcium pulses are present in the growing myotubes, whereas no calcium pulses are present in the myocells. (time scale: hh: mm). Scale bar = 50 μm.
Fig. 5A-5I are a series of images, charts and blots showing that CaMKII is necessary for effective muscle regeneration.
(5A) Western blot indicating protein analysis in muscle after Cardiotoxin (CTX) induced muscle injury. The line represents where lanes were deliberately removed. (5B) Schematic representation of a satellite cell specific double CaMKII KO mouse model. (5C) Schematic drawing of a timeline depicting a repeat injury experiment design. (5D) Western blot validated for CaMKII depletion (depletion) in WT or scDKO primary myoblasts isolated 2 weeks after initial injury. (5E) Immunofluorescent staining of WT or scDKO primary myoblasts 24 hours after ERKi-induced fusion following treatment. The inset is enlarged on the right side. (5F) Fusion index comparison between WT (n=4) and scDKO (n=4) primary myoblasts, stratified by the number of nuclei per fiber. Total number of cores detected n= 12,743. (5G) Representative visual field of WT and scDKO muscles 14 days after CTX-induced re-injury. (5H) Quantification of muscle fiber cross-sectional area in WT (n=4) and scDKO (n=4) mice 14 days after the re-injury. (5I) Average percentage of central nuclei in WT (n=4) and scDKO (n=4) mice 14 days after re-injury. For 5H and 5I, at least 9,000 fibers were measured per mouse. Error bars represent SEM. All scales, 100 μm;
FIGS. 6A-6C are schematic illustrations of ERK1/2-CaMKII myotube driven secondary fusion pathways.
Schematic representation of ERK-CaMKII pathway during myoblast differentiation and fusion: 6A) In proliferating myoblasts, ERK1/2 inhibits MYOG and p21/p27 activation. 6B) After ERK1/2 inhibition, p21/p27 expression, cells exit the cell cycle; at the same time, MYOG was upregulated and cells differentiated. 5C) During differentiation, ERK1/2 inhibition results in transactivation of RXR, resulting in upregulation of RYR1/3 and accumulation in the SR of early myotubes, ultimately resulting in asymmetric fusion of calcium-dependent CaMKll activation and CaMKll-dependent myotube driving.
Figures 7A-7E are a series of images, blots and charts showing the criticality of Ca-dependent CaMKII activation of multi-nuclei myotube development.
(7A) Representative western blots showed CaMKII activation of myoblasts treated with DMSO (control), 1 μm ERK inhibitor SCH772984 (ERKi), caMKII inhibitor KN93 μm (CaMKIIi) or co-treated with ERKi and CaMKII 24 hours post treatment. (7B) pH3 positive quantification after 24 hours post treatment with DMSO (control), 1 μm SCH772984 (ERKi), KN93 μm (CaMKIIi) or co-treatment with ERKi and CaMKIIi. (7C) Cell motility quantification of myoblasts in 24 hours treated with DMSO (control), 1 μm SCH772984 (ERKi), KN93 μm (CaMKIIi), or co-treated with ERKi and CaMKIIi. (7D) Representative IF images of myoblasts infected with control virus or virus expressing myoplasmin (Myomaker) and treated with DMSO (control), 1. Mu.M SCH772984 (ERKi), KN93 5. Mu.M (CaMKII), or treated with ERKi and CaMKII for 18 hours. (7E) Quantification of the average number of nuclei per myhc+ cell in 7D. All data represent at least 3 biological replicates. Error bars represent SEM.
FIG. 8 is an assessment of gene expression of several maturation markers in mouse myoblasts treated with SCH772984 at 24 hours post-treatment compared to conventional differentiation media. qRT-PCR analysis of gene expression of Myh1, myh2 and Tnt 3 between myoblasts grown in proliferation medium (CTRL), treated with μM ERK inhibitor, SCH772984 (ERKi) or conventional Differentiation Medium (DM) was compared. Gene expression was normalized to the internal housekeeping gene Hprt and showed fold change with CTRL.
FIGS. 9A-9C show that ERK inhibition induced the excessive differentiation and fusion phenotype of chicken myoblasts. (9A) Time course experiments with chicken-derived primary myoblasts (Time-course experiment) showed the effectiveness of ERKi treatment (1 μm SCH772984, ERKi) in proliferation medium compared to conventional Differentiation Medium (DM). Myofibers are indicated by myosin heavy chain staining (red), nuclear staining is indicated by DAPI (blue). (9B) Quantification of the fusion index 72 hours after treatment indicated a near 4-fold increase in myoblast fusion after ERKi treatment compared to DM. (9C) qRT-PCR analysis of gene expression of various differentiation markers over a 72 hour period showed that ERKi treatment and DM induced differentiation, but ERKi effect was more pronounced than DM.
FIGS. 10A-10B show that ERKi induced chicken muscle fiber differentiation more effectively than conventional DM. (10A) qRT-PCR analysis of gene expression of transcription factor mrf and myoglobin heavy chain (myh, myh 2) and troponin (tnnnnt 3) of sarcomere gene demonstrated significantly improved expression after ERKi treatment compared to DM. (10B) Immunofluorescent staining of the sarcomere proteins of ERKi-treated chicken myoblasts, including alpha actin, filiform actin (phalloidin) and troponin, 48 hours after treatment, indicated classical streaks of mature sarcomere. At this point, no comparison with DM fibers was possible, as they had not yet formed (demonstrating the early phenotype obtained by ERKi).
FIGS. 11A-11D show a quantitative analysis of the effect of ERKi on muscle tissue yield. (11A) The surface area covered by the ERKi treated fibers was significantly greater than the fibers induced in DM. (11B) Relative mass evaluation of muscle product 72 hours after treatment with 1. Mu. MSCH772984 (ERKi) compared to DM. Briefly, the same number of cells were treated with either condition. After 72 hours, the tissue culture plates were scraped, and cells were collected and centrifuged. The wet weight of the particles was measured. ERKi treatment resulted in an approximately 40% increase in product mass at 72 hours post-treatment. (11C) The number of starting cells required to reach 1 kg of end product 72 hours after treatment with ERKi or DM was determined from the cell pellet data of 11A. (11D) The relative protein production of ERKi or DM treated products was measured 72 hours post-treatment, indicating that ERKi-induced myogenesis resulted in a 4-fold increase in total protein production.
FIG. 12 shows the conserved phenotype obtained after ERKi treatment in bovine myoblasts compared to conventional differentiation media. Immunofluorescence images and fusion index quantification of bovine myoblasts were performed after 72 hours of Proliferation Medium (PM), differentiation Medium (DM) or treatment with 0.5uM SCH772984 (ERKi). ERKi resulted in nearly 8-fold increase in fusion compared to DM.
Fig. 13 demonstrates that ERKi-induced bovine myotubes showed earlier maturation than bovine myotubes treated with DM. Immunofluorescent staining of the sarcomere components of myosin heavy chain (MyHC), alpha-actin and troponin T96 hours after treatment with Proliferation Medium (PM), differentiation Medium (DM) or 1uM SCH772984 (ERKi) is shown. Although myotubes were still present after 96 hours of treatment with DM, the levels of these sarcomere markers were higher in the ERKi-induced myotubes as demonstrated by quantification of the relative intensities of fluorescent signals.
Fig. 14A and 14B are a series of images and graphs showing that various ERK inhibitors induce myoblast fusion. Representative images (fig. 14A) and fusion indices (fig. 14B) of primary bovine myoblasts treated with ERK inhibitors SCH772984, AZD0364, BVD523, DEL22379, FR180204, GDC0994, KO947 and LY3214996 (all 1 uM) in proliferation medium showed similar myoblast differentiation and fusion levels for all ERK inhibitors. Samples were fixed 72 hours after treatment and immunostained for alpha rhabdomyoactin (red) and nuclei were stained with DAPI (cyan). Error bars represent SEM. The scale bar is 100 μm.
Fig. 15A and 15B are a series of images and graphs showing the effect of calcium ionophore on ERK inhibitor-induced myoblast fusion. Representative images (FIG. 15A) and fusion index (FIG. 15B) of primary chicken myoblasts treated with ERK inhibitors (SCH 772984 1uM, SCH) alone or in combination with various calcium ionophores (ionomycin-2 uM, calcmycin (calcymemicin) -1uM and calcium ionophore I-2 uM) in proliferation media demonstrate synergistic effects of combined administration of ERK inhibitors and calcium ionophores. Samples were fixed 48 hours after treatment, and the myosin heavy chain (MF 20, red) was immunostained and nuclei were DAPI stained (cyan). Error bars represent SEM. The scale bar is 100 μm.
FIGS. 16A and 16B are a series of images and graphs showing the effect of retinoid-X receptor (RXR)/ranitidine (RAR) agonists on ERK inhibitor-induced myoblast fusion. Representative images (FIG. 16A) and fusion index (FIG. 16B) of primary chicken myoblasts treated with ERK inhibitors (SCH 772984 uM, SCH) alone or in combination with various RXR/RYR agonists (9-cis retinoic acid, 9-cis RA-200nM, AM80-200 nM, AM580-100 nM and CH55-200 nM, TTNPB 200nM, fenretinide 200 nM) in proliferation media demonstrated synergistic effects of combined administration of ERK inhibitors and RXR/RYR agonists. Samples were fixed 48 hours after treatment, and the myosin heavy chain (MF 20, red) was immunostained and nuclei were DAPI stained (cyan). Error bars represent SEM. The scale bar is 100 μm.
FIGS. 17A and 17B are a series of images and graphs showing the effect of an ranitidine (RYR) agonist on ERK inhibitor-induced myoblast fusion. Representative images (FIG. 17A) and fusion index (FIG. 17B) of primary chicken myoblasts treated with ERK inhibitors (SCH 772984 1uM, SCH) alone or in combination with various RYR agonists (caffeine-2 mM and suramin-10 uM) in proliferation media demonstrated synergistic effects of combined administration of ERK inhibitors and RYR agonists. Samples were fixed 48 hours after treatment, and the myosin heavy chain (MF 20, red) was immunostained and nuclei were DAPI stained (cyan). Error bars represent SEM. The scale bar is 100 μm.
Fig. 18A and 18B are a series of images and graphs showing that MEK inhibition has an effect on the myoblast fusion phenotype that is superior to ERK inhibition. Representative images (fig. 18A) and fusion index (fig. 18B) of primary chicken myoblasts treated with ERK inhibitor (SCH 772984 1 or 10 uM) alone compared to myoblasts treated with MEK inhibitor (U0126 or 10 uM) in Proliferation Medium (PM) or Differentiation Medium (DM) demonstrate that ERK inhibition achieves better myoblast fusion, particularly in Proliferation Medium (PM). Samples were fixed 48 hours after treatment, and the myosin heavy chain (MF 20, red) was immunostained and nuclei were DAPI stained (cyan). Error bars represent SEM. The scale bar is 100 μm.
Detailed Description
The present invention relates in some embodiments to methods of differentiating myogenic progenitor cells, and more particularly, but not exclusively, to cultured meat and cultured meat products.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by the examples. The invention is capable of other embodiments or of being practiced or of being carried out in various ways.
Current methods for culturing muscle cells to produce cultured meats (e.g. "in vitro meats", "laboratory meats") require lengthy (bovine species require up to 14 days) differentiation steps to induce myotubes from amplified muscle stem/progenitor cells, thereby increasing production costs and duration. The present inventors have disclosed methods for significantly increasing the degree and rate of myoblast-multinuclear myotube transition, increasing the production efficiency of cultured meat, and reducing costs.
The inventors have shown that by inhibiting or reducing ERK1/2, cultured myogenic precursors can be induced to form large polynuclear myotubes (see, e.g., fig. 1A, 1B), and that myogenic precursor-myotube transitions and asymmetric fusions are associated with an increase in intracellular ca2+ (see, e.g., fig. 3E and 3F). Furthermore, the inventors have shown that enhancement of myoblast differentiation and fusion can be achieved with a variety of ERK inhibitors (example 10), and that manipulation of ERK1/2 downstream factors by calcium ionophores (example 11), RXR/RAR agonists (example 12) and RYR agonists (example 13) can effectively enhance the efficacy of ERK inhibition.
The inventors demonstrate the superiority of ERK inhibition (ERKi) compared to conventional methods for culturing meat (referred to as "DM" in some embodiments of the invention). Specifically, as demonstrated by myogenesis in chickens in tissue culture: ERKi enhances the differentiation transcription process, leading to early formation of myotubes; ERKi enhances fusion, resulting in a significantly larger myotube; and ERKi enhances the maturation of muscle fibers by increasing the expression of maturation markers, leading to early formation of sarcomere structure (see, e.g., example 7). Furthermore, the inventors demonstrate that this effect is conserved and pronounced in at least 2 other species (cattle and sheep). Similarly, data from bovine myoblasts indicate that ERKi-induced fibers mature faster than those obtained with DM. Overall, treatment of myoblasts with ERKi can achieve earlier differentiation and stronger fusion, resulting in earlier myotube maturation, ultimately increasing the production efficiency of the cultured meat by increasing the overall mass, area coverage of the meat product, and ultimately increasing total protein production.
Thus, in some embodiments, a method of inducing myotube formation is provided, the method comprising contacting myogenic precursor cells from a cultured animal with an extracellular regulated signaling kinase (ERK 1/2) inhibitor and/or an up-regulating factor of intracellular ca2+.
In other embodiments, a method of inducing multinuclear myotube formation is provided, the method comprising contacting a myogenic precursor cell from a farmed animal with an extracellular regulated signaling kinase (ERK 1/2) inhibitor and/or an up-regulating factor for intracellular ca2+, wherein when the myogenic precursor cell is a chicken myogenic precursor cell, the contacting is performed in the presence of the extracellular regulated signaling kinase (ERK 1/2) inhibitor and the up-regulating factor for intracellular ca2+.
As used herein, the term "myogenic precursor" or "myogenic precursor cell" refers to any cell that can differentiate into a muscle cell. Myogenic precursors are critical for muscle regeneration. Although the most naturally abundant animal myogenic precursors are satellite cells found on the serosal surface of muscle fibers, other cells with myogenic potential have been identified and may be suitable for use in the methods of the invention. These cells include myoblasts of mesodermal origin, myosided crowd (mSP) cells located in the stroma, stem cells of muscle origin (MDSC) and myoepithelial cells from endothelial-related myofibers (myo-endothesial cells), mesodermal pericytes and mesodermal angioblasts, and mesodermal cd133+ progenitor cells.
Different myogenic precursor cells can be characterized by a cell marker profile, such as myod+ and desmin+ of myoblasts; cd34+/-, ckit-and CD 45-of Msp; cd56+ and cd29+ of muscle precursor; cd133+ and cd34+/-, of cd133+ mesodermal progenitor cells.
As used herein, the term "polynuclear myotube" refers to a fusion myogenic precursor (e.g., fused into myocytes) having 3 or more nuclei. A precursor of mononuclear or binuclear myogenesis is not considered to be a "polynuclear myotube" even if it expresses a myogenic differentiation marker.
As used herein, the term "multinuclear myotube" is equivalent to the terms "multinuclear myoblasts", "multinuclear muscle fibers (multinucleated muscle fibers or multinucleate muscle fibers)", "multinuclear syncytia (multinucleated syncitia or multinucleate syncitia)", "multinuclear muscle syncytia (multinucleated muscle syncitium or multinucleate muscle syncitium)", and are used interchangeably herein.
In some embodiments, the multi-core myotubes each have 4-10,000, 10-8,000, 20-500, 15-250, 50-1000, 100-800, 60-2000, 70-4000, 80-6000, 90-5000 cores. In specific embodiments, the multinuclear myotube has 10 to 100, 10 to 500, or 10 to 1000 nuclei. Thus, in some embodiments, the multi-core myotube comprises at least 3 cores, at least 10 cores, at least 50 cores, or at least 100 cores.
The nuclei can be identified and quantified by a number of techniques, including but not limited to immunofluorescence, flow cytometry, and immunohistological techniques. Common nuclear stains include DAPI (fluorescence), hematoxylin (cytological stains), hoechst33258 and 33342 (fluorescence), methyl blue (cytological stains), safranin (cytology). In specific embodiments, the nuclei are labeled with Hoechst 3342 (Thermo-Fisher) or DAPI (Sigma) and observed by fluorescence microscopy. In some embodiments, in contrast to multinuclear myotubes having four (3) or more nuclei, multinuclear myotube formation is quantified by layering cells into single-and double-nuclear cells.
In addition to developing polymorphous nuclei, myogenic precursor cells that are induced to form multinuclear myotubes are expanded by fusion with differentiated myogenic cells. While putting the invention into practice, the inventors have shown that myogenic precursor-myotube formation includes "asymmetric fusion", i.e., the fusion according to the methods of the invention is primarily myoblast to myotube fusion ("secondary fusion", "asymmetric fusion") rather than myoblast to myoblast enhanced fusion ("primary fusion"). Thus, according to some embodiments of the invention, multinuclear myotube formation includes mononuclear myocell-myotube fusion and/or binuclear and trinuclear myotube expansion into large multinuclear fibers.
In addition, in some embodiments, myogenic precursor cells may be embryonic stem cells (ESC, totipotent cells) and Induced Pluripotent Stem Cells (iPSC). ipscs can be produced from adult fibroblasts by inducible expression of reprogramming factors, have unlimited replicative capacity in vitro, and can differentiate into myoblast-like cells (see, e.g., roca et al, journal of clinical medicine (j. Clin. Med) 2015).
The phrase "embryonic stem cells" refers to embryonic cells that are capable of differentiating into cells of all three embryonic germ layers (i.e., endoderm, ectoderm and mesoderm) or remain in an undifferentiated state. The phrase "embryonic stem cells" may include cells obtained from embryonic tissue (e.g., blasts) formed after gestation of an embryo prior to implantation (i.e., pre-implantation embryo blasts), extended Blasts (EBCs) obtained from post-implantation/pre-gastrulation blasts (see WO 2006/040763), embryonic Germ (EG) cells obtained from reproductive tissue of a fetus, and cells produced in unfertilized eggs stimulated by parthenogenesis (parthenogenesis activated embryos).
Induced pluripotent stem cells (iPS; embryonic-like stem cells) are cells obtained by dedifferentiation of adult cells endowed with pluripotency (i.e., capable of differentiating into three embryonic germ cell layers, i.e., endoderm, ectoderm and mesoderm). According to some embodiments of the invention, such cells are obtained from differentiated tissue (e.g., somatic tissue, such as skin) and dedifferentiated by genetic manipulation that reprograms the cells to obtain embryonic stem cell characteristics.
In some embodiments, the myogenic precursor cells may be induced muscle progenitor cells obtained by manipulating small molecules in a medium and/or forcing MyoD expression in non-muscle cells to directly transform and differentiate non-muscle tissue (e.g., fibroblasts) into muscle progenitor cells. U.S. patent application US2019/061731 to hochellinger et al discloses a method for producing induced muscle progenitor cells (ifcs) with satellite cell phenotype from fibroblasts without going through the iPS cell stage. Bin Xu et al (Nature Research, scientific report) DOI 10.1038/s41598-020-78987-8,2020) disclose transdifferentiation of fibroblasts by forced induction of MyoD. As used herein, "transdifferentiation" refers to the process by which a somatic cell is transformed into another somatic cell without undergoing an intermediate pluripotent state or progenitor cell type.
The phrase "adult stem cells" (also referred to as "tissue stem cells" or stem cells from somatic tissue) refers to any stem cells derived from somatic tissue [ post-natal or prenatal animals (especially humans) ]. Adult stem cells are generally considered to be multipotent stem cells capable of differentiating into a variety of cell types. The adult stem cells may be derived from any adult, neonatal or fetal tissue, such as adipose tissue, skin, kidney, liver, prostate, pancreas, intestine, bone marrow, and placenta.
Hematopoietic stem cells, which may also be referred to as adult tissue stem cells, include stem cells obtained from blood or bone marrow tissue of an individual of any age or from umbilical cord blood of a neonatal individual. Placenta and cord blood stem cells may also be referred to as "young stem cells".
Mesenchymal stem cells are multipotent stromal cells that can differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (chondryte or cartilage cells), muscle cells (muscle cells), and adipocytes (adipocytes that produce bone marrow adipose tissue). The term includes multipotent cells derived from bone marrow and other non-bone marrow tissues such as placenta, umbilical cord blood, adipose tissue, adult muscles, corneal stroma, or dental pulp of deciduous teeth. These cells do not have the ability to reconstruct the entire organ.
The myogenic precursor cells may be freshly isolated cells, cells cultured in primary culture from living tissue, or cells of an isolated myogenic cell line developed from repeated serial passages of primary muscle cells. U.S. patent application publication No. US2021/037870 to Kreiger et al discloses exemplary animal cell lines suitable for use in foods containing cultured animal cells. In some embodiments, myogenic precursor cells may be genetically modified, for example, for enhanced proliferation or for expression of tissue-specific factors (see, e.g., U.S. patent application publication No. US2020/0140821 to Elfenben et al).
According to some embodiments of the invention, the initial phase of myoblast enrichment is performed when freshly obtained from a tissue biopsy or primary culture. Specifically, cells are cultured on non-coated dishes that allow preferential adhesion of fibroblasts. Myoblasts predominantly remaining in suspension were collected and re-plated to remove fibroblasts and obtain an enriched culture of myoblasts. This process is referred to as "preplating". This process may be repeated as desired (e.g., 2-4 times). The presence of fibroblasts on the culture dish can be monitored by microscopy.
Thus, in some embodiments, the myogenic precursor cells are selected from the group consisting of myoblasts, satellite cells, myosided group (mSP) cells, myogenic stem cells (MDSCs), mesenchymal Stem Cells (MSCs), myogenic pericytes, embryonic Stem Cells (ESCs), and induced pluripotent stem cells (ipscs).
Recent reports show that: establishment of stem cell lines from domesticated ungulates, for example (challenge and prospect of establishing domesticated ungulate embryonic stem cell lines (Challenges and prospects for the establishment of embryonic stem cell lines of domesticated ungulates), animal propagation science (Anim Reprod sci.) 2007;98 (1-2): 147-168.doi:10.1016/j. Anireprosci.2006.10.009, which is incorporated herein by reference). Bach et al (muscular tissue engineering (Engineering of muscle tissue), plastic surgery clinical (Clin Plast surg.) 2003;30 (4): 589-599.Doi:10.1016/S0094-1298 (03) 00077-4) suggest muscle satellite cells as a preferred source of primary myoblasts because they re-myoblast more closely than immortalized muscle cell lines. Optimization of myotube formation from bovine (Dodson et al, bovine satellite cell origin) (Optimization of bovine satellite cell derived myotube formation in vitro), tissue cell (Tissue cell.) 1987;19 (2): 159-166.Doi:10.1016/0040-8166 (87) 90001-2), chicken (Yablonka-Reuvini et al, development biology (Dev biol.) 1987;119 (1): 252-259.Doi:10.1016/0012-1606 (87) 90226-0), fish (Powell et al, culture and differentiation (Cultivation and differentiation of satellite cells from skeletal Muscle of the rainbow trout Salmo gairdneri) of rainbow trout skeletal Muscle satellite cells, journal of experimental animal (J Exp zoo.) 1989;250 (3): 333-338), lamb (Dodson et al, isolation of sheep skeletal Muscle satellite cells (Isolation of satellite cells from ovine skeletal muscles), journal of Tissue culture methods (JTissue methods) 1986;10 (4): 233-237.Doi:10.1007/BF 01404483), pig (Blanton et al, isolation of two myoblasts of pig skeletal Muscle cells (Isolation of two populations of myoblasts from porcine skeletal Muscle), isolation of Muscle and nerve (Muscle Neve) 1999 (1): 43-50; 35:35:35:35:35) of human, isolation of human Tissue culture methods (JTissue knife methods) 1986; 10.1007-237.1007/BF 01404483), isolation of human skeletal Muscle cells of pig skeletal Muscle groups (35:35:35-35:35:35, etc., journal of Cell biochemistry (J Cell biochem.) 2008;105 (5) 1228-1239), and turkeys (McFarland et al, proliferation of turkey myogenic satellite cells in serum-free medium (Proliferation of the turkey myogenic satellite cell in a serum-free medium), comparison of biochemistry and physiology (Comp Biochem Physiol.) 1991;99 (1-2) isolation and characterization of muscle satellite cells from skeletal muscle tissue of 163-167.Doi:10.1016/0300-9629 (91) 90252-8). Porcine muscle progenitor cells have the potential to differentiate multiple lineages into adipogenic, osteogenic and chondrogenic lineages, which may play a role in the development of co-cultures (Wilschut et al, 2008, supra).
Alternatively, as described above, adult stem cells from farmed animal species may be used. For example, muscle satellite cells are an adult stem cell type with multilineage potential (Asakura et al, differentiation) 2001;68 (4-5): 245-253.Doi: 10.1046/j.1432-0436.2001.680412). These cells also have the ability to differentiate into skeletal muscle cells. A rare multipotent cell population found in Adipose tissue, termed Adipose tissue-derived adult stem cells (ADSCs), is another relevant cell type for in vitro meat production (Gimble et al, adipose-derived stem cells for regenerative medicine (Adipose-derived stem cells for regenerative medicine), flow-through research (circle Res.) 2007;100 (9): 1249-1260.doi:10.1161/01.RES. 0000265074.83288.09), which can be obtained from subcutaneous fat and subsequently transformed into myogenic, osteogenic, chondrogenic or Adipose cell lineages (Kim et al, muscle regeneration by Adipose tissue-derived adult stem cells attached to injectable PLGA spheres (Muscle regeneration by Adipose tissue-derived adult stem cells attached to injectable PLGA spheres), biochemical and biophysical research communication (Biochem Biophys Res Commun.) 2006;348 (2): 386-392.doi: 10.1016/j.bbrc.2006.07.063).
Matsumoto et al (J Cell Physiol.) 2007;215 (1): 210-222) report that mature adipocytes can be dedifferentiated in vitro into a multipotent preadipocyte line, called dedifferentiated adipocyte (DFAT) cells, and that the terminally differentiated cells are reversed into multipotent Cell types. These DFAT cells are capable of transdifferentiating into skeletal muscle cells (Kazama et al, mature adipocyte-derived dedifferentiated adipocytes can be transdifferentiated in vitro into skeletal muscle cells (Material ado-derived dedifferentiated fat cells can transdifferentiate into skeletal myocytes in vitro), biochemical and biophysical research communication (Biochem Biophys Res Commun.) 2008;377 (3): 780-785.Doi:10.1016/j. Bbrc. 2008.10.046), an attractive alternative to stem cells.
In a specific embodiment, the myogenic precursor is a myoblast.
Myogenic precursors can be characterized by the expression levels of certain cell markers, such as, but not limited to, ATP-binding cassette transporter G2 (ABCG 2), M Cadherin/Cadherin 15 (MCadherin/Cadherin 15), caveolin-1, CD34, foxK1, integrin α7, integrin α7β1, MYF-5, myoD (MYF 3), myogenin (MYF 4), neural cell adhesion molecule 1[ ncam1 (CD 56) ], CD82, CD318Pax3, and Pax7. In some embodiments, the myogenic precursor cells are cells that express significant levels of: at least one of MyoD, pax3 and Pax7, or a corresponding, species-appropriate, interspecies homologous gene thereof. In other embodiments, the myogenic precursor cells express MyoD and at least one of Pax3 and Pax7, or their corresponding, interspecies homologous genes of a suitable species. In particular embodiments, myogenic precursor cells express all MusD, pax3 and Pax7, or their corresponding, interspecies homologous genes of the appropriate species.
Once the myogenic precursor cells are obtained, they can be grown in culture to expand their mass, then form multinucleated myotubes, which then form the cultured meat composition. Culturing the cells includes providing a culture system; transferring the basal medium or basal medium supplemented with serum, serum substitutes and/or other components that may be required for efficient growth of the cells into a culture vessel; adding cells and culturing cells. Basal media (e.g., dulbecco's modified Eagle medium, DMEM) can include water, salts, vitamins, minerals, amino acids, and carbon sources such as glucose. In some embodiments of the invention, the basal medium comprises growth factors of animal origin. In other embodiments, the basal medium comprises a growth factor of non-animal origin.
In some embodiments of the invention, the basal medium comprises serum of animal origin. In other embodiments of the invention, the basal medium of the invention does not include serum of animal origin, such as fetal bovine serum, calf serum or horse serum. As used herein, "excluding animal serum" or "animal serum free" means that the medium contains less than about 1% or less than about 0.5% or less than about 0.1% or less than about 0.01% or zero animal derived serum, based on the total weight of the medium. In some embodiments of the invention, it is contemplated that the serum-free medium may contain growth factors and other substances, but not any substances derived from animals.
According to some embodiments, the culturing is performed in the presence of serum at a level that is not considered starvation conditions that prevent cell proliferation. For example, greater than 2% serum (e.g., 3-25%). According to some embodiments, the conditions comprise 5-25%, 10-25% serum, e.g. 15-25% serum, about 20% serum. These conditions are provided in the examples section below. Thus, according to one embodiment, the medium is BIO-AMF TM -2 medium (e.g. obtainable from Biological Industries) comprising basal medium supplemented with Fetal Calf Serum (FCS), steroids, basic fibroblast growth factor, insulin, glutamine and antibiotics.
According to some embodiments, the culturing of myogenic precursor or progenitor cells and the culturing of multinucleated myotubes are performed in media having generally recognized safety (GRAS) and/or "xeno-free" ingredients and components. In some embodiments, the culture medium comprises certified GRAS and/or xeno-free ingredients and/or components. In other embodiments, the culture medium comprises certified GRAS and non-heterogeneous ingredients and/or components. In other embodiments, the culture medium consists of certified GRAS and/or xeno-free ingredients and/or components. In other embodiments, the culture medium consists of certified GRAS and non-heterogeneous ingredients and/or components.
A list of media components used in meat production and their worst case exposure estimates and associated authority limits or published toxicology/safety data supporting their use are listed in table 1.
Table 1: exemplary list of cell culture medium components, risk classification and safety information for use in meat production processes
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A1 =sufficient intake
ADI = daily acceptable intake
FNB-IOM = food and nutrition committee of medical institute
FIDT = highest dose tested
N/a = inapplicable
Nr=unreported
NS = unspecified since there is no evidence that excessive intake is known to produce toxicity
NOAEL = no adverse reaction level was observed
UL = upper allowable limit
OSL = observed safety level
* There is no upper limit in providing as dietary protein
* Maximum meal exposure estimated using the conservative assumption (weight/weight basis) that the medium components were completely transferred to the finished product.
Category 1: these cell culture media components are GRAS food ingredients/additives or food ingredients/additives that federal regulations allow unrestricted use. Exemplary compounds of this class include innocuous ingredients such as sugars, pH buffers, water-soluble vitamins, and common antioxidants (e.g., tocopherols).
Category 2: these cell culture media components are common dietary nutrients and are expected to have a GRAS status, useful for food use or allowed by regulations to be added to food products. Examples of such compounds include most inorganic salts and macronutrients present in the cell culture medium. If these compounds are allowed to be added directly to food products, the level of use is comparable to the expected concentration that might reasonably be expected in cell-based meat products, no safety issues are expected. Most of the nutrients present in poultry cell based meat can be easily measured using the general validation method of the food composition test. A batch analysis of the multiple batches of finished products may be obtained to verify the assumptions described above. In some cases, security may be supported in view of established security levels (e.g., ADI, UL) obtained from related authorities (e.g., u.s.fda, EFSA, JECFA, FSANZ, u.s.epa). If a comparison of the expected meal intake to the authoritative reference intake is used, the additive intake for all meal sources may be considered. NOAEL published in animal toxicology studies can be used to evaluate safety in the absence of an authoritative reference intake value using standard scientific procedures for food safety evaluation. An exposure boundary value (MoE) between NOAEL and estimated meal intake for food exposure of 100 times or greater is generally considered sufficient to support safety. In the case of MoE < 100-fold, additional measures may need to be taken to further reduce the medium composition, or it may be necessary to further characterize the intra/inter-species differences in metabolism. These situations also require careful consideration of regulatory conditions (e.g., need for pre-market approval as a food additive or need for GRAS assessment) depending on the particular situation.
Category 3: these cell culture medium components have not been previously used in food production (e.g., no federal regulation or previous GRAS status), but there is sufficient information to conclude that the intended use of these compounds in food production does not pose a risk. For example, the compound is not detected in the finished product or is present at the same level in the comparative food, is thermally unstable and is digestible during cooking, and/or is expected to be digestible to harmless compounds after ingestion. Examples of compounds that meet the above conditions include recombinant growth factors and serum components. For class 3 substances, the final consideration in the safety assessment process may involve potentially harmful characterization of the substance that produces toxic biological effects beyond the endpoint measured in the subclinical rat toxicity study. A biologically active substance may require other hazard characterizations related to reproductive and developmental toxicity or immunotoxicity. Sensitization, human biological effects (e.g., effects on blood pressure) and synergistic effects with other media components can also be assessed. Such studies may preferably be evidence-based (i.e., there are clinical trials demonstrating that a substance affects blood pressure) rather than theoretical (i.e., based on a putative mechanism of action). Similar to the class 2 substance, the regulatory status of the class 3 component requires a case-by-case assessment of the regulatory status of the compound (e.g., requiring pre-market approval or GRAS assessment). Examples of class 3 components include recombinant proteins and animal serum.
According to some embodiments, the components of the medium are certified as "generally recognized as safe" (GRAS) components (e.g., some of class 1 and class 2 of table 1). As used herein, authentication of GRAS status is granted by a recognized regulatory agency, such as the USFDA. FDA GRAS certification may be awarded (or denied) based on use in food prior to 1958, or for other substances, based on security analysis files made by the manufacturer and reviewed by the FDA. According to some embodiments, the composition of the medium is an authenticated "xeno-free" composition. As used herein, the term "xeno-free" medium refers to a cell culture medium that does not contain components derived from species other than the cultured cells. In some embodiments, the term "xeno-free" refers to "non-human" or the absence of a component derived from a species other than human.
Cells for expansion in cell culture may be obtained from living farmed animals by biopsy, for example from fish, pigs, cattle, chickens, turkeys, sheep, goats, etc.
As used herein, the term "farmed animal" refers to any animal that is grown (cultivated) for agricultural purposes, particularly meat, in order to provide a cost of consumption. Thus, farmed animals include, but are not limited to, avian species, mammalian species, invertebrates (e.g., shellfish), reptiles (e.g., alligators, crocodiles, snakes, tortoises, etc.), and amphibians (e.g., frog). The farmed animals include domesticated species (e.g., cattle, chickens, pigs, ducks, sheep, etc.) and non-domesticated species (trout, salmon, lobster, shrimp, etc.). Examples of avian species suitable for use in the methods of the present invention include, but are not limited to, geese, ducks, chickens, broilers, pheasants, turkeys, bezoans, quails, partridges, pigeons, emus, ostrich, caponies, turkeys, swans, white pigeons, pimples, stone chickens, and sand cone birds. Examples of farmed aquatic species suitable for use in the methods of the invention include, but are not limited to, carp, tilapia, salmon, fish of the order of the shade, trout, bream, snakehead, eel, catfish, molitor in south asia, halibut, sea bass, cod, rabbit, shrimp, crayfish, lobster, crab, oyster and clams. Examples of cultured mammalian species suitable for use in the methods of the invention include, but are not limited to, cattle, bison, buffalo, yaks, dromedaries, llamas, goats, sheep, elks, deer, elk, reindeer, cats, dogs, donkeys, horses, rabbits, kangaroos, guinea pigs, and wild pigs. In a specific embodiment, the myogenic precursor cells are from a farmed animal selected from the group consisting of pigs, cattle, sheep, fish, chickens, ducks and shellfish. As used herein, the term "animal cell" refers to "non-human cells".
In some embodiments, the myogenic precursor cells are obtained by muscle biopsy, e.g., gastrocnemius muscle of a mammal or pectoral muscle of a bird. The biopsy tissue may then be dissociated into cells by enzymatic and/or mechanical means.
Enzymatic cleavage can be achieved by protein digestion (e.g., trypsin, pronase digestion) alone or In combination with collagenase and/or DNase (see, e.g., miesch et al, in vit. Cell and Dev Biol-Animal,54:406-412,2018). In specific embodiments, biopsies are dissociated by incubation with 0.25% trypsin (e.g., trypsin B). The trypsinized tissue may then be further dissociated by mechanical means. In particular embodiments, the enzymatically dissociated tissue is mechanically dissociated using a blunt instrument such as a serum pipette. Individual cells can be obtained by filtering the supernatant, gently centrifuging to pellet the dissociated cells, and re-suspending the pellet in a propagation (e.g., growth) medium. In some embodiments, dissociated muscle tissue is "pre-plated" on an uncoated plate to reduce the number of fibroblasts.
Before induction of multinucleated myotube formation, myogenic precursor cells (whether dissociated myogenic precursor cells from a biopsy or otherwise, such as embryonic stem cells or iPSCs/imscs) are typically cultured in a proliferation medium without inducing differentiation to greatly increase the number of cells useful in the methods of the invention. Culturing of myogenic precursors may include optimizing growth conditions in each culture vessel independently or throughout the system using a gas. Suitable gases include, but are not limited to, oxygen, carbon dioxide, and the like. In addition, salts are used to optimize the growth conditions of the cells. Suitable salts include, but are not limited to, sodium, potassium, calcium, and the like. The amount of salt used is consistent with ranges known in the tissue or cell culture arts. Cells need nutrients to grow; the nutrients provide a carbon source. Suitable carbon sources include, but are not limited to, glucose, glycerol, galactose, hexose, fructose, pyruvic acid, glutamine, and the like. The amount of carbon source used is consistent with the ranges known in the tissue or cell culture arts. The basal medium may also include buffers such as Phosphate Buffered Saline (PBS), TRIS (hydroxymethyl) aminomethane (TRIS), phosphate-citrate buffer, soranson phosphate buffer, sodium citrate buffer, 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid (HEPES), and the like. Alternatively, carbon dioxide may be added to the medium to control the pH. The pH is maintained at about 5.5 to about 7.5. Vitamins are used to optimize the growth conditions of the cells. Suitable vitamins include, but are not limited to folic acid, nicotinamide, riboflavin, B12, and the like. The amount and concentration of vitamins used are in accordance with ranges known in the tissue or cell culture arts. Thus, as described above, local culture conditions can be independently controlled to optimize the growth of cells within each culture vessel.
The culture conditions may be further controlled by temperature. Although mammalian cells are typically cultured at body temperature (i.e., 37 ℃), depending on the cell type, it may sometimes be desirable to deviate from this temperature. Thus, the temperature of the culture vessel can be independently controlled in the range of 20-38 ℃ (e.g., from room temperature to near body temperature). The culture temperature may also be adjusted according to the source of the myogenic precursor cells (mammal, reptile, bird, etc.). Further control and optimization of the culture can be achieved by adjusting the perfusion, its speed, pressure and its pulse frequency and intensity in the case of pulsating flow.
In some embodiments, the proliferation medium contains basal medium with or without additional growth/development factors. In a specific embodiment, the proliferation medium is a basal medium such as BioAmf-2 medium (Biological Industries, israel) supplemented with antibiotics (e.g., gentamicin), mammalian serum (e.g., bovine or fetal bovine serum), and L-glutamine. In some embodiments, the proliferation medium is a basal medium supplemented with growth factors that allow cells to continue growing in culture without transforming into differentiation (e.g., "no differentiation proliferation" through at least 3-5 passages). Growth factors for use in the proliferation medium include, but are not limited to, FGF2, IL-6, IGF1, VEGF, HGF, PDGF-BB, growth hormone, TGF-. Beta.1, nodal collagenase, MMP1, and forskolin. Thus, in some embodiments, the proliferation medium comprises one or more of FGF2, IL-6, IGF1, VEGF, HGF, PDGF-BB, growth hormone, TGF-. Beta.1, nodal collagenase, MMP1, and forskolin.
According to a preferred embodiment, the medium comprises serum or serum substitutes or other defined factors that can be used to promote cell proliferation.
As used herein, the phrase "serum replacement" refers to a defined formulation that replaces the function of serum by providing the cells with components required for growth and survival.
Various serum replacement formulations are known in the art and are commercially available.
For example, GIBCO TM Knockout TM The serum replacement (Gibco-Invitrogen Corporation, grandide island, N.Y., U.S.A., catalog No. 10828028) is a defined serum-free formulation that is optimized to grow cells in culture. Note that GIBCO TM Knockout TM Formulations of serum substitutes include Albumax (lipid-rich bovine serum albumin) from animal sources (Price, p.j. Et al, international patent publication No. WO 98/30679). However, recently a paper published by rook et al in 2007 (rook JM. et al, 2007, cell Stem Cell (Cell) 1:490-494) describes the use of FDA approved clinical-grade foreskin fibroblasts in the Knockout manufactured by cGMP TM Six clinical-grade hESC lines generated in serum substitutes (Invitrogen Corporation, USA, catalog No. 04-0095).
According to some embodiments of the invention, GIBCO TM Knockout TM The concentration of serum replacement in the medium was about 3% [ volume/volume (v/v)]To about 50% (v/v), such as about 5% (v/v) to about 40% (v/v), such as about 5% (v/v) to about 30% (v/v), such as about 10% (v/v) to about 25% (v/v), such as about 10% (v/v) to about 20% (v/v), such as about 10% (v/v), such as about 15% (v/v), such as about 20% (v/v), such as about 30% (v/v).
Another commercial serum replacement is a vitamin A-free B27 supplement, available from Gibco-Invitrogen, catalog No. 12587-010, of Greendeland, N.Y.. B27 supplement is a serum-free formulation comprising D-biotin, fatty acid free component V Bovine Serum Albumin (BSA), catalase, L-carnitine hydrochloride, corticosterone, ethanolamine hydrochloride, D-galactose (anhydrous), glutathione (reduced), recombinant human insulin, linoleic acid, linolenic acid, progesterone, putrescine-2-HCl, sodium selenite, superoxide dismutase, T-3/albumin complex, DL alpha-tocopherol, and DL alpha-tocopheryl acetate.
Thus, in some embodiments, the myogenic precursor cells are undifferentiated myogenic precursor cells that are cultured in a proliferation medium prior to inducing multinuclear myotube formation. In particular embodiments, the proliferation medium lacks factors active in inducing the formation of polynuclear myotubes. In some embodiments, the proliferation medium lacks one or more of EGF1, a p38 agonist and a TGFB inhibitor.
The inventors have shown that the addition of one or more selected from the group consisting of the following to myogenic precursors in differentiation medium can also greatly promote the transition to multinucleated myotubes (see, e.g., fig. 18A and 18B): an extracellular regulated signal kinase (ERK 1/2) inhibitor, a mitogen activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranialkali receptor (RYR 1, RYR 3) agonist, a ranialkali receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, a calcium ionophore, and a calmodulin-dependent protein kinase II (CaMKII) activator. Thus, in some embodiments, the method comprises contacting myogenic precursor cells (which may or may not have been previously cultured in proliferation medium) that have been or are being cultured in differentiation medium with one or more of the at least one molecule of the invention, thereby inducing or enhancing multinuclear myotube formation.
The expansion of myogenic precursor cells may be performed in a culture plate (e.g., a culture dish, "2D culture"), a culture vessel, a bioreactor, or the like. In some embodiments, myogenic precursor cells are expanded on a coated plate or container, e.g., coated with a recombinant substrate membrane (e.g., matrigel). In other embodiments, myogenic precursor cells are expanded on a substrate or scaffold for "3D culture".
In some cases, the cells are cultured in suspension in a cell culture flask. Cell culture flasks are optionally stacked and/or arranged side-by-side with the 2D surface cell culture. Cells in suspension culture are typically non-adherent cells. However, in some cases, adherent cells are cultured on scaffolds in suspension. Scaffolds provide structural support and physical environment for cell attachment, growth and migration. In addition, stents generally have mechanical properties such as elasticity and tensile strength. Typically, 3D scaffolds are used to culture adherent cells to enable 3D growth of the cells. Scaffolds sometimes have a specific shape or size to guide the growth of cultured cells. In some cases, the stent is composed of one or more different materials. Some stents are solid stents, while others are porous stents. Porous scaffolds allow cells to migrate or permeate into the pores. Scaffolds are typically composed of biocompatible materials to induce proper recognition of cells. Furthermore, the scaffold is made of a material with mechanical properties and degradation kinetics suitable for the desired tissue type produced by the cells. In some cases, the scaffold comprises a hydrogel, a biological material such as extracellular matrix molecules (ECM) or chitosan, or a biocompatible synthetic material (such as polyethylene terephthalate). ECM molecules are typically proteoglycans, non-proteoglycan polysaccharides, or proteins. Possible ECM molecules for scaffolds include collagen, elastin, heparan sulfate, chondroitin sulfate, keratan sulfate, hyaluronic acid, laminin and fibronectin. Sometimes, plant-based scaffolds are used for 3D culture.
Normal cells in culture tend to proliferate until confluence, at which point contact inhibition prevents further division. Thus, in some embodiments, the cells are cultured in proliferation medium until confluence. In other embodiments, the expansion is prolonged by partial depletion of cells, transfer to a more spacious culture vessel, or by preventing confluence, e.g., spin flasks, bioreactors.
In some embodiments, amplification is performed for about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 days, 1.5 weeks, 2.0 weeks, or longer. In a specific embodiment, myogenic precursor cells are expanded in proliferation medium for 24 hours.
The mode of operation, whether batch, fed-batch or continuous, affects bioreactor size and media requirements. From a large scale point of view, it is often preferred to feed the medium in a batch or continuous manner. A variety of configurations may operate in these modes. Stirring vessels are currently the most common in the biotechnology industry. They provide a time-averaged, uniform, well-mixed environment by convective mixing induced by mechanical, pneumatic or hydraulic agitation, such as the rocking motion in impeller-driven stirred tank bioreactors (STR), rotating Wall Bioreactors (RWB) and wave bioreactors. Other bioreactor configurations may enable continuous perfusion operations such as Packed Bed Bioreactors (PBB), fluidized Bed Bioreactors (FBB) and membrane bioreactors such as Hollow Fiber Bioreactors (HFB). For non-perfusion reactors such as STR, continuous (perfusion) operation requires connecting the bioreactor to an internal or external cell retention device on the circulation line by centrifugation, sedimentation, ultrasonic separation or microfiltration with a rotary filter, alternate Tangential Flow (ATF) filtration or Tangential Flow Filtration (TFF). See also:
Clincke,M.F.、C.、Samani,P.K.、Lindskog,E.、/>E. Walsh, K.et al, (2013 a), very high density of Chinese hamster ovary cells in perfusion by alternating tangential flow or tangential flow filtration in WAVE bioreactorTM-part II: applications for antibody production and cryopreservation.Biotechnol. Prog.29,768-777.Doi:10.1002/btpr.1703;
Clincke,M.F.、C. zhang, y., lindskog, e., walsh, k, and choteteau, v. (2013 b) Very high density of CHO cells in perfusion by ATF or TFF in WAVE bioreactor TM :Part I:Effect of the cell density on the process.Biotechnol.Prog.29,754-767.doi:10.1002/btpr.1704。
Allen et al, 6.12, describe bioreactors commonly used for muscle cell expansion, ront. www.doi.org/10.3389/fsufs.2019.00044.
The decisions regarding the type, size and number of bioreactors are affected by many factors including passage, which are within the skill of the ordinarily skilled artisan and are described in a non-limiting manner in: allen et al, ront. Sustain. Food syst., 6 months 12 days 2019:www.doi.org/10.3389/fsufs.2019.00044. Passaging, in the form of sequential transfer to reactors of increasing size, as seen in seed culture, requires that minimum and maximum cell densities be met. Microcarrier culture and bead-to-bead transfer capability of cell lines (Verbruggen et al 2017, ytotechnology,1-10.Doi:10.1007/s 10616-017-0101-8) can be increased in surface area by adding microcarriers without increasing vessel size. The comparison of bioreactors is typically based on achievable final cell density, rather than volume, which is an arbitrary concept without background, such as seeding density and final cell number or density and passaging steps. The achievable cell densities will be different for suspension systems using microcarriers for anchorage dependent cells than for single cell suspensions.
After expansion of the myogenic precursor cells, the cells are washed and the proliferation medium is replaced with a medium having a reduced amount of proliferation-inducing factors compared to the concentration in the proliferation medium and comprising factors that support myogenesis, or in other embodiments, the medium is supplemented with molecules for inducing the formation of polynuclear myotubes from the myogenic precursor.
The inventors have shown that ERK1/2 is a key factor in maintaining myogenic precursor characteristics of precursor cells, and that inhibition of ERK1/2 can induce the formation of polynuclear myotubes from cultured myogenic precursors (see, e.g., fig. 1A and 1E, in particular fig. 10). Furthermore, the inventors have shown that additional factors constitute regulatory effects in the transition of myogenic precursors to fusion multinuclear myotubes. In general, other factors that may be added to induce the transition of myogenic precursors to the fused polymyotubes include inhibitors of the regulatory function upstream of ERK1/2 and activators/agonists of the regulatory function downstream of ERK 1/2.
Thus, in some embodiments, there is provided a method of inducing myotube formation in a polynuclear, the method comprising contacting myogenic precursor cells from a farmed animal with at least one molecule selected from the group consisting of: an extracellular regulatory signal kinase (ERK 1/2) inhibitor, a mitogen-activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranialkali receptor (RYR 1, RYR 3) agonist, a ranialkali receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, and a calmodulin-dependent protein kinase II (CaMKII) activator.
In other embodiments, a method of inducing myotube formation is provided, the method comprising contacting myogenic precursor cells from a farmed animal with at least one molecule selected from the group consisting of: an extracellular regulated signal kinase (ERK 1/2) inhibitor, a mitogen activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranialkali receptor (RYR 1, RYR 3) agonist, a ranialkali receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, and a calmodulin-dependent protein kinase II (CaMKII) activator, wherein when the myogenic precursor cells are chicken myogenic precursor cells, the contacting is performed in the presence of the extracellular regulated signal kinase (ERK 1/2) inhibitor and the up-regulator of intracellular ca2+.
Myogenesis is the entire process involving differentiation and fusion, and maturation of muscle fibers. Differentiation is a unique stage of this process. Undifferentiated and quiescent muscle progenitor/satellite cells express markers such as Pax3/Pax7. Progenitor cells are activated and begin to proliferate signaling early differentiation into myoblasts. Myoblasts may express residues of Pax3/Pax7 but characteristically begin to express markers Myf1 and Myf2. When the myocytes exit the cell cycle, late differentiation is marked by the expression of markers such as MEF2 protein and myogenin (MyoG) and MRF 4. Positive expression of these specific proteins/RNAs indicates their activation and typing phase (stage of commitment). Traditionally, the marker used to assess typing and differentiation is MyoG. Once differentiated into myoblasts of MyoG, the cells also begin to express myosin heavy chains (MyHC) before fusing with other myoblasts, and are considered "differentiated" and "fusion-competent" at this stage. However, differentiated cells may also remain unfused as mononuclear myocytes (myosin heavy chain positive and MyoG positive). It will be appreciated that cell culture is a population of cells and maturation is a dynamic process-thus, although the expression of markers in the culture is subject to statistical distribution and you may find that cells in transitional phases in the culture express markers at different phases simultaneously, culture conditions such as those of the present invention may reproducibly provide a population of cells enriched in polynuclear myotubes expressing characteristic high levels of maturation markers compared to cells found only in differentiated cells.
In some embodiments, at least one molecule is an ERK1/2 inhibitor. ERK1/2 inhibitors suitable for use IN the methods of the invention include, but are not limited to MK-8353 (SCH 900353), SCH772984, CC-90003, dehydrogambir, ERK1/2 inhibitor 1, magnolin, ERK IN-1, ERK IN-2, ERK IN-3, LY3214996, ravoxertiinib, hydrochloric acid Ravoxertinib, VX-11e, FR180204, ulitinib (Ulixertiinib), ulitinib hydrochloride, ADZ0364, KO947, FRI-20 (ON-01060), bromoacetoxycalcitol (B3 CD), BVD523, DEL22379, FR180204, GDC0994, KO947, AEZ-131 (AEZS-131), AEZS-136, AZ-13767370, BL-EI-001, LTT, ASTX-029, TCS ERK 11e, and CAY10561. In some embodiments, the ERK inhibitor is selected from the group consisting of peptide inhibitor EPE, ERK activation inhibitor peptide I (ERK inhibitor IV), and ERK activation inhibitor peptide II (ERK inhibitor V). In a specific embodiment, ERK1/2H is SCH772984.
In some embodiments, at least one molecule is an inhibitor of an ERK1/2 upstream regulator, including but not limited to a MEK1 inhibitor. MEK1 inhibitors suitable for use in the methods of the present invention include, but are not limited to, trametinib, PD98059, U0126 (U0126-EtOH), PD0325901, semetinib (AZD 6244), cobratinib (GDC-0973, RG 7420), bimetinib (MEK 162), CI-1040 (PD 184352), refatinib (BAY 869766; RDEA119), pimatinib (AS 703026), semetinib (AZD 6244), cobratinib hemi-fumarate (Cobimetinib hemifumarate), GDC-0623 (RG 7421), RO 4987550, AZD8330 (ARRY-424704), SL327, MEK inhibitors, PD318088, cobratinib racemate (GDC-0973 racemate; XL518 racemate), PD198306, AS-703026, ADZ8330 and EBI-1051.
In some embodiments, at least one molecule is an inhibitor of a mitogen such as FGF1 and its receptor, including but not limited to FGF inhibitors. FGF inhibitors suitable for use in the methods of the present invention include, but are not limited to, delazantinib, PD161570, SSR 128129E, CH5183284, PD166866, and pemitinib.
In some embodiments, at least one molecule is an inhibitor of tgfβ and its receptor, including but not limited to tgfβ inhibitors. TGF-beta inhibitors suitable for use in the methods of the invention include, but are not limited to, SD208, LY364947, repSox, SB 525334, R268712, and GW 788388.
In some embodiments, at least one molecule is a molecule that activates or is an agonist of a retinoid-X receptor (RXR) and/or a retinoid-X receptor (RXR), including, but not limited to, RXR/RAR agonists. RXR/RAR agonists suitable for use in the methods of the present invention include, but are not limited to, CD3254, docosahexaenoic acid, LG100268, SR11237, AC261066, AC55649, adapalene, BMS961, CD1530, CD2314, CD437, BMS453, EC23, all-trans retinoic acid, all-trans-4-hydroxy retinoic acid, all-trans retinoic acid-D5, cyantraniliprole, vitamin A, all-trans retinol, LG100754, beta-carotene, beta-apo-13-carotene ketone, lycopene, all-trans-5, 6-epoxyretinoic acid, all-trans-13, 14-dihydroretinol, retinol acetate, and honokiol, valeric acid, HX630, HX600, LG101506, 9cUAB30, AGN194204, LG101305, UVI3003, net-4IB, CBt-PMN, XCT0135908, PA024, methoprenoic acid, 9-cis-AM 80, LGCH 55, LG-13, and (alopram) and the combination thereof, and the combination thereof (including the combination of the above) and the following, and the combination of the following (including the combination of the above) and the following).
In some embodiments, at least one molecule is a molecule that increases expression of or acts as an agonist of the ranibase receptors (RYR 1 and RYR 3), including, but not limited to, RYR agonists. RYR agonists suitable for use in the methods of the invention include, but are not limited to, caffeine, chlorocresol, CHEBI:67113, chlorantraniliprole, S107 hydrochloride, JTV519, trifluoperazine (TFP), xanthine, suramin, sodium suramin, NAADP tetrasodium salt, S100A1, cyclic ADP-ribose (ammonium salt), theobromine, 4-chloro-3-methylphenol (4-chloro-m-cresol), tetrazolium chlorantraniliprole, trifluoperazine (TFP), cycloartemia and cyantraniliprole. In a specific embodiment, the RYR agonist is methylxanthine. In a specific embodiment, the RYR agonist is caffeine.
In some embodiments, at least one molecule is a molecule that activates or acts as an agonist of the cytoplasmic level of ca2+ or that upregulates the cytoplasmic level of ca2+, including but not limited to an upregulating factor of intracellular ca2+. Ca2+ up-regulating factors suitable for use in the methods of the invention include, but are not limited to, NAADP tetrasodium salt, cyclic ADP-ribose, 4-bromo A23187, ionomycin, A23187, and isoprenaline.
In some embodiments, at least one molecule is an agonist of CaMKII or a molecule that is an agonist of CaMKII, including but not limited to an agonist of CaMKII. CaMKII agonists suitable for use in the methods of the invention include, but are not limited to, calcium, calmodulin, CALP1 and CALP3. In other embodiments, at least one molecule is a molecule that modulates a target downstream of CaMKII. Such molecules that modulate downstream targets of CaMKII and are suitable for use in the present invention include, but are not limited to IRSP53, RAC1, CDC42, SRF, CREB, actin, human potassium protein-7 (Kalirin-7), synGap, sarcoplasmic proteins, and transfer 1 protein (Tiam 1).
In other embodiments, at least one of the molecules is a molecule that upregulates the cytoplasmic level of ca2+ and is a calcium ionophore. Suitable calcium ionophores for use in the present invention include, but are not limited to, ionomycin, calicheamicin, calcium ionophore I (CA 1001; ETH 1002), beauvericin, lyxotrope (Laidlomycon), lasaloxil, salinomycin, and domicile (Semduramycin).
Intramuscular calcium-ATPase (Sarcoendoplasmic calcium-ATPase) is an intracellular membrane transporter that actively transports Ca2+ ions from the cytoplasm to the lumen of the muscular (endo) plasma compartment. Inhibiting SERCA channel activity may enhance cytoplasmic calcium retention. Thus, in other embodiments, at least one molecule is an inhibitor of the enzyme intramuscular calcium-atpase (SERCA). SERCA inhibitors suitable for use in the present invention include, but are not limited to, cyclopeanic acid, 2, 5-di-tert-butylhydroquinone, (DBHQ), ruthenium red, tert-butylhydroquinone (t-butyl hyroquinone), gingerol (gineerol), CPG 37157, thapsigargin and muscarinic (Paxilline).
These molecules may be contacted with the myogenic precursor alone or in combination with other suitable molecules. In specific embodiments, the myogenic precursor is contacted with an ERK1/2 inhibitor or an up-regulator of intracellular Ca2+, or with both an ERK1/2 inhibitor and an up-regulator of intracellular Ca2+. In particular embodiments, when the myogenic precursor is derived (e.g., derived) from chicken, the contacting is performed in the presence of ERK1/2N and an up-regulating factor for intracellular ca2+.
The culturing of myogenic precursors for inducing the formation of polynuclear myotubes may be performed in a vessel or plate or bioreactor, as described for the expansion of myogenic precursor cells with proliferation medium. In short, induction of multinuclear myotube formation may be performed in culture plates (e.g., petri dishes, "2D culture"), culture vessels, bioreactors, and the like. In some embodiments, myogenic precursor cells are expanded on a coated plate or container, e.g., coated with a recombinant substrate membrane (e.g., matrigel). In other embodiments, myogenic precursor cells are expanded on a substrate or scaffold for "3D culture".
Notably, the use of 3D scaffolds may be effective because the cultured multinuclear myotubes may be incorporated into cultured meats or cultured muscle compositions. Scaffolds sometimes have a specific shape or size for guiding the growth of cultured cells. The scaffold is made of a material with mechanical properties and degradation kinetics appropriate for the desired tissue type produced by the cells. In some cases, the scaffold comprises a degradable material to enable remodeling and/or elimination of the scaffold in the cultured food product. For example, in some cases, a 3D stent that shapes a cultured myotube into a patties shape may biodegrade after the myotube is expanded to fill the interior space of the stent. In other cases, the scaffold comprises material that remains in the cultured food product. For example, at times, at least a portion of the collagen scaffold that provides support for the cultured muscle cells remains to provide texture and continuous structural support in the cultured food product. In some cases, the scaffold comprises a hydrogel, a biological material such as extracellular matrix molecules (ECM) or chitosan, or a biocompatible synthetic material (such as polyethylene terephthalate).
The formation of multinuclear myotubes may be accompanied by an increase in expression or activity of differentiation-related factors. Skeletal muscle markers include, but are not limited to, alpha-, beta-, and epsilon-muscle glycans, calpain inhibitors, creatine kinase MM/CKMM, elF5A, enolase 2/neuron-specific enolase, FABP3/H-FABP, GDF-8/myosin (Myosttin), GDF-11/GDF8, MCAM/CD146, myoD, myogenin, myosin light chain kinase inhibitors, troponin 1/Tnn13. Thus, in some embodiments, the myogenic precursor is cultured in a medium comprising at least one of the following: an extracellular regulated signal kinase (ERK 1/2) inhibitor, a mitogen activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranine receptor (RYR 1, RYR 3) agonist, a ranine receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, a SERCA inhibitor, and a calmodulin-dependent protein kinase II (CaMKII) activator, resulting in increased expression of myogenic differentiation factors including, but not limited to MyoD, myoG, mymk, mymx, troponin (tnn 3), myofestival gene myosin heavy chains 1 and 2 (hc 1, hc 2) and actin. In some embodiments, inducing the multinuclear myotubes results in an increase in the fraction of MYOG-positive nuclei in the cultured myogenic precursor compared to nuclei of the myogenic precursor cultured in serum-depleted differentiation medium (serum-depleted differentiation medium) lacking or lacking at least one of the following: an extracellular regulatory signal kinase (ERK 1/2) inhibitor, a mitogen-activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranialkali receptor (RYR 1, RYR 3) agonist, a ranialkali receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, and a calmodulin-dependent protein kinase II (CaMKII) activator.
Thus, according to some aspects of the present invention there is provided a cultured meat composition derived from myogenic precursor or progenitor cells, characterized by an enhancement of the myogenic marker compared to the same cells cultured for the same time in the absence of at least one of the following substances of the invention: an extracellular regulatory signal kinase (ERK 1/2) inhibitor, a mitogen-activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranialkali receptor (RYR 1, RYR 3) agonist, a ranialkali receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, and a calmodulin-dependent protein kinase II (CaMKII) activator.
In some embodiments, the cultured meat compositions are characterized by the presence of abundant mature myofibers and characteristic fringes of actin, troponin and phalloidin signals already after a short 24 hour period of culture according to the methods of the present invention. Such streaks indicate that the polynuclear myotubes form a sarcomere structure. In other embodiments, the cultured meat composition is characterized by increased expression and activation of the camkl and Ryodine receptor (RYR) as compared to the same cells cultured for the same time in the absence of at least one of the following: an extracellular regulatory signal kinase (ERK 1/2) inhibitor, a mitogen-activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranialkali receptor (RYR 1, RYR 3) agonist, a ranialkali receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, and a calmodulin-dependent protein kinase II (CaMKII) activator.
In other embodiments, the cultured meat composition is characterized by increased expression of the myogenic marker as compared to the same cells cultured for the same time without at least one of the following of the invention: an extracellular regulatory signal kinase (ERK 1/2) inhibitor, a mitogen-activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranialkali receptor (RYR 1, RYR 3) agonist, a ranialkali receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, and a calmodulin-dependent protein kinase II (CaMKII) activator, including, but not limited to, myosin heavy chain (MyHC), myoG, myo-wire protein (desmin), dystrophin, and laminin.
In other embodiments, the cultured meat composition is characterized by an increased presence of polyrhachis tube (indicating a higher fusion index) as compared to the same cells cultured for the same time without at least one of the following of the invention: an extracellular regulatory signal kinase (ERK 1/2) inhibitor, a mitogen-activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranialkali receptor (RYR 1, RYR 3) agonist, a ranialkali receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, and a calmodulin-dependent protein kinase II (CaMKII) activator.
In some embodiments, the duration of the incubation (e.g., prior to comparing muscle maturation characteristics) is 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64. 66. 68, 70 or 72 hours or more, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14 or more days. In some embodiments, the myogenic characteristics of the cultures can be compared after 1, 2, 4, 6, 8, 12, 16, 20, 24, 36, 48, or 72 hours.
The inventors have found that culturing myogenic precursors with the indicated molecules results in rapid and powerful fusion, effectively producing multi-core myotubes in surprisingly short time (on the order of hours rather than days) (see, e.g., fig. 4A-4E). Thus, in some embodiments, contacting the myogenic precursor with at least one molecule of the invention is performed for 10-96 hours, 12-72 hours, 12-48 hours, 18-36 hours, 24-28 hours, 16-48 hours, or 16-24 hours. In specific embodiments, contacting the myogenic precursor with at least one molecule of the invention is performed for 12-48 hours, or 16-24 hours.
Notably, the methods of the present invention result in simultaneous transition from mononucleated and binuclear myogenic precursors to multinucleated myotubes, as well as significantly improved efficiency in forming multinucleated myotubes from myogenic precursors. Thus, in some embodiments, a myogenic precursor is contacted with at least one molecule of the invention until 30%, 40%, 50%, 60% or more of the nuclei in the culture are from polynuclear myogenic precursors. In other embodiments, the myogenic precursor is contacted with at least one molecule of the invention until at least 50% of the nuclei in the culture are from polynuclear myogenic precursors.
It will be appreciated that myogenic precursor or progenitor cells cultured according to the methods disclosed herein can have a characteristic and/or unique gene expression pattern or temporal pattern, which can be different from those that were not cultured according to the methods of the invention.
Thus, in some embodiments, a myogenic precursor or progenitor cell cultured according to the methods of the invention is characterized by at least one of a gene expression profile, an RNA profile (e.g., transcript), and/or a protein profile (e.g., proteasome) that is different from a myogenic precursor or progenitor cell not cultured according to the methods of the invention. Such a map can be obtained by using a commercially available map (e.g., affymetrix Gene ) Or custom arrays.
The method of the invention can be used to produce multinuclear myotubes suitable for use as cultured meat.
The teachings of the present invention are particularly valuable for the meat industry where large numbers of cells are required to be produced at minimal commodity cost.
As described above, and further described in the examples section that follows, the inventors were able to demonstrate improved yields in terms of fiber yield, protein yield, and cell weight yield (see fig. 11A-D). Finally, this means that less growth medium (and general resources) is needed to produce the same amount of product in the same time; thus reducing the overall cost of the manufacturing process.
According to some embodiments of the invention, an exemplary procedure for obtaining myotubes is described in the examples section below. Briefly, a muscle biopsy is performed. The primary culture is subjected to 1 or more (e.g., 2-3) pre-plating steps in the presence of proliferation medium (a medium known to those skilled in the art that allows proliferation) to remove fibroblasts and enrich myoblasts. The molecules as described herein, e.g., ERKi, RXR/RAR agonists, ranin base receptor agonists (RYR), caMKII inhibitors, calcium ionophores, or combinations thereof, are then added to the culture for a predetermined period of time, after which the cells are washed and re-cultured in the absence of the molecules in the presence of proliferation medium.
It will be appreciated that molecules ("at least one of … …") as described herein may also be used to obtain multinuclear myotubes from myogenic precursors or progenitor cells when cultured in differentiation medium to enhance (e.g., increase or accelerate) fusion and development of multinuclear myotubes.
When a sufficient amount of cells is obtained, a culture of polynuclear myogenic precursors may be harvested to provide biomass for culturing the meat composition. In some embodiments, the culture is harvested prior to "maturation" (less than 100% of the cells are multinucleated), while in other embodiments, the culture is harvested at "maturation", i.e., substantially all of the cells are multinucleated.
At any stage (i.e., myoblasts to myotubes), the cells can be collected and stored for further use.
Thus, according to some embodiments, there is provided a cultured meat composition comprising multinuclear myotubes produced by the methods of the invention described herein.
In the production of the cultured meat composition, the desired biomass of the multinucleated myotubes may be the biomass that is reached when the cells are no longer able to proliferate, or may be the maximum biomass that the cells are able to reach at a given culture scale and culture conditions. In some embodiments, the biomass of the polynuclear myotubes is the biomass of at least 50%, 60% or more of the nuclei in the culture from polynuclear myogenic precursors. Alternatively, the desired biomass may be biomass that has produced enough cells to form a cultured meat composition.
The cultured meat composition, cultured meat product, meat analogue composition or product, and cultured meat composition or product refer to meat compositions or products that contain animal cells that are grown in plates, containers, flasks, bioreactor systems or other similar production systems outside the animal body. The cultured meat compositions or products may take a variety of forms and be used in different ways. The artificial or cultured animal cells may be used as ingredients of foods containing a high percentage of plant material, or they may be produced with sufficient biomass as the major ingredient in foods. The cultured meat composition or product may also contain other ingredients or additives including, but not limited to, preservatives.
Thus, in some embodiments, a food is provided comprising the cultured meat composition of the present invention. As used herein, the term "food" refers to a food product, an edible item. In a specific embodiment, the cultured meat composition or the food comprising the cultured meat composition is suitable for human or animal consumption.
The cultured meat compositions or foods of the present invention may include tissue engineering products, cultured animal cells mixed with plant-based proteins, or pure animal cell products. In some embodiments, the cultured meat composition or food includes cultured animal cells, which may or may not be combined with plant-based proteins or other food additives or ingredients, may produce unstructured minced meat products, such as minced beef, or may be tissue engineered/synthesized into structured tissue, such as bacon or steak. In some embodiments, the cultured meat or food may comprise additional cells, including but not limited to adipocytes, muscle cells, blood cells, chondrocytes, bone cells, connective tissue cells, fibroblasts, and/or cardiomyocytes, and/or additional food products of plant or animal origin, in addition to the multinucleated myotubes. The cultured meat composition may be configured as living tissue that can mature in a bioreactor, or as non-living tissue as the final product.
In some cases, the food of the present invention may be combined with or consist essentially of plant matter. Plant sources that may be used include, but are not limited to, peas, chickpeas, mung beans, kidney beans, broad beans, soybeans, cowpeas, pine nuts, rice, corn, potatoes, and sesame. An exemplary method for producing a hybrid food composition comprising a culture meat composition and a plant-based or plant-derived component (e.g., a plant protein) is described in detail in U.S. patent application publication US 20200100525.
The food comprising the cultured meat composition of the present invention may have an increased meat-like flavor, aroma or color compared to a cultured meat product comprising the same number of unmodified cells of the same type. The food of the present invention comprising both the plant-based product and the cultured meat composition of the present invention may have an increased meat-like flavor, aroma or color compared to a plant-based product without the cultured meat composition of the present invention.
When desired, the meat compositions and foods may be enriched to some extent with additives to protect or alter their flavor or color, improve their tenderness, juiciness or adhesiveness, or to aid in their preservation. Thus, the cultured meat additives may include salts and other substances that impart flavor and inhibit microbial growth, extend product shelf life, and aid in emulsifying finely processed products (e.g., sausage). Nitrite can be used to marinate meat to stabilize the color and flavor of the meat and to inhibit the growth of spore-forming microorganisms (e.g., botulinum). The phosphate used in meat processing is typically an alkaline polyphosphate, such as sodium tripolyphosphate. Erythorbate or its equivalent ascorbic acid (vitamin C) can be used to stabilize the color of salted meat. Sweeteners such as sugar or corn syrup impart sweetness, bind water, and promote surface browning during cooking in the maillard reaction. Flavoring agents impart or change flavor. They include spices or oleoresins, herbs, vegetables and essential oils extracted from them. Flavoring agents such as monosodium glutamate impart or enhance a particular flavor. The tenderizer breaks down collagen, making the meat more palatable. They include proteolytic enzymes, acids, salts and phosphates. Specific antibacterial agents include lactic acid, citric acid and acetic acid, sodium diacetate, acidified sodium chloride or calcium sulfate, cetylpyridinium chloride, activated lactoferrin, sodium lactate or potassium lactate, or bacteriocins such as nisin. Antioxidants include a variety of chemicals that limit lipid oxidation, which can create an undesirable "off-flavor" in precooked meat products. Acidulants, most commonly lactic acid or citric acid, impart a strong or acidic flavor note, extend shelf life, tenderize fresh meat or aid in protein denaturation and moisture release in dried meat. They replace the natural fermentation process of acidifying some meat products such as hard salami or smoked ham.
Thus, within the scope of the present invention, wherein the food or cultured meat composition additionally comprises an acidity regulator, alkalinity regulator, anti-caking agent, defoamer, natural and other antioxidants, bulking agent, food colorant, color retention agent, emulsifier, flavor, flavoring agent, flour treating agent, polish, humectant, trace gas, preservative, probiotic microorganism, stabilizer, sweetener, thickener, and any mixture thereof. In a specific embodiment, the additive is an authenticated GRAS additive.
In another embodiment of the invention, the food or cultured meat composition has the final organoleptic properties of a meat product, in particular a product (or products) selected from the group consisting of: beef, beef heart, beef liver, beef tongue, bone soup from allowed meats, buffalo, bison, calf liver, reindeer, goat, ham, horse, kangaroo, mutton, bone marrow soup, camel, mutton, chinchilla, viscera, pork, bacon, rabbit, snake, squirrel, omnice, beef tripe, turtle, veal, venison, chicken liver, kang Nixi young chicken, duck liver, emu, gizzard, goose liver, turkey, guinea fowl, liver, ostrich, partridge, pheasant, quail, squab, and turkey.
According to one embodiment, the food or cultured meat composition has enhanced meat feel characteristics or meat nutritional characteristics over a cultured meat composition that does not contain the multinucleated myoblasts cultured according to the disclosed methods. As used herein, organoleptic properties refer to aspects of food (or other substances) that are experienced by the sense of the sense, including taste, vision, smell, and touch. Exemplary organoleptic properties include, but are not limited to, taste, smell, texture, and color. Methods of sensory detection are well known in the art, some of which are described below.
Sensory (or sensor) evaluations are a common and very useful tool in the quality evaluation of processed food (e.g., meat, cultured meat) products. It utilizes sensory attributes to evaluate the overall acceptability and quality attributes of the product. Detection typically uses specialized panelists and/or manual means.
The test methods commonly used in sensory evaluation are: 1. for a pair-wise comparison test for simple differences, two coded samples were provided to panelists for simple difference evaluation. 2. Three coded samples are provided simultaneously, two of which are identical, the third of which is different, and panellists are required to identify the different types of samples. 3. A preference rating test (Hedonic scale rating test) or acceptability test, samples are tested to determine their acceptability or preference.
Sensory testing (chewing) is often sufficient to test the tenderness/toughness or uniformity/fiber structure of meat and meat products. If more objective results are desired, special instruments can be used to measure texture. Such devices are commonly used to measure the shear force required to cut meat/meat products. The comparative texture measurement (comparative texture measurement) is typically taken from the same tissue or product that has been subjected to different treatments (e.g., maturation, cooking, etc.).
The list of relevant sensory attributes comprises the following three main groups, individually tailored to the type of product: appearance: surface color, internal color, texture (roughness, uniformity), overall rating related to the type of product tested. Texture: hardness/softness, juiciness/dryness, cohesiveness, chewiness, fat/oil mouthfeel, overall assessment. Taste and flavor (possible list of positive and negative characteristics of aroma and taste): meat flavor, cooked chicken, roast chicken, bouillon-like (broth), greasy, burnt, sweet, bitter, rancid, overall assessment.
The present invention also provides a method of producing a food product or food product comprising the steps of: a. providing a cultured meat composition or food as described herein, and b, forming the cultured meat composition or food into a desired form. Further steps may include adding components for nutrition, flavor, taste, texture, color, odor, shelf life, etc.
According to some embodiments, the food or food product of the present invention comprises a range of cultured meat compositions or foods disclosed herein: about 1% to about 99%, about 2% to about 95%, about 3% to about 92%, 4% to about 90%, about 5% to about 87%, about 6% to about 85%, about 7% to about 82%, about 8% to about 80%, about 9% to about 77%, about 10% to about 75%, about 12% to about 70%, about 13% to about 65%, about 15% to about 60%, about 18% to about 55%, about 20% to about 50%, about 23% to about 45%, about 25% to about 43%, about 30% to about 40%. In other embodiments, the food or food product of the invention comprises a range of cultured meat compositions or foods disclosed herein: about 1% to about 10%, about 10% to about 20%, about 20% to about 30%, 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 99%, or 100%.
It is included in any method known in the art for cooking, sterilizing, pasteurizing, packaging, and storing food or food products.
A method of providing nutrition to a subject in need thereof is also provided. The method comprises providing to the subject an amount of a food product comprising a cultured meat composition or food to enhance nutrition of the subject. According to a specific embodiment, the subject is at risk for nutritional deficiency. According to one embodiment, the subject is a healthy subject (e.g., does not have a disease associated with nutrition/absorption).
According to a specific embodiment, the subject suffers from malnutrition. According to a specific embodiment, the subject suffers from a disease associated with nutrition/absorption, such as hypocobalamia, iron deficiency anemia, zinc deficiency and vitamin D deficiency, fatty acid deficiency.
As mentioned above, the formation of polynuclear myotubes from myogenic precursors is a critical stage of muscle tissue regeneration. Thus, the methods disclosed herein for enhancing multi-core myotube formation may be used to treat muscle injuries, where muscle regeneration is desired.
Thus, according to some embodiments, there is provided a method of treating muscle damage in a farm animal, the method comprising contacting damaged muscle tissue of the farm animal with at least one molecule selected from the group consisting of: an extracellular regulatory signal kinase (ERK 1/2) inhibitor, a mitogen-activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranialkali receptor (RYR 1, RYR 3) agonist, a ranialkali receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, and a calmodulin-dependent protein kinase II (CaMKII) activator, thereby inducing myotube regeneration and treating muscle injury, wherein the contacting occurs in the presence of extracellular regulatory signal kinase (ERK 1/2) inhibitor and an up-regulator of intracellular ca2+ when the myogenic precursor cell is a myogenic precursor cell of a chicken.
In particular embodiments, the method comprises contacting the damaged muscle tissue with an ERK1/2 inhibitor and an up-regulating factor for intracellular ca2+.
In some embodiments, the muscle injury may be, but is not limited to, an abrasion, a laceration, a contusion (contusion), a pathologic degenerative process, an inflammation, an ischemic injury, an autoimmune injury, or a bacterial, parasitic, or viral infection.
As used herein, the term "treating" refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the alleviation, alleviation or regression of a pathology. Those skilled in the art will appreciate that a variety of methods and assays can be used to assess pathology development, and similarly, a variety of methods and assays can be used to assess alleviation, alleviation or regression of a pathology.
As used herein, the term "preventing" refers to preventing a disease, disorder, or condition from occurring in a subject that may be at risk of having the disease but has not yet been diagnosed with the disease.
As used herein, the term "subject" includes any farmed animal of any age suffering from a pathology. Preferably, the term encompasses individuals at risk of developing the pathology. The disclosed treatment method is only used for breeding animals and specifically excludes treatment of humans.
As used herein, the phrase "treatment regimen" refers to a treatment plan that specifies the type, dosage, schedule, and/or duration of treatment provided to a subject in need thereof (e.g., a subject diagnosed with a pathology). The treatment regimen selected may be a positive treatment regimen that is expected to produce the best clinical outcome (e.g., complete cure of the pathology), or a milder treatment regimen that may alleviate symptoms of the pathology but result in incomplete cure of the pathology. It will be appreciated that in some cases, a more aggressive treatment regimen may be associated with some discomfort or adverse side effects (e.g., damage to healthy cells or tissue) of the subject. Types of treatment may include surgical intervention (e.g., excision of a lesion, diseased cells, tissue, or organ), cell replacement therapy, administration of therapeutic agents (e.g., receptor agonists, antagonists, hormones, chemotherapeutic agents) in a local or systemic mode, radiation treatment using an external source (e.g., external beam) and/or an internal source (e.g., brachytherapy), and/or any combination thereof. The dosage, schedule, and duration of treatment may vary depending on the severity of the pathology and the type of treatment selected, and one skilled in the art can adjust the type of treatment based on the dosage, schedule, and duration of treatment.
It is expected that during the life of a patent from the beginning of this application many relevant methods of culturing myoplasma will be found and the scope of the term cultured meat is intended to include all such new techniques a priori.
As used herein, the term "about" refers to ± 10%.
The terms "including (comprises, comprising, includes, including)", "having (having)" and their cognates mean "including but not limited to".
The term "consisting of … …" is intended to be "including and limited to".
The term "consisting essentially of … …" means that the composition, method, or structure can include additional ingredients, steps, and/or portions, provided that the additional ingredients, steps, and/or portions do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.
As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual values within the range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within the range, e.g., 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, is applicable.
Whenever numerical ranges are indicated herein, it is intended to include any reference number (fractional or integer) within the indicated range. The expressions "a range between the first indicator number and the second indicator number" and "a range from the first indicator number to the second indicator number" are used interchangeably herein and are meant to include the first indicator number and the second indicator number and all numbers and integers therebetween.
As used herein, the term "method" refers to means, techniques, and procedures for accomplishing a given task including, but not limited to, those means, techniques, and procedures known to, or readily developed from, practitioners of the chemical, pharmacological, biological, biochemical, and medical arts.
As used herein, the term "treating" includes cancelling, substantially inhibiting, slowing or reversing the progression of a disorder, substantially ameliorating the clinical or aesthetic symptoms of a disorder, or substantially preventing the appearance of the clinical or aesthetic symptoms of a disorder.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered as essential features of such embodiments unless the embodiment is not functional without such elements.
Various embodiments and aspects of the invention as described above and as claimed in the claims section below are experimentally supported in the following examples.
Examples
Reference is now made to the following examples, which together with the above description illustrate some embodiments of the invention in a non-limiting manner.
Materials and methods
All experiments in mice were approved by the committee for animal care and use of the institute of science and the Weitman, utility, IACUC application # 00720120-4. To generate satellite cell-specific and tamoxifen-induced CaMK2δ/γ double KO mice, pax7-Cre was used ERT Mice (Murphy et al 2011) Jackson laboratories, stock No. 017763) and double flow (double floxed mice) CaMK2 delta fl/fl /γ fl/fl Mice (Kreuser et al, 2014), pax7 CreERT/+ ；CaMK2δ fl/fl /γ fl/fl (scDKO) or Pax7 +/+ ；CaMK2δ fl/fl /γ fl/fl (WT) littermates. Wild-type c57/BL6 mice were purchased from ENVIGO. Nuclear and membrane reporter gene mice pass nTnG +/+ And mTMG +/+ Mice (jackson laboratories, stock numbers 023537, 007576, respectively) were hybridized and grown internally. Actin/nuclear reporter mice were treated with nTnG by a life act-GFP mouse (Riedl et al 2008) +/+ Mice were bred internally by hybridization and the calcium reporter mice were bred by Pax7-Cre ERT+/+ (Jackson laboratories, stock number 017763) and GCaMP6s flstop/flstop (Jackson laboratories, stock number 028866) mice were crossed and incubated internally, tdTomato reporter mice were crossed by Pax7-Cre ERT+/+ With tdTomato flstop/flstop Hybridization and internal incubation. Genotyping was performed on litters.
Isolation and treatment of primary myoblasts. Primary mouse myoblasts were isolated from gastrocnemius muscle using trypsin-tissue dissociation. Briefly, muscle tissue was incubated in trypsin B (0.25%, biological Industries) and mechanically dissociated with a serum pipette. The supernatant was filtered and centrifuged. The pellet was resuspended in BioAmf-2 medium (Biological Industries, israel) and grown at 37℃and 5% CO 2 The lower plate was plated on 10% matrigel (BD Biosciences) coated plates. For all in vitro experiments, the proliferation medium was Bio-Amf2 (Biological Industries, israel) and the Differentiation Medium (DM) was DMEM 2% Horse Serum (HS) containing 1% penicillin/Streptococcus. For the fusion assay, cells were trypsinized with trypsin C (0.05%, biological Industries) and two rounds of pre-plating on uncoated plates to reduce the number of fibroblasts. The cells were grown at 8X 10 3 Density of wells/wells were plated in proliferation medium in 10% matrigel coated 96 well plates for 24 hours. The next day, the proliferation medium was replaced with proliferation medium or DM containing DMSO (control) or 1. Mu.M ERK1/2 inhibitor (ERKi; SCH772984, cayman Chemicals), 20. Mu.M RXR antagonist (RXRI; HX-531,Cayman Chemicals), 50. Mu.M Raney receptor agonist (RYRi; dantroene, cayman Chemicals), 5. Mu.M CaMKII inhibitor (CaMKII; KN93, cayman Chemicals), or DM was used as control.
Immunofluorescent staining. First generation primary myoblasts isolated from various strains (as shown in the legend) were plated in 96-well plates or chamber slides and treated as described previously. Cells were fixed with ice-cold 4% PFA in PBS for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 6 min, and blocked with PBS containing 0.025% tween, 10% normal horse serum, and 10% normal goat serum for 1 hour at room temperature. The primary antibody was incubated in blocking buffer at 4 ℃ for one night using the following antibodies: myosin heavy chain (MyHC, MF20, DSHB hybridoma supernatant 1:10 or MY-32ABCAM ab51263 1:400), myogenin (MYOG sc-13137SCBT 1:200), pHistone 3 (PH 3, ab47297 ABCAM 1:1000), ki-67 (Cell Marque # 275R), RYR (ab 2868 ABCAM 1:100) and pCaMKII (SIGMA SAB4504356 1:100). Cells were washed 3 times in PBS containing 0.025% tween and then incubated with the appropriate secondary antibody in PBS for 1 hour. If desired, the nuclei are labeled with DAPI (SIGMA D9542,5 ug/ml) or Hoechst 33342 (Thermo scientific #62249, 1:2000). Fixed cells 24 hours after treatment with the indicated inhibitors were imaged using a Nikon Eclipse Ti2 microscope (further described in the microscopy section). All analyses were performed on at least 1000 nuclei. For cells fixed with ERKi or DM over time (fig. 1B), images were captured with an inverted Olympus IX83 microscope (see microscopy section for more details). All imaging assays were performed on at least 1000 cells.
Retrovirus production and transduction for live cell imaging. 24 hours prior to transfection, 3X 10 6 Individual Platinum E cells (Cell Biolabs) were seeded into 100-mm dishes. 10. Mu.g of retroviral plasmid DNA was transfected using FuGENE 6 (Roche). Virus suspension was collected from conditioned medium 48 hours post-transfection. The medium was centrifuged (2500 RPM/10 min) to remove cell debris. The clarified virus suspension was used to transduce primary myoblasts. Briefly, 30,000 primary myoblasts were seeded per well in 6-well plates using polybrene (6. Mu.g/mL) (Merck: #TR 1003-G) as a transduction reagent 48 hours prior to transduction. 1.5 hours after infection, the virus suspension was removed, the cells were washed with PBS, and fresh Bioamf-2 medium was added to the cells. 24 hours after transfection, cells were trypsinized and seeded at a density of 20,000 per well in 8-chamber slides (Ibidi # 80826) and allowed to adhere. The next day, proliferation medium was replaced with appropriate treatment conditions and imaging was started (start time and duration are shown in the legend).
Microscopy
Rotating disc confocal microscopy: live cell imaging (37 ℃ C., 5% CO) using an Olympus IX83 fluorescence microscope controlled by VisiView (Visitron Systems GmbH) software 2 ) The microscope was equipped with a CoolLED pE-4000 light source (CoolLED ltd., UK), PLAPON60XOSC2 NA 1.4 oil immersion objective lensAnd Prime 95B SCMOS cameras (photo metrics). Fluorescence excitation and emission were detected using filter sets 488nm and 525/50nm, 561nm and 609/54nm for mCherry for GFP.
Cell discover 7-Zeiss: fixed samples were imaged using a wide field mode inverted Cell discover 7-Zaiss with an s CMOS 702 camera Carl Zeiss ltd (fig. 1B). Images were acquired using a ZEISS Plan-apochomat 20x/0.95Autocorr objective. Image acquisition was performed using Zen blue software 3.1, myHC signals were acquired using AF647, and nuclei were acquired using DAPI. Brightness and contrast were linearly adjusted using ImageJ v1.52 software if necessary (Schneider et al 2012).
Nikon Eclipse Ti2 microscope: fixed samples (FIGS. 2A-J and 3A-M) were imaged using a Nikon Eclipse Ti2 microscope and NIS-Elements imaging software 5.11.00 version, using a 10-fold objective to obtain MyHC, MYOG, KI-67, pH3 and DAPI staining. The brightness and contrast are linearly adjusted using Photoshop, if necessary. Myoblasts (not shown) expressing tdTomato were imaged in real time using a Nikon Eclipse Ti2 microscope and NIS-elements software using a 10-fold objective lens. Brightness and contrast were adjusted linearly using ImageJ v1.52 software (Schneider et al 2012).
Quantification of fusion index. Following immunostaining and imaging, the fusion index is quantified by manually identifying nuclei found in MyHC positive cells with at least 2 nuclei. These values are then expressed as a percentage of total nuclei per field of view. Briefly, in a graph in which fusion indices are stratified into subgroups by fiber size, the number of nuclei in MyHC positive cells is quantified artificially in a given field of view and stratified into single-nuclear, dual-nuclear myotubes, myotubes with 3-10 nuclei and groups of myotubes with more than 10 nuclei. For myotube growth curves, lifeAct-EGFP; NTNG reporter primary myoblasts were imaged for a delay of up to 23 hours starting 8 hours after treatment. The visual field was analyzed per hour, and the nuclei of each cell were quantified and stratified into single, double, triple, and cells with ≡4 nuclei.
Data-driven cell fusion simulation. For each experiment we defined a matching "shadow" simulation, realThe kinetics of fusion was compared to the scenario where cell fusion occurred randomly. The input for the "shadow" simulation is the distribution of multinucleated cells observed in each time frame. This includes the number of cells with single, paired, triple or quadruple or more nuclei that are manually annotated with a time resolution of 60 minute intervals between successive measurements. The estimated number of fusion events per time interval is calculated as the difference between the weighted cumulative number of multinucleated cells Where i is the number of nuclei in the multinucleated cells, t is the time interval, ct (i) is the number of cells having i nuclei at time interval t. We hypothesize that the number of cells remains constant throughout the experiment. The inputs to the simulation included (1) the number of nuclei determined at the beginning of the N-experiment, where each cell had exactly one nucleus, and (2) a list of N fusion (N fusion) -estimated fusion events per time interval. For each time interval t, we simulated the n_fusion (t) fusion event by randomly selecting two cells and fusing them, generating one cell with a combined number of nuclei for the next round of simulation. For each time interval we record the probability that a core becomes part of a 4-core cell, i.e. what the proportion of cores in a multi-core cell comprising 4 or more cores is. This ratio is used as a measure to compare the experiment with the simulation. Due to the limitations of the annotation we considered multinucleated cells containing 4 nuclei. This means that multinucleated cells with more than 4 nuclei are annotated as 4-nucleated cells. On the one hand, this limitation affects the calculation of the estimated fusion number—this is the lower limit on the true number of fusion events. On the other hand, the calculated probability of the nuclei participating in 4-nuclear cells is also the lower limit of the true probability. This double lower limit effect is expected to cancel each other out and also occurs only later in the experiment.
Statistical significance for each experiment was calculated using the bootstrap method (Bootstrapping approach). For each experiment we performed 1000 simulations. For each time interval in each simulation we recorded whether the probability of nuclei in 4-multinuclear cells was equal to or exceeded experimental observations. The p-value is defined as the probability of modeling more than an experiment using the measurement. We use a cutoff threshold of 0.05 (50 out of 1000 simulations per experiment) to negate the null hypothesis of random fusion. Importantly, this assessment provides a P value for each time interval in each experiment. As a more realistic scenario we consider the possibility that the probability of selecting cells for fusion is proportional to the number of nuclei in them. This follows a simple assumption that the area of n-nucleated cells is n times that of monocytes. Thus, the situation of random fusion of one cell is simulated, but the chance that it collides and fuses to another cell depends on its area.
Real-time fluorescent quantitative PCR (qRT-PCR). Tri-Reagent (SIGMA) was used to isolate total RNA according to the manufacturer's instructions. cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer's instructions. qRT-PCR was performed using a StepOneGlus real-time PCR system (Applied Biosystems) using SYBR green PCR Master Mix (Applied Biosystems). Values for specific genes were normalized to Gapdh or Hprt housekeeping controls, as shown in the legends. Expression was calculated using the ddCT method.
Western blot analysis. Cultured cells and whole tissue extracts were prepared with RIPA buffer supplemented with protease inhibitor cocktail (SIGMA P8340) and phosphatase inhibitor cocktails (SIGMA P5726 and P0044). Western blotting was performed using Mini-PROTEANtetra cell electrophoresis system and transferred onto PVDF membrane. The following primary antibody concentrations were used: p-CAMKII 1:1000 (Abcam ab 182647), caMKII 1:1000 (Cell Signaling 3362), GAPDH 1:10,000 (Abcam ab 181602), p-ERK 1/2:20,000 (SIGMA M9692), ERK 1/2:40,000 (SIGMA), myosin heavy chain (MF-20 DSHB hybridoma supernatant 1:100), horseradish peroxidase conjugated secondary anti-mouse, anti-rabbit or anti-goat were used to detect proteins (Jackson immunology). Western blots were imaged using Chemidoc Multiplex system (Bio-rad).
Co-immunoprecipitation (Co-IP). Primary myoblasts from gastrocnemius were pooled from 10 mice, plated on 15cm dishes, and allowed to adhere for 24 hours. The following day, the Bio-amf2 medium was replaced with Bio-amf2 medium supplemented with DMSO or 1. Mu.M SCH 772984. Cells were treated for 4 hours and then nuclear lysates were prepared according to the instructions of Universal Magnetic Co-IP KIT (Active Motif cat # 54002). ERK1/2 was immunoprecipitated with 1mg protein using 2. Mu.g ERK1/2 antibody (Sigma M7927). Rabbit IgG was used as a control. The reaction was resuspended in 2 Xsample buffer containing DTT and loaded onto a 12% Tris-glycine SDS-page gel. 1% of the original volume of lysate loaded into the IP reaction was loaded into the gel as input control. The membranes were blotted with RXR antibody (SCBT sc-553).
Cloning and expression of CaMKII adenovirus for fusion assays. CaMKII-delta cDNA was PCR amplified from mouse primary myoblasts using primers CAMK2D-F and CAMK2D-R designed for the published CaMKII-delta sequence, ligated into the PGEM-T-easy cloning system (Promega), and the sequences verified. T287V mutations were introduced by PCR assembly. The primers XhoI-FLAG-CAMK2D-F and CAMK2D-T287V-IN-R were used to amplify a 909bp upstream PCR fragment with a primer sequence designed to incorporate an XhoI site and FLAG tag at the N-terminus of CAMK2D and a T287V mutation. The 640bp downstream PCR fragment was similarly amplified with primers CAMK2D-T287V-IN-F and CAMK2D-BamHI-R to introduce T287V mutations and BamHI sites. The two PCR fragments were used as templates for assembly PCR reactions with XhoI-FLAG-CAMK2D-F and CAMK2D-BamHI-R primers, yielding 1525bp products, which were ligated back into PGEM. Similarly, WT CAMK2D was amplified with the same primers to incorporate FLAG tag and ligated back into PGEM. 1525bp FLAG-CAMK2D was digested from PGEM with BamHI and XhoI WT And FLAG-CAMK2D T287V Fragments and ligated into pEGFP-C1 (Clontech). Digestion of 2865bp product EGFP-FLAG-CAMK2D using KPNI and ECORV WT Or EGFP-FLAG-CAMK2D T287V And inserted into readtrackcmv (adedge plasma # 50957). RedTrack-CMV-EGFP-FLAG-CAMK2D WT (Ad-CaMK2D WT )、RedTrack-CMV-EGFP-FLAG-CAMK2D T287V (Ad-CaMK2D T287V ) And empty RedTrack-CMV (Ad-control) vectors were used as templates to grow adenovirus using the Adeasy system, as described previously (Luo et al, 2007). Myoblasts were infected with crude adenovirus lysate with MOI of 100 when plated in BioAmf2 medium (counter infection). After one night of incubation, use of temperatureThe cells were washed once in DM and incubated in DM for 72 hours, and the number of nuclei per fiber was quantified.
Myomaker plasmid construction and over-expression fusion assay. Retroviral vectors (pBabe-puro) and pBabe-GFPfarn plasmids were purchased from Addgene (plasmids #1764 and #21836, respectively). pBabe-CFPnls is produced by replacing Puro in pBabe-Puro R Constructed in which the DNA sequence encoding the CFP is fused to two tandem repeats of the nuclear input signal. The pBabe-dsRed plasmid was constructed in a similar manner. To generate pBabe-Mymk-CFPns, the CDS sequence of murine MMK (Millay et al 2013) was subcloned into the MCS region of the pBabe-CFPns plasmid using unlimited cloning. Retrovirus was produced as described above. At a rate of 7X 10 per well of 96 wells 3 Myoblasts were seeded. The next morning, the virus preparation supernatant was infected with polybrene (1 mL/. Mu.L) for 1 hour, then replaced with fresh growth medium, and after 8 hours the medium was replaced according to the indicated conditions. Cells were fixed and stained 18 hours after treatment.
CTX-induced damage. All experiments were approved by the committee for animal care and use of the institute of science and research with wettman (IACUC application # 00720120-4). Pax7 CreERT/+ ；CaMK2δ fl/fl /γ fl/fl (scDKO) or Pax7 +/+ ；CaMK2δ fl/fl /γ fl/fl (WT) intraperitoneal administration of tamoxifen was received 6 consecutive days from weaning (4 weeks of age), followed by weekly booster doses up to 12 weeks of age. Mice were anesthetized with isoflurane and CTX in PBS (latoxan) was injected into the right gastrocnemius muscle at a concentration of 10. Mu.M using a Hamilton syringe at 10 sites (3. Mu.l per site). All lesions were performed on female mice. For mice receiving repeated injury: after the first injury, the mice were maintained for an additional 8 weeks and then were again injured in the right gastrocnemius muscle as described above.
Histological and CSA quantification. 14 days after CTX induction of the re-injury, the muscles were excised and fixed in 4% PFA, embedded in paraffin, and sectioned. The muscle was cut transversely from the center and cut into serial sections at 0.3mm intervals. To analyze myofiber cross-sectional area (CSA), sections were permeabilized and stained with WGA and DAPI. The whole muscle cross section of WT and scDKO mice taken at the same location in the muscle was imaged at 10-fold using Nikon. CSA was quantified using the semiautomatic analysis tool Open-CSAM of FIJI (Desgeorges et al 2019). The accuracy of each field of view was evaluated and manually corrected. At least 9,000 fibers/mouse were measured.
And (5) statistics. The sample size was chosen based on experience from previous evaluations of experimental variability. In general, all experiments were repeated with n.gtoreq.3 organisms. The number of animals or cells analyzed for each group is depicted in the graphical illustration. All animals were matched for age and sex and cells were collected from mice of similar age. Animals were genotyped before and after the completion of the experiment and they were related together and treated in the same manner. Statistical analysis was performed using Prism software. The data were analyzed using a two-tailed student t-test each time the two conditions were compared. If more than two conditions are compared, an ANOVA analysis of multiple comparisons is performed. In all figures, the measurement results are reported as an average of multiple biological replicates, error bars represent SEM unless otherwise indicated in the figure legends. Throughout the study, the statistical significance threshold was considered to be represented by one asterisk (x) for p.ltoreq.0.01, two asterisks (x) for P <0.001, three asterisks (x) for p.ltoreq.0.001, and four asterisks (x) for p.ltoreq.0.001.
Example 1
ERK1/2 inhibition induces myoblast differentiation and hyperfusion
The inventors hypothesize that ERK1/2 prevents myogenesis not only by maintaining myoblast proliferation, but also by actively inhibiting the myogenic process, by inhibiting gene expression by various nuclear targets (Michailovici et al, 2014; yohe et al, 2018). To examine the role of ERK1/2 in myoblast differentiation and fusion, the specific ERK1/2 inhibitor FSCH772984 (ERKi) was applied to primary myoblasts of mouse origin of the first generation in growth medium, resulting in robust formation of myotubes compared to conventional serum reduced Differentiation Medium (DM) (90.5% in ERKi, 11.6% in DM after 24 hours) (fig. 1A-B). Differentiation and fusion factors MyoD, myoG, mymk and Mymx were up-regulated in ERKi treated cells compared to DM, and the proportion of MYOG positive nuclei was significantly higher at 24 hours (fig. 1C-E). In addition, immunofluorescent staining of ERKi-treated cultures with proliferation markers KI-67 (fig. 1F and 1G) and phosphorylated histone 3 (pH 3, fig. 1H and 1I) demonstrated myoblasts to undergo cell cycle arrest. Taken together, these results indicate that ERKi induces a more intense differentiation and fusion response than myoblasts cultured in normal DM, resulting in myotube hypertrophy.
Example 2
ERK1/2 inhibition initiates RXR/RYR dependent fusion reactions
The inventors hypothesize that ERK1/2 inhibits downstream targets, which drive fusion processes leading to myofiber growth. In cancer cell lines, ERK1/2 phosphorylates RXR (nuclear retinoid-X receptor), thereby inhibiting its transactivating potential (Macoritto et al, 2008; matsushima-Nishiwaki et al, 2001). RXR activity promotes myogenesis primarily by modulating Myod expression and acting as a MYOG cofactor (Alric et al, 1998; froeschle et al, 1998; khilji et al, 2020; leMay et al, 2011; zhu et al, 2009). Thus, RXR is assumed to be a nuclear ERK1/2 target in myoblasts. In myoblasts RXR immunoprecipitated with ERK1/2 and this interaction was attenuated when treated with ERKi (fig. 2A). Furthermore, co-treatment of myoblasts with ERKi and specific RXR antagonist HX531 (20 um, rxri) inhibited fusion at 24 hours by 47% (fig. 2B and 2C) without affecting differentiation, as measured by the percentage of nuclei positive for MYOG staining (fig. 2B and 2D). These results indicate that RXR is activated and promotes myoblast fusion following ERK1/2 inhibition.
Next, studies showed that ERKi-treated myoblast cultures up-regulated Ryr1 and Ryr3 expression, as well as Ca 2+ Sensing channels such as SERCA1/2 (Atp a1 and Atp a 2), orai1/2 and STIM1/2 (FIG. 2E). Interestingly, co-treatment of myoblasts with ERKi and RXRi resulted in downregulation of Ryr and Ryr3 mRNA expression 24 hours after treatment (fig. 2F). The lanine receptor (RYR 1-3) is a channel that mediates release of ca2+ storage from SR to the cytoplasm during excitation-contraction coupling in heart and skeletal muscle cells. However, myoblast fusion precedes myoma due to the hierarchical structure of molecular eventsMeat contracts, so we want to know cytoplasmic Ca 2+ Whether elevated levels play a role in myoblast fusion as suggested previously (Shannberg et al, 1969). Co-treatment of the cultures with ERKi and RYR specific antagonist dantrolene (50. Mu.M, RYRi) reduced fusion by 60% (FIGS. 2B and 2G) without affecting differentiation, as determined by MYOG staining (FIGS. 2B and 2H). Likewise, myoblasts co-treated with ERKi and the calcium chelator BAPTA-AM showed a 81% reduction in fusion (fig. 2B and 2I) without affecting early differentiation (fig. 2B and 2J). Taken together, these results demonstrate Ca 2+ Is essential for myoblast fusion, RXR-transactivation Ryr1 and Ryr3 expression following ERK1/2 inhibition, which may promote Ca 2+ Release from the SR results in myofiber fusion and growth.
Example 3
Calcium dependent CaMKII activation promotes myotube fusion of myoblasts and growth
Next, ca is determined 2+ Whether or not dependent phosphorylation and activation of cellular kinases are likely to be involved in the observed growth of myofibers through myoblast fusion. By monitoring Ca 2+ The level of the dependent enzyme p-CaMKII T287 was found to be activated when myoblasts were treated with ERKi (FIG. 3A), and its activation was dependent on RXR, ryR and Ca 2+ Upstream activity of (a) in the (B) cell (FIGS. 3B-D). Remarkably, co-treatment with the CaMKll inhibitor KN93 (5 uM; caMKII) inhibited CaMKII activation (FIG. 7A) and formation of multinucleated myotubes, while maintaining binuclear and trinuclear MyHC+ cells (FIGS. 3E and 3F). In contrast to ERKi alone (fig. 3H), co-treatment of ERKi with CaMKIIi did not affect cell cycle arrest, as measured by pH3 staining (fig. 7B) or expression of cell cycle inhibitors p21 and p 27. Similarly, co-treatment with CaMKIIi did not affect the initiation of myogenic procedures, as neither the percentage of MYOG positive nuclei (fig. 3E and 3G) nor the expression of differentiation markers was affected (fig. 3H). The lack of change in cell motility with the ERKi and CaMKIIi co-treatment compared to the ERKi treated cells demonstrated that the fusion failure was not due to effects on cell migration (not shown). These results indicate that CaMKII activation is necessary for myoblasts to myotube fusion, but not for myoblasts to myoblasts Necessary. Thus, in the presence of CaMKIIi, binuclear and trinuclear myotubes are formed, but these myotubes cannot be amplified into large polynuclear fibers.
After ERKi treatment, the expression of Mymk and Mymx is increased; however, when co-treated with ERKi and CaMKIIi, only Mymk expression was elevated, whereas Mymx expression was partially inhibited (fig. 3H). Thus, it was examined whether the decrease in fusion following CaMKII inhibition was attributable to a decrease in Mymk expression. To assess this, MYMK was overexpressed in primary myoblasts by retroviral transduction and treated with ERKi and camkili. As a result, ERKi-dependent fusion was found to be enhanced after MYMK overexpression, since the average number of nuclei per myotube was almost doubled. Interestingly, this effect was completely dependent on CaMKII activity, as after co-treatment with CaMKIIi, the large myotubes were lost and the accumulation of mononuclear and binuclear cells was similar to that of cells transduced with control retroviruses. These experiments showed that CaMKII acts downstream or in parallel with MYMK.
To examine whether CaMKII activation was sufficient to induce myoblast fusion with myotubes, empty adenovirus vector (Ad-control), caMK2 delta WT Or phosphate null Ad-CAMK2 delta T287V Primary myoblasts were transduced and induced to differentiate in DM for 72 hours. Discovery of exogenous camk2δ WT Activated by phosphorylation in DM, while CAMK2. Delta T287V Failing to undergo activation (fig. 3I). Furthermore, it was observed that, in comparison to the control, caMK2δ was found to be WT Expression of (c) enhances the formation of binuclear and polynuclear MyHC positive cells, but CAMK2δ T287V But did not inhibit the growth of multinucleated cells (fig. 3J). The results indicate that CaMKII activation is sufficient to promote secondary (myoblast to myotube) fusion.
Activation of CaMKII is an advanced event that occurred 16 hours after ERKi treatment, concurrent with an increase in MyHC and MEF2C levels (fig. 3K). Interestingly, ryR and activated CaMKII were localized primarily to myotubes rather than mononuclear myhc+ cells following ERK inhibition (fig. 3L and 3M). Taken together, these results indicate that Ca 2+ Dependent CaMKII activation is a downstream event of RXR and RYR activation, and CaMKII activity is mediated fusion of myoblasts to myotubes for myotubesAmplification of the complex is of paramount importance.
Example 4
Myotubes grow asymmetrically by recruiting mononuclear myoblasts at fusion synapses
As observed by the present inventors, the co-treatment of ERKi with CaMKII did not show complete fusion loss, and RYR and activated CaMKII proteins were located only in myotubes and not in differentiated MyHC+ myoblasts under ERKi treatment, thus assuming whether myoblast fusion occurred mainly between mononuclear myoblasts and myofibers, as previously described in Drosophila and Renkawitz-Pohl, 2009). To further investigate this concept, live cell imaging was performed with calculated hourly fusion index over a period of 8-23 hours after treatment with ERKi. After the initial formation of binuclear and trinuclear cells, these cells were found to expand rapidly by growing at the expense of monocytes (fig. 4A, not shown). In order to verify that the expansion of the fiber is a regulated phenomenon, a data-driven simulation was performed. The inventors considered that the greater probability of the multinucleated cells interacting with and fusing with their neighboring cells was higher, indicating that myotubes attracted neighboring myoblasts fused (fig. 4B). None of the simulations re-state the current results, which means that fiber growth is a regulated process.
This behavior is also evident in high resolution time-lapse microscopy of myoblasts expressing membrane-targeted GFP and cytoplasmic dsRed. The fibers spread rapidly through several fusion events that occur at about the same time (fig. 4C, not shown). The inventors also observed that myoblasts showed consistent collective movement and increased actin-rich membrane processes after ERKI treatment (not shown). Furthermore, live cell imaging showed that fusion occurred at protrusions extending from only one of the fusion partners (observed in 85% of fusion events; n=46; fig. 4D, not shown). To visualize Ca during ERKi-induced myogenesis 2+ Dynamic, from GCaMP6 Ca 2+ Imaged myoblasts were harvested in the report mice.These experiments revealed that Ca in the myotubes of the new born muscle 2+ The pulse precedes the myotube rapid growth phase, which supports the idea: ca released from SR in early myotubes 2+ Is responsible for CamKII activation, which mediates an asymmetric fusion reaction between myoblasts and myotubes (fig. 4E, not shown). Taken together, these results indicate that myoblast fusion in mammals is triggered by the production of multinucleated producer cells (2-3 nuclei) that expand by "attacking" myoblasts to "accepting" myotube fusion, and this phenomenon is mediated by CaMKII activity in the myotubes.
Example 5
CaMKII is essential for efficient muscle regeneration to examine the role of CaMKII in the in vivo muscle regeneration process, wild type mice were subjected to Cardiotoxin (CTX) induced injury and tissues were collected continuously on the day of injection and on days 2-8 after injury. 2 days after CTX injury, acute activation of ERK1/2 was evident, possibly associated with increased myoblast proliferation (FIG. 5A). On day 3 after injury, caMKII levels in regenerated muscle increased and remained higher for 8 days examined; 5 days after injury, caMKII activation peaked (fig. 5A). Based on these promising results, the inventors sought to examine the need for CaMKII during muscle regeneration. To achieve this, the inventors generated tamoxifen-inducible and satellite cell-specific conditional double knockout CaMKII delta and gamma isotype mice (fig. 5B and 5C).
CaMKII protein levels in resting satellite cells were found to be highly stable and were not effectively reduced 3 months after tamoxifen administration. To overcome this problem, the present inventors implemented a repetitive damage model. It is reasonable to think that the first round of regeneration is needed to drastically reduce the level of highly stable CaMKII protein in the satellite cell pool (in order to evaluate the function in vitro) and allow partial knockdown of regenerated muscle fibers, since the DNA content fused into muscle cells will now be integrated. Thus, at 4 weeks of age, satellite cell double knockout (scDKO) Pax7 CreERT/+ ；CaMK2δ fl/fl /γ fl/fl Or Pax7 +/+ ；CaMK2δ fl/fl /γ fl/fl (WT) tamoxifen was administered to induce Cre/Lox based gene disruption. When mice were 12 weeks old, the mice were allowed to fully regenerate for 8 weeks after administration of Cardiotoxin (CTX). 8 weeks after injury, mice were sacrificed to harvest primary myoblasts from the injured leg (to assess function in vitro), or a second CTX injury was performed, and the mice were sacrificed 14 days after injury for histological analysis. The reduction in CaMKII levels was indeed demonstrated in scDKO myoblasts harvested 8 weeks after the first injury (fig. 5D). The fusion index demonstrated that this scDKO myoblast exhibited significant defects in ERKi-induced fusion compared to myoblasts isolated from wild-type littermates (fig. 5E and 5F). Specifically, scDKO myoblasts exhibited the loss of the over-fused myotubes observed in WT cultures, instead of accumulated mononuclear myhc+ cells and neomyotubes (fig. 5E and 5F). These results were matched with and restated the observations made on myoblast cultures treated with CaMKIIi. Furthermore, the fiber cross-sectional area (851.4 μm2±37.5) was significantly smaller (fig. 5G and 5H) and had a tendency to tilt toward the more central nuclei (fig. 5I) in the scDKO mice that suffered repeated injury compared to WT mice (975 μm2±25). Taken together, the genetic deletion of camk2δ/γ is sufficient to impair myoblast fusion and muscle regeneration.
In general, and without being bound by theory, the inventors have described a signaling pathway leading to CaMKII activation that mediates myotube-driven asymmetric myoblast fusion. After inhibition of ERK1/2, myoblast proliferation was prevented and the differentiation procedure was initiated. ERK1/2 inhibition results in RXR activation and induction of RYR expression in the newly myogenic duct, ca leading to CaMKII in the myogenic duct 2+ Dependent activation and ultimately, fusion of CaMKII dependent asymmetric myoblasts with myotubes (fig. 6A-6C).
Example 6
ERK inhibition promotes maturation of mouse myoblast myofibers
Myoblasts from the first generation mice were seeded in the same number in proliferation medium in 12-well tissue culture plates pre-coated with 10% matrigel solution. After 24 hours, the medium was allowed to fully attach, washed and replaced with fresh Proliferation Medium (PM), PM supplemented with 1 μm SCH 772984 (ERKi) or Differentiation Medium (DM). After 24 hours, the cells were lysed and RNA was collected. Gene expression analysis was performed using SYBR Green qRT-PCR analysis.
Results
As shown in fig. 8, the ERKi-induced mouse myotubes had stronger expression of the maturation markers myosin heavy chain and troponin, a component of the sarcomere structure necessary for muscle contraction, compared to DM, suggesting that the ERKi-induced fibers may reach maturation earlier and may have the ability to contract before those obtained using DM.
Example 7
ERK inhibition promotes early induction of differentiation, fusion and myotube formation in chicken myoblasts
Using chicken primary-derived myoblasts as a model, the efficiency of SCH772984 as ERKi was evaluated for muscle (meat) production under tissue culture conditions as compared to conventional Differentiation Medium (DM). Chicken myoblasts were isolated from chicken embryos and expanded in proliferation medium. When a sufficient number of cells were obtained, the cells were seeded onto tissue culture plates and allowed to adhere for 24 hours. The medium was then replaced with ERKi-supplemented PM or DM. 24 hours after treatment, cells receiving ERKi were supplemented with fresh PM (no more ERKi was added) and DM-treated cells were supplemented with fresh DM. The medium was changed daily over 72 hours.
Muscle fiber formation was assessed by fixing cells and staining the expression of myosin heavy chain (fig. 9A). The time course demonstrated that fiber formation was significantly enhanced after ERKi treatment; early myotubes consisting of 2-3 nuclei were evident 24 hours after treatment, which continued to grow for the remaining time of 72 hours. However, conventional DM treatments only begin forming fibers 72 hours after treatment. Fusion index was quantified 72 hours after treatment (fig. 9B), where there was a percentage of total nuclei present in myotubes (MyHC positive cells with 2 or more nuclei). However, although the fusion index of conventional DM was 15%, the ERKi-induced fusion index was 62%.
Taken together, the results show that ERKi is more effective in inducing myogenesis in chicken myoblasts than DM treatment. This is supported by the fact that: muscle fibers began to form 48 hours earlier than DM and at 72 hours post-treatment, when ERKi reached its maximum effect, the fusion index increased 4-fold over DM.
By assessing gene expression of various transcription factors indicative of the myoblast differentiation process, both ERKi and DM treatments were observed to induce a differentiated transcription program, as evidenced by a decrease in Pax7 RNA expression. Although the effect of ERKi on upregulation of MyoD expression may occur before the 24 hour time point of collection, its final downregulation occurs before DM, indicating its early regulation. The effect of ERKi treatment on Myf5 expression down-regulation is more pronounced. Although both DM and ERKi treatment resulted in Myf5 down-regulation, the effect of ERKi was stronger at all time points. Similar to MyoD, the greatest effect of ERKi on Myog expression may occur before 24 hours, since both DM and ERKi increased Myog expression at 24 hours, although in DM, expression was continuously increased over 72 hours, while the ERKi level of Myog decreased at 48 hours post-treatment, which corresponds to massive fiber formation (fig. 9C).
Interestingly, differentiation, fusion and myotube formation were induced earlier and myofibers were also matured earlier after treatment of myoblasts with ERKi compared to DM, as can be seen from the elevated gene expression of various maturation/differentiation markers including transcription factor mrf, and myosin heavy chains 1 and 2 (myh and myh 2) and troponin 3 (tnnt 3) (fig. 10A). At 48 hours post treatment, sarcomere structure has become apparent by ERKi-induced immunofluorescent staining of myotubes, as demonstrated by classical ladder-like stripes of actin and troponin signals and phalloidin staining representing actin filaments (fig. 10B).
Example 8
ERK inhibition increases the yield of the produced fibers
For surface area coverage-equal amounts of chicken primary myoblasts were seeded in 96-well plates and the next day treated with PM or Differentiation Medium (DM) supplemented with 1 μm SCH 772984 (ERKi). Media was supplemented daily with fresh PM or DM (ERKi was no longer added). Plates were fixed 72 hours after treatment and immunostained for myosin heavy chain. Images were captured using ImageJ software and the area coverage of the red signal was analyzed compared to the total area of each field.
For cell pellet weight-equal amounts of chicken primary myoblasts were inoculated into 10cm dishes, the next day treated with PM or differentiation medium supplemented with 1uM SCH 772984 (ERKi). Medium was supplemented daily with fresh PM or DM (ERKI was no longer added). 72 hours after treatment, the treatment medium was aspirated and 1ml of PBS was placed in each dish and the cells were scraped with a rubber spatula. Before collection, each empty individual collection tube was weighed on an analytical scale. The cell suspension was then collected and centrifuged in a cooled centrifuge. The supernatant was gently aspirated with a hand-held pipette and the wet pellet and collection tube were re-weighed. The weight of the wet precipitate was determined by subtracting the original weight of the empty relevant tube. Each treatment was repeated 6 times.
For protein production-equal amounts of chicken primary myoblasts were inoculated in 12-well plates, the next day treated with PM or Differentiation Medium (DM) supplemented with 1. Mu.M SCH 772984 (ERKi). Media was supplemented daily with fresh PM or DM (ERKi was no longer added). At 72 hours post-treatment, the treatment medium was aspirated and 200 μl RIPA buffer supplemented with protease inhibitor cocktail was added to each well, the cells scraped off and lysates incubated at 4 ℃ for 30 minutes. The lysate was then centrifuged to remove insoluble material and the supernatant was evaluated for total protein by BCA method.
Results
In order to evaluate the parameters of the total yield, i.e. the total amount of all products after the process, several methods have been used so far by the industry: first, assessing the volume or surface area coverage of the muscle fibers produced; second, the quality (weight) of the product produced is evaluated; third, the total protein contained in the final product was assessed by Bradford or BCA assay.
By assessing the total surface area coverage of the muscle fibers obtained from either ERKi or DM treatment, it was observed that the ERKi-derived fibers covered 45% of the surface area at 48 hours post-treatment, while DM fibers covered only 7% and increased 6-fold (fig. 11A). When comparing the total cell pellet weight obtained 72 hours after treatment, the cell pellet weight of ERKi was increased by 40% compared to DM (fig. 11B).
Prior to the experiment, the cell population underwent several rounds of pre-plating to eliminate as many fibroblasts as possible; however, during the 72 hour period, the minority fibroblasts remaining in the starting culture did proliferate and contributed to the total mass and protein yield at the end point. However, it was estimated that the initial cell number required to produce 1kg of product 72 hours after treatment with ERKi was approximately 9700 ten thousand cells, whereas DM required approximately 1300 ten thousand cells (fig. 11C). Finally, the total protein mass of ERKi treated cells was increased by nearly 4-fold compared to DM treated cells (fig. 11D).
Example 9
ERK inhibition improves fibrogenesis in bovine and ovine myoblasts
Equal amounts of bovine myoblasts were seeded in 96-well plates and the next day were treated with PM, PM supplemented with 0.5. Mu.M SCH 772984 (ERKi), or Differentiation Medium (DM). Media was supplemented daily with fresh PM or DM (ERKi was no longer added). At 72 hours post-treatment, cells were fixed and stained for myosin heavy chain, and several fields of view were imaged under each condition. The nuclei of each fiber are quantified at each field of view to produce a fusion index. For sheep myoblasts, an equal amount of cells was seeded in 8-well chamber slides for 24 hours. The following day, cells were treated with 1. Mu.M SCH 772984 (ERKi) or Differentiation Medium (DM) supplemented with 100nM Sir-Actin reagent, then incubated for 4 hours, and then slides were transferred to heated CO 2 In a room microscope device. Several fields of view are selected for real-time imaging by capturing images of each field every 7 minutes. A series of images processed each time over a period of 31 hours were stacked into a movie file using ImageJ.
Results
Furthermore, the inventors sought to demonstrate the applicability of ERKi to myoblasts from other species. Bovine myoblasts were harvested and grown. The effect of ERKi was found to be conserved across different species. After a single administration of 0.5 μm ERKi treatment, bovine myoblasts showed a 6-fold increase in fusion index compared to DM at 72 hours post treatment.
Mouse and chicken data indicate that ERKi-induced myotubes have increased maturation markers at 96 hours post-treatment compared to DM. Despite the fact that myotubes were present in DM 96 hours ago, the expression of sarcomeric proteins MyHC, actin and troponin was significantly lower than that induced by ERKi treatment, as evident by evaluation of the relative signal intensities of immunofluorescent staining (fig. 13).
Finally, the inventors demonstrated that ERKi was equally effective on sheep-derived myoblasts, as 1uM treatment induced significantly more fusion and myotube formation than treatment with DM (not shown).
Materials and methods of examples 10-15
Isolation and treatment of primary chicken myoblasts: chicken myoblasts were isolated from chest and leg muscles of day 18 broiler embryos using trypsin B. After tissue dissociation, the cell suspension was grown on 10% matrigel coated plates for 3-4 days. At the first passage, cells were lifted and pre-plated twice for 30 minutes each to enrich for myoblasts and reduce the number of fibroblasts. Cells were then seeded at 8,000 per well in proliferation medium in optical 96-well plates. 24 hours after plating, the medium was aspirated and replaced with the treatment conditions specified in the proliferation medium or differentiation medium (as shown). After 24 hours of treatment, the medium was aspirated and all wells were replenished with fresh proliferation medium or differentiation medium without any treatment. This operation was repeated daily. Cells were fixed 48 hours after treatment. Compounds were purchased from Cayman Chemicals.
Isolation and treatment of primary bovine myoblasts: bovine myoblasts were isolated from freshly slaughtered bovine muscle using collagenase type II. After tissue dissociation, the cell suspension was grown on 10% matrigel coated plates for 3-4 days. At the first passage, cells were lifted and pre-plated twice for 30 minutes each to enrich for myoblasts and reduce the number of fibroblasts. Cells were then seeded at 8,000 per well in proliferation medium in optical 96-well plates. 24 hours after plating, the medium was aspirated and replaced with the treatment conditions specified in the proliferation medium. After 24 hours of treatment, the medium was aspirated and all wells were replenished with fresh proliferation medium or differentiation medium without any treatment. This operation was repeated daily. Cells were fixed 72 hours after treatment. All ERK inhibitors were purchased from Cayman Chemicals.
Microscopy: fixed samples were imaged using a Nikon Eclipse Ti2 microscope and NIS-Elements imaging software version 5.11.00, and MyHC, a-sarcomere actin and DAPI staining were collected using a 10-fold objective. If necessary, the brightness and contrast are linearly adjusted using Photoshop.
Immunofluorescent staining: cells were fixed with ice-cold 4% PFA in PBS for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 6 min, and blocked with PBS containing 0.025% Tween, 10% normal horse serum, and 10% normal goat serum for 1 hr at room temperature. The primary antibody was incubated in blocking buffer at 4 ℃ for one night using the following antibodies: myosin heavy chain (MyHC, MF20, DSHB hybridoma supernatant 1:10, alpha-actin (SIGMA A A7811)). Cells were washed 3 times in PBS containing 0.025% tween and then incubated with the appropriate secondary antibody in PBS for 1 hour. Nuclei were labeled with DAPI (SIGMA D9542, 5. Mu.g/ml). All fusion indices and imaging analysis were repeated at least 1000 times per technique.
Example 10
Various ERK inhibitors induce differentiation and fusion in primary bovine myoblasts
Several ERK inhibitors other than SCH772984 (AZD 0364, BVD523, DEL22379, FR180204, GDC0994, KO947 and LY 3214996) were compared for their ability to induce differentiation and fusion in primary isolated bovine myoblasts and compared to treatment with 1 μm SCH 772984. Quantification of the fusion index (fig. 14B) of the fixed and stained α -sarcomere actin and DAPI cells 72 hours after treatment (fig. 14A) indicated that all the ERK inhibitors tested had similar ability to induce differentiation and fusion in primary myoblasts when added to proliferation medium at the same concentration as SCH772984 (1 uM).
Example 11
Calcium ionophore enhances ERK-inhibitor induced primary chicken myoblast differentiation and fusion
The calcium ionophore may be used to increase the calcium ionophore required for CaMKII activation. Thus, the effect of calcium ionophores on ERKi-induced differentiation and fusion phenotypes was studied. The addition of three different calcium ionophores (ionomycin, calicheamicin, calcium ionophore I (AKA CA1001 or ETH 1002)) to the ERKi administration significantly increased the fusion index of primary chicken myoblasts (in proliferation medium) from 62% in the ERKi treatment alone to 89%, 94% and 89% of ionophores, respectively (fig. 15A and 15B).
Example 12
RXR/RAR agonists enhance ERK-inhibitor induced differentiation and fusion of primary chicken myoblasts
retinoid-X receptor (RXR) activation is associated with CaMKII signaling pathways. The effect of RXR and related Retinoic Acid Receptor (RAR) agonists on ERKi-induced differentiation and fusion phenotypes in myoblasts was investigated. Primary chicken myoblasts treated with ERK inhibitors (SCH 772984 um, SCH) alone or in combination with various RXR/RAR agonists (9-cis retinoic acid, 9-cis RA-200nM, AM80-200 nM, AM580-100 nM and CH55-200nM, TTNPB 200nM, fenretinide 200 nM) in proliferation medium. The combination of RXR/RAR agonists with ERKi inhibitors significantly increased the fusion index of primary myoblasts (fig. 16A and 16B).
Example 13
RYR agonists enhance ERK-inhibitor induced differentiation and fusion of primary chicken myoblasts
Raney (RYR) activation is associated with the CaMKII signaling pathway. The effect of RYR agonists on ERKi-induced myoblast differentiation and fusion phenotype was investigated. Primary chicken myoblasts treated with ERK inhibitors (SCH 772984 um, SCH) alone or in combination with various RYR agonists (caffeine-2 mM and suramin-10 um) in proliferation medium. The combination of RYR agonists with ERKI inhibitors significantly increased the fusion index of primary myoblasts (fig. 17A and 17B).
Example 14
Effect of ERK and MEK inhibition on primary chicken myoblast differentiation and fusion
Mitogen activated protein kinase (MEK) and ERK are both important components of the MAPK pathway. Comparing the effect of SCH772984 and MEK inhibitor (U0126, MEKi) at 1 and 10 μm on primary chicken myoblasts in proliferation or differentiation medium, ERK inhibition (SCH 772984) was clearly observed to be superior to MEK inhibition (U0126) on primary chicken myoblast differentiation and fusion: in proliferation medium. 48 hours after treatment, a 1. Mu.M dose of SCH772984 induced 59% fusion, whereas a similar dose of MEKi induced only 16% fusion, a 10. Mu.M dose of SCH772984 induced 69% fusion, and 10. Mu.M MEKi induced less fusion (7%) than 1. Mu.M MEKi (16%). In differentiation medium, 1. Mu.M SCH772984 induced 46% fusion, while 1. Mu.M MEKi induced only 29% fusion. At 10 μm, SCH772984 induced 61% fusion, whereas MEKi induced only 47% fusion (see fig. 18A and 18B).
Example 15
Combination of ERK inhibitor, ranibase receptor agonist, RXR/RAR agonist and calcium ionophore: effects on fusion phenotype induction in myoblasts compared to ERK inhibitor treatment alone
Interactions of various agents (i.e., RXR/RAR agonists, RYR agonists, and calcium ionophores) capable of modulating the effect of ERK inhibition on myoblast fusion were studied. The different molecules identified from each class were combined and tested for their ability to further enhance myoblast fusion or shorten the time required to reach comparable fusion levels.
The method comprises the following steps:
treatment of myoblasts with a combination of molecules: bovine and chicken myoblasts were isolated and cultured as described above. Cells were seeded at 8,000 per well in proliferation medium of an optical 96-well plate. 24 hours after plating, the medium was aspirated and replaced with the treatment conditions specified in the proliferation medium or differentiation medium. Using RXR/RAR, RYR agonists and calcium ionophores, it was identified that fusion could be enhanced when co-treated with ERK inhibitors, and various combinations of 3-4 different compounds tested at different doses. After 24 hours of treatment, the medium was aspirated and all wells were replenished with fresh proliferation medium without any treatment. The protocol was repeated daily. Cells were fixed for evaluation 72 hours after treatment.
Microscopy: fixed samples were imaged using a Nikon Eclipse Ti2 microscope and NIS-Elements imaging software version 5.11.00, and MyHC, a-sarcomere actin and DAPI staining were collected using a 10-fold objective. If necessary, the brightness and contrast are linearly adjusted using Photoshop.
Immunofluorescent staining: cells were fixed with ice-cold 4% PFA in PBS for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 6 min, and blocked with PBS containing 0.025% Tween, 10% normal horse serum, and 10% normal goat serum for 1 hr at room temperature. The primary antibody was incubated in blocking buffer at 4 ℃ for one night using the following antibodies: myosin heavy chain (MyHC, MF20, dev Stud Hyridoma Bank hybridoma supernatant 1:10). Cells were washed 3 times in PBS containing 0.025% tween and then incubated with the appropriate secondary antibody in PBS for 1 hour. Nuclei were labeled with DAPI (SIGMA D9542, 5. Mu.g/ml). All fusion indices and imaging analysis were repeated at least 1000 times per technique.
Results: the combination of agents selected to provide a significant increase in fusion index at a particular time after exposure, or to significantly reduce incubation time to achieve a specified level of myoblast fusion. Synergistic combinations are of particular interest.
Example 16
Effect of sequential treatment of bovine myoblasts on fusion phenotype with ERKi, RXR/RAR agonist, RYR agonist and calcium ionophore
The significance of the time sequence and timing of the addition of agents that enhance the induction of myoblast differentiation and fusion by ERK-inhibitors was investigated. Since the natural timeline for bovine myoblasts to differentiate and fuse is longer than chicken or mouse myoblasts, sequential administration of RXR/RAR agonists, RYR agonists and calcium ionophores with ERKi was tested. ERK inhibitor is administered at t0 followed by administration of RXR/RAR agonist, RYR agonist and calcium ionophore alone or in combination to myoblasts 24, 48 or 72 hours after initial ERK inhibitor treatment.
The method comprises the following steps:
isolation and treatment of primary bovine myoblasts: bovine myoblasts were isolated from freshly slaughtered bovine muscle using collagenase type II. After tissue dissociation, the cell suspension was grown on 10% matrigel coated plates for 3-4 days. At the first passage, cells were lifted and pre-plated twice for 30 minutes each to enrich for myoblasts and reduce the number of fibroblasts. Cells were then seeded at 8,000 per well in proliferation medium in optical 96-well plates. 24 hours after plating, the medium was aspirated and replaced with ERKi treatment in proliferation medium or differentiation medium. Using RXR/RAR, RYR agonists and calcium ionophores, it was identified that fusion could be enhanced when co-treated with ERK inhibitors, and various combinations of 3 to 4 different compounds were tested at different doses 24, 48 and 72 hours after initial treatment with ERK inhibitors. All wells were replenished daily with fresh proliferation or differentiation medium and given treatments were performed in the given cases. The protocol was repeated daily and cells were fixed 72 hours after treatment.
Microscopy: fixed samples were imaged using a Nikon Eclipse Ti2 microscope and NIS-Elements imaging software version 5.11.00, and MyHC, a-sarcomere actin and DAPI staining were collected using a 10-fold objective. If necessary, the brightness and contrast are linearly adjusted using Photoshop.
Immunofluorescent staining: cells were fixed with ice-cold 4% PFA in PBS for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 6 min, and blocked with PBS containing 0.025% Tween, 10% normal horse serum, and 10% normal goat serum for 1 hr at room temperature. The primary antibody was incubated in blocking buffer at 4 ℃ for one night using the following antibodies: myosin heavy chain (MyHC, MF20, DSHB hybridoma supernatant 1:10, alpha-actin (SIGMA A A7811)). Cells were washed 3 times in PBS containing 0.025% tween and then incubated with the appropriate secondary antibody in PBS for 1 hour. Nuclei were labeled with DAPI (SIGMA D9542, 5. Mu.g/ml). All imaging analyses were repeated at least 1000 times per technique.
Results: the sequence of the combination agents is selected to provide a significant increase in fusion index at a particular time after exposure, or the incubation time can be significantly reduced to achieve a combination of agents for a specified level of myoblast fusion. For different schemes, indications of differences in the effective temporal order of the combination are of particular interest.
Example 17
Effect of inhibition of SERCA channels or other calcium modulators on myoblast fusion: combinations with ERK inhibitors and/or with RXR/RAR agonists, RYR agonists or calcium ionophores
Previous results indicate that ERKi treatment in myoblasts, in addition to activating RYR and inducing calcium release and CaMKII activation, increases flow through SERCA channels and activates other calcium modulators. Without wishing to be limited to a particular hypothesis, it is believed that their upregulation may be a compensatory mechanism within the cell to balance the amount of available calcium and return the calcium to the ER. Further inhibition of the affected channels may promote long-term accumulation of intracellular calcium, activation of CaMKII is stronger, even to a greater extent enhancing fusion than ERKi treatment alone or in combination with RXR/RAR agonists and/or RYR agonists and/or calcium ionophores.
The method comprises the following steps:
isolation and treatment of primary chicken myoblasts: chicken myoblasts were isolated from chest and leg muscles of day 18 broiler embryos using trypsin B. After tissue dissociation, the cell suspension was grown on 10% matrigel coated plates for 3-4 days. At the first passage, cells were lifted and pre-plated twice for 30 minutes each to enrich for myoblasts and reduce the number of fibroblasts. Cells were then seeded at 8,000 per well in proliferation medium in optical 96-well plates. 24 hours after plating, the medium is aspirated and replaced with proliferation medium or differentiation medium (containing SCH772984 alone, or SCH772984 in combination with SERCA inhibitor and/or other calcium reuptake modulator, or the latter in combination with RXR/RAR agonist, and/or RYR agonist and/or calcium ionophore). After 24 hours of treatment, the medium was aspirated and all wells were replenished with fresh proliferation medium or differentiation medium without any treatment. This operation was repeated daily. Cells were fixed 24, 48, 72 and 96 hours after treatment.
Isolation and treatment of primary bovine myoblasts: bovine myoblasts were isolated from freshly slaughtered bovine muscle using collagenase type II. After tissue dissociation, the cell suspension was grown on 10% matrigel coated plates for 3-4 days. At the first passage, cells were lifted and pre-plated twice for 30 minutes each to enrich for myoblasts and reduce the number of fibroblasts. Cells were then seeded at 8,000 per well in proliferation medium in optical 96-well plates. 24 hours after plating, the medium was aspirated and replaced with the treatment conditions specified in the proliferation medium or differentiation medium. Using RXR/RAR, RYR agonists and calcium ionophores, it was identified that fusion could be enhanced when co-treated with ERK inhibitors, and various combinations of 3 to 4 different compounds were tested at different doses 24, 48 and 72 hours after initial treatment with ERK inhibitors. All wells were replenished daily with fresh proliferation or differentiation medium and given treatments were performed in the given cases. The protocol was repeated daily, fixing cells 24, 48 and 72 and 96 hours after treatment.
Microscopy: fixed samples were imaged using a Nikon Eclipse Ti2 microscope and NIS-Elements imaging software version 5.11.00, and MyHC, a-sarcomere actin and DAPI staining were collected using a 10-fold objective. If necessary, the brightness and contrast are linearly adjusted using Photoshop.
Immunofluorescent staining: cells were fixed with ice-cold 4% PFA in PBS for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 6 min, and blocked with PBS containing 0.025% Tween, 10% normal horse serum, and 10% normal goat serum for 1 hr at room temperature. The primary antibody was incubated in blocking buffer at 4 ℃ for one night using the following antibodies: myosin heavy chain (MyHC, MF20, DSHB hybridoma supernatant 1:10, alpha-actin (SIGMA A A7811)). Cells were washed 3 times in PBS containing 0.025% tween and then incubated with the appropriate secondary antibody in PBS for 1 hour. Nuclei were labeled with DAPI (SIGMA D9542, 5. Mu.g/ml). All imaging analyses were repeated at least 1000 times per technique.
Example 18
Effect of ERK inhibition on teleost (fish) derived myoblast differentiation and fusion alone or in combination with RXR/RAR or RYR agonists, calcium ionophores and SERCA/calcium reuptake channel inhibitors
The ability of ERK inhibitors alone and in combination with other molecules affecting the ERK-CaMKII signaling pathway to induce teleosts (fish) myoblast fusion was studied and compared/contrasted with their effects on mouse, chicken and bovine myoblast development.
The method comprises the following steps:
Isolation and treatment of primary trout and zebrafish myoblasts: myoblasts of trout and zebrafish were isolated from freshly killed mature fish using trypsin B or collagenase digestion. After tissue dissociation, the cell suspension was grown on 10% matrigel coated plates for 3-4 days. At the first passage, cells were lifted and pre-plated twice for 30 minutes each to enrich for myoblasts and reduce the number of fibroblasts. Cells were then seeded at 8,000 per well in proliferation medium in optical 96-well plates. 24 hours after plating, the medium was aspirated and replaced with proliferation medium or differentiation medium containing SCH77s984 alone, or SCH772984 was co-treated with various SERCA inhibitors and/or calcium reuptake modulators, RXR/RAR agonists and/or RYR agonists, and/or various combinations/doses of calcium ionophores (as shown). After 24 hours of treatment, the medium was aspirated and all wells were replenished with fresh proliferation medium or differentiation medium without any treatment. The protocol was repeated daily and cells were fixed 24, 48, 72 and 96 hours after treatment.
Microscopy: fixed samples were imaged using a Nikon Eclipse Ti2 microscope and NIS-Elements imaging software version 5.11.00, and MyHC, a-sarcomere actin and DAPI staining were collected using a 10-fold objective. If necessary, the brightness and contrast are linearly adjusted using Photoshop.
Immunofluorescent staining: cells were fixed with ice-cold 4% PFA in PBS for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 6 min, and blocked with PBS containing 0.025% Tween, 10% normal horse serum, and 10% normal goat serum for 1 hr at room temperature. The primary antibody was incubated in blocking buffer at 4 ℃ for one night using the following antibodies: myosin heavy chain (MyHC, MF20, DSHB hybridoma supernatant 1:10, alpha-actin (SIGMA A A7811)). Cells were washed 3 times in PBS containing 0.025% tween and then incubated with the appropriate secondary antibody in PBS for 1 hour. Nuclei were labeled with DAPI (SIGMA D9542, 5. Mu.g/ml). All imaging analyses were repeated at least 1000 times per technique.
While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
It is intended that all publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. Furthermore, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. As for the chapter titles used, they should not be construed as necessarily limiting. In addition, the entire contents of any one or more priority files of the present invention are incorporated herein by reference in their entirety.

Claims (45)

1. A method of inducing myotube formation in a polynuclear, the method comprising contacting a myogenic precursor cell from a cultured animal with an extracellular regulated signal kinase (ERK 1/2) inhibitor and/or an up-regulator of intracellular ca2+.
2. A method of inducing myotube formation in a polynuclear, the method comprising contacting myogenic precursor cells from a farmed animal with at least one molecule selected from the group consisting of: an extracellular regulated signal kinase (ERK 1/2) inhibitor, a mitogen activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranialkali receptor (RYR 1, RYR 3) agonist, a ranialkali receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, a calcium ionophore, and a calmodulin-dependent protein kinase II (CaMKII) activator.
3. The method of claim 1 or 2, wherein the ERK1/2 inhibitor is selected from the group consisting of: MK-8353 (SCH 900353), SCH772984, CC-90003, rhynchophylline, ERK1/2 inhibitor 1, magnolipids, ERKIN-1, ERKIN-2, ERKIN-3, LY3214996, ravoxertinib, ravoxertinib, VX-11e, FR180204, ulitinib hydrochloride, ADZ0364, KO947, FRI-20 (ON-01060), bromoacetoxycalcitol (B3 CD), BVD523, DEL22379, FR180204, GDC0994, KO947, AEZ-131 (AEZS-131), AEZS-136, AZ-13767370, BL-EI-001, LTT, ASTX-029, TCS ERK 11e and CAY10561.
4. The method of claim 1 or 2, wherein the MEK1 inhibitor is selected from the group consisting of: trametinib, PD98059, U0126 (U0126-EtOH), PD0325901, semetinib (AZD 6244), cobratinib (GDC-0973, RG 7420), bimetanib (MEK 162), CI-1040 (PD 184352), remiratinib (BAY 869766; RDEA119), pimozide (AS 703026), semetinib (AZD 6244), cobratinib hemi-fumarate, GDC-0623 (RG 7421), RO 498755, AZD8330 (ARRY-424704), SL327, MEK inhibitors, PD318088, cobratinib racemate (GDC-0973 racemate; XL518 racemate) and EBI-1051.
5. The method of claim 1 or 2, wherein the FGF inhibitor is selected from the group consisting of delazantinib, PD 161570, SSR 128129E, CH5183284, PD 166866, and pemitinib.
6. The method of claim 1 or 2, wherein the TGF- β1 is selected from the group consisting of SD208, LY364947, repox, SB 525334, R268712 and GW 788388.
7. The method according to claim 1 or 2, wherein the RXR/RAR agonist is selected from the group consisting of: CD3254, docosahexaenoic acid, LG100268, SR11237, AC261066, AC55649, adapalene, BMS961, CD1530, CD2314, CD437, BMS453, EC23, all-trans retinoic acid, all-trans-4-hydroxy retinoic acid, all-trans retinoic acid-D5, cyantraniliprole, vitamin A, all-trans retinol, LG100754, beta-carotene, beta-apo-13-carotene ketone, lycopene, all-trans-5, 6-epoxyretinoic acid, all-trans-13, 14-dihydroretinol retinyl acetate, honokiol, valeric acid, HX630, HX600, LG101506, 9cUAB30, AGN194204, LG101305, UVI3003, net-4IB, CBt-PMN, XCT0135908, PA024, methoprene acid, 9-cis retinoic acid, AM80, AM580 and CH55, TTNPB and fenretinide, LG-100064, flubensalrotene (compound 20), bexarotene (LGD 1069), bexarotene D4, NBD-125 (B-12), LGD1069D4 and 9-cis retinoic acid (ALRT 1057).
8. The method of claim 1 or 2, wherein the RYR1, RYR3 agonist is selected from the group consisting of: caffeine, chlorocresol, CHEBI 67113, chlorantraniliprole, S107 hydrochloride, JTV519, trifluoperazine (TFP), xanthine, suramin, sodium suramin, NAADP tetrasodium salt, S100A1, cyclic ADP-ribose (ammonium salt), hexidine, 4-chloro-3-methylphenol (4-chloro-m-cresol), tetrazolium carboxamide, trifluoperazine (TFP), cyclic bromaromide and cyantraniliprole.
9. The method of claim 1 or 2, wherein the intracellular ca2+ up-regulating factor is selected from the group consisting of NAADP tetrasodium salt, cyclic ADP-ribose, 4-bromo a23187, ionomycin, a23187, and isoprenaline.
10. The method of claim 1 or 2, wherein the CaMKII agonist is selected from the group consisting of calcium, calmodulin, CALP1 and CALP 3.
11. The method of any one of claims 1 to 10, wherein the myogenic precursor cells are selected from the group consisting of: myoblasts, satellite cells, myoside group (mSP) cells, myogenic stem cells (MDSCs), mesenchymal Stem Cells (MSCs), myogenic pericytes, embryonic Stem Cells (ESCs), induced muscle progenitor cells (iccs), and induced pluripotent stem cells (ipscs).
12. The method of any one of claims 1 to 10, wherein the myogenic precursor cells express MyoD, pax3 and Pax7, or their corresponding interspecies homologous genes.
13. The method of any one of claims 1 to 10, wherein the myogenic precursor cells are myoblasts.
14. The method of any one of claims 1 to 13, wherein the myogenic precursor cells are from a biopsy of the farmed animal.
15. The method of claim 14, wherein the biopsy is a muscle biopsy.
16. The method of claim 14 or 15, wherein the myogenic precursor cells are isolated from the biopsy by enzymatic and/or mechanical dissociation.
17. The method of any one of claims 1 to 12, wherein the myogenic progenitor cells are undifferentiated myogenic precursor cells cultured in proliferation medium prior to inducing formation of the multinucleated myotubes.
18. The method of claim 17, wherein the proliferation medium is free of molecules selected from the group consisting of: an extracellular regulated signal kinase (ERK 1/2) inhibitor, a mitogen activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranialkali receptor (RYR 1, RYR 3) agonist, a ranialkali receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, a calcium ionophore, and a calmodulin-dependent protein kinase II (CaMKII) activator.
19. The method of any one of claims 1 to 12, wherein the myogenic progenitor cells are myogenic precursor cells that are cultured in a differentiation medium prior to inducing formation of the multinucleated myotubes.
20. The method of any one of claims 1 to 17, performed in a single vessel.
21. The method according to any one of claims 17 to 20, performed by supplementing the culture medium with any of the molecules.
22. The method according to any one of claims 1 to 21, carried out in the presence of serum or serum substitutes in an amount that allows cell proliferation and/or under normoxic conditions.
23. The method of any one of claims 1 to 22, wherein the farmed animal is selected from the group consisting of mammals, birds, fish, invertebrates, reptiles and amphibians.
24. The method of any one of claims 1 to 23, wherein the multi-core myotube comprises at least three cores.
25. The method of any one of claims 1 to 24, wherein the multinuclear myotube expresses myogenic differentiation and fusion factors selected from the group consisting of MyoD, myoG, mymk and Mymx.
26. The method of any one of claims 1 to 25, wherein the inducing multinuclear myotubes results in a partial increase in MYOG positive nuclei compared to nuclei of myogenic progenitor cells cultured in differentiation medium without the at least one molecule.
27. The method of any one of claims 1 to 26, wherein the induction of multinuclear myotube formation results in classical ladder-like streaks of actin and troponin signals and/or phalloidin staining representing actin filaments.
28. The method of any one of claims 1 to 27, wherein the multinuclear myotube formation comprises mononuclear myocell-myotube fusion and/or binuclear and trinuclear myotube expansion into large multinuclear fibers.
29. The method of any one of claims 1 to 28, wherein the contacting the myogenic precursor cells is performed for 12-48 hours.
30. The method of claim 29, wherein said contacting said myogenic precursor cells is performed for 16-24 hours.
31. A cultured meat composition comprising the multinuclear myotubes produced by the method of any one of claims 1-30.
32. A food comprising the cultured meat composition of claim 31.
33. The food of claim 32 processed to impart a organoleptic sensation and texture to the meat.
34. The food of claim 33, further comprising a food of vegetable and/or animal origin.
35. The food according to any one of claims 32 to 34, further comprising adipocytes, muscle cells, blood cells, chondrocytes, bone cells, connective tissue cells, fibroblasts and/or cardiomyocytes.
36. The food of any one of claims 32 to 35, further comprising a plant-based protein.
37. A method of producing a food product, the method comprising combining the cultured meat composition of claim 31 or the food of any one of claims 32-36 with an edible composition for human or animal consumption.
38. A method of treating muscle damage in a farm animal, the method comprising contacting damaged muscle tissue with at least one molecule selected from the group consisting of: an extracellular regulated signal kinase (ERK 1/2) inhibitor, a mitogen activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranialkali receptor (RYR 1, RYR 3) agonist, a ranialkali receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, and a calmodulin-dependent protein kinase II (CaMKII) activator, thereby inducing myotube regeneration and treating the muscle injury.
39. At least one molecule for use in inducing myotube regeneration and treating muscle injury in a farmed animal, the molecule selected from the group consisting of: an extracellular regulatory signal kinase (ERK 1/2) inhibitor, a mitogen-activated protein kinase 1 (MEK 1) inhibitor, a Fibroblast Growth Factor (FGF) inhibitor, a transforming growth factor-beta (TGF-beta) inhibitor, a retinoid-X receptor (RXR) agonist, a retinoid-X receptor (RXR) activator, a Retinoic Acid Receptor (RAR) agonist, a Retinoic Acid Receptor (RAR) activator, a ranitidine receptor (RYR 1, RYR 3) agonist, a ranitidine receptor (RYR 1, RYR 3) activator, an up-regulator of intracellular ca2+, a calmodulin-dependent protein kinase II (CaMKII) agonist, a calmodulin-dependent protein kinase II (CaMKII) activator.
40. A cell culture medium for preparing multinuclear myotubes from myogenic precursor cells, the medium comprising a basal medium and an extracellular regulated signal kinase (ERK 1/2) inhibitor.
41. The cell culture medium of claim 40, further comprising at least one of: mitogen-activated protein kinase 1 (MEK 1) inhibitors, fibroblast Growth Factor (FGF) inhibitors, transforming growth factor-beta (TGF-beta) inhibitors, retinoid-X receptor (RXR) agonists, retinoid-X receptor (RXR) activators, retinoic Acid Receptor (RAR) agonists, retinoic Acid Receptor (RAR) activators, ranitidine receptor (RYR 1, RYR 3) agonists, ranitidine receptor (RYR 1, RYR 3) activators, intracellular ca2+ upregulation factors, calmodulin-dependent protein kinase II (CaMKII) agonists, calcium ionophores, and calmodulin-dependent protein kinase II (CaMKII) activators.
42. The cell culture medium of claim 40 or 41, consisting of a composition certified as a generally recognized safety (GRAS).
43. The cell culture medium of any one of claims 40 to 42, wherein the medium is a serum-free medium.
44. The cell culture medium of claim 43, wherein the medium comprises a serum replacement component.
45. The cell culture medium of any one of claims 40-44, consisting of a composition that is certified as xeno-free.
CN202280046185.2A 2021-05-06 2022-05-05 Method for inducing hypertrophic muscle fibers for industrial meat production Pending CN117580943A (en)

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