Muscle cells differentiated from pluripotent cells, methods for producing same, and uses thereof

Abstract

The present invention relates to the field of production of bioengineered tissues comprising skeletal muscle cells, and in particular to compositions and methods for producing a plurality of skeletal muscle committed progenitor cells from pluripotent stem cells and skeletal muscle cells differentiated therefrom. The present invention further provides skeletal muscle committed progenitor cell masses and / or skeletal muscle cell masses, engineered tissues comprising same, and uses thereof.

Description

[Technical field]

[0001] The present invention relates to the field of production of bioengineered tissues comprising skeletal muscle cells, and in particular to compositions and methods for producing a plurality of skeletal muscle committed progenitor cells from pluripotent stem cells and differentiated skeletal muscle cells therefrom. The present invention further provides masses of skeletal muscle committed progenitor cells and / or masses of skeletal muscle cells, engineered tissues comprising same, and uses thereof. [Background technology]

[0002] Animal meat is a good nutritional source of protein and contains all the essential amino acids in the proper ratios required for the growth and maintenance of the human body. However, the modern practice of growing livestock for food has harmful effects on air and water quality and requires large land areas and energy investments. It also raises moral questions, as livestock are typically raised in crowded habitats and sometimes in unsuitable conditions that cause the livestock subjects to suffer. For this reason, cell-cultured meat products may also be consumed by people who abstain from meat for humanitarian reasons.

[0003] Among other things, the challenge in producing cultured meat products is the texture and mouthfeel, which cannot replicate that of comparable slaughtered meat products. Cultured meat containing only cultured cells is typically in the form of ground meat, which significantly limits the types of food that can be offered to consumers. Hybrid products containing cultured cells and plant-based proteins form one potential solution, but there remains a need to obtain cell-based parts that mimic slaughtered meat, which is primarily composed of muscle.

[0004] Attempts to differentiate muscle in vitro have been primarily performed in the areas of basic science research and therapeutic drug development. The skeletal muscle lineage originates from the embryonic paraxial mesoderm (PM), which also gives rise to the axial skeleton, dermis, brown fat, meninges, and endothelial cells. Experimental strategies have recently been reported to recapitulate myogenesis from mouse and human pluripotent stem cells (embryonic stem cells or induced pluripotent stem cells) in vitro, all of which rely on the early activation of Wnt signaling substances (such as CHIR) at the epiblast stage. This induces neuro-mesodermal progenitors (NMPs) that can then be directed to PM fates and skeletal muscles. These protocols can efficiently produce fetal muscle fibers and immature satellite cells. Until recently, the only efficient protocols allowing differentiation of reasonably mature muscle cells relied on overexpression of transcription factors such as MyoD or Pax3 / 7 followed by cell isolation of induced progenitor bodies (Pourquie O et al., 2018. Curr Top Dev Biol. 129:123-142. doi:10.1016 / bs.ctdb.2018.03.003).

[0005] Previous studies have shown that Activin / Nodal / TGFβ, BMP, FGF, and WNT broadly induce mesoderm from pluripotent stem cells (Kyle ML et al., 2016. Cell 166(2):451-467). FGF and Activin were shown to be essential for cardiac differentiation (Shen M et al., 2021. Circulation Research 128:670-686; Sasano Y et al., 2020. Journal of Bioscience and Bioengineering, 129(6):749-755), while Activin A was shown as a negative regulator of muscle mass (Latres E et al., 2017. Nat Commun 8:15153, DOI:10.1038 / ncomms15153). Targeting (inhibiting) Activin A signaling pathway has been shown to have a significant beneficial effect in protecting against both muscle and bone loss in microgravity, suggesting that this strategy may be effective in preventing or treating muscle and bone loss (Lee Se-Jin et al., 2020. PNAS 117(38):23942-23951, doi.org / 10.1073 / pnas.2014716117). The TGFβ inhibitor SB431542, a known somatic mesoderm-like cell inducer, was shown to enhance myotube generation in association with PAX7-induced myogenic differentiation (Selvaraj S et al., 2019. eLife 8:e47970, DOI:10.7554 / eLife.47970).

[0006] US Patent Application Publication No. 2012 / 0164731 discloses a method for producing skeletal muscle progenitor cells using pluripotent stem cells, particularly induced pluripotent cells, which comprises culturing pluripotent stem cells in the presence of activin A to obtain PDGFRα-positive mesodermal cells, and then culturing the obtained mesodermal cells under serum-free conditions in the presence of a Wnt signal inducer to differentiate the cells into skeletal muscle progenitor cells. Also disclosed are a cell population containing skeletal muscle progenitor cells obtained by the method, as well as a skeletal muscle regeneration promoter and a therapeutic agent for muscle diseases such as muscular dystrophy, the promoter containing skeletal muscle progenitor cells as an active ingredient.

[0007] U.S. Patent Application Publication No. 2019 / 0010460 generally discloses a method for producing bioengineered heart muscle (BHM) from pluripotent stem cells, including inducing mesodermal differentiation, cardiac differentiation, and cardiac maturation by directed tissue formation.

[0008] Most of the current methods for differentiating muscle cells from pluripotent cells require the use of numerous components, including expensive growth factors and media, and further require long culture periods.

[0009] There is a need for simple and economical compositions and methods for obtaining muscle cells that can be used in the industry of cell culture products, including cultivated meat products. Summary of the Invention

[0010] The present invention addresses the above-mentioned need for masses of skeletal muscle cells that can form part of an engineered tissue or cultured meat product, and provides compositions and methods for producing masses of skeletal muscle-committed progenitor cells that can be more readily differentiated into muscle cells.

[0011] The present invention is based in part on the unexpected discovery that pluripotent stem cells (PSCs) cultured in a medium containing a combination of an activator of the TGF-beta (TGF-β) signaling pathway, particularly activin A, and an inhibitor of the Glycogen synthase kinase-3 (GSK3) signaling pathway, particularly CHIR-99021, produce a plurality of cells including skeletal muscle committed progenitor cells. The muscle committed progenitor cells then differentiate into muscle cells. Advantageously, the plurality of cells further includes additional lineage committed cells that can also readily differentiate into extracellular matrix (ECM) producing cells and adipocytes.

[0012] Furthermore, the teachings of the present invention are advantageous over previously known methods for producing skeletal muscle committed progenitor cells from PSCs in that the lineage committed differentiation process requires only media supplemented with nutrients as well as a combination of at least one TGF-β activator and at least one GSK3 inhibitor. The method of the present invention also allows for the shortening of the production of muscle cells from PSCs to a time frame of several days, allowing the entire process of producing bioengineered tissues containing skeletal muscle cells to be completed in about 2 to about 4 weeks. Furthermore, the method is easily scalable to reactors of tens of liters volume, which allows for its use in the food industry at a cost comparable to that of conventional processes in the food industry.

[0013] According to certain aspects, the present invention provides a method for producing a plurality of cells comprising skeletal muscle committed progenitor cells, the method comprising culturing a plurality of pluripotent stem cells (PSCs) in a culture medium comprising a combination of (i) at least one activator of the TGF-beta (TGF-β) signaling pathway and (ii) at least one inhibitor of the GSK3 signaling pathway, thereby producing a plurality of cells comprising skeletal muscle committed progenitor cells.

[0014] According to certain embodiments, the medium is a serum-free medium.

[0015] According to certain embodiments, the culturing is performed under three-dimensional (3D) culture conditions. According to certain exemplary embodiments, the 3D culture is a suspension culture. According to certain embodiments, the suspension culture is devoid of adhesive material and / or support matrix. According to these embodiments, the cells self-assemble to form at least one cell aggregate. According to certain embodiments, the cell aggregate is in a form selected from the group consisting of clusters, spheroids, organoids, and the like.

[0016] According to further additional or alternative embodiments, the suspension culture comprises at least one adhesive material and / or support matrix. According to certain exemplary embodiments, the 3D culturing is performed in a container.

[0017] According to certain alternative embodiments, the culturing is performed under two-dimensional (2D) culture conditions. According to some embodiments, the 2D culture comprises at least one adhesive material and / or support matrix.

[0018] Any adhesive material or support matrix known or that becomes known in the art that allows for and supports cell culture, particularly the culture of PSCs and cells differentiated therefrom, can be used in accordance with the teachings of the present invention. According to certain embodiments, the support matrix is ​​a semi-solid matrix. According to some embodiments, the support matrix is ​​solid.

[0019] According to certain embodiments, the at least one activator of the TGF-β signaling pathway is selected from the group consisting of activin A, TGF-β, BMP2, BMP7, GDF9, NODAL, and any combination thereof, each possibility representing a separate embodiment of the present invention.

[0020] According to certain embodiments, the inhibitor of the GSK3 signaling pathway is CHIR-99021 (C22H18Cl2N8) or a salt thereof, SB 216763, LY2090314, TWS119, Tideglusib, GSK-3β inhibitor 1, GSK-3β inhibitor 2, GSK-3β inhibitor 3, AR-A014418, TDZD-8, Kenpaullone, GSK3 inhibitor IX, Cromolyn sodium, CHIR-98014, AZD1080, SB 415286, IM-12, 9-ING-41, indirubin-3'-monoxime, 1-azakempauron, BRD0705, AZD2858, CP21R7, BIO-acetoxime, bikinin, VP3.15, VP3.15 dihydrobromide, GNF4877, KY19382, SAR502250, A 1070722, (R)-BRD3731, BRD3731, BIP-135, 5-iodo-indirubin-3'-monoxime, BRD5648, GSK3 inhibitor 1, GSK3 / CDK5 / CDK2-IN-1, indirubin-3'-monoxime-5-sulfonic acid, GSK3β inhibitor flavonoids, lithium, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0021] According to certain embodiments, the CHIR-99021 salt is selected from the group consisting of CHIR-99021 monohydrochloride and CHIR-99021 trihydrochloride, Each possibility represents a separate embodiment of the present invention.

[0022] According to certain embodiments, the GSK3β inhibitory flavonoid is selected from the group consisting of luteolin, apigenin, quercetin, myricetin, and any combination thereof, each possibility representing a separate embodiment of the present invention.

[0023] According to certain embodiments, the medium lacks growth factors other than those that activate the TGF-β pathway. According to some embodiments, the medium lacks bFGF.

[0024] According to certain exemplary embodiments, the combination comprising at least one activator of the TGF-β signaling pathway and at least one inhibitor of the GSK3 signaling pathway comprises Activin A and CHIR-99021. According to certain embodiments, the combination consists of Activin A and CHIR-99021.

[0025] According to certain embodiments, at the time of seeding, the medium further comprises an inhibitor of Rho-associated protein kinase (Rock).

[0026] According to certain embodiments, the culturing is carried out for a period of time that allows reaching about 10% to about 90% skeletal muscle committed progenitor cells out of the total number of cells.

[0027] According to certain embodiments, culturing the plurality of PSCs is performed continuously in a medium comprising a combination of at least one activator of the TGF-β signaling pathway and at least one inhibitor of the GSK3 signaling pathway.

[0028] According to certain additional or alternative embodiments, culturing the plurality of PSCs is performed in cycles, and the medium comprising a combination of at least one activator of the TGF-β signaling pathway and at least one inhibitor of the GSK3 signaling pathway is replaced after each cycle. The combination of at least one activator of the TGF-β signaling pathway and at least one inhibitor of the GSK3 signaling pathway in each cycle may be the same or different.

[0029] According to certain embodiments, the plurality of cells produced further comprises at least one additional lineage committed progenitor cell. According to certain embodiments, the at least one additional lineage committed progenitor cell is selected from stromal committed progenitor cells, adipocyte committed progenitor cells, and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0030] According to certain embodiments, the plurality of PSCs are of an origin selected from the group consisting of non-human animals and humans. According to certain embodiments, the non-human animals are selected from the group consisting of ungulates, poultry, aquatic animals, invertebrates, and reptiles. According to certain embodiments, the ungulates are selected from the group consisting of bovines, sheep, goats, buffaloes, equines, pigs, giraffes, camels, deer, hippos, or rhinos. According to certain exemplary embodiments, the ungulates are bovines.

[0031] According to certain embodiments, the PSCs are selected from the group consisting of induced PSCs (iPSCs), embryonic stem cells (ESCs), and non-embryonic stem cells.

[0032] According to certain embodiments, the PSCs are not genetically modified. According to certain embodiments, the PSCs are genetically modified.

[0033] According to another aspect, the present invention provides a method for producing a plurality of skeletal muscle cells, the method comprising: - depositing a plurality of cells, including skeletal muscle committed progenitor cells produced by the method of the invention as described herein above, on an adhesive material and / or support matrix; - culturing the plurality of cells in a differentiation medium that promotes differentiation of the skeletal muscle committed progenitor cells into skeletal muscle cells; thereby producing a plurality of differentiated cells including skeletal muscle cells.

[0034] In the method of the present invention, various differentiation medium types can be used. Nevertheless, the present invention now demonstrates that serum-free medium supplemented with nutrients and, optionally, certain hormones is sufficient to obtain the differentiation of muscle-committed progenitor cells produced by the method of the present invention into skeletal muscle cells. According to certain embodiments, the differentiation medium lacks activators of the TGF-β signaling pathway and inhibitors of the GSK3 signaling pathway. According to certain exemplary embodiments, the medium lacks activin A and CHIR-99021.

[0035] According to certain embodiments, the cells are cultured in the differentiation medium for a period of about 3 days to about 30 days. According to certain embodiments, the cells are cultured in the differentiation medium for a period of about 3 days to about 10 days. According to certain embodiments, the cells are cultured in the differentiation medium for a period of about 5 days to about 8 days. According to certain embodiments, the total period for obtaining a plurality of differentiated cells including skeletal muscle cells is about 6 days to about 30 days. According to certain embodiments, the total period for obtaining a plurality of differentiated cells including skeletal muscle cells is about 6 days to about 12 days.

[0036] According to certain embodiments, the plurality of differentiated cells comprises about 10% to about 90% skeletal muscle cells of the total number of cells. According to certain embodiments, the skeletal muscle cells are viable.

[0037] The present invention further provides a plurality of cells comprising skeletal muscle committed progenitor cells produced by the methods of the present invention. According to certain embodiments, the plurality of skeletal muscle committed progenitor cells are essentially devoid of PSCs. As used herein, the term "essentially devoid" with respect to PSCs refers to some PSCs that are not detectable by standard methods currently known in the art.

[0038] According to certain embodiments, skeletal muscle committed progenitor cells are generated from non-human animal PSCs. According to certain embodiments, skeletal muscle committed progenitor cells are generated from bovine PSCs.

[0039] The invention further encompasses engineered tissues comprising differentiated skeletal muscle cells produced by the methods of the invention described herein. According to certain embodiments, the differentiated muscle skeletal cells are produced from non-human animal PSCs. According to certain embodiments, the differentiated muscle cells are produced from bovine PSCs.

[0040] According to certain embodiments, the plurality of differentiated cells comprises about 10% to about 90% differentiated skeletal muscle cells of the total number of cells. According to certain embodiments, the plurality of differentiated cells further comprises at least one of differentiated stromal cells, differentiated adipocytes, and combinations thereof. According to certain embodiments, the differentiated stromal cells comprise about 10% to about 90% of the total number of cells. According to certain embodiments, the differentiated adipocytes comprise about 10% to about 90% of the total number of cells. According to certain embodiments, the stromal cells are ECM producing cells. According to certain embodiments, the plurality of differentiated cells is devoid of cardiac muscle cells. The plurality of differentiated cells and / or engineered tissues comprising skeletal muscle cells of the present invention can be used in a variety of applications, depending primarily on the source and type of PSCs from which the skeletal muscle cells are differentiated.

[0041] According to certain exemplary embodiments, the plurality of differentiated cells and / or engineered tissues comprising skeletal muscle cells of the present invention are used for the production of cultured foods, in particular cultured meat.

[0042] According to still further certain aspects, the present invention provides a plurality of in vitro grown cells comprising skeletal muscle committed progenitor cells, the skeletal muscle committed progenitor cells characterized by expression of at least one mesodermal marker and / or at least one early myogenic marker.

[0043] According to certain embodiments, the at least one mesoderm marker is selected from the group consisting of TBXT, TBX6, MSGN1, Pax3, and any combination thereof, and the at least one early myogenic marker is Six1. Each possibility represents a separate embodiment of the present invention.

[0044] According to certain embodiments, progenitor cells committed to skeletal muscle are characterized by expression of MSGN1 and Six1.

[0045] According to certain embodiments, the plurality of in vitro expanded cells further comprises at least one additional lineage committed cell selected from the group consisting of stromal committed progenitor cells, adipocyte committed progenitor cells, and combinations thereof, each possibility representing a separate embodiment of the present invention.

[0046] According to certain embodiments, the plurality of in vitro grown cells comprises at least one GSK3β inhibitory flavonoid and / or a metabolite thereof.

[0047] According to certain additional aspects, the present invention provides a plurality of in vitro grown cells comprising differentiated skeletal muscle cells, the skeletal muscle cells being characterized by expression of at least one myogenic marker.

[0048] According to certain embodiments, the at least one myogenic marker is selected from the group consisting of Myf5, Pax7, MEF2C, SIX1, NYOD1, MYOG, MYH3, MYH7, NYH8, MB, MYMK, and combinations thereof, each possibility representing a separate embodiment of the present invention.

[0049] According to certain embodiments, the plurality of in vitro grown cells further comprises at least one of stromal cells, adipocytes, or a combination thereof, Each possibility represents a separate embodiment of the present invention.

[0050] According to certain embodiments, the stromal cells are extracellular matrix (ECM) producing cells.

[0051] According to certain embodiments, the cells in the plurality of in vitro grown cells are non-human animal cells.

[0052] The present invention further includes an engineered tissue comprising a plurality of in vitro grown cells, the cells comprising skeletal muscle cells.

[0053] According to certain embodiments, the in vitro grown differentiated cells, including skeletal muscle cells, are non-human animal cells. According to these embodiments, the plurality of cells, or engineered tissues comprising same, form a cultured food product, particularly a cultured meat product.

[0054] It is to be understood that any combination of each of the aspects and embodiments disclosed herein is expressly encompassed within the present disclosure.

[0055] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating exemplary embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0056] [Figure 1] Representative brightfield images of skeletal muscle committed progenitor cells derived from bovine pluripotent stem cells after 4 days of incubation in serum-free growth medium containing a combination of Activin A and CHIR-99021 in suspension. Magnification x4. [Diagram 2] Representative RT-PCR analysis of markers of differentiation after 4 days starting from bovine PSCs incubated in serum-free growth medium containing activin A and CHIR-99021 is shown. [Figure 3A]Representative immunofluorescence staining (using MF-20 antibody) of myosin heavy chain after a total of 11 days of differentiation starting from bovine PSCs is shown. Bovine PSCs were cultured for the first 4 days in 3D suspension (Figure 3A-C) or in 2D culture on supports (Figure 3D) in serum-free growth medium containing CHIR-99021 alone (Figure 3A), in serum-free growth medium containing CHIR-99021 in combination with Activin A (Figure 3B and Figure 3D), or in serum-free growth medium containing CHIR-99021 in combination with TGFβ (Figure 3C). After 4 days, cells / cell aggregates were deposited on 2D supports and cultured for an additional 7 days in serum-free medium lacking Activin A, CHIR-99021, and TGFβ. Grey - DAPI / nuclei, white - MyhC positive cells. Magnification x10. [Figure 3B] Representative immunofluorescence staining (using MF-20 antibody) of myosin heavy chain after a total of 11 days of differentiation starting from bovine PSCs is shown. Bovine PSCs were cultured for the first 4 days in 3D suspension (Figure 3A-C) or in 2D culture on supports (Figure 3D) in serum-free growth medium containing CHIR-99021 alone (Figure 3A), in serum-free growth medium containing CHIR-99021 in combination with Activin A (Figure 3B and Figure 3D), or in serum-free growth medium containing CHIR-99021 in combination with TGFβ (Figure 3C). After 4 days, cells / cell aggregates were deposited on 2D supports and cultured for an additional 7 days in serum-free medium lacking Activin A, CHIR-99021, and TGFβ. Grey - DAPI / nuclei, white - MyhC positive cells. Magnification x10. [Figure 3C]Representative immunofluorescence staining (using MF-20 antibody) of myosin heavy chain after a total of 11 days of differentiation starting from bovine PSCs is shown. Bovine PSCs were cultured for the first 4 days in 3D suspension (Figure 3A-C) or in 2D culture on supports (Figure 3D) in serum-free growth medium containing CHIR-99021 alone (Figure 3A), in serum-free growth medium containing CHIR-99021 in combination with Activin A (Figure 3B and Figure 3D), or in serum-free growth medium containing CHIR-99021 in combination with TGFβ (Figure 3C). After 4 days, cells / cell aggregates were deposited on 2D supports and cultured for an additional 7 days in serum-free medium lacking Activin A, CHIR-99021, and TGFβ. Grey - DAPI / nuclei, white - MyhC positive cells. Magnification x10. [Figure 3D] Representative immunofluorescence staining (using MF-20 antibody) of myosin heavy chain after a total of 11 days of differentiation starting from bovine PSCs is shown. Bovine PSCs were cultured for the first 4 days in 3D suspension (Figure 3A-C) or in 2D culture on supports (Figure 3D) in serum-free growth medium containing CHIR-99021 alone (Figure 3A), in serum-free growth medium containing CHIR-99021 in combination with Activin A (Figure 3B and Figure 3D), or in serum-free growth medium containing CHIR-99021 in combination with TGFβ (Figure 3C). After 4 days, cells / cell aggregates were deposited on 2D supports and cultured for an additional 7 days in serum-free medium lacking Activin A, CHIR-99021, and TGFβ. Grey - DAPI / nuclei, white - MyhC positive cells. Magnification x10. [Figure 4] Representative RT-PCR analysis of myogenic markers Myf5, Pax7, Mef2C, Six1, MyoD1, MyoG, MYH3, MYH7, MYH8, myoglobin (MB), and myomarker (Myomaker (MYMK)) after a total of 11 days of differentiation starting from bovine pluripotent stem cells incubated in serum-free growth medium containing only Activin A and CHIR-99021 for the first 4 days during the commitment phase. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] The present invention provides a method for producing skeletal muscle cells from pluripotent stem cells (PSCs), which is suitable for large-scale, cost-effective production. The present invention is based in part on the unexpected discovery that by culturing PSCs in the presence of a combination of activin A, an activator of the TGF-β signaling pathway, previously known as a negative regulator of muscle mass, and CHIR 99021, an inhibitor of the GSK3 signaling pathway, a significant number of PSCs can be converted into mesoderm lineage cells, which can then differentiate into skeletal muscle cells. Furthermore, the culture medium required throughout the process of producing a plurality of skeletal muscle cells and / or engineered tissue masses containing same is simple to produce, contains a minimal number of growth factors and small molecules, and the entire growth cycle is short compared to previously known protocols, allowing for large-scale production at economical costs, even if the product is to be used in the food industry.

[0058] The invention further provides skeletal muscle cells produced by the methods of the invention, which can be used for therapeutic applications in the form of a plurality of cells or engineered tissues comprising same, hi certain embodiments, the skeletal muscle cells of the invention and engineered tissues comprising same are tailored for the food industry, in particular for the production of cultivated meat.

[0059] definition As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0060] The terms "comprise," "comprising," "include," "including," "having" and their conjugations mean "including but not limited to."

[0061] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0062] Whenever a numerical range is given herein, it is intended to include any recited numbers (fractional or integer) within the given range. The phrases "ranging / ranges" from a first denoted number to a second denoted number and "ranging / ranges" "from" a first denoted number to a second denoted number are used interchangeably herein and are intended to include the first and second denoted numbers and all fractional and integer numbers therebetween.

[0063] As used herein, the term "about" refers to a variation of the numerical designation of +10% or -10% of the numerical designation. Furthermore, all numerical ranges herein should be understood to include each whole integer within the range.

[0064] As used herein, the term "plurality", particularly with respect to PSCs, refers to "at least 2", particularly at least 2 cells, at least 5 cells, at least 10 cells, or at least 100, or at least, or at least 1,000, or at least 10,000 cells.

[0065] As used herein, the term "pluripotent stem cells (PSCs)" refers to cells that can proliferate indefinitely and give rise to all other cell types in the body. The term explicitly includes both naive and primed pluripotent stem cells.

[0066] As used herein, the term "induced pluripotent stem cells (iPSCs)" refers to a type of pluripotent stem cell that can be generated directly from a somatic cell.

[0067] As used herein, the term "embryonic stem cell (ESC)" refers to a type of pluripotent stem cell derived blastocyst.

[0068] As used herein, the term "engineered tissue" as used herein refers to an association of cells in the X and Y planes that is a plurality of cells thick and forms at least one layer. In some embodiments, the engineered tissue comprises one layer. In other embodiments, the engineered tissue comprises multiple layers. In some embodiments, the layer forms a continuous, substantially continuous, or discontinuous cell layer of cells. In some aspects, the engineered tissue or its layer comprises a plurality of cells in the X, Y, and Z axes. The engineered tissue according to the teachings of the present invention may or may not include a cell adhesive material and / or a support matrix. According to some embodiments, the cell adhesive material and / or the support matrix forms a nanocarrier, a microcarrier, a macrocarrier, or a combination thereof. According to some embodiments, the cell adhesive material and / or the support matrix forms a scaffold.

[0069] The term "cultured meat" is used herein to describe meat grown from in vitro non-human animal cell culture, as distinguished from meat from slaughtered animals. Additional terms that can be used in the art to describe meat grown from in vitro non-human animal cell culture include cell-cultured meat, cultured meat, clean meat, laboratory-grown meat, test tube meat, in vitro meat, tube steak, synthetic meat, tissue-engineered meat, engineered meat, artificial meat, and artificial meat.

[0070] According to certain aspects, the present invention provides a method of producing a plurality of cells, including muscle-committed progenitor cells, comprising culturing a plurality of pluripotent stem cells (PSCs) in a culture medium comprising a combination of (i) at least one activator of the TGF-beta (TGF-β) signaling pathway and (ii) at least one inhibitor of the GSK3 signaling pathway, thereby producing a plurality of muscle-committed progenitor cells.

[0071] It should be clearly understood that both at least one activator of the TGF-β signaling pathway and at least one inhibitor of the GSK3 signaling pathway are present in the culture medium during the entire incubation time until skeletal muscle committed progenitor cells are formed.

[0072] TGF-β signaling is involved in many cellular functions, including cell growth, cell fate, and apoptosis. Signaling typically begins with the binding of TGF-β superfamily ligands to type II receptors, which recruit and phosphorylate type I receptors. Type I receptors then phosphorylate the SMAD family of transcription factors, which act as transcription factors in the nucleus and regulate target gene expression. TGF-β superfamily ligands include bone morphogenic proteins (BMPs), growth and differentiation factors (GDFs), anti-Mullerian hormone (AMH), activin, nodal, and TGF-β. In general, Smad2 and Smad3 are phosphorylated by ALK4, 5, and 7 receptors in the TGF-β / activin pathway. In contrast, Smad1, Smad5, and Smad8 are phosphorylated as part of the bone morphogenic protein (BMP) pathway. Although there is some crossover between the pathways, in the context of the present invention, activators of the TGF-β signaling pathway are preferably activators of the TGF-β pathway that act through Smad2 and Smad3.

[0073] According to certain embodiments, the activator of TGF-β signaling pathway is selected from the group consisting of, but not limited to, activin A, TGF-β, BMP2, BMP7, GDF9, NODAL, and any combination thereof. Each possibility represents a separate embodiment of the present invention. Any other activator of TGF-β signaling pathway suitable for the method of the present invention can also be applied.

[0074] According to certain exemplary embodiments, the activator of the TGF-β signaling pathway is activin A.

[0075] Glycogen synthase kinase 3 (GSK3) is highly conserved from yeast to mammals. Mammals express two GSK3 isoforms, α (51 kDa) and β (47 kDa), which are encoded by distinct genes and share 97% amino acid sequence identity within their catalytic domains. However, their sequences differ significantly outside of kinase domain 2. Both GSK3 isoforms appear to be ubiquitously expressed, and while they appear to be functionally redundant in some signaling pathways, including Wnt-β-catenin signaling, they perform distinct functions in other signaling pathways. Many studies have linked GSK3 dysregulation, particularly hyperactivation, to various pathological conditions, including diabetes mellitus, obesity, inflammation, neuropathy, and tumorigenesis.

[0076] According to certain embodiments, the inhibitor of the GSK3 signaling pathway is CHIR 99021 (C22H18Cl2N8) or a salt thereof, SB 216763, LY2090314, TWS119, tideglusib, GSK-3β inhibitor 1, GSK-3β inhibitor 2, GSK-3β inhibitor 3, AR-A014418, TDZD-8, kenpaullone, GSK3 inhibitor IX, cromolyn sodium, CHIR-98014, AZD1080, SB 415286, IM-12, 9-ING-41, indirubin-3'-monoxime, 1-azakempauron, BRD0705, AZD2858, CP21R7, BIO-acetoxime, bikinin, VP3.15, VP3.15 dihydrobromide, GNF4877, KY19382, SAR502250, A 1070722, (R)-BRD3731, BRD3731, BIP-135, 5-iodo-indirubin-3'-monoxime, BRD5648, GSK-3 inhibitor 1, GSK-3 / CDK5 / CDK2-IN-1, indirubin-3'-monoxime-5-sulfonic acid, GSK3β inhibitor flavonoid, lithium, and any combination thereof. Each possibility represents a separate embodiment of the present invention. Any other GSK3 inhibitor suitable in the methods of the present invention can also be applied.

[0077] According to certain embodiments, the CHIR-99021 salt is selected from the group consisting of CHIR-99021 monohydrochloride and CHIR-99021 trihydrochloride, Each possibility represents a separate embodiment of the present invention.

[0078] According to certain exemplary embodiments, the inhibitor of the GSK3 signaling pathway is CHIR-99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile, C 22 H 18 C 12 N8).

[0079] Flavonoids have been found to have inhibitory activity against GSK3β (e.g., Johnson LJ, et al., 2011. J Med Food 14(4):325-333; Jung Y et al., 2017. Appl Biol Chem 60(3):227-232). According to certain embodiments, the inhibitor of the GSK3 signaling pathway according to the teachings of the present invention is a GSK3β inhibitory flavonoid. According to some embodiments, the flavonoid is selected from the group consisting of luteolin, apigenin, quercetin, myricetin, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0080] According to certain exemplary embodiments, a culture medium for forming multiple skeletal muscle committed progenitor cells comprises a combination of Actin A as an activator of the TGF-β signaling pathway and CHIR-99021 as an inhibitor of the GSK3 signaling pathway.

[0081] It will be understood by those skilled in the art that the concentration of the effective amount of the activator of the TGF-β signaling pathway and the inhibitor of the GSK3 signaling pathway will depend on the specific type of agent used. Typically, the culture medium for forming multiple skeletal muscle committed progenitor cells lacks growth factors other than the growth factors that activate the TGF-β signaling pathway and the growth factors that inhibit the GSK3 signaling pathway. In alternative embodiments, such additional growth factors may be added to the medium for a limited time at a certain growth stage of the cells.

[0082] According to certain embodiments, the culture medium is serum-free. As used herein, the term "serum-free" with respect to medium refers to a medium that does not contain animal serum.

[0083] According to certain embodiments, the culture medium is free of animal-derived components. As used herein, the term "animal-derived component-free" in reference to a medium refers to a medium that does not contain any components of animal origin, in particular a medium that does not contain any components of mammalian origin.

[0084] According to certain embodiments, the culturing is performed under three-dimensional (3D) culture conditions. 3D cell culture is an artificially created environment in which biological cells can grow or interact with their surrounding cells. 3D cell culture allows the formation of self-assembled cell aggregates or clusters in in vitro growth that mimics the earliest in vivo developmental steps. According to certain exemplary embodiments, 3D culture typically includes cells grown in liquid suspension in a container.

[0085] The terms "vessel" or "tissue culture vessel" are used interchangeably herein and refer to any receptacle in which cells can be grown in suspension. Receptacles can be of various sizes, ranging from the few milliliter range (e.g., non-adherent plates or Erlenmeyer flasks) to the thousands of liter range (e.g., bioreactors or culture bags).

[0086] According to certain embodiments, suspension cultures are devoid of adhesive material and / or support matrix and the cells and / or cell clusters are freely suspended / floating in a liquid. According to certain embodiments, suspension cultures are maintained in vessels having walls of a material to which cells do not adhere.

[0087] When cells are grown in suspension culture without an adhesive material / support matrix, they tend to cluster with other cells to form cellular aggregates, which may be in the form of clusters, spheroids, organoids, etc.

[0088] According to certain embodiments, culturing is performed under conditions that form an adherent monolayer, also referred to herein as "two-dimensional (2D) culture." Any adhesive substrate / support matrix known to be used in cell culture can be used in accordance with the teachings of the present invention. Examples include growth plates coated with adhesive substances (e.g., inactivated feeder cells, organic extracellular matrices such as Matrigel or Vitronectin, or feeder cell conditioned medium), or hydrogels.

[0089] Pluripotent stem cells from any source known or to become known in the art can be used according to the teachings of the present invention. According to certain embodiments, the PSCs are derived from a human. According to additional or alternative embodiments, the PSCs are derived from a non-human animal. According to certain embodiments, the non-human animal is selected from the group consisting of an ungulate, a poultry, an aquatic animal, an invertebrate, and a reptile. According to certain embodiments, the ungulate is selected from the group consisting of a bovine, a sheep, a goat, an equine, a pig, a giraffe, a camel, a deer, a hippopotamus, or a rhinoceros. Each possibility represents a separate embodiment of the present invention. According to certain exemplary embodiments, the ungulate is a bovine.

[0090] According to some embodiments, the PSCs are embryonic stem cells (ESCs).

[0091] According to some embodiments, the PSCs are non-embryonic stem cells (ESCs).

[0092] According to certain embodiments, the PSCs are induced PSCs (iPSCs) reprogrammed from somatic cells.

[0093] According to certain embodiments, the PSCs are induced PSCs (iPSCs) reprogrammed from non-ESC-containing somatic cells.

[0094] Reprogramming of cells to produce iPSCs can be performed by any method known in the art, including, for example, those described in Bessi et al., 2021. Cells 10(6):1531; Kawaguchi et al., 2015. PLoS One 10(8):e0135403; Zhao et al., 2021. PNAS118(15):e2018505118; Poleganov et al., Hum. Gene Ther. 2015;26:751-766).

[0095] According to certain embodiments, isolation and / or culture of PSCs, in particular bovine PSCs, and / or reprogramming of cells to produce iPSCs can be performed by the methods described in International (PCT) Application Publication No. 2020 / 230138 to the applicant of the present invention.

[0096] Commercial preparations of bovine-derived PSCs are also available, including, for example, blastocyst-derived PSCs.

[0097] According to certain embodiments, at the time of seeding, the medium further comprises an inhibitor of Rho-associated protein kinase (Rock). Any Rock inhibitor currently known in the art or developed in the future can be used according to the teachings of the present invention. According to certain embodiments, the Rock inhibitor is selected from the group consisting of Thiazovivin, Fasudil, Ripasudil, Netarsudil, RKI-1447, Y-27632, GSK429286A, Y30141. Each possibility represents a separate embodiment of the present invention. According to certain exemplary embodiments, the Rock inhibitor is Y-27632 dihydrochloride (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide).

[0098] According to certain embodiments, culturing the plurality of PSCs is performed continuously in a medium comprising a combination of at least one activator of the TGF-β signaling pathway and at least one inhibitor of the GSK3 signaling pathway.

[0099] According to certain additional or alternative embodiments, culturing the plurality of PSCs is performed in cycles, and the medium comprising a combination of at least one activator of the TGF-β signaling pathway and at least one inhibitor of the GSK3 signaling pathway is replaced after each cycle. The combination of at least one activator of the TGF-β signaling pathway and at least one inhibitor of the GSK3 signaling pathway in each cycle may be the same or different.

[0100] According to certain embodiments, the culturing is performed for a period of time that allows for reaching about 10% to about 90% skeletal muscle committed progenitor cells out of the total number of cells.

[0101] According to certain embodiments, the time period that allows for reaching about 10% to about 90% skeletal muscle committed progenitor cells of the total number of cells is about 3 days to about 7 days. According to certain embodiments, the time period is about 3 days to 6 days, 3 days to 5 days, or 3 days to 4 days. Each possibility represents a separate embodiment of the invention. According to certain embodiments, the time period that allows for reaching about 10% to about 90% skeletal muscle committed progenitor cells of the total number of cells is 4 days.

[0102] This short time frame, especially of cultivation in a bioreactor, together with the option of using 3D culture, is a significant advantage of the method of the present invention over previously known methods for producing progenitor cells committed to skeletal muscle, enabling the use of the methods in large-scale production facilities, and in particular their use in the food industry for producing cultivated meat products, the costs of which must be reduced to allow the replacement of slaughtered meat.

[0103] According to certain embodiments, the present invention provides a plurality of cells comprising skeletal muscle committed progenitor cells produced as described herein above.

[0104] According to certain embodiments, the plurality of cells comprises about 10% to about 90% skeletal muscle committed progenitor cells of the total number of cells. According to some embodiments, the plurality of cells comprises about 15% to about 90%, about 25% to about 90%, about 30% to about 90%, about 40% to about 90%, about 45% to about 90%, or about 50% to about 90% skeletal muscle committed progenitor cells of the total number of cells.

[0105] According to still further certain embodiments, the plurality of cells further comprises at least one additional lineage committed progenitor cell. According to certain embodiments, the lineage committed progenitor cell is selected from the group consisting of stromal committed progenitor cells, adipocyte committed progenitor cells, and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0106] According to certain embodiments, the plurality of cells comprises between about 10% and about 90% stromal committed progenitor cells of the total number of cells. According to certain embodiments, the plurality of cells comprises between about 10% and about 90% adipocyte committed progenitor cells of the total number of cells.

[0107] According to certain embodiments, muscle-committed progenitor cells produced by the methods of the invention comprise at least one mesoderm marker selected from the group consisting of TBXT, TBX6, MSGN1, Pax3, and any combination thereof, and the at least one early myogenic marker is Six1. Each possibility represents a separate embodiment of the invention. According to certain embodiments, skeletal muscle-committed progenitor cells are characterized by expression of MSGN1 and Six1. Each possibility represents a separate embodiment of the invention.

[0108] According to certain embodiments, the plurality of cells comprising skeletal muscle committed progenitor cells essentially lacks PSCs. According to some embodiments, the plurality of cells comprising skeletal muscle committed progenitor cells essentially lacks PSCs.

[0109] According to certain embodiments, the plurality of cells, including skeletal muscle committed progenitor cells, is from a non-human animal. According to these embodiments, the plurality of cells is generated from non-human animal PSCs. The non-human animal is as described herein above. According to certain exemplary embodiments, the non-human animal is a bovine animal.

[0110] According to certain embodiments, the plurality of cells, including skeletal muscle committed progenitor cells, comprises at least one GSK3β inhibitory flavonoid or metabolite thereof. According to certain embodiments, the plurality of cells, including skeletal muscle committed progenitor cells, comprises at least one flavonoid selected from the group consisting of luteolin, apigenin, quercetin, myricetin, their metabolites, and any combination thereof. Each possibility represents a separate embodiment of the present invention. The amount of the at least one flavonoid or metabolite thereof can vary from one to a few nanomolar to hundreds of micromolar. According to some embodiments, the amount of the at least one flavonoid or metabolite thereof is 1 nM to 100 μM, or 1 nM to 10 μM, or 1 nM to 1 μM. Each possibility represents a separate embodiment of the present invention.

[0111] According to certain aspects, the present invention provides a method for producing a plurality of differentiated cells, including skeletal muscle cells, comprising: a) culturing a plurality of pluripotent stem cells (PSCs) in a culture medium comprising a combination of (i) at least one activator of the TGF-beta (TGF-β) signaling pathway and (ii) at least one inhibitor of the GSK3 signaling pathway, thereby forming a plurality of cells comprising skeletal muscle committed progenitor cells; b) depositing a plurality of cells, including skeletal muscle committed progenitor cells obtained in step (a), onto an adhesive material and / or a support matrix; c) culturing the plurality of cells in a differentiation medium that promotes differentiation of the skeletal muscle committed progenitor cells into skeletal muscle cells; thereby producing a plurality of differentiated cells including skeletal muscle cells.

[0112] The PSC, the activator of the TGF-β signaling pathway, the inhibitor of the GSK3 signaling pathway, and the culture medium, as well as the process of steps (a) and (b) are as described herein above.

[0113] According to certain embodiments, the plurality of cultured PSCs are not genetically modified. The method of the present invention allows for the production of a plurality of differentiated cells, including skeletal muscle cells, without any genetic manipulation throughout the entire process. According to these embodiments, the non-genetically modified skeletal muscle cells and tissues containing the same may have advantages in certain applications in the pharmaceutical and food industries.

[0114] According to certain alternative embodiments, the plurality of cultured PSCs are genetically modified. Using genetic engineering techniques can facilitate the formation of induced PSCs (iPSCs) from somatic cells, and can have the advantage of being embryonic stem cell independent, for example, without the need for specific embryonic mammalian stem cells.

[0115] Any method known or that becomes known in the art for depositing a plurality of cells, including skeletal muscle committed progenitor cells, onto an adhesive material and / or support matrix can be used in accordance with the teachings of the present invention.

[0116] According to certain embodiments, the adhesive material is selected from the group consisting of inactivated feeder cells, organic extracellular matrices such as Matrigel or Vitronectin, or feeder cell conditioned medium, each possibility representing a separate embodiment of the present invention.

[0117] According to certain embodiments, the adhesive material or support matrix is ​​in a form selected from the group consisting of nanocarriers, microcarriers, macrocarriers, scaffolds, tissue culture plates, tissue culture vessels, etc. Each possibility represents a separate embodiment of the present invention.

[0118] According to certain embodiments, the support matrix is ​​in a form selected from a semi-solid form and a solid form.

[0119] According to certain embodiments, deposition is performed by bioprinting using a suitable printer known in the art. According to some embodiments, bioprinting is performed as described in International (PCT) Application Publication No. 2022 / 162662.

[0120] Any differentiation medium known or to become known in the art for promoting differentiation of muscle-committed progenitor cells into skeletal muscle cells can be used in accordance with the teachings of the present invention. Advantageously, the present invention now discloses that a serum-free differentiation medium supplied with nutrients including, for example, vitamins, inorganic salts, amino acids, antioxidants, sugars, etc., and certain hormones, such as insulin, is sufficient for differentiation without the need for multiple expensive growth factors.

[0121] According to certain exemplary embodiments, the differentiation medium lacks activators of the TGF-β signaling pathway and inhibitors of the GSK3 signaling pathway. According to further exemplary embodiments, the differentiation medium lacks activin A and CHIR 99021.

[0122] According to certain embodiments, the cells are cultured in the differentiation medium for about 3 days to about 27 days. According to some embodiments, the cells are cultured in the differentiation medium for about 3 days to about 26 days, about 25 days, about 24 days, about 23 days, about 22 days, about 22 days, about 20 days, about 19 days, about 18 days, about 17 days, about 16 days, about 15 days, about 14 days, about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, or about 6 days. Each possibility represents a separate embodiment of the invention. According to some embodiments, the cells are cultured in the differentiation medium for about 3 days to about 15 days, or about 5 days to about 10 days, or about 6 days to about 9 days. According to some embodiments, the cells are cultured in the differentiation medium for about 7 days.

[0123] Regarding the time required to obtain skeletal muscle-differentiated progenitor cells according to the present invention, the time to differentiate progenitor cells into muscle cells is also shortened compared to previously known methods. According to certain embodiments, the total time to obtain engineered tissue containing muscle cells is about 6 to about 30 days. According to certain embodiments, the total time to obtain engineered tissue containing muscle cells is about 7 to about 25 days, about 8 to about 24 days, about 9 to about 23 days, about 10 to about 22 days, about 10 to about 21 days, about 10 to about 21 days, about 10 to about 21 days, about 10 to about 20 days, about 10 to about 19 days, about 10 to about 18 days, about 10 to about 17 days, about 10 to about 16 days, or about 10 to about 15 days.

[0124] According to certain embodiments, the total time to obtain engineered tissue containing muscle cells is about 11 to about 14 days.

[0125] This overall period, up to about 30 days, typically 11-14 days, is highly advantageous in the large-scale production of engineered tissues, including muscle cells.

[0126] According to certain embodiments, the plurality of cells deposited on the adhesive material and / or support matrix further comprises at least one additional lineage committed progenitor cell. According to some embodiments, the lineage committed progenitor cells are selected from stromal committed progenitor cells, adipocyte committed progenitor cells, and combinations thereof. Each possibility represents a separate embodiment of the invention. According to these embodiments, the formed plurality of cells further comprises at least one of stromal cells and adipocytes.

[0127] According to some embodiments, the stromal cells are extracellular matrix (ECM) producing cells.

[0128] According to certain embodiments, the method of the present invention further comprises separating the plurality of differentiated cells, including muscle cells and optionally at least one of stromal cells and adipocytes, from the adhesive material and / or support matrix.

[0129] According to certain embodiments, the present invention further provides a plurality of differentiated cells, including differentiated skeletal muscle cells, produced by the methods of the present invention.

[0130] According to certain embodiments, the differentiated skeletal muscle cells produced by the methods of the invention are characterized by expression of at least one myogenic marker selected from the group consisting of Myf5, Pax7, MEF2C, SIX1, NYOD1, MYOG, MYH3, MYH7, NYH8, MB, MYMK, and any combination thereof, each possibility representing a separate embodiment of the invention.

[0131] According to certain embodiments, the plurality of differentiated cells comprises about 10% to about 90% skeletal muscle cells of the total number of cells. According to some embodiments, the plurality of differentiated cells comprises about 15% to about 90%, about 25% to about 90%, about 30% to about 90%, about 40% to about 90%, about 45% to about 90%, or about 50% to about 90% skeletal muscle cells of the total number of cells. According to certain embodiments, the plurality of differentiated cells comprises about 30% skeletal muscle cells of the total number of cells. According to certain embodiments, the skeletal muscle cells are viable cells.

[0132] According to certain embodiments, the plurality of differentiated cells comprises about 10% to about 90% stromal cells of the total number of cells. According to some embodiments, the plurality of differentiated cells comprises about 15% to about 90%, about 25% to about 90%, about 30% to about 90%, about 40% to about 90%, about 45% to about 90%, or about 50% to about 90% stromal cells of the total number of cells. According to certain embodiments, the stromal cells are viable cells.

[0133] According to certain embodiments, the plurality of differentiated cells comprises about 10% to about 90% adipocytes of the total number of cells. According to some embodiments, the plurality of differentiated cells comprises about 15% to about 90%, about 25% to about 90%, about 30% to about 90%, about 40% to about 90%, about 45% to about 90%, or about 50% to about 90% adipocytes of the total number of cells. According to certain embodiments, the adipocytes are viable cells.

[0134] According to certain embodiments, the plurality of differentiated cells comprises a detectable amount of at least one GSK3β inhibitory flavonoid or metabolite thereof, as described herein above.

[0135] According to certain embodiments, the plurality of differentiated cells, including muscle cells and optionally at least one of stromal cells and adipocytes, form an engineered tissue. According to certain embodiments, the engineered tissue comprises an adhesive material and / or a support matrix used in the production of the plurality of differentiated cells. According to certain additional or alternative embodiments, the engineered tissue is devoid of an adhesive material and / or a support matrix used in the production of the plurality of differentiated cells.

[0136] According to still further certain aspects, the present invention provides a plurality of in vitro grown cells comprising skeletal muscle committed progenitor cells, the skeletal muscle committed progenitor cells characterized by expression of at least one mesodermal marker and / or at least one early myogenic marker.

[0137] According to certain embodiments, the at least one mesoderm marker is selected from the group consisting of TBXT, TBX6, MSGN1, Pax3, and any combination thereof, and the at least one early myogenic marker is Six1. Each possibility represents a separate embodiment of the present invention.

[0138] According to certain embodiments, skeletal muscle committed progenitor cells are characterized by expression of MSGN1 and Six1. Each possibility represents a separate embodiment of the present invention.

[0139] According to certain embodiments, the plurality of in vitro grown cells comprising skeletal muscle committed progenitor cells further comprises at least one additional lineage committed cell selected from the group consisting of stromal committed progenitor cells, adipocyte committed progenitor cells, and combinations thereof, each possibility representing a separate embodiment of the present invention.

[0140] According to certain embodiments, the plurality of in vitro grown cells comprising skeletal muscle committed progenitor cells comprises at least one GSK3β inhibitory flavonoid and / or metabolite thereof, the flavonoid and mount being as described herein above.

[0141] According to certain additional aspects, the present invention provides a plurality of in vitro grown differentiated cells comprising differentiated skeletal muscle cells, the differentiated skeletal muscle cells being characterized by expression of at least one myogenic marker.

[0142] According to certain embodiments, the at least one myogenic marker is selected from the group consisting of Myf5, Pax7, MEF2C, SIX1, NYOD1, MYOG, MYH3, MYH7, NYH8, MB, MYMK, and combinations thereof, each possibility representing a separate embodiment of the present invention.

[0143] According to certain embodiments, the plurality of in vitro grown and differentiated cells further comprises at least one of stromal cells, adipocytes, or a combination thereof, Each possibility represents a separate embodiment of the present invention.

[0144] According to certain embodiments, the stromal cells are extracellular matrix (ECM) producing cells.

[0145] According to certain embodiments, the cells in the plurality of in vitro grown and differentiated cells are non-human animal cells.

[0146] The present invention further includes an engineered tissue comprising a plurality of in vitro grown and differentiated cells, including skeletal muscle cells.

[0147] The muscle-committed progenitor cells, differentiated cells, and engineered tissues containing them produced by the methods of the present invention are suitable for a variety of uses, including therapeutic applications and use as foods.

[0148] As described herein above, the media used, the time required to obtain a plurality of differentiated cells, including skeletal muscle cells, and the overall effectiveness make the method of the present invention highly suitable for use in the food industry, and in particular for use in the technology of producing cell-cultured meat. Thus, the present invention specifically encompasses cultured meat and cultured meat products comprising the skeletal muscle cells of the present invention and / or engineered tissues comprising same.

[0149] According to these embodiments, the engineered tissue or cultured food optionally further comprises at least one plant protein. According to certain embodiments, the engineered tissue and / or cultured meat further comprises at least one additional food-safe supplement. Suitable additional supplements include, but are not limited to, saturated and / or unsaturated fatty acids, lipids, flavorings, colorants, texturants, edible fibers, and the like. Each possibility represents a separate embodiment of the present invention.

[0150] The following examples are presented to more fully illustrate some embodiments of the present invention. However, they should in no way be construed as limiting the broad scope of the present invention. Those skilled in the art can easily devise many variations and modifications of the principles disclosed herein without departing from the scope of the present invention. EXAMPLES

[0151] Materials and Methods RNA extraction and RT-PCR analysis: For gene expression analysis, RNA was extracted from bovine pluripotent stem cells, bovine committed cells, and skeletal muscle cells using the NucleoSpin® RNA Purification Kit (Macherey-Nagel), and RNA concentrations were determined using Nanodrop One C (ThermoFisher). Purified RNA was reverse transcribed using the ReverseAid First strand cDNA Synthesis Kit (ThermoFisher) and a Thermal Cycler SimpliAmp device (ThermoFisher). cDNA was subjected to RT-PCR analysis using TaqMan Fast Advanced Master Mix (ThermoFisher) and the subsequent list of primers listed in Table 1 using a QuantStudio 5 real-time PCR device (ThermoFisher). [Table 1]

[0152] Immunofluorescence Bovine skeletal muscle cells in 2D culture were subjected to immunofluorescence analysis as follows: cells were permeabilized using 4% paraformaldehyde (PFA; Santa Cruz Biotechnology), 0.5% Triton X-100 (Sigma) and blocked using 5% BSA (MP biomedicals). Cells were incubated with the primary antibody, anti-myosin MF-20 (DSHB) 1:75, followed by secondary goat anti-mouse IgG H&L Alexa Fluor 594) antibody (Abcam ab150116) 1:500. Nuclei were stained using 0.5 μg / ml DAPI (Sigma). Imaging was performed using EVOS FL Auto 2 Fluorescent Microscopy (ThermoFisher).

[0153] Example 1: Generation of multiple skeletal muscle committed cells Bovine pluripotent stem cells (PSCs) were grown in 2D or 3D culture. Cells were harvested, dissociated, and resuspended in serum-free growth medium as a control or in serum-free growth medium containing Activin A (20 ng / ml) and CHIR-99021 (10 μM) (skeletal muscle progenitor medium) for 4 days. Rock inhibitor (10 μM) was added during growth on day 1 under both control and assay conditions. Figure 1 shows representative brightfield images of skeletal muscle committed progenitor cells obtained from bovine PSCs after 4 days.

[0154] After 4 days, cells were harvested and subjected to RT-PCR analysis as described herein above to examine the expression of mesodermal markers TBXT, TBX6, MSGN1, and Pax3, and the early myogenic marker Six1.

[0155] As shown in Figure 2, we observed a marked decrease in expression of pluripotency markers OCT4 and Nanog, and a marked increase (>10-fold compared to PSCs) of mesodermal markers TBXT, TBX6, MSGN1, and Pax3 and the early myogenic marker Six1. There was no significant upregulation of fully differentiated muscle satellite markers Myf5 and Pax7 at this early time point.

[0156] These results clearly demonstrate that culturing PSCs, specifically bovine PSCs, in basal serum-free medium supplemented with a combination of a TGF-β activator (Activin A) and a GSK3 inhibitor (CHIR-99021) induced the differentiation process into skeletal muscle cells. After a relatively short period of time, about 4 days, the cells expressed mesodermal and early myogenic markers at levels that identified the cells as cells committed to skeletal muscle.

[0157] Example 2: Differentiation of bovine pluripotent stem cells into skeletal muscle cells Bovine pluripotent stem cells (PSCs) were grown in 2D and / or 3D culture. Cells were dissociated and resuspended in serum-free growth medium containing Activin A (20 ng / ml) and CHIR99021 (10 μM) (skeletal muscle progenitor medium), in the same serum-free growth medium containing only CHIR99021 (10 μM), or in the same serum-free growth medium containing TGFβ (2 ng / ml) and CHIR-99021 (10 μM) and cultured under suspension culture conditions. Rock inhibitor (RI, 10 μM) was added to all media types.

[0158] Suspension culture conditions ("3D culture conditions") included ultra-low adherent (ULA) 6-well plates (Corning) placed under rotation at 38.5°C, 5% CO2 and >75% humidity. Skeletal muscle progenitor medium (no RI) was used to refresh the medium and the formed aggregates were allowed to differentiate for a total of 4 days.

[0159] Aggregates containing cells committed to skeletal muscle were harvested and seeded onto tissue culture vitronectin (0.005 mg / ml) pre-coated plates in serum-free growth medium ("2D conditions"). Plates were incubated at 38.5°C, 5% CO2 and >75% humidity for an additional 7 days (total process 11 days), with medium changed on days 1 and 3 (no RI).

[0160] Representative immunofluorescence staining of myosin heavy chain (using MF-20 antibody as described herein above) after a total of 11 days of differentiation showed the presence of skeletal muscle cells in cultures subjected to medium containing a combination of Activin A and CHIR-99021 (FIG. 3B), and to a somewhat lesser extent in cultures subjected to medium containing a combination of TGFβ, an additional activator of the TGF-beta (TGF-β) signaling pathway, and CHIR-99021 (FIG. 3C). In contrast, no myosin-positive cells were observed in cultures subjected to medium containing only CHIR-99021 (FIG. 3A).

[0161] These results were further supported by RT-PCR analysis of myogenic markers Myf5, Pax7, Mef2C, Six1, MyoD1, MyoG, MYH3, MYH7, MYH8, myoglobin (MB), and myomarker (Myomaker, MYMK) after a total of 11 days of differentiation. All markers were upregulated, indicating differentiation into skeletal muscle (Figure 4).

[0162] In further experiments, PSCs were resuspended in serum-free growth medium containing activin A (20 ng / ml) and CHIR99021 (10 μM) and cultured in 2D culture conditions in tissue culture plates placed at 38.5 °C, 5% CO2 and >75% humidity. The medium was refreshed using skeletal muscle precursor medium (without RI) and the formed aggregates were allowed to differentiate for a total of 4 days. After 4 days, cells containing cells committed to skeletal muscle were harvested and seeded in 2D culture conditions as described for the aggregates obtained from the 3D culture conditions above. Figure 3D shows that there is no significant difference between culturing PSCs in 2D or 3D culture conditions at the commitment induction stage with regard to the further differentiation of committed cells into skeletal muscle cells.

[0163] The foregoing description of the specific embodiments fully reveals the general nature of the present invention, so that others can easily modify and / or adapt such specific embodiments to various uses by applying current knowledge without undue experimentation and without departing from the general concept, and therefore such adaptations and modifications should be understood within the meaning and scope of equivalents of the embodiments of the present disclosure, and are intended to be so. It should be understood that the expressions or terms used herein are for the purpose of description and not for the purpose of limitation. The means, materials, and steps for carrying out the various functions of the present disclosure may take various alternative forms without departing from the present invention.

Claims

1. 1. A method for producing a plurality of cells comprising skeletal muscle-committed progenitor cells, the method comprising: culturing a plurality of pluripotent stem cells (PSCs) in a culture medium comprising a combination of (i) at least one activator of the TGF-beta (TGF-β) signaling pathway and (ii) at least one inhibitor of the GSK3 signaling pathway, thereby producing a plurality of cells comprising skeletal muscle-committed progenitor cells.

2. Culturing is performed under three-dimensional (3D) culture conditions; 2. The method of claim 1, wherein the 3D culture is selected from the group consisting of a 3D culture lacking an adhesive material and / or a support matrix and a 3D culture comprising at least one adhesive material and / or a support matrix.

3. 2. The method of claim 1, wherein the culturing is performed under two-dimensional (2D) culture conditions comprising at least one adhesive material and / or support matrix.

4. 2. The method of claim 1, wherein the at least one activator of the TGF-β signaling pathway is selected from the group consisting of activin A, TGF-β, BMP2, BMP7, GDF9, NODAL, and any combination thereof.

5. The at least one inhibitor of the GSK3 signaling pathway is selected from the group consisting of CHIR-99021 (C22H18Cl2N8) or a salt thereof, SB 216763, LY2090314, TWS119, tideglusib, GSK-3β inhibitor 1, GSK-3β inhibitor 2, GSK-3β inhibitor 3, AR-A014418, TDZD-8, kenpaullone, GSK3 inhibitor IX, cromolyn sodium, CHIR-98014, AZD1080, SB 415286, IM-12, 9-ING-41, indirubin-3'-monoxime, 1-azakempaulon, BRD0705, AZD2858, CP21R7, BIO-acetoxime, bikinin, VP3.15, VP3.15 dihydrobromide, GNF4877, KY19382, SAR502250, A 1070722, (R)-BRD3731, BRD3731, BIP-135, 5-iodo-indirubin-3'-monoxime, BRD5648, GSK-3 inhibitor 1, GSK-3 / CDK5 / CDK2-IN-1, indirubin-3'-monoxime-5-sulfonic acid, GSK3β inhibitor flavonoids, lithium, and any combination thereof.

6. 2. The method of claim 1, wherein the combination comprises activin A and CHIR-99021. (i) culturing the plurality of PSCs continuously in the medium containing the combination of at least one activator of the TGF-β signaling pathway and at least one inhibitor of the GSK3 signaling pathway; and (ii) culturing the plurality of PSCs is performed in cycles, and the medium containing the combination of at least one activator of the TGF-β signaling pathway and at least one inhibitor of the GSK3 signaling pathway is replaced after each cycle; The method of claim 1 , wherein at least one of

8. 10. The method of claim 1, wherein the plurality of cells produced further comprises at least one additional lineage-committed progenitor cell.

9. 10. The method of claim 1, wherein the PSCs are of an origin selected from the group consisting of a non-human animal and a human.

10. 1. A method for producing a plurality of differentiated cells comprising skeletal muscle cells, said method comprising: a. depositing a plurality of cells, including skeletal muscle committed progenitor cells produced by the method of claim 1, onto an adhesive material and / or support matrix; b. culturing the plurality of cells in a differentiation medium that promotes differentiation of the skeletal muscle-committed progenitor cells into skeletal muscle cells; thereby producing a plurality of differentiated cells comprising skeletal muscle cells.

11. The method of claim 10, wherein the differentiation medium lacks activators of the TGF-β signaling pathway and inhibitors of the GSK3 signaling pathway.

12. 11. The method of claim 10, wherein the total time period for obtaining the plurality of differentiated cells comprising skeletal muscle cells from PSCs is from about 6 days to about 30 days.

13. 11. The method of claim 10, wherein the plurality of differentiated cells further comprises at least one additional cell type selected from the group consisting of stromal cells and adipocytes.

14. 10. A plurality of cells comprising skeletal muscle committed progenitor cells produced by the method of claim 1.

15. 15. The plurality of cells of claim 14, wherein the skeletal muscle-committed progenitor cells are produced from non-human animal PSCs.

16. 11. A plurality of differentiated cells comprising skeletal muscle cells produced by the method of claim 10.

17. 17. The plurality of differentiated cells of claim 16, wherein the plurality of differentiated cells further comprises at least one type of cell selected from the group consisting of stromal cells, adipocytes, and combinations thereof.

18. 18. The plurality of differentiated cells of claim 17, wherein the stromal cells comprise collagen-producing cells.

19. 17. An engineered tissue comprising a plurality of cells according to claim 16.

20. 17. A cultured food product comprising a plurality of differentiated cells of claim 16 and / or engineered tissues comprising the same.

21. 21. The cultured food product of claim 20, wherein the cultured food product is cultured meat.

22. A plurality of in vitro grown cells comprising skeletal muscle committed progenitor cells, wherein said skeletal muscle committed progenitor cells are characterized by expression of at least one mesodermal marker and / or at least one early myogenic marker.

23. 23. The plurality of in vitro grown cells of claim 22, wherein the plurality of cells comprises at least one GSK3β-inhibiting flavonoid and / or metabolite thereof.

24. A plurality of in vitro grown and differentiated cells comprising skeletal muscle cells, said skeletal muscle cells characterized by expression of at least one myogenic marker.

25. 25. The plurality of in vitro grown and differentiated cells of claim 24, wherein the plurality of cells further comprises at least one of stromal cells, adipocytes, or a combination thereof.

26. 23. The plurality of in vitro grown and differentiated cells of claim 22, wherein the cells are non-human animal cells.

27. 25. An engineered tissue comprising a plurality of in vitro grown and differentiated cells according to claim 24.

28. 27. A cultured food product comprising a plurality of in vitro grown and differentiated cells of claim 26 and / or engineered tissues comprising same.

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