BACKGROUND

[0001] The disclosure is directed to methods, and compositions for accelerating muscle formation. More specifically, the disclosure is directed to methods and compositions for accelerating the formation of myotube by bovine satellite cells.

[0002] In an effort to reduce the impact of animal agriculture and to improve people's nutrition, as well as for various other incentives, there is a need for alternatives to animal meat for development of novel protein sources containing viable cells culture(s) that correspond to the three-dimensional (3D) tissue, for instance, muscle tissue.

[0003] In-vivo, skeletal muscle differentiation is a highly coordinated multi-step process in which mononucleated myoblasts first withdraw from the cell cycle upon extracelluar cues, differentiate into post-mitotic myocytes (early differentiation), and subsequently fuse into multinucleated myotubes (late differentiation), which finally bundle to form mature muscle fibers (terminal differentiation).

[0004] However, ex-vivo (and/or in-vitro), the interrelationship between cell isolation and characterization, bioreactor design and cell culture scale-up, growth media optimization, three- dimensional structure of the cultured muscle and sensory and nutritional parameters for forming muscle still remain largely unknown.

[0005] Accordingly, there is a need for reliable and commercially practicable methods of forming muscle tissue in-vitro.

SUMMARY

[0006] Disclosed, in various implementations, are methods and compositions for accelerating the formation of myotube by bovine satellite cells.

[0007] In an exemplary implementation provided herein is a method of accelerating myotube formation, comprising: accelerating myodifferentiation and proliferation of bovine satellite cells (BSCs) to myoblast cells; and accelerating myotube formation of the myoblasts.

[0008] In another exemplary implementation, accelerating BSCs differentiation to myoblast cells comprises contacting a predetermined density of BSCs population with a differentiation medium composition comprising Insulin, Transferrin, Sodium Selenite and Ethanolamine (ITS-X), at a predetermined concentration, frequency and duration; while accelerating myotube formation of the myoblast cells, comprises contacting the myoblast cells population with a composition of predetermined concentration (w/w growth medium), comprising at least one of: a myostatin (MSTN) inhibitor, a myostatin downregulator, and a follistatin upregulator.

BRIEF DESCRIPTION OF THE FIGURES

[0009] For a better understanding of the method and compositions for accelerating the formation of myotube(s) by bovine satellite cells, with regard to the implementations thereof, reference is made to the accompanying examples and figures, in which:

[00010] FIG. 1, is a schematic illustrating the simultaneous acceleration of differentiation and formation of myotubes using the compositions disclosed;

[00011] FIG. 2, is an image depicting the SCs before myotube formation using the methods disclosed;

[00012] FIG.s 3-8, are image depicting the effect of pretreatment time (right, left) between various medium components (top and bottom);

[00013] FIG.s 9-11, are images depicting the effect of myostatin inhibitors on the myotube growth in immortalized Bovine Satellite Cells after 10 passes;

[00014] FIG. 12 illustrates the effective range of MSTNi;

[00015] FIG. 13, depicts the difference in several parameters between growth media and differentiation media;

[00016] FIG.s 14-15, depicts the difference in several parameters between control and differentiation media of the immortalized cell line, using different concentration of MSTNi;

[00017] FIG.s 16-18, depicts the effect of myostatin inhibitors at various concentration on myofiber formation; and

[00018] FIG. 19, depicts the difference in several parameters between growth media and differentiation media when myostatin inhibitors is added to the growth media;

DET AIEED DESCRIPTION

[00019] Provided herein are implementations of methods and compositions for simultaneously accelerating the differentiation of satellite cells to myoblasts and their fusion to form myotubes. [00020] Several stem cell types can be utilized for in-vitro culturing of muscle (hence meat). These are, for example, embryonic stem cells (ESCs), mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), with the most reliable being satellite cells (SCs).

[00021] Satellite cells (SCs) are adult stem cells located between the sarcolemma and the basal lamina of skeletal muscle fibers, typically dormant until activation by, e.g., muscle damage, releasing a number of myogenic regulatory factors (MRFs), which in turn initiate proliferation, differentiation to, e.g., myoblasts which can then fuse to form myotubes.

[00022] Accordingly and in an exemplary implementation, provided herein is a method of accelerating myotube formation, comprising: accelerating myodifferentiation and proliferation of bovine BSc to myoblasts; and accelerating myotube (referring to a multi-nucleated fiber that is formed from the fusion of a plurality of myoblasts and/or myocytes) formation of the myoblast cells. In the context of the disclosure, the term “myotube formation” means a process in which myoblasts fuse into multi-nucleated fibers myotube (see e.g., FIG 21, left column).

[00023] In an exemplary implementation, the step of accelerating BSCs differentiation to myoblast cells comprises contacting a predetermined BSCs’ population density (see e.g., FIG. 2) as well as triggering myoblast fusion in underconfluent cells (about 40% confluence), with a differentiation medium composition comprising Insulin, Transferrin, Sodium Selenite and Ethanolamine (ITS-X), at a predetermined concentration, frequency and duration. As used herein, the term “contacting” (i.e., contacting the predetermined BSCs’ population density) is intended to include incubating the differentiation medium and/or ITS-X and the BSCs together in vitro (e.g., adding the differentiation medium or ITS-X to cells in culture). In certain exemplary implementations, “contacting” does not include the in vivo exposure of cells to the compounds as disclosed herein that may occur naturally in a subject (i.e., exposure that may occur as a result of a natural physiological process). The step of contacting at least one myoblast or the BSCs’ precursor thereof with the differentiation medium can be conducted in any suitable manner. For example, the cells may be treated in adherent culture, or in suspension culture. In some exemplary implementations, the cells are treated in conditions that promote the formation of myotubes. The disclosure contemplates any conditions which promote the formation of skeletal muscle organoids, such as myotubes, but not limited to myotubes. Examples of conditions that promote the formation of skeletal muscle organoids can be, for example, suspension culture in low attachment tissue culture plates, spinner flasks, aggrewell plates. In some exemplary implementations, the differentiation media further contain about 20% (w/w final medium content) serum (e.g., heat inactivated fetal bovine serum).

[00024] In another exemplary implementation, the BSCs contacted with a differentiation medium can also be, but not necessarily, simultaneously or subsequently contacted with another agent, such as other differentiation agents or environments to stabilize the cells, or to differentiate the cells further. For example, contacting the differentiated myoblasts at a predetermined period, with a predetermined concentration of ERK inhibitor (ERKi).

[00025] Another agent used to increase the differentiation efficiency, can be a specific mT0RC2 activator. That activator can be, for example, specific activator of mammalian target of Rapanycin complex 2 (mT0RC2). mT0RC2 is a complex of four subunits; kinase catalytic subunit mTOR, mammalian lethal with SEC13 protein 8 (mLST8), rapamycin-insensitive companion of mTOR (RICTOR), and mammalian stress-activated Map kinase-interacting 1 (mSINl), where RICTOR mainly has a scaffolding role, and mSIN 1 likely contains the substrate binding site and determines subcellular localization of mT0RC2. Specific activators of mT0RC2 can be, for example, PI(3,4,5)P3. Turning to FIG. 1, and in an exemplary implementation, considering growth factor mediated Akt phosphorylation, PI3K inhibition abolishes Akt phosphorylation at both Thr3O8 and Ser473 (which is an mT0RC2 target site). Not wishing to be bound by theory, upon growth factor (GF) stimulation, PI3K phosphorylates PI(4,5)P2 at the plasma membrane to produce phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), which is counteracted by phosphatase and tension homolog (PTEN). Akt and its activating kinase PDK1 specifically bind to PI(3,4,5)P3 via their PH domains, and are recruited to the plasma membrane, leading to phosphorylation of Akt by PDK1 at Thr3O8 forming e.g., PKCa affecting cytoskeleton reorganization and the formation of myotubes.

[00026] In another exemplary implementation, the differentiation medium is at least one of serum free medium, and low- serum containing medium. The differentiation medium may include a nutrient medium (e.g., DMEM), non-essential amino acids, and glutamax. In certain exemplary implementations, the myotubes are further cultured in differentiation medium for at least 1 day, at least 5 days, at least 10 days, 1 to 10 days, 1 to 30 days, 10 to 30 days, 10 to 20 days, or in some exemplary implementations, for 10 days. During culture with the differentiation medium the proliferative myogenic cells present in the myotube quiesce and return to a satellite cell state. The satellite cells may then be further isolated from the medium. For example, 8 generations of functional (spontaneously contracting) myofiber formation were obtained over a period of 52 days both in 2D and 3D from the same culture of SCs.

[00027] In certain exemplary implementations, the BSCs are expandable in culture and are further contacted with a composition operable to increase BSCs proliferation. Method of inducing, enhancing or increasing BSCs proliferation can be, for example, contacting the BSCs with, for example (see e.g., FIG. 1), kinase inhibitors, G protein coupled receptor (GPCR) modulators, epigenetic modifiers, histone deacetylases (HD AC) modulators, hedgehog signaling pathway modulators, neuropeptides, dopamine receptor modulators, serotonin receptor modulators, histamine receptor modulators, adenosine receptor agonists, ionophores, ion channel modulators, gamma- secretase modulators, corticosteroids, and any combinations thereof.

[00028] As indicated, the step of contacting the predetermined density of bovine myoblast cell population with the ITS-X composition is carried out at a frequency of once every 24 hours (e.g., once a day), for a period of at least 4 days, wherein the concentration of each of the ITS-X components is 1:100 (w/w) of the differentiation medium. For example, between about O.Olg/L and about 2.0g/L Insulin, between about 0.05g/L and about 1.0 g/L Transferrin, between about O.OOOlg/L and about 0.001 g/L Sodium Selenite and between about O.Olg/L and about l.Og/L Ethanolamine, while each component is then reconstituted to be 1:100 in the final differentiation medium. Typically, myodifferentiation can be induced by adding ITS-X only once, when the cells are 70-85% confluent OR once a week OR daily, for 4 days in a row. In other words, every ITS-X addition to the BSCs culture triggers the process of differentiation within 3-6 days post-treatment.

[00029] In an exemplary implementation, the methods and compositions for simultaneously accelerating the differentiation of satellite cells to myoblasts and their subsequent fusion to form myotubes, further comprises replacing a portion of the differentiation medium prior to the introduction of the ITS-X composition, the portion being between about 10% and about 90%, or between about 25% and 75%, for example the portion of differentiation medium replaced can be between 40% and 60%. It is noted, that replacing only a portion of the differentiation medium is beneficial for preserving trophic factors, cytokines, micro-RNA molecules (miRs), small RNA molecules, extracellular matrix (ECM)- forming factors, and the like, secreted by the differentiating cells.

[00030] In another exemplary implementation, and as indicated, the differentiated myoblasts are optionally, though not necessarily contacted at a predetermined period, with a predetermined concentration of ERK inhibitor (ERKi), configured to maximize myoblasts fusion at the shortest time. The predetermined period for introducing the ERKi, is in an exemplary implementation, on day 3 of the replacement protocol as an additional differentiation agent. Furthermore, the concentration of ERKi can be between about 0.05 pM and about 10.0 pM in the differentiation medium, for example, between about 0.25pM and about 7.5 pM, or between about 0.25pM and about 1.50 pM, for example, between about 0.5pM and about 1.0 pM.

[00031] In another exemplary implementation, the methods and compositions for simultaneously accelerating the differentiation of satellite cells to myoblasts and their subsequent fusion to form myotubes, further comprise contacting the myoblast cells population with a composition of predetermined concentration (w/w growth medium), comprising at least one of: a myostatin (MSTN) inhibitor (MSTNi), a myostatin downregulator, and a follistatin upregulator.

[00032] In the context of the disclosure, “inhibitor of myostatin” and “myostatin inhibitor” are intended to be interchangeable herein. The myostatin inhibitor may be a protein or may be an oligonucleotide (RNA or DNA). Myostatin inhibitor proteins may be, for example, peptides or polypeptides. The proteins may inhibit myostatin by binding myostatin [McPherson et al., Nature, 387(6628): 83-90 (1997)] or by binding the myostatin receptor activin lib [McPherron et al., Nat. Genet., 22(3): 260-264 (1999)]. Examples of proteins that inhibit myostatin by binding to myostatin are myostatin propeptide, follistatin, other follistatin-like proteins, protein fragments or chimeric (i.e., fusion) proteins.

[00033] Myostatin inhibitor oligonucleotides as used herein, may be antisense oligonucleotides [Eckstein, Antisense Nucleic Acid Drug Dev., 10: 117-121 (2000); Crooke, Methods Enzymol., 313: 3-45 (2000); Guvakova et al., J. Biol. Chem., 270: 2620-2627 (1995); Manoharan, Biochim. Biophys. Acta, 1489: 117-130 (1999); Baker et al., J. Biol. Chem., 12-. 11994-12000 (1997); Kurreck, Eur. J. Biochem., 270: 1628-1644 (2003); Sierakowska et al., Proc. Natl. Acad. Sci. USA, 93: 12840-12844 (1996); Marwick, J. Am. Med. Assoc. 280: 871 (1998); Tomita and Morishita, Curr. Pharm. Des., 10: 797-803 (2004); Gleave and Monia, Nat. Rev. Cancer, 5: 468-479 (2005) and Patil, AAPS J. 7: E61-E77 (2005], triplex oligonucleotides [Francois et al., Nucleic Acids Res., 16: 11431-11440 (1988) and Moser and Dervan, Science, 238: 645-650 (1987)], ribozymes/deoxyribozymes (DNAzymes) [Kruger et al., Tetrahymena. Cell, 31: 147-157 (1982); Uhlenbeck, Nature, 328: 596-600 (1987); Sigurdsson and Eckstein, Trends Biotechnol., 13 286-289 (1995); Kumar et al., Gene Ther., 12: 1486-1493 (2005); Breaker and Joyce, Chem. Biol., 1: 223-229 (1994); Khachigian, Curr. Pharm. Biotechnol., 5: 337-339 (2004); Khachigian, Biochem. Pharmacol., 68: 1023-1025 (2004) and Trulzsch and Wood, J. Neurochem., 88: 257-265 (2004)], small-interfering RNAs/RNAi [Fire et al., Nature, 391: 806-811 (1998); Montgomery et al., Proc. Natl. Acad. Sci. U.S.A., 95: 15502-15507 (1998); Cullen, Nat. Immunol., 3: 597-599 (2002);

Hannon, Nature, 418: 244-251 (2002); Bernstein et al., Nature, 409: 363-366 (2001); Nykanen et al., Cell, 107: 309-321 (2001); Gilmore et al., J. Drug Target., 12: 315-340 (2004); Reynolds et al., Nat. Biotechnol., 22: 326-330 (2004); Soutschek et al., Nature, 432173-178 (2004); Ralph et al., Nat. Med., 11: 429-433 (2005); Xia et al., Nat. Med., 10816-820 (2004) and Miller et al., Nucleic Acids Res., 32: 661-668 (2004)], aptamers [Ellington and Szostak, Nature, 346: 818-822 (1990); Doudna et al., Proc. Natl. Acad. Sci. U.S.A., 92: 2355-2359 (1995); Tuerk and Gold, Science, 249: 505-510 (1990); White et al., Mol. Ther., 4: 567-573 (2001); Rusconi et al., Nature, 419: 90-94 (2002); Nimjee et al., Mol. Ther., 14: 408-415 (2006); Gragoudas et al., N. Engl. J. Med., 351: 3805- 2816 (2004); Vinores, Curr. Opin. Mol. Ther., 5673-679 (2003) and Kourlas and Schiller et al., Clin. Ther., 28 36-44 (2006)] or decoy oligonucleotides [Morishita et al., Proc. Natl. Acad. Sci. U.S.A., 92: 5855-5859 (1995); Alexander et al., J. Am. Med. Assoc., 294: 2446-2454 (2005); Mann and Dzau, J. Clin. Invest., 106: 1071-1075 (2000) and Nimjee et al., Annu. Rev. Med., 56: 555-583 (2005). The myostatin inhibitor oligonucleotides inhibit the expression of myostatin or expression of its receptor activin lib. The foregoing documents are hereby incorporated by reference in their entirety herein, with particular emphasis on those sections of the documents relating to methods of designing, making and using oligonucleotides.

[00034] Downregulation of MSTN is done in certain exemplary implementation using over expression (upregulation) of follistatin, as well as through introduction of mRNA that interferes with the expression of MSTN. Additionally, or alternatively, using gene editing tools (e.g., CRISPR- cas system), expression of MSTN can be eliminated or substantially diminished.

EXAMPLES

EXAMPLE I - Growth

Cells culture:

[00035] In-house isolated and immortalized bovine Satellite cells were cultured for one passage after thawing and seeded for the experiment at 5000cells/cm2.All the cells exhibited similar proliferation rates. All the results are based on comparison to growth medium or/and basis of differentiation medium controls.

[00036] The cells were grown in BioAmf2 medium (growth medium) until reaching the confluence of 65-75% before treatment administration. On day 0 of treatment, growth medium was discarded from the plates and low-serum (2% horse serum) medium enriched with Insulin- Transferrin-Selenium + Ethanolamide (ITS-X) was added. Low-serum medium without ITS or growth medium were used as controls. Differentiation protocol lasted for up to 9 days, with differentiation medium change on day 3.

Treatments:

[00037] mT0R2 activator (mT0R2 act) WHY 1485 was added at two concentrations (2uM and lOuM as pre-treatment for 0.5hr or 2hrsbefore the experiment start. After pre-treatment, the medium was changed to differentiation medium (low-serum medium) as described before.

[00038] ERK inhibitors (ERKi) -SCH772984 and PD0325901 were used at predetermined concentrations as temporary treatment. The medium was refreshed once every 24hrs after administration.

[00039] Metformin was used at 0.5- ImM. Once added to the medium, no medium change was necessary.

[00040] Myostatin inhibitor effective timing: D(-l) -day before differentiation start, D3.

WHY1485 mT0RC2 activator And Metformin

[00041] Turning now to FIG.s 3-8, depicting the effect of pretreatment time (right, left) between various medium components (top and bottom) on myofiber formation. As shown in FIG. 3, pretreatment of immortalized BSCs at pass 10, with mTOR activator (4,6-Di-4-morpholinyl-N-(4- nitrophenyl)-l,3,5-triazin-2-amine) (WHY1485) and mT0RC2 act at 2 pM for 0.5 hrs. (top left) and for 2.0 hrs, (top right) on day 1, as compared to day 8 of the same medium using ITS enriched growth media. As illustrated, massive myofiber network formation is evident on day 8 of diff protocol, without ERKi and using Only mTORC2+ITS.

[00042] As is also evident, 2hrs pre-treatment with WHY 1485 promotes myoblast fusion and results in wider myofibers.

[00043] FIG. 4, depict myo fiber formation and is identical to the treatment in FIG. 3, only ERKi PD0325901 is used for the pretreatment. As shown, ERKi PD0325901 does not provide significant improvement. [00044] FIG. 5, depict myofiber formation and is identical treatment to the treatment in FIG.4, measured at Day 11. As shown, ERKi PD0325901 does not provide significant improvement or shows a significant effect.

[00045] FIG.s 6 and 7, depict myofiber formation and is identical to the treatment in FIG. 3, only Metformin is used in addition for the pretreatment, and the measurement was done on day 6. As shown, Metformin addition significantly increases myogenic differentiation rate and quality.

[00046] FIG. 8, depict myofiber formation and is identical to the treatment in FIG. 7, only ERKi is used in addition for the pretreatment, and the measurement was done on day 7. As shown, combinations of ITS + WHY 1485 or Metformin + WHY 1485 Result in better myogenic differentiation compared to regular differentiation protocol (ITS alone - control) or ERK inhibitors.

Myostatin Inhibition

[00047] Turning now to FIG.s 9 -10, depicting the effect of myostatin inhibiotors on myofiber differentiation and growth. As illustrated, using Immortalized Satellite cells at pass 13, imaged on day 7, growth control of bovine satellite cells (A), with ACE-083 (a Follistatin-based ligand trap) used as myostatin inhibitor (B), compared with ITS -differentiated control (C) and with ACE-083 (D) are depicted on day 7 (FIG. 9) and day 8 (FIG. 10). As illustrated, in FIG. 10D, ITS and ACE- 083 produces larger and thicker myofiber.

[00048] FIG. 11, illustrates the effect of a different myostatin inhibitor, ACE-031 (a fusion protein of activin receptor type IIB and IgGl-Fc,), compared to ACE-083. As shown, ACE-083 is more effective. In certain implementations, either or both are used.

[00049] FIG. 12, illustrates the effective range of MSTNi inhibitors in promoting myofibril formation.

[00050] FIG.s 13-18 depicts the effect of MSTNi ACE-083 on formation of various components within the myofibrils, namely, from left to right - Phalloidin - fActin; MyHC - Myosin Heavy Chain; DAPI - Nuclei; and merged image, where the top row is growth media, and bottom row, is differentiation media. FIG. 13, illustrates results without any MSTNi, while FIG. 14, illustrates the effect of 1 pM ACE-083 on these parameters, and FIG. 15, illustrates the effect of 10 pM of ACE-083. FIG. 16 is an isolation of the merged image in FIG. 15 on one side, while FIG.s 17 shows the other side, and 18 is an enlarged image from FIG. 16, showing visible fiber of about 300 pm. As illustrated, MSTNi is beneficial in forming myofiber in growth media across the measured range.

[00051] Turning now to FIG. 19, depicting the effect of both the treatment components and the MSTNi on the formation of myofibers. It is clear that MSTNi in addition to ITS promotes better myofiber alignment and higher fiber density in 3D hydrogel.

[00052] The term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

[00053] All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other. “Combination” is inclusive of blends, mixtures, alloys, reaction products, and the like. The terms “a”, “an” and “the” herein do not denote a limitation of quantity, and are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The suffix “(s)” as used herein is intended to include both the singular and the plural of the term that it modifies, thereby including one or more of that term (e.g., the cell(s) includes one or more cell). Reference throughout the specification to “one implementation”, “another implementation”, “an implementation”, “an exemplary implementation” and so forth, when present, means that a particular element (e.g., feature, structure, and/or characteristic) described in connection with the implementation is included in at least one implementation described herein, and may or may not be present in other implementations. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various implementations.

[00054] All ranges disclosed herein are inclusive of the endpoints, and the endpoints are independently combinable with each other. Furthermore, the term "about" means that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is "about" or "approximate" whether or not expressly stated to be such. For example, “about” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% or at least ±10% of the modified term if this deviation would not negate the meaning of the word it modifies.

[00055] Accordingly, provided herein is a method of accelerating myotube formation, comprising: accelerating myodifferentiation and proliferation of bovine satellite cells (BSCs) to myoblast cells; and accelerating myotube formation of the myoblasts, wherein (i) the step of accelerating BSCs differentiation to myoblast cells comprises contacting a predetermined density of BSCs population with a differentiation medium composition comprising Insulin, Transferrin, Sodium Selenite and Ethanolamine (ITS-X), at a predetermined concentration, frequency and duration, (ii) the step of accelerating myotube formation of the myoblast cells, comprises contacting the myoblast cells population with a composition of predetermined concentration (w/w growth medium), comprising at least one of: a myostatin (MSTN) inhibitor, a myostatin downregulator, and a follistatin upregulator, wherein (iii) contacting the predetermined density of bovine myoblast cell population with the ITS-X composition is carried out at a frequency of 1/24 hrs, for a period of between 4-13 days, wherein (iv) the concentration of each of the ITS-X components is 1:100 (w/w) of the differentiation medium, the composition (v) further comprising replacing a portion of the differentiation medium, the portion being between 40% and 60%, the method (vi) further comprising contacting the differentiated myoblasts at a predetermined period, with a predetermined concentration of ERK inhibitor (ERKi), configured to maximize myoblasts fusion at the shortest time, (vii) the concentration of ERKi is between about 0.25pM and about 7.5 pM in the differentiation medium, (viii) e.g., the concentration of ERKi is between about 0.5pM and about 1.0 pM, wherein (ix) the step of contacting the differentiated myoblasts with ERKi is done on the third day, the method (x) further comprising contacting the differentiated myoblasts with a specific mT0RC2 activator, and wherein (xi) the specific mT0RC2 activator is phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3).

[00056] Although the foregoing disclosure for methods and compositions for simultaneously accelerating the differentiation of satellite cells to myoblasts and their subsequent fusion to form myotubes, has been described in terms of some implementations, other implementations will be apparent to those of ordinary skill in the art from the disclosure herein. Moreover, the described implementations have been presented by way of example only, and are not intended to limit the scope of the inventions. Indeed, the novel methods, systems and compositions described herein may be embodied in a variety of other forms without departing from the spirit thereof. Accordingly, other combinations, omissions, substitutions and modifications will be apparent to the skilled artisan in view of the disclosure herein.