A method for the self-organized production of vascularized skeletal muscle from ungulate embryonic stem cells

Abstract

The present invention relates to an in vitro method for the production of ungulate myocytes and muscle fibers from suitable ungulate stable ESCs based on an improved protocol in a suitable culture medium free from fetal bovine serum. The method further provides a 3D culture with self-organization of muscle fibers and vascular-like networks with innervation. The present invention furthermore relates to the method for producing a comestible meat product, a comestible meat product produced according to the invention, and respective uses thereof.

Description

[0001] A METHOD FOR THE SELF-ORGANIZED PRODUCTION OF VASCULARIZED

[0002] SKELETAL MUSCLE FROM UNGULATE EMBRYONIC STEM CELLS

[0003] The present invention relates to an in vitro method for the production of ungulate myocytes and muscle fibers from suitable ungulate stable ESCs based on an improved protocol in a suitable culture medium free from fetal bovine serum. The method further provides a 3D culture with self-organization of muscle fibers and vascular-like networks with innervation. The present invention furthermore relates to the method for producing a comestible meat product, a comestible meat product produced according to the invention, and respective uses thereof.

[0004] Background of the invention

[0005] Cultured meat, also referred to as cell-based meat, lab-grown meat, or clean meat, is produced through culturing animal cells in vitro, and it has the potential to address ethical, environmental, and health concerns associated with conventional meat production from livestock (1-4).

[0006] Current practices in cultured beef production predominantly rely on muscle satellite cells and mesenchymal stem cells (MSCs) isolated from adult bovine tissue biopsy or bone marrow samples (4-6).

[0007] The proliferation capacity of these adult stem cells diminishes overlong-term culture. Although they can be immortalized (7), the genetic modifications required for immortalization may present a challenge in obtaining food authorization. The differentiation capacity of adult stem cells is also limited. For instance, satellite cells are committed to muscle fate; although MSCs can form osteoblasts, adipocytes, and chondrocytes in addition to myocytes, the differentiation trajectory of MSCs is still restricted and dependent on the tissue source from which they are isolated. By contrast, pluripotent stem cells, namely embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) proliferate indefinitely, offering a more stable cell source for cultured meat. Pluripotent stem cells can also differentiate into cells of all three germ layers: mesoderm, ectoderm, and endoderm. Unmodified ESCs are more favorable for food production than iPSCs that need genetic reprogramming. The derivation of stable bovine ESCs was historically challenging but recently achieved (8), and a large-scale culture of bovine ESCs in stirred bioreactors has been reported (9).

[0008] Bogliotti, Y. S. etal. (in: Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts. Proc. Natl. Acad. Sci. 115, 2090-2095 (2018)) discloses embryonic stem cells (ESCs) that are derived from the inner cell mass of preimplantation blastocysts. From agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates is important for genomic testing and selection, genome engineering, and modeling human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors. To date, however, it remains a challenge to produce stable pluripotent bovine ESC lines. Employing a culture system containing fibroblast growth factor 2 and an inhibitor of the canonical Wnt-signaling pathway, the inventors derived pluripotent bovine ESCs (bESCs) with stable morphology, transcriptome, karyotype, population-doubling time, pluripotency marker gene expression, and epigenetic features. Under this condition bESC lines were efficiently derived (100% in optimal conditions), were established quickly (3-4 wk), and were simple to propagate (by trypsin treatment). When used as donors for nuclear transfer, bESCs produced normal blastocyst rates, thereby opening the possibility for genomic selection, genome editing, and production of cattle with high genetic value.

[0009] Another challenge in the cultured meat field is how to produce structured steaks instead of ground meat. The incorporation of diverse cell types within the muscle tissue holds the key to more accurate recreation of steaks (10, 11). For in vitro tissues to keep growing, oxygen and nutrient supply through blood vessels or alternative porous structures is also crucial (12). The current manufacturing strategy for cultured beef steaks involves the separate preparation of distinct cell types followed by their assembly. This assembly process necessitates advanced engineering techniques, such as bioprinting purified cells with a bioink, co-culturing cells on edible porous scaffolds, and stacking myocyte-laden hydrogel modules (13-16).

[0010] Additionally, electrical or mechanical stimuli are often applied to facilitate muscle maturation and improve the texture (16-21). While these engineering approaches are promising and evolving rapidly, they add extra manufacturing steps to cultured steak production. A potential alternative approach is utilizing pluripotent stem cells and leveraging their differentiation and self-organization abilities that have been demonstrated by the development of a variety of complex organoids in recent years (22-28). WO 2013 / 016547A2 relates to engineered meat products formed as a plurality of at least partially fused layers, wherein each layer comprises at least partially fused multicellular bodies comprising non-human myocytes and wherein the engineered meat is comestible. Also provided are multicellular bodies comprising a plurality of non-human myocytes that are adhered to and / or cohered to one another; wherein the multicellular bodies are arranged adjacently on a nutrient- permeable support substrate and maintained in culture to allow the multicellular bodies to at least partially fuse to form a substantially planar layer for use in formation of engineered meat. Further described are methods of forming engineered meat utilizing said layers.

[0011] EP2736357B9 relates to a method of forming engineered meat, the method comprising: preparing a plurality of multicellular bodies comprising a plurality of non-human myocytes cohered to one another; laying more than one multicellular body adjacently onto a planar support substrate; fusing said multicellular bodies at least partially together to form a first layer; stacking more than 50 additional layers onto the first layer; fusing the stacked layers to form a volume of engineered meat; and culturing the stacked layers to fuse the layers while the layers in an inner region of the volume die such that the majority of cells in the volume have died after fusion between the layers is at least partially complete, and wherein the engineered meat is comestible.

[0012] WO 2019 / 140260 provides ungulate embryonic stem cells (uESCs) derived from the inner cell mass of pre-implantation blastocysts or pluripotent cells from embryos. From an agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates is important for genomic testing and selection, genetic engineering, and providing an experimental tool for studying human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors. Specifically, provided is a method for producing stable uESC, comprising culturing an ungulate blastocyst cell or a pluripotent cell isolated from an embryo in a cell culture media comprising: (i) inactivated feeder cells; (ii) an effective amount of Fibroblast Growth Factor 2 (FGF2) or an equivalent thereof; and (iii) an effective amount of one or more inhibitors of Wnt signaling, such as IWR1.

[0013] WO 2020 / 230138A1 relates to producing aggregates of non-genetically modified non-human- animal- derived pluripotent stem cells (PSCs) comprising the steps of: seeding at least one PSC in an expansion medium, the expansion medium is a serum-free liquid medium comprising a combination of the growth factor bFGF and at least one of (i) at least one additional growth factor and (ii) at least one small molecule selected from the group consi sting of an inhibitor of the Wnt-P-catenin signaling pathway, to form a seeding suspension culture; and growing the suspension culture under conditions enabling aggregate formation and aggregate expansion, thereby forming homogenous aggregates of said PSCs. These aggregates of bovine-derived pluripotent stem cells are used in cell grown meat cultures and in production of cell-based meat products.

[0014] Wu J, et al. (An alternative pluripotent state confers interspecies chimaeric competency. Nature. 2015 May 21;521(7552):316-21. doi: 10.1038 / naturel4413. Epub 2015 May 6. PMID: 25945737; PMCID: PMC5278765) discloses that pluripotency, the ability to generate any cell type of the body, is an evanescent attribute of embryonic cells. Transitory pluripotent cells can be captured at different time points during embryogenesis and maintained as embryonic stem cells or epiblast stem cells in culture. Since ontogenesis is a dynamic process in both space and time, it seems counterintuitive that these two temporal states represent the full spectrum of organismal pluripotency. Here the inventors show that by modulating culture parameters, a stem-cell type with unique spatial characteristics and distinct molecular and functional features, designated as region-selective pluripotent stem cells (rsPSCs), can be efficiently obtained from mouse embryos and primate pluripotent stem cells, including humans. The ease of culturing and editing the genome of human rsPSCs offers advantages for regenerative medicine applications. The unique ability of human rsPSCs to generate post-implantation interspecies chimaeric embryos may facilitate the inventor’s understanding of early human development and evolution.

[0015] WO 2019 / 016795 Al is directed to a method for producing an edible composition, comprising incubating a three-dimensional porous scaffold and a plurality of cell types comprising: myoblasts or progenitor cells thereof, at least one type of extracellular (ECM)-secreting cell and endothelial cells or progenitor cells thereof, and inducing myoblasts differentiation into myotubes. Thus, multiple cell types are combined in order to produce a steak-like tissue.

[0016] WO 2013 / 030243A1 relates to an ex vivo method for preparing induced paraxial mesoderm progenitor (iPAM) cells, said method comprising the step of culturing pluripotent cells in an appropriate cul ture m edium comprising an effective am ount of an acti vator of the Wnt signali ng pathway and an effective amount of an inhibitor of the Bone Morphogenetic Protein (BMP) signaling pathway. The disclosure seeks to develop better ES and iPS cell differentiation strategies to produce muscle cells and paraxial mesoderm derived lineages for the development of applications in regenerative medicine.

[0017] David, S., et al. (in: Co-culture approaches for cultivated meat production. Nat Rev Bioeng 1, 817-831 (2023). https: / / doi.org / 10.1038 / s44222-023-00077-x) outline how co-culture approaches commonly used in biomedical tissue engineering can be applied to produce cultured meat. They discuss the relevant cell types and cell sources and examine different co-culture approaches for skeletal muscle and adipose tissue engineering. Finally, they discuss the application of such approaches for animal-free meat production, highlighting their potential to reduce cultured meat production costs, improve the organoleptic properties of cultured meat and increase tissue thickness.

[0018] Guo, X., et al. (in: 3D Bioprinting of Cultured Meat: A Promising Avenue of Meat Production. Food Bioprocess Technol (2023). https: / / doi.org / 10.1007 / sl l947-023-03195-x) summarize existing methods of cultured meat production, especially 3D bioprinting of cultured meat, which is an emerging approach with unique advantages.

[0019] Bomkamp C, et al. (in: Scaffolding Biomaterials for 3D Cultivated Meat: Prospects and Challenges. Adv Sci (Weinh). 2022 Jan;9(3):e2102908. doi: 10.1002 / advs.202102908. Epub 2021 Nov 16. PMID: 34786874; PMCID: PMC8787436) discusses the properties of vertebrate skeletal muscle that will need to be replicated in a successful product and the current state of scaffolding innovation within the cultivated meat industry, highlighting promising scaffold materials and techniques that can be applied to cultivated meat development.

[0020] Conventional cultured beef uses satellite cells isolated from adult bovine biopsy tissues. Although those bovine satellite cells proliferate, their proliferation is not infinite. Also, the differentiation capacity of satellite cells is limited - they don’t differentiate into blood vessels (that may help make larger tissues) or neurons (that potentially stimulate the maturation of muscle tissues).

[0021] In contrast, pluripotent stem cells, namely ESCs and induced pluripotent stem cells (iPSCs), can proliferate indefinitely, offering a more stable cell source for cultured meat. Pluripotent stem cells can also differentiate into almost all cell types theoretically, including blood vessel cells and neuronal cells. ESCs are suitable for food production as they are not genetically modified, unlike iPSCs.

[0022] The induction of muscle fibers from bovine satellite cells in 2D culture is already well established. Those muscle fibers can be used as ‘ground meat’ but not as a structured beef ‘steak’. To create a more realistic meat tissue, several groups co-cultured satellite cells and other cell types isolated from adult bovine tissues in 3D structures using either bioprinting or artificial extracellular matrices (Zagury Y. et al. Engineered marble-like bovine fat tissue for cultured meat. Commun Biol 5, 927 (2022). https: / / doi.org / 10.1038 / s42003-022-03852-5; Furuhashi, M. et al. Formation of contractile 3D bovine muscle tissue for construction of millimetre-thick cultured steak, npj Sci Food 5, 6 (2021). https: / / doi.org / 10.1038 / s41538-021- 00090-7; Tanaka, Ri. et al. Production of scaffold-free cell-based meat using cell sheet technology, npj Sci Food 6, 41 (2022). https: / / doi.org / 10.1038 / s41538-022-00155-l).

[0023] Another group recently reported an even more realistic steak-like tissue (Dong-Hee Kang et al. Engineered whole cut meat-like tissue by the assembly of cell fibers using tendon-gel integrated bioprinting. Nat Commun 12, 5059 (2021). https: / / doi.org / 10.1038 / s41467-021-25236-9) by bioprinting three cell types (satellite cells, adipose-derived stem cells, and vascular epithelial cells differentiated from adipose-derived stem cells) simultaneously. However, these printed meat tissues need multiple cell sources. And they were artificially assembled while in natural development different types of cells self-organize into a tissue.

[0024] There seems to be no scientific publication where bovine ESCs were used for skeletal muscle cell induction or cultured meat. An Israel-based company, Aleph farms, published the large- scale culture method of bovine ESCs (WO 2020 / 230138A1; Zehorai E. From fertilised oocyte to cultivated meat - harnessing bovine embryonic stem cells in the cultivated meat industry. Reproduction, Fertility, and Development (2023) https: / / d / oi.org / 10.1071 / RD23169, no muscle differentiation reported) and claims that they actually use bovine ESCs to make cultured steakcuts (Aleph Farms: https: / / aleph-farms.com / the inventor’ s-recipe / ).

[0025] In summary, still the problems associated with conventional methods for cultured beef are: 1) living bovine tissues are necessary for the isolation of satellite cells or other cell types, 2) satellite cells cannot differentiate into broad cell types such as blood vessels, and 3) different cell types need to be artificially printed / assembled to form a tissue-like structure.

[0026] It is therefore an object of the present invention to provide a method for providing cultured meat, such as beef that overcomes these problems. Other objects and advantages will readily become apparent for the person of skill from studying the following more detailed description and examples.

[0027] In a first aspect of the present invention, the problem of the present invention is solved by providing an in vitro method for the production of ungulate myocytes and muscle fibers, comprising the steps of i) providing a suitable ungulate stable-ESC line in a suitable culture medium free from fetal bovine serum; ii) contacting the uESC line with suitable concentrations of at least one WNT inducer / activator, bFGF, at least one TGFb inhibitor, and at least one BMP inhibitor for about 2 days to induce presomitic mesoderm cells (PSMs); iii) contacting the PSMs with suitable concentrations of HGF, IGF, and bFGF for about 4 days to induce myoblasts and iv) contacting the myoblasts with suitable concentrations of HGF and IGF for about 9 days to induce myocytes and muscle fibers. Preferred is a method according to the present invention, wherein the overall method does not exceed about 15 days from the start of the contacting in step ii).

[0028] In the context of the present invention, the inventors developed a skeletal muscle induction protocol from ungulate, such as bovine, ESCs by modifying and combining elements from a human muscle induction protocol. Nevertheless, the combination of the growth factors, small compounds, administration timings lead to completely new and surprising effects.

[0029] In a second aspect of the present invention, the problem of the present invention is solved by the method according to the present invention, wherein the myocytes and muscle fibers form a tissue furthermore comprising neuronal cells derived from neuromesodermal progenitors (NMPs), such as neurons.

[0030] In a third aspect of the present invention, the problem of the present invention is solved by the method according to the present invention, further comprising the co-production of endothelial cells, by further contacting the PSMs in step iii) with suitable concentrations of Forskolin and VEGF to induce endothelial cells, and further contacting the endothelial cells in step iv) with suitable concentrations of VEGF, to thus further induce endothelial cells.

[0031] In an important fourth aspect of the present invention, the problem of the present invention is solved by a method according to the present invention wherein the production provides a 3D culture, further comprising adding suitable low concentrations of a scaffold / matrix material, such as Matrigel, TrueGel3D™ and / or Cultrex to the PSMs in step iii) after aggregate formation, preferably at the end of step ii), and shaking the culture at about 50 to 100 rpm, preferably about 75 rpm after ECM addition in step iii) and in step iv), thereby inducing a 3D culture.

[0032] In a fifth aspect of the present invention, the problem of the present invention is solved by a method according to the present invention, wherein the method comprises the self-organization of myocytes and muscle fibers with innervation, such as neurons. In a sixth aspect of the present invention, the problem of the present invention is solved by a method according to the present invention, wherein the method comprises wherein the method comprises forming of regular endothelial cellular networks inside muscle tissues, such as a vascular-like network, and / or the self-organization of myocytes and muscle fibers with innervation, such as neurons, and / or vascularization, for example derived from ESCs.

[0033] Preferred is the method according to the present invention, wherein the method is devoid of genetic manipulation or genetic modification of the cells.

[0034] In a seventh aspect of the present invention, the problem of the present invention is solved by a method for producing a comestible meat product, comprising the method according to the present invention to produce a 3D skeletal muscle tissue, in particular a self-organized 3D tissue, and / or further comprising the step of suitably combining at least two or more of the 3D tissues as produced to obtain a comestible meat product.

[0035] In an eighth aspect of the present invention, the problem of the present invention is solved by a 3D culture, such as a 3D skeletal muscle tissue, in particular a self-organized 3D tissue produced according to the present invention, or a comestible meat product produced according to the present invention. Finally, in a ninth first aspect of the present invention, the problem of the present invention is solved by the use of a 3D culture, such as a 3D skeletal muscle tissue, in particular a selforganized 3D tissue produced according to the present invention to make a comestible meat product.

[0036] As mentioned above, the present invention, in a first aspect thereof, relates to an in vitro method for the production of ungulate myocytes and muscle fibers. The method comprises the steps of first providing a suitable ungulate stable, such as a bovine (bESC) embryonic stem cell line (uESC or ESC line). After years of research, derivation of stable bESCs was only recently reported, and preferred are CTFR-bESCs, isolated according to Bogliotti et al. (Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts. Proc Natl Acad Sci U S A. 2018 Feb 27;115(9):2090-2095. doi: 10.1073 / pnas. l716161115. Epub 2018 Feb 9. PMID: 29440377; PMCID: PMC5834688). Nevertheless, other ungulate stem cells can be used as well, as they fulfill the requirements to differentiate in accordance with the present protocol (see, for example, Lazaro J, et al. A stem cell zoo uncovers intracellular scaling of developmental tempo across mammals. Cell Stem Cell. 2023 Jul 6;30(7):938-949.e7. doi: 10.1016 / j.stem.2023.05.014. Epub 2023 Jun 20. PMID: 37343565; PMCID: PMC10321541).

[0037] Preferred is therefore a method according to the present invention wherein the ungulate stable- ESC line is selected from CTFR-bESC and NBFR-bESC (see, for example, Soto, D.A., et al. Simplification of culture conditions and feeder-free expansion of bovine embryonic stem cells. Sci Rep 11, 11045 (2021). https: / / doi.org / 10.1038 / s41598-021-90422-0).

[0038] According to the present invention, the cells are provided / grown in a suitable culture medium free from fetal bovine serum. Examples are mTeSR medium or StemFit medium as the maintenance medium, or CTFR medium or NBFR medium as described herein. The medium can be replaced as needed, for example, about every 24 hours with fresh medium.

[0039] The uESC or bESC line is then in a second step ii) contacted with suitable concentrations of at least one WNT inducer / activator, bFGF, at least one TGFb inhibitor, and at least one BMP inhibitor in order to induce PSMs (see Fig. 1). The factors are added for about 2 days.

[0040] In the next step iii), the PSMs are then contacted with suitable concentrations of the factors HGF, IGF, and bFGF to induce myoblasts. The factors are added for about 4 days. Finally, in step iv), the myoblasts are contacted with suitable concentrations of the factors HGF and IGF to induce myocytes and muscle fibers. The factors are added for about 9 days.

[0041] Preferred is a method according to the present invention wherein the overall method does not exceed about 15 days from the start of the contacting in step ii). Further preferred is the protocol according to the present invention according to the schematic overview in Fig. 1 a).

[0042] The inventors developed induction protocols for skeletal muscle and endothelial cells a skeletal muscle induction protocol from ungulates, such as bovine, uESCs by modifying and combining a human muscle induction protocol (Chai, J. et al. Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy. Nat Biotechnol 33, 962-969 (2015). https: / / doi.org / 10.1038 / nbt.3297), and human endothelial induction protocol (Olmer R. et al. Differentiation of Human Pluripotent Stem Cells into Functional Endothelial Cells in Scalable Suspension Culture. Stem Cell Reports, 10(5): 1657-1672, (2018). https: / / doi.Org / 10.1016 / j.stemcr.2018.03.017). Nevertheless, the combination of the growth factors, small compounds, and administration timings surprisingly provided the surprising results according to the present invention, that uESCs developed into the desired cells and self- organized 2D and 3D cultures as described herein.

[0043] According to the present invention, any suitable factor can be used as indicated. Preferred is the method according to the present invention wherein the WNT inducer / activator is selected from a GSK-3 inhibitor, such as CHIR, e.g. CHIR 90221, CHIR 90221, the TGFb inhibitor is selected from SB431542, and the BMP inhibitor is selected from DMH1 (4-(6-(4- isopropoxyphenyl)pyrazolo(l,5-a)pyrimidin-3-yl)quinoline).

[0044] In the context of the present invention, it was surprisingly found that the induced muscle cells and fibers showed calcium pulses in addition to sarcomere structures (myofilaments). Neuronal cells were detected in the induced tissue, causing the calcium pulses in muscle cells (see, for example, Rosenberg SS, Spitzer NC. Calcium signaling in neuronal development. Cold Spring Harb Perspect Biol. 2011 Oct l;3(10):a004259. doi: 10.1101 / cshperspect.a004259. PMID: 21730044; PMCID: PMC3179332). Therefore, the method according to the present invention furthermore provides for myocytes and muscle fibers that form a tissue in addition comprising neuronal cells derived from NMPs, such as neurons. The muscle stimulation by neurons may help to achieve muscle maturation in the culture or tissues in the future.

[0045] The present invention, in a second aspect thereof, then relates a method according to the present invention further comprising the co-production of endothelial cells. Endothelial cells (the main cell type to form blood vessels) are co-induced together with muscle cells by adding forskolin and VEGF (see preferred method in Fig. 3). Having endothelial cells may help make a vascularized tissue to make a larger beef steak in the future. This method comprises further contacting the PSMs in step iii) with suitable concentrations of forskolin and VEGF to induce endothelial cells, and further contacting the endothelial cells in step iv) with suitable concentrations of VEGF in order to further induce endothelial cells.

[0046] The present invention, in a third aspect thereof, relates to the production of the cells as a 3D culture. This 3D culture provides the basis for tissues and larger pieces of meat based on the methods according to the present invention as described herein.

[0047] Provided is therefore a method according to the present invention, wherein the production leads to a 3D culture, further comprising adding suitable low concentrations of a substrate, scaffold and / or matrix material, such as Matrigel, TrueGel3D™ and / or Cultrex to the PSMs in step iii) after aggregate formation, preferably at the end of step ii). Furthermore, the culture is gently shaken, for example at about 50 to 100 rpm, preferably about 75 rpm after ECM addition in step iii) and in step iv), thereby inducing a 3D culture.

[0048] The inventors also induced all three cell types (muscle, endothelial cells, and neuronal cells) in 3D cultures to produce a self-organizing tissue (Fig. 5). For this, the inventors made cell aggregates in the middle of the protocol, added low concentration of Matrigel to the media, and cultured the 3D tissues on a rotary shaker. The co-induced endothelial cells formed interconnected networks inside the 3D skeletal muscle tissue.

[0049] In the context of the present invention, a “low” concentration of Matrigel, TrueGel3D™ or Cultrex is between about 0.5 and 5%, preferably at between about 1 and 3%, more preferably at about 2%. Another suitable substrate, scaffold and / or matrix material is branched poly(Arg, epsilon-Lys)-hyaluronic acid hydrogel.

[0050] In the context of the present invention, any suitable uESCs can be used. Preferred are cells from an ungulate selected from pigs, peccaries, hippopotamuses, antelopes, deer, giraffes, camels, llamas, alpacas, sheep, goats, and cattle, and in particular bovines, pigs or sheep. The person of skill in the art will be readily able to adjust the protocols as presented here for bovine ESCs to other uESCs.

[0051] It was surprisingly found that the method according to the present invention provides / leads to / comprises the self-organization of myocytes and muscle fibers with innervation, such as neurons. Furthermore, the method according to the present invention even provides / leads to / comprises the wherein the method comprises forming of regular endothelial cellular networks inside muscle tissues, such as a vascular-like network (see for example Fig. 5), and / or self-organization of myocytes and muscle fibers with innervation, such as neurons, and / or vascularization, for example derived from endothelial cells.

[0052] Although a previous study showed that the co-induction of muscle and certain blood vessel cells from human pluripotent stem cells seems to require the overexpression of master regulator genes (Dailamy A. et al. Programmatic introduction of parenchymal cell types into blood vessel organoids. Stem Cell Reports, 16-10, 2432-2441, (2021). https : / / doi . org / 10.1016 / j . sterner.2021.08.014) the inventors co-induced multiple cell types from bovine ESCs and make a structured muscle tissue through self-organization based on a protocol that does not require genetic modification. Therefore, preferred is the method according to the present invention wherein the method is devoid of genetic manipulation or genetic modification of the cells.

[0053] In the context of the present invention, any suitable concentration of the factors as added to the culture(s) may be used. Nevertheless, preferred is the method according to the present invention wherein the suitable concentration of SB431542 is at about 10 pM, of DMH1 is at about 2 pM, of CHIR is about 10 pM, bFGF is at about 20 ng / ml, HGF is at about 10 ng / ml, IGF (e.g. IGF- 1) is at about 2 ng / ml, forskolin is at about 2 pM, and VEGF, such as rhVEGFies, is at about 100 to about 200 ng / ml. In the context of the present invention, any suitable conditions for the cell or tissue culture may be used that leads to the induction and growth of the cells as desired. Nevertheless, preferred is the method according to the present invention wherein the method is performed in mTeSRl medium, StemFit medium, CTFR or NBFR medium at about 37 °C, and at about 5% CO2.

[0054] In another aspect thereof, the method according to the present invention further comprises the detection of at least one cell specific marker selected from the group consisting of NANOG, 0(474, S0X2, BRAGHYURY, TBX6, HES7, MYF5, MYODI, MYOG, MYH4, MYF6, MYH7, SAA, MHC, TUJ1, and SMI-32. Using these markers, the progress and stability of the induction and differentiation of the cells as well as the composition can be detected or monitored. Respective assays are known to the person of skill, and include qRT-PCR, antibodies, fluorescent labels, and luciferase constructs.

[0055] In another aspect thereof, the present invention provides a method for producing a comestible meat product, comprising the method according to the present invention suitably adjusted to produce a 3D skeletal muscle tissue, in particular a self-organized 3D tissue, and / or further comprising the step of suitably combining at least two or more of the 3D tissues as produced to obtain a comestible meat product. Methods for combining may comprise the use of suitable scaffold materials, microcarriers, hydrogels, and / or 3D printing.

[0056] In another aspect thereof, the present invention then provides a 3D culture, such as a 3D skeletal muscle tissue, in particular a self-organized 3D tissue produced according to the present invention or a comestible meat product produced according to the present invention, preferably comprising regular endothelial cellular networks inside muscle tissues, such as a vascular-like network and / or vascularization, and / or self-organized myocytes and muscle fibers with innervation, such as neurons. Preferred examples are beef steak or burgers.

[0057] The method according to the present invention is advantageously able to provide a 3D-culture comprising vascular-like networks (endothelial networks). This leads to perfusing the 3D muscle tissue with oxygen and a nutrient supply. This, in turn, will for the first time allow to generate substantially sized steak tissues.

[0058] In another aspect thereof, the present invention then provides the use of a 3D culture, such as a 3D skeletal muscle tissue, in particular a self-organized 3D tissue produced according to the present invention to make a comestible meat product. Preferred examples are beef steak or burgers.

[0059] In the context of the present invention, the term “about” shall mean to include a deviation of + / - 10% from the given value, unless indicated otherwise.

[0060] In the context of the present invention, the inventors utilized bovine ESCs to co-induce skeletal muscle, neuronal, and endothelial cells, fostering forming of regular endothelial cellular networks inside muscle tissues, such as a vascular-like network, and / or the self-organization of muscle tissues with innervation and vascularization.

[0061] To address these issues, the inventors used bovine ESCs that were originally established by Bogliotti et al. (Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts. PNAS 115, (2018). https: / / doi.org / 10.1073 / pnas.1716161115). Briefly, bovine ESCs were treated with CHIR (WNT activator), bFGF, SB (TGFb inhibitor), and DMH1 (BMP inhibitor) to induce presomitic mesoderm cells, then treated with HGF, IGF, and bFGF to induce myoblasts, and finally treated with HGF and IGF to induce myocytes and muscle fibers (Fig. 1). The protocol takes only ~15 days.

[0062] The induced muscle fibers showed sarcomere structures and calcium pulses. Neuronal cells were detected in the induced tissue, causing the calcium pulses in muscle cells (Fig. 2). The muscle stimulation by neurons may help muscle maturation in the future. The inventors then co-induced endothelial cells (the main cell type to form blood vessels) together with muscle cells by adding forskolin and VEGF (Fig. 3). Having endothelial cells may help make a vascularized tissue to make a larger beef steak in the future. The inventors also induced these three cell types (muscle, endothelial cells, and neuronal cells) in 3D cultures to make a selforganizing tissue (Fig. 5, Fig. 6). For this, the inventors made cell aggregates in the middle of the protocol, added low concentration Matrigel to the media, and cultured the 3D tissues on a rotary shaker. The co-induced endothelial cells formed interconnected networks inside the 3D skeletal muscle tissue.

[0063] The methods of the present invention have several advantages. For example, ungulate, such as bovine, ESCs proliferate stably and theoretically indefinitely, making large-scale production feasible. Bovine ESCs are used to generate muscle fibers instead of traditional adult satellite cells. The inventor’s bovine ESC maintenance and differentiation protocols do not need fetal bovine serum. No genetic modification is needed, in contrast to published muscle induction protocols. All protocols are rather quick (~15 days).

[0064] Furthermore, endothelial cells and neurons can be co-induced from bovine ESCs together with muscle cells, and the inventor’s co-induction protocol(s) enable(s) the preparation of multiple cell types (skeletal muscle, spinal neurons, and endothelial cells) from a single cell source (i.e., bovine ESCs). This is much simpler than isolating multiple types of adult stem cells from bovine biopsy tissues and then inducing individual cell types separately.

[0065] Finally, in addition to two-dimensional (2D) cell cultures, three-dimensional (3D) cultures can also be used to make a tissue that includes neuromuscular junctions and endothelial cell networks from bovine ESCs. The inventor’s 3D induction protocol enables the selforganization of steak-like tissues. The co-induced neurons formed neuromuscular junctions whereas the endothelial cells formed uniform networks, both of which may help develop larger, mature beef pieces, like steaks, in the future.

[0066] In the context of the present invention, the inventors demonstrated the potential of utilizing bovine ESCs as a stable cell source for cultured beef and steaks. The induction of bovine myocytes and muscle fibers from indefinitely proliferative stem cells should make cultured beef production more scalable and robust. The induction of distinct cell types, such as endothelial cells and neurons, from the same cell source can also simplify the production process. All the protocols described in this invention are serum-free and can be completed within 15 days. The rapid induction should be advantageous for potential applications.

[0067] Moreover, the inventors generated a bovine 3D tissue containing skeletal muscle, spinal neurons, and endothelial cell networks. Several 3D skeletal muscle organoids were previously developed from human pluripotent stem cells, but not from bovine stem cells (28, 54-56). Even though vascularized skeletal muscle tissues were previously engineered, the muscle cells and endothelial cells were separately prepared and assembled (13, 56-58), or the cells were induced through the overexpression of master regulators (59). Thus, the co-induction of bovine myocytes and endothelial cells without genetic modification as well as the subsequent selforganization of muscle fibers and endothelial networks inside the tissue represent a crucial step toward the production of structured beef steaks. The stimulation of skeletal muscle by spinal neurons will also facilitate the maturation of the muscle tissue. As the lumen formation in blood vessels is stimulated by intraluminal flow, the next crucial step should involve establishing the connection between the endothelial networks within the muscle aggregates and in vitro perfusable vasculatures (60-63). Perfusion through blood vessels is vital for nurturing steaksized tissues, and perfusion with blood substitutes will improve the flavor of cultured steaks. As the blood vessel formation is also heavily influenced by stroma cells, such as pericytes and fibroblasts, co-inducing bovine stroma cells together with endothelial cells will prove beneficial (26, 64, 65).

[0068] Despite the remaining challenges, the inventors propose the co-induction of multiple cell types and self-organization of tissues as feasible directions in the production of cultured beef steaks. The inventor’s approach will also be complementary to engineering approaches: co-induced cells can be mixed with a bioink and readily used for bioprinting, while muscle aggregates featuring endothelial networks can serve as building blocks for complex tissue assembly. Diverse types of cultured beef will offer novel food options.

[0069] The present invention relates to the following items:

[0070] Item 1. An in vitro method for the production of ungulate myocytes and muscle fibers, comprising the steps of i) providing a suitable ungulate stable-ESC line in a suitable culture medium free from fetal bovine serum; ii) contacting the uESC line with suitable concentrations of at least one WNT inducer / activator, bFGF, at least one TGFb inhibitor, and at least one BMP inhibitor for about 2 days to induce presomitic mesoderm cells (PSMs); iii) contacting the PSMs with suitable concentrations of HGF, IGF, and bFGF for about 4 days to induce myoblasts; and iv) contacting the myoblasts with suitable concentrations of HGF and IGF for about 9 days to induce myocytes and muscle fibers, and wherein preferably the overall method does not exceed about 15 days from the start of the contacting in step ii).

[0071] Item 2. The method according to Item 1, wherein the WNT inducer / activator is selected from a GSK-3 inhibitor, such as CHIR, e.g. CHIR 90221, the TGFb inhibitor is selected from SB431542, and the BMP inhibitor is selected from DMH1.

[0072] Item 3. The method according to Item 1 or 2, wherein the myocytes and muscle fibers form a tissue furthermore comprising neuronal cells derived from neuromesodermal progenitors (NMPs), such as neurons. Item 4. The method according to any one of Items 1 to 3, further comprising the co-production of endothelial cells, comprising further contacting the PSMs in step iii) with suitable concentrations of forskolin and VEGF to induce endothelial cells, and further contacting the endothelial cells in step iv) with suitable concentrations of VEGF to further induce endothelial cells.

[0073] Item 5. The method according to any one of Items 1 to 4, wherein the production provides a 3D culture, further comprising adding suitable low concentrations of Matrigel, TrueGel3D™ and / or Cultrex to the PSMs in step iii) after aggregate formation, preferably at the end of step ii), and shaking the culture at about 50 to 100 rpm, preferably about 75 rpm after ECM addition in step iii) and in step iv), thereby inducing a 3D culture.

[0074] Item 6. The method according to Item 5, wherein the low concentration of Matrigel, TrueGel3D™ or Cultrex is between about 0.5 and 5%, preferably at between about 1 and 3%, more preferably at about 2%.

[0075] Item 7. The method according to any one of Items 1 to 6, wherein the ungulate stable-ESC line is selected from CTFR-uESC and NBFR-uESC, or CTFR-bESC and NBFR-bESC.

[0076] Item 8. The method according to any one of Items 1 to 7, wherein the ungulate is selected from pigs, peccaries, hippopotamuses, antelopes, deer, giraffes, camels, llamas, alpacas, sheep, goats, and cattle, and in particular bovines.

[0077] Item 9. The method according to any one of Items 1 to 8, wherein the method comprises the self-organization of myocytes and muscle fibers with innervation, such as neurons.

[0078] Item 10. The method according to any one of Items 2 to 9, wherein the method comprises forming of regular endothelial cellular networks inside muscle tissues, such as a vascular-like network, and / or the self-organization of myocytes and muscle fibers with innervation, such as neurons, and / or vascularization, for example derived from endothelial cells.

[0079] Item 11. The method according to any one of Items 1 to 10, wherein the method is devoid of genetic manipulation or genetic modification of the cells. Item 12. The method according to any one of Items 1 to 11, wherein the suitable concentration of SB431542 is at about 10 pM, of DMH1 is at about 2 pM, of CHIR is at about 10 pM, bFGF is at about 20 ng / ml, HGF is at about 10 ng / ml, IGF (e.g. IGF-1) is at about 2 ng / ml, forskolin is at about 2 gM, and VEGF, such as rhVEGFies, is at about 100 to 200 ng / ml.

[0080] Item 13. The method according to any one of Items 1 to 12, wherein the method is performed in mTeSRl medium, StemFit medium, CTFR or NBFR medium at about 37 °C, and at about 5% CO2.

[0081] Item 14. The method according to any one of Items 1 to 13, further comprising the detection of at least one cell specific marker selected from the group consisting of NANOG, OCT4, SOX2, BRACHYURY, TBX6, HES7, MYF5, MYODI, MYOG, MYH4, MYF6, MYH7, SAA, MHC, SMI-32 and TUJ1.

[0082] Item 15. A method for producing a comestible meat product, comprising the method according to any one of Items 5 to 14 to produce a 3D skeletal muscle tissue, in particular a self-organized 3D tissue, and / or further comprising the step of suitably combining at least two or more of the 3D tissues as produced to obtain a comestible meat product.

[0083] Item 16. A 3D culture, such as a 3D skeletal muscle tissue, in particular a self-organized 3D tissue produced according to any one of Items 5 to 14, or a comestible meat product produced according to Item 15.

[0084] Item 17. Use of a 3D culture, such as a 3D skeletal muscle tissue, in particular a self-organized 3D tissue produced according to any one of Items 5 to 14 to make a comestible meat product.

[0085] The invention will now be described further in the following examples with reference to the accompanying figures, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited are incorporated by reference in their entireties.

[0086] Figure 1 shows the induction of PSM and muscle fibers from bovine ESCs. a, Induction protocol for bovine myocytes and muscle fibers from bovine ESCs. PSM cells were initially induced from ESCs using CHIR, bFGF, SB, and DMH1. Then myocytes were induced from PSM using HGF, IGF, and bFGF. Maintenance: Maintenance medium, ESC: Embryonic stem cell, NMP: Neuromesodermal progenitor, PSM: Presomitic mesoderm. Typical bright field images are also shown (bottom), b, IHC images of the day-2 culture. TBX6 is a PSM marker. SOX2- and BRA-double positive cells are NMPs. Three independent experiments showed similar staining patterns, c, The oscillatory activity of the HES7 promoter-luciferase reporter in PSM. Raw (top) and detrended (bottom) signals. The vertical lines indicate the timings of medium change, which resets the oscillation phase. Three independent experiments showed similar oscillation patterns, d, qRT-PCR quantification of MYOG expression. The average value of ESC sample was set to 1. Mean ± SEM. N = Three from three independent experiments. e,f, IHC images of the day- 15 cultures. MYOG and SAA are myocyte / muscle fiber markers. Four independent experiments showed similar staining patterns, g, Percentage of the SAA-positive area in the field of view. Mean ± SEM. N = 14 (day 10), 25 (day 13), and 42 (day 15) from three-nine independent experiments, h, Sarcomeric structures on SAA-positive muscle fibers (arrowheads in the left pictures) on day 15. The sarcomere length was defined as the distance between intensity peaks (right). Mean ± SEM. N = 88 from four independent experiments. Scale bars: 150 pm (a,b,e), 300 pm (f), and 30 pm (h). Microscopes: 1X73 (a) and FV3000 confocal (b,e,f,h).

[0087] Figure 2 shows the co-induction of bovine muscle and spinal neurons, a, Fluo-8 calcium signal of the day- 18 culture, b, Calcium level fluctuations in the absence (blue) and presence (orange) of 10 pM curare, an acetylcholine receptor blocker. Regions of interest (ROIs) are marked in a. c, Quantification of the total number of calcium oscillation peaks within 2 min in the absence and presence of curare. Mean ± SEM. N = 23 (control) and 23 (curare) from three independent experiments. P-value is from paired t-test. d-g, IHC images of the day- 15 cultures. TUJ1 and SMI-32 are neuronal markers. SAA and MHC are skeletal muscle markers. Three-eight independent experiments showed similar staining patterns. e,g, Enlarged images of d and f, respectively. Scale bars: 150 pm (a,d,f) and 50 pm (e,g). Microscopes: Thunder (a) and FV3000 confocal (d-g).

[0088] Figure 3 shows the co-induction of bovine muscle and endothelial cells, a, Co-induction protocol for bovine muscle fibers and endothelial cells. Forskolin and VEGF were added to induce endothelial cells from bovine PSM cells, b, IHC images over the time course. VE-cad is an endothelial cell marker. Three-six independent experiments showed similar staining patterns, c, Characterization of the endothelial cell networks. After the IHC images were masked and segmented, the vessel segments (red edges) and branching points (white nodes) were identified and quantified using the connectivity analysis pipeline. The vessel segments that do not connect two points (green edges) were not counted. The vessel area, the total number of vessel segments, the total number of branching points, and the mean number of vessel segments per branching point were quantified. Mean ± SEM. N = 15 (day 8), 25 (day 10), 12 (day 12), 12 (day 14), and 23 (day 15) from three-six independent experiments, d, IHC images of the day- 15 culture showing endothelial and skeletal muscle domains. Six independent experiments showed similar staining patterns, e, Enlarged image of d. f, Size of skeletal muscle domains. The area, major axis length, and minor axis length of VE-cad-negative domains were quantified. Mean ± SEM. N = 39 (day 12), 19 (day 14), and 30 (day 15) from three-four independent experiments. Scale bars: 300 pm (b,d) and 150 pm (e). Microscope: FV3000 confocal.

[0089] Figure 4 shows the induction of bovine muscle fibers in 3D cultures, a, 3D induction protocol for bovine muscle fibers and spinal neurons. The aggregate of 20,000 bovine PSM cells was made on day 2. Matrigel was added to the medium on day 3. Aggregates were cultured on a rotary shaker after day 6. b,c, Characterization of aggregates over the time course. The area, major axis length, and minor axis length of the aggregates were quantified using 2D bright field images. Mean ± SEM. N = 15 (day 3), 93 (day 6), 90 (day 10), and 48 (day 15) from three independent experiments, d, IHC images of the day- 15 aggregate. SAA and TUJ1 are muscle fiber and neuronal markers, respectively. Six independent experiments showed similar staining patterns, e, Enlarged image of d. f,g, Effects of aggregate size on cell fates. The aggregates were made from 10,000-30,000 PSM cells on day 2, and the percentages of the SAA- and TUJ1- positive volumes were quantified in the day-15 aggregates. N = 57 from seven independent experiments. Scale bars: 300 pm (b), 150 pm (d) and 50 pm (e). Microscopes: 1X73 (b) and Light-sheet (d,e).

[0090] Figure 5 shows the co-induction of bovine muscle and endothelial networks in 3D. a, 3D coinduction protocol for bovine muscle fibers and endothelial cell networks. The aggregate of 20,000 bovine PSM cells was made on day 2. Forskolin and VEGF were added to induce endothelial cells from PSM cells. b,c, Characterization of aggregates over the time course. The area, major axis length, and minor axis length of the aggregates were quantified using 2D bright field images. Mean ± SEM. N = 15 (day 3), 94 (day 6), 93 (day 10), and 65 (day 15) from three independent experiments, d-h, IHC images of the day-15 aggregates, d, SAA and VE-cad are muscle fiber and endothelial cell markers, respectively. Ten independent experiments showed similar staining patterns, e, Enlarged image of d. f, Max projection images of d. The max projection of SAA used all Z-stacks. The max projection of VE-cad used the Z-stacks within the 40 pm range around the central slice, g, ZO-1 is a tight junction marker. Max projection used the Z-stacks within the 40 pm range around the central slice. Three independent experiments showed similar staining patterns, h, Enlarged image of g. i,j, Effects of aggregate size on cell fates. The aggregates were made from 10,000-30,000 PSM cells on day 2, and the percentages of the VE-cad- and SAA-positive volumes were quantified in the day-15 aggregates. Mean ± SEM. N = 31 from seven independent experiments. Scale bars: 300 pm (b), 150 pm (d,f,g), and 50 pm (e,h). Microscopes: 1X73 (b) and Light-sheet (d-h).

[0091] Figure 6 shows single-cell RNA sequencing analyses of individual induction protocols, a, Uniform manifold approximation and projection (UMAP) plot of the integrated data from 4 samples shown in b colored by 3 clusters, b, Example bright field images of the 4 samples corresponding to individual induction protocols on day 15 (top). Scale bars: 300 pm. Split UMAP plots of individual samples (bottom). N = 8562 cells (Muscle 2D protocol), 10294 cells (Muscle / EC 2D protocol), 8247 cells (Muscle 3D protocol; 30 aggregates), and 7676 cells (Muscle / EC 3D protocol; 30 aggregates) from a single experiment. EC: Endothelial cell, c, Proportions of the muscle, neural, and endothelial cell clusters in individual samples, d, UMAP plots colored by the expression of selected marker genes for somite, muscle, endothelial, and neural cells.

[0092] Figure 7 shows the induction of PSM and muscle fibers from bovine ESCs. a, Bright field images over the time course of the muscle 2D protocol. The enlarged views of these images are shown in Fig. la. b, i.e. IHC images of the day-2 culture. Scale bars: 300 pm (a) and 50 pm (b).

[0093] Figure 8 shows qRT-PCR measurements. Relative mRNA levels of selected marker genes in bovine ESCs and the time course of the muscle 2D protocol. The values were normalized to GAPDH expression, and the average values of ESCs were set to 1. Mean ± SEM. N = 3 from three independent experiments, a, Pluripotency markers, b, PSM markers, c, Myoblast markers, d, Myocyte markers.

[0094] Figure 9 shows the co-induction of endothelial and neural cells from bovine ESCs. IHC images of the day-10 culture of the muscle / EC 2D protocol. Scale bar: 150 pm. TUJ1 and VE-cad are neuronal and endothelial cell markers, respectively. Three independent experiments showed similar staining patterns.

[0095] Figure 10 shows the effects of Matrigel on aggregates. Bright field images of the muscle aggregates made without or with (2% or 5%) Matrigel in the medium. 2% is the standard concentration. Three independent experiments showed similar patterns. Scale bars: 300 pm.

[0096] Figure 11 shows the co-induction of muscle, neural, and endothelial cells in 3D. a, IHC images of the day- 15 aggregate of the muscle / EC 3D protocol. SAA and TUJ1 are muscle fiber and neuronal markers, respectively. Three independent experiments showed similar staining patterns. The max projection was shown, b, IHC images of the day-15 aggregates. The aggregate made with the standard PSM medium with DMH1 (the BMP inhibitor) showed a uniform VE-cad-positive endothelial network throughout the tissue (top), while the one made without DMH1 showed local endothelial domains (bottom). The max projection of VE-cad used the Z-stacks within the 40 pm range around the central slice. Five-ten independent experiments showed similar patterns. Scale bars: 150 pm (a) and 200 pm (b).

[0097] Figure 12 shows the scRNA-seq analyses of four induction protocols, a, UMAP plot of the integrated data colored by the 4 samples corresponding to individual induction protocols, b, Heatmap of 3 identified clusters with the scaled expression of top marker genes, c, Heatmap of the expression of selected marker genes for muscle differentiation steps. Color range indicates pseudo-loglO expression levels of SCTransform values, d, UMAP plots colored by the expression of selected marker genes for neuromuscular junctions.

[0098] Figure 13 shows the subset analyses of the muscle cluster, a, Heatmap of 2 identified subclusters of the muscle cluster with the scaled expression of top marker genes, b, UMAP plot of the integrated data colored by 4 samples (left) and 2 muscle subclusters (right), c, Split UMAP plots of the 4 samples corresponding to individual induction protocols, d, Proportions of the 2 muscle subclusters in individual samples, e-j, UMAP plots colored by the expression of selected marker genes for skeletal muscle (e), smooth muscle (f), fibroblast (g), adipocyte (h), pericyte (i), and osteoblast (j).

[0099] Figure 14 shows the subset analyses of the neural cluster, a, Heatmap of 2 identified subclusters of the neural cluster with the scaled expression of top marker genes, b, UMAP plot of the integrated data colored by 4 samples (left) and 2 neural subclusters (right), c, Split UMAP plots of the 4 samples corresponding to individual induction protocols, d, Proportions of the 2 neural subclusters in individual samples, e-i, UMAP plots colored by the expression of selected marker genes for neural progenitor (e), neuron (f), astrocyte (g), oligodendrocyte (h), and spinal neuron (i).

[0100] Examples

[0101] Methods

[0102] Bovine ESC culture

[0103] The bovine ESC line was obtained from Dr. Juan Carlos Izpisua Belmonte (8). The HES7 promoter-luciferase reporter bovine ESC line was described previously (30). The ESCs were maintained without feeder cells and cultured on Matrigel (Coming, 35623 l)-coated dishes. Either mTeSRl medium (STEMCELL Technologies) or StemFit medium (Ajinomoto, StemFit BasicO4CT) supplemented with 20 ng / ml bFGF (PeproTech, AF-100-18B), 20 ng / ml Activin A (R&D, 338-AC), and 2.5 pM IWR1 (Tocris, 3532) was used as the maintenance medium. The ESCs were passaged every other day. The cells were trypsinized into single cells by TrypLE solution (Gibco, A1285901) and 0.5 mM ethylenediaminetetraacetic acid (EDTA) in phosphate-buffered saline (PBS) (1 : 1 mixture) at room temperature for 1 min. Then 2.3 x 105- 2.8 x io5cells were seeded on a Matrigel-coated 3.5 cm dish in the maintenance medium containing 5 pM ROCK inhibitor, Y27632 (Sigma, Y0503). The cells were maintained at 37 °C in a humidified atmosphere of 5% CO2. The medium was changed the next day into the maintenance medium without Y27632.

[0104] Induction media

[0105] From day 0 to day 2: PSM medium

[0106] The PSM medium is CDMi containing 10 pM CHIR99021 (Sigma, SML1046), 20 ng / ml bFGF, 10 pM SB431542 (Sigma, S4317), and 2 pM DMH1 (Sigma, D8946) as previously described (28, 33, 66).

[0107] From day 2 to day 6: Muscle medium l

[0108] The basal medium is DMEM / F12 (Gibco, 11320033) containing 15% Knockout Serum Replacement (Gibco, 10828028), 0.1 mM nonessential amino acids (Gibco, 11140-035), 50 mM 2-mercaptoethanol (Gibco, 31350-010), and 0.5x penicillin / streptomycin (Gibco, 15140- The muscle medium l is the basal medium supplemented with 10 ng / ml HGF (R&D, 2207- HG-025 / CF), 2 ng / ml IGF (R&D, 791-MG-050), and 20 ng / ml bFGF.

[0109] From day 2 to day 6: Muscle / Endothelial Cells (EC) medium l

[0110] The muscle / EC medium l is the basal medium supplemented with 10 ng / ml HGF, 2 ng / ml IGF, 20 ng / ml bFGF, 2 pM forskolin (R&D, 2207-HG-025 / CF), and 200 ng / ml VEGF165 (R&D, 2207-HG-025 / CF).

[0111] From day 6 to day 15: Muscle medium l

[0112] The muscle medium ! is the basal medium supplemented with 10 ng / ml HGF and 2 ng / ml IGF.

[0113] From day 6 to day 15: Muscle / EC medium l

[0114] The muscle / EC medium ! is the basal medium supplemented with 10 ng / ml HGF, 2 ng / ml IGF, and 100 ng / ml VEGF165.

[0115] Bovine muscle induction protocol in 2D

[0116] One day before the induction protocol started, 2.3 x 104- 2.6 x io4 / cm2bovine ESCs were seeded on Matrigel-coated dishes or plates in the maintenance medium without IWR1 and with 5 pM Y27632. The maintenance medium was changed to the PSM medium the next day (defined as day 0). The cells were incubated in the PSM medium for 2 days, and the medium was changed every day. On day 2, the PSM medium was switched to the muscle medium l, and the cells were incubated for 4 days with daily medium changes. On day 6, the muscle medium l was switched to the muscle medium !, and the cells were incubated until day 15 with medium changes every other day.

[0117] Bovine muscle and endothelial cell co-induction protocol in 2D

[0118] Bovine PSM cells were induced as described above. During days 2-6, the cells were incubated in the muscle / EC medium l with daily medium changes. During days 6-15, the cells were incubated in the muscle / EC medium ! with medium changes every other day.

[0119] Bovine muscle induction protocol in 3D

[0120] Bovine PSM cells were induced as described above. On day 2, PSM cells were trypsinized into single cells using TrypLE solution at 37 °C for 3 min. The cells were mechanically dissociated by pipetting and transferred into 1 ml of the muscle medium l containing 5 pM Y27632. The cell suspension was centrifuged at 258 xg for 3 min and resuspended into 1 ml of the muscle medium l containing Y27632. After one more wash with the muscle medium l containing Y27632, the supernatant was completely removed. The cell pellet was resuspended into 600- 800 pl of the muscle medium l containing Y27632. The cell concentration was adjusted to 133.3 cells / jul with a sufficient amount of the pre-warmed muscle medium l containing Y27632. Then 150 pl of the cell suspension (= 20,000 cells) was aliquoted into each well of a U-Shaped-Bottom, 96-well-plate (96-well Clear Round Bottom Ultra-Low Attachment Microplate, Coming, 7007) by using a multichannel pipette. The 96-well plate was centrifuged at 258 xg for 2 min for the cells to settle down on the bottom of the plate. On day 3, 100 pl of the medium was carefully removed and replaced with 150 pl of the fresh muscle medium l containing 2% Matrigel (growth factor reduced, Corning, 356231). After day 4, the plate was placed on a rotary shaker (Celltron, INFORS HT) with the rotation speed set at 75 r.p.m. The medium was not changed until day 6. On day 6, the aggregates were collected using wide-bore tips and transferred into a 6-well plate containing 3 ml of the muscle medium_2. The aggregates were incubated in the muscle medium_2 until day 15 with medium changes every 3 days.

[0121] Bovine muscle and endothelial cell co-induction protocol in 3D

[0122] The aggregate of 20,000 PSM cells was made as described above using the muscle / EC medium l instead of the muscle medium l. During days 6-15, the aggregates were incubated in the muscle / EC medium_2 instead of the muscle medium_2.

[0123] Immunohistochemistry (IHC) for 2D cell cultures

[0124] Cells in 2D cultures were washed twice with PBS and fixed in 300 pl of 4% paraformaldehyde (PF A) at room temperature for 15 min. The cells were washed twice with PBSTw (0.2% Tween20 in PBS) for 5 min each, and then treated with the blocking buffer (PBS containing 3% Bovine Serum Albumin (BSA) and 0.2% Tween20) at room temperature for a few hours or at 4°C overnight. The cells were then incubated with the following primary antibodies in the blocking buffer at 4°C overnight: anti-TBX6 antibody (1 / 300 dilution, Abeam, ab38883), anti- SOX2 antibody (1 / 300 dilution, R&D, MAB2018), anti -BRACH YURY antibody (1 / 300 dilution, R&D, AF2085), anti-SARCOMERIC ALPHA ACTININ (SAA) antibody (1 / 1000 dilution, Abeam, ab9465), anti-BETA III TUBULIN (TUJ1) antibody (1 / 400 dilution, Abeam, abl8207), anti- MYOGENIN antibody (1 / 1000 dilution, Abeam, abl24800), anti-VE- CADHERIN antibody (1 / 300 dilution, Cell Signaling Technologies, 2500S), anti- Neurofilament heavy polypeptide (SMI-32) antibody (1 / 300 dilution, Abeam, ab8135), or anti- Myosin heavy chain (MHC) antibody (1 / 300 dilution, R&D, MAB4470). The next day, the cells were washed 3 times with PBSTw for 15 min each. Then the cells were incubated with the following secondary antibodies at a 1 / 500 dilution together with DAPI (1 / 500 dilution, Invitrogen, 62247) in PBSTw at 4°C overnight: Alexa Fluor 594 Goat anti-Rabbit IgG (H+L) (Invitrogen, A-l 1037), Alexa Fluor 647 Donkey anti-Goat IgG (H+L) (Invitrogen, A32849), or Alexa Fluor 488 Goat anti -Mouse IgG (H+L) (Invitrogen, A-l 1029). The cells were washed 3 times with PBSTw for 15 min each, and images were taken with an FV3000 confocal microscope (Olympus, FV3000 Fluoview RS software). Tiled images were stitched together using Imaged Grid / Collection stitching. The fluorescent signal-positive area was measured using Imaged thresholding.

[0125] IHC for 3D aggregates

[0126] Aggregates in 3D cultures were collected using wide-bore tips into 2 ml Eppendorf tubes. After 2 washes with PBS, the aggregates were fixed in 500 pl of 4% PF A at 4°C overnight. They were washed 3 times with PBSTt (0.5% Triton X-100 in PBS) for 15 min each, and treated with the blocking buffer (PBS containing 5% Normal Goat Serum and 0.5% Triton X-100) at 4°C overnight on a shaker. Then the aggregates were incubated with the primary antibodies described above in the blocking buffer at 4°C for 2 days on a shaker. They were washed 3 times with PBSTt for 20 min each and incubated with the secondary antibodies described above in PBSTt at 4°C overnight on a shaker. After 3 washes with PBSTt for 20 minutes each, the aggregates underwent a clearing treatment. They were washed once with MilliQ and then embedded in 1% Agarose (low gelling temperature, Sigma, A9414) using a handmade rectangular box. The aggregates were then incubated with 100% Methanol in a glass bottle at room temperature for 2 days in the dark. The Methanol was changed daily. Then the Methanol was replaced with BABB (a mixture of Benzyl benzoate and Benzyl alcohol at a 1 : 1 ratio), and the aggregates were incubated at room temperature for 2 days in the dark. The BABB solution was changed daily. Images were taken using a MuVi-SPIM Light-Sheet Microscope with a clear chamber and BABB (Z = 2 pm, XY = 0.65 pm). Tiled images were stitched together using the MuVi-SPIM Light-Sheet Microscope’s software. The volume of the fluorescent signalpositive region was measured using the ilastik software with a pixel configuration and a custom- written Python script. Once the masked images of Z-stacks were generated by ilastik, the positive pixel values within each mask were counted, and these pixel counts were converted into a volume.

[0127] Time-lapse imaging and quantification of HES7 oscillation

[0128] The HES7 promoter-luciferase reporter ESCs were incubated in the PSM medium containing

[0129] 0.5 mM luciferin. The medium was changed daily. The luciferase activity was recorded using a Kronos Dio Luminometer (Atto, Kronos control software v2.3) every 10 min with 1 min exposure. The detrended signal was calculated by pyBOAT (67).

[0130] Calcium oscillation measurement

[0131] Calcium activity was visualized with Fluo-8 AM (Abeam, abl 12129) following the manufacturer’s protocol. Briefly, cells were washed twice with PBS, and incubated with a mixture of Component A, B, and C at 37 °C for 30 min with or without 10 pM Tubocurarine Chloride Pentahydrate (Curare, Sigma, T2379). After 2 washes with the muscle medium_2, the Fluo-8 signals were recorded in the absence or presence of curare using the Thunder Imager Live Cell & 3D assay (Leica, LAS X software) every 100 msec.

[0132] For analyses, the Fluo-8 intensity from a randomly selected cell was measured using ImageJ. The signal was denoised using the Savitzky-Golay filter with a 50 window size using Python. The amplitudes between a signal peak and its adjacent two valleys were measured, and the peak was counted only if the smaller amplitude was larger than half of the larger amplitude. The total number of oscillation peaks in 2 min was manually counted.

[0133] Quantitative RT-PCR (qRT-PCR)

[0134] Total RNA was extracted with RNeasy kit (Qiagen, 74104), and 1 pg RNA was reverse- transcribed with QuantiTect Reverse Transcription kit (Qiagen, 205311) to generate cDNA. The cDNA level was measured by a LightCycler 480 II (Roche) with LightCycler 480 SYBR Green I Master (Roche, 4707516001). The expression levels of the target genes were normalized by the GAPDH level.

[0135] Endothelial network connectivity analysis

[0136] Endothelial network connectivity was analyzed with VascuMap software using a UNet architecture with a mixed vision transformer encoder (68). The model, trained on endothelial cell images co-cultured with bovine muscle, labeled manually on an iPad 11 Pro with Photoshop and binarized via Python script. Images were divided into training and validation sets (70% / 30%) with a fixed seed of 42. Training involved 200 epochs, batch size 16, RAdam optimizer, initial learning rate 0.001, step scheduler for rate reduction, and augmentations like flips and rotations. The loss function combined binary cross-entropy and dice coefficient, with the best loU metric model on the validation set retained. For unseen data, VascuMap's probability map was binarized using hysteresis thresholding (thresholds: 0.15, 0.5). The characterization of the binary masks returned the vessel area, total number of vessel segments, total number of branching points, and mean number of vessel segments per branch point.

[0137] Single-cell RNA sequencing (scRNA-seq)

[0138] Cell preparation for 2D cultures

[0139] Cells were induced with individual protocols (Muscle 2D or Muscle / EC 2D) for 15 days using a Matrigel-coated 12-well plate. After 2 washes with PBS, the samples were trypsinized into single cells through the treatment with the TrypLE solution and 0.5 mM EDTA in PBS (1 :1 mixture) at 37°C for 10 min. The cells were mechanically dissociated by pipetting and transferred into PBS containing 0.1% bovine serum albumin (BSA). The cell suspension was centrifuged at 160 xg for 3 min and resuspended into 1 ml of 0.1% BSA in PBS. After one more wash with 0.1% BSA in PBS, the supernatant was completely removed. The cell pellet was resuspended in Cell Prep Buffer (PBS with 0.1% poly(vinyl alcohol) (PVA) and 1 mM EDTA) and then filtered through a 35-pm cell strainer.

[0140] Cell preparation for 3D aggregates

[0141] 3D aggregates were induced with individual protocols (Muscle 3D or Muscle / EC 3D) for 15 days. 30 aggregates were collected with wide-bore tips into 2 ml Eppendorf tubes. After two washes with PBS, the samples were trypsinized into single cells through the treatment with 0.25 mM Trypsin-EDTA at 37°C for 10 min. The cells were mechanically dissociated by pipetting and transferred into of 0.1% BSA in PBS. The dissociated cells were washed twice with 0.1% BSA in PBS and resuspended in Cell Prep Buffer and then filtered through a 35-pm cell strainer.

[0142] Barcoding and sequencing

[0143] Transcripts of individual cells were barcoded, and dual-indexed cDNA libraries were generated with the Chromium Single Cell G Chip Kit and Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (lOx Genomics) by using the Chromium Controller (lOx Genomics, firmware version 4.00) according to the manufacturer's protocol. The finished cDNA libraries were sequenced with NextSeq2000 (Illumina). 28, 10, 10, and 90 base pairs were read for lOx barcodes and unique molecular identifiers (UMIs), for the i7 index, for the i5 index, and for fragmented cDNA, respectively.

[0144] Read alignment Sequenced reads were aligned to the bovine genome (ARS-UCD1.2 Ensembl release 110) and counted to generate the feature-barcode matrices with the CellRanger pipeline (version 7.0.1, lOx Genomics). The reference index for the pipeline was prepared with the cellranger mkref command in the same CellRanger package, based on the aforementioned genome reference and the gene annotation file from ARS-UCD1.2 Ensembl release 106. The reads containing the same UMIs were collapsed as a single count. The basic statistics of sequencing results are shown in Supplementary Table 3.

[0145] Using R version 4.3.1 and Seurat version 5.1.0, the single-cell gene expression matrices underwent quality control based on the following criteria: (1) the number of detected genes exceeded 1,500 across all 4 samples; (2) the total number of detected RNA molecules was greater than 50,000 in the Muscle 2D sample and greater than 40,000 in the other samples; and (3) the proportion of detected mitochondrial genes was less than 31% in the Muscle / EC 2D sample, less than 32% in the Muscle / EC 3D sample, and less than 33% in the Muscle 2D and Muscle 3D samples.

[0146] The gene expression matrices from the four samples corresponding to individual protocols were integrated into a single Seurat object using sctransform normalization, implemented in Seurat. For dimensionality reduction and clustering, the integrated dataset was analyzed using the fastMNN method, considering the first 20 principal components (PCs) from PCA. Clustering was performed at a resolution of 0.003, identifying muscle, neural, and endothelial cell clusters. Subsequently, the muscle and neural cell clusters were extracted into separate Seurat objects. Each subset was further analyzed by applying fastMNN for dimensionality reduction and clustering, with subclusters generated at resolutions of 0.01 for muscle cells and 0.03 for neural cells.

[0147] Sample definition

[0148] All measurements were taken from distinct experiments (biological replicates).

[0149] Results

[0150] Induction of PSM and muscle fibers from bovine ESCs

[0151] The inventors first established a protocol to induce muscle fibers from bovine ESCs (Fig. la, Fig. 7). As skeletal muscle is generated from presomitic mesoderm (PSM) through transient structures of somites and dermomyotomes in embryonic development (29), the inventor’s stepwise protocol comprised the induction of PSM followed by the induction of myoblasts and myocytes. Based on the PSM induction protocol the inventors previously reported (30-33), bovine ESCs were treated with CHIR (WNT signaling activator), bFGF, SB431542 (TGFb signaling inhibitor), and DMH1 (BMP signaling inhibitor) for 2 days. The expression levels of pluripotency markers, NANOG, OCT4, and SOX2, decreased before day 2 (Fig. 8a) while those of a mesodermal marker BRACHYURY (BRA, also known as T) and a PSM marker TBX6 increased (Fig. 8b). Most cells were TBX6-positive on day 2, suggesting efficient PSM induction (Fig. lb). Since the remarkable characteristic of PSM cells is the segmentation clock, the oscillatory gene expressions that regulate the timing of somite formation, the inventors monitored the expression pattern of HES7, a core gene of the molecular oscillator (34, 35). A HES7 promoter-luciferase reporter (30-33) showed an elevated expression around day 2 (Fig. 1c, top), simultaneously displaying clear oscillations of the segmentation clock (Fig. 1c, bottom). Consistent with the previous report (30), the oscillation period of the bovine segmentation clock was ~4 hours. These results confirmed the efficient induction of bovine PSM from bovine ESCs.

[0152] To further induce skeletal muscle from bovine PSM, the inventors adapted the muscle induction protocol previously reported for mouse and human cells (36-38). Bovine PSM cells were treated with HGF, IGF, and bFGF for 4 days before further treatment with HGF and IGF for 9 days (Fig. la). The expression levels of myoblast markers, MYF5 andMYODl, increased around day 6 (Fig. 8c). The expressions of myocyte markers, MYOG, MYF6, MYH7, and MYH4, were induced around day 10 (Fig. Id, Fig. 8d). The MYOG- and sarcomeric a-actinin (SAA)-positive muscle fibers first appeared around day 10 and peaked around day 14 (Fig. le-g). These bovine muscle fibers displayed sarcomeric structures, and their average sarcomere length was 2.1 pm (Fig. Ih), similar to previously reported values for in vivo bovine skeletal muscles (39, 40). The ~15-day duration of bovine myocytes and muscle fiber induction was slightly shorter than the one described in the human skeletal muscle induction protocol (37), potentially reflecting the faster pace of bovine embryonic development compared with human (30). These results collectively established the induction protocol for bovine myocytes and muscle fibers from bovine PSM. Note that the protocol does not require serum.

[0153] Co-induction of bovine muscle and spinal neurons

[0154] The induced bovine myocytes and muscle fibers displayed spontaneous fluctuations of intracellular calcium levels (Fig. 2a, b). The calcium pulses were effectively suppressed by the treatment with curare (Fig. 2b, c), an acetylcholine receptor blocker that is used to inhibit neuromuscular junctions (28, 41, 42). This suggested the presence of neuromuscular junctions connecting skeletal muscles and spinal neurons. Indeed, the inventors detected TUJ1 -positive neurons intermingling with SAA-positive muscle fibers in the day- 15 muscle culture (Fig. 2d,e). A neurofilament maker SMI-32 and another muscle marker, myosin heavy chain (MHC), showed similar staining patterns of aligned neurons and muscle fibers (Fig. 2f,g). The expression of a neuronal marker S0X2 also increased around day 10-13 (Fig. 8). These neurons could be derived from neuromesodermal progenitors (NMPs), given that the day -2 PSM culture contained a fraction of SOX2- and BRA-double positive NMPs (Fig. lb, Fig. 7b). These results suggested that spinal neurons were co-induced from bovine ESCs, transmitting a signal to the skeletal muscles through neuromuscular junctions. The stimulation from neurons may be beneficial for muscle maturation, potentially offering an alternative to the external electrical and mechanical stimuli currently employed in the muscle bioengineering field (16-21).

[0155] Co-induction of bovine muscle and endothelial cells

[0156] The inventors next developed a protocol to co-induce skeletal muscle and endothelial cells from bovine ESCs (Fig. 3a). The lack of blood vessels is a major challenge that currently hampers the development of large, mature organoids in the stem cell biology field. As blood vessels are first formed by endothelial cells, the inventors prioritized the induction of bovine endothelial cells. Note that while endothelial cells arise primarily from lateral plate mesoderm in embryonic development (43, 44), a contingent of endothelial cells are derived from PSM and somites (44- 49). The inventors thus hypothesized that endothelial cells and myocytes could be co-induced from the same PSM cell population. The BMP inhibitor DMH1 that prevents lateral plate mesoderm differentiation was kept in the protocol (Fig. 3a). On day 2, the inventors added the angiogenic factors VEGF and forskolin (cAMP signaling activator) to the bovine PSM culture and initiated the induction of endothelial cells, adapting the reported protocols for human endothelial cells (50-52). The elongated cells, positive for an endothelial marker VE-cadherin (VE-cad, also known as CDH5), appeared around day 8 and peaked around day 14 (Fig. 3b). The induced bovine endothelial cells formed interconnected vessel-like networks: the numbers of vessel segments and branching points increased during days 8-15 (Fig. 3c). The endothelial networks and muscle fibers tended to form small individual domains (Fig. 3d,e). The skeletal muscle domains encased by endothelial cells measured 200-300 pm in diameter on day 15 (Fig. 3f), which surpassed the intercapillary distance of approximately 100 pm in tissues (53), yet remained within a comparable range. TUJ1 -positive neurons were also co-induced under this protocol (Fig. 9). These results demonstrated the co-induction of skeletal muscle and endothelial cells from bovine PSM and the self-organization of vascular networks in 2- dimensional (2D) cultures.

[0157] Induction of bovine muscle fibers in 3D cultures

[0158] With the ultimate goal of self-organizing beef steaks in mind, the inventors applied our bovine ESC differentiation protocols to 3-dimensional (3D) cultures. The inventors developed a protocol to induce bovine muscle fibers in 3D cell aggregates (Fig. 4a). Bovine PSM cells were induced according to our 2D protocol, and on day 2, a 3D aggregate was created out of 20,000 PSM cells (Fig. 4b), which contained a small fraction of NMPs. The 2% Matrigel was added to the medium to bolster the structural integrity of aggregates: without Matrigel, the aggregates gradually fell apart, and a higher concentration of Matrigel triggered the separation of two aggregate domains (Fig. 10). The PSM cell aggregates were treated with the skeletal muscleinducing cocktail, and after day 6, they were cultured on a rotary shaker that facilitated the diffusion of oxygen and nutrients (Fig. 4a). The aggregates were spherical and -600 pm in diameter (Fig. 4c), and the size did not dramatically change over the time course, probably reflecting a declined cell proliferation during the muscle differentiation phase. SAA-positive muscle fibers were induced around day 10 (Fig. 4d). Consistent with the results from our 2D cultures, TUJ1 -positive neurons were co-induced in aggregates, forming neuronal domains next to muscle domains (Fig. 4d,e). To assess the effects of aggregate size on cell fates, the inventors prepared diverse sizes of aggregates by changing the initial PSM cell number. The proportions of the skeletal muscle and spinal neurons gradually decreased as the aggregate size increased (Fig. 4f,g). These results established the induction protocol for a bovine 3D tissue comprising skeletal muscle and spinal neurons from bovine ESCs.

[0159] Co-induction of bovine muscle and endothelial networks in 3D

[0160] Finally, the inventors developed a protocol to co-induce muscle fibers and endothelial cell networks from bovine ESCs in 3D cultures (Fig. 5a). The aggregates of bovine PSM cells were created on day 2, and then treated with the induction cocktail for skeletal muscle and endothelial cells. The aggregate size was again -600 pm in diameter over the entire time course (Fig. 5b, c). VE-cad-positive endothelial cells emerged around day 10, forming interconnected vessel-like networks adjacent to the domains of muscle fibers inside the aggregates (Fig. 5d-f). ZO-1 staining unveiled similarly connected networks, suggesting the presence of tight junctions in the endothelial networks (Fig. 5g, h). Note that the endothelial networks displayed a regular pattern throughout an aggregate, penetrating deeply into the tissue. The abundant and relatively uniform distribution of vasculature will be advantageous for facilitating oxygen and nutrient delivery throughout the muscle aggregates, even though vascular connectivity via intraluminal flow remains to be seen. TUJ1 -positive neurons were also co-induced in the aggregates, forming neural domains (Fig. I la). The endothelial network formation depended on the induction protocols: when PSM cells were induced without the BMP inhibitor DMH1, the subsequent endothelial cells did not form uniform networks, and the shape of the aggregates was no longer spherical (Fig. 11b). Unlike the aggregates that lack endothelial cells (Fig. 4g), the proportions of the muscle domain and the endothelial networks slightly increased or remained constant as the aggregate size increased (Fig. 5i,j). These results underscored the feasibility of generating vascularized skeletal muscle tissues from bovine ESCs according to the invention.

[0161] Cell types induced from bovine ESCs

[0162] The inventor’s single-cell RNA sequencing (scRNA-seq) analyses validated the presence of muscle and neural cell clusters (Fig. 6a; Fig. 12). The endothelial cell (EC) cluster was exclusively detected when using the EC co-induction protocols (Fig. 6b, c). Muscle cells were further classified into skeletal and smooth muscle subclusters (Fig. 6d; Fig. 13a-f), with the 3D induction protocols yielding much higher proportions of skeletal muscle compared to the 2D protocols. Both skeletal and smooth muscle subclusters contained fibroblast-like cells (Fig. 13g). While mature fat cells were not explicitly identified, several adipocyte progenitor markers were detected, particularly under the EC co-induction protocols (Fig. 13h). Similarly, the expression of pericyte and osteoblast markers was more effectively induced under the EC coinduction protocols (Fig. 13i,j). The neural cell cluster showed more effective induction under the 3D induction protocols compared to the 2D protocols (Fig. 6b, c; Fig. 14a-c). Neural cells were further classified into neural progenitors and mature neural cell subclusters, with both subclusters expressing markers for spinal neurons and glial cells (Fig. 14d-i), suggesting the heterogeneous nature of neural subtypes. Consistent with the observed calcium pulses and their suppression by the acetylcholine receptor blocker (Fig. 2b, c), neuromuscular junction markers MUSK and ACHE were detected in the muscle and neural clusters (Fig. 12d). These results affirm the effectiveness of our induction protocols and highlight their potential to co-induce an even broader range of cell types from bovine ESCs. However, they also present the challenges of precisely controlling cell differentiation processes: variations in protocols, such as 2D versus 3D cultures, can lead to unexpected differences in cell type composition. References as cited

[0163] 1. Bhat, Z. F. & Fayaz, H. Prospectus of cultured meat — advancing meat alternatives. J. Food Sci. Technol. 48, 125-140 (2011).

[0164] 2. Post, M. J. Cultured meat from stem cells: Challenges and prospects. Meat Sci. 92, 297-301 (2012).

[0165] 3. Ben- Arye, T. & Levenberg, S. Tissue Engineering for Clean Meat Production. Front. Sustain. FoodSyst. 3, 46 (2019).

[0166] 4. Melzener, L., Verzijden, K. E., Buijs, A. J., Post, M. J. & Flack, J. E. Cultured beef: from small biopsy to substantial quantity. J. Sci. FoodAgric. 101, 7-14 (2021).

[0167] 5. Reiss, J., Robertson, S. & Suzuki, M. Cell Sources for Cultivated Meat: Applications and Considerations throughout the Production Workflow. Int. J. Mol. Sci. 22, 7513 (2021).

[0168] 6. Ozhava, D., Bhatia, M., Freman, J. & Mao, Y. Sustainable Cell Sources for Cultivated Meat.

[0169] J. Biomed. Res. Environ. Sci. 3, 1382-1388 (2022).

[0170] 7. Stout, A. J. et al. Immortalized Bovine Satellite Cells for Cultured Meat Applications. ACS Synth. Biol. 12, 1567-1573 (2023).

[0171] 8. Bogliotti, Y. S. et al. Efficient derivation of stable primed pluripotent embryonic stem cells from bovine blastocysts. Proc. Natl. Acad. Sci. 115, 2090-2095 (2018).

[0172] 9. Zehorai, E. et al. From fertilised oocyte to cultivated meat - harnessing bovine embryonic stem cells in the cultivated meat industry. Reprod. Fertil. Dev. 36, 124-132 (2023).

[0173] 10. Messmer, T. etal. Single-cell analysis of bovine muscle-derived cell types for cultured meat production. Front. Nutr. 10, 1212196 (2023).

[0174] 11. Wang, L. et al. A single-cell atlas of bovine skeletal muscle reveals mechanisms regulating intramuscular adipogenesis and fibrogenesis. J. Cachexia Sarcopenia Muscle 14, 2152-2167 (2023).

[0175] 12. Folkman, J. & Hochberg, M. SELF -REGULATION OF GROWTH IN THREE DIMENSIONS. J. Exp. Med. 138, 745-753 (1973).

[0176] 13. Ben- Arye, T. et al. Textured soy protein scaffolds enable the generation of three- dimensional bovine skeletal muscle tissue for cell-based meat. Nat. Food 1, 210-220 (2020).

[0177] 14. Kang, D.-H. etal. Engineered whole cut meat-like tissue by the assembly of cell fibers using tendon-gel integrated bioprinting. Nat. Commun. 12, 5059 (2021).

[0178] 15. Zagury, Y., lanovici, I., Landau, S., Lavon, N. & Levenberg, S. Engineered marble-like bovine fat tissue for cultured meat. Commun. Biol. 5, 927 (2022). 16. Furuhashi, M. et al. Formation of contractile 3D bovine muscle tissue for construction of millimetre-thick cultured steak. Npj Sci. Food 5, 6 (2021).

[0179] 17. Powell, C. A., Smiley, B. L., Mills, J. & Vandenburgh, H. H. Mechanical stimulation improves tissue-engineered human skeletal muscle. Am. J. PhysioL-Cell Physiol. 283, C1557- C1565 (2002).

[0180] 18. Edelman, P. D., McFarland, D. C., Mironov, V. A. & Matheny, J. G. Commentary: In Vitro -Cultured Meat Production. Tissue Eng. 11, 659-662 (2005).

[0181] 19. Langelaan, M. L. P. et al. Advanced maturation by electrical stimulation: Differences in response between C2C12 and primary muscle progenitor cells. J. Tissue Eng. Regen. Med. 5, 529-539 (2011).

[0182] 20. Rangarajan, S., Madden, L. & Bursae, N. Use of Flow, Electrical, and Mechanical Stimulation to Promote Engineering of Striated Muscles. Ann. Biomed. Eng. 42, 1391-1405 (2014).

[0183] 21. Ito, A. etal. Induction of functional tissue-engineered skeletal muscle constructs by defined electrical stimulation. Sci. Rep. 4, 4781 (2014).

[0184] 22. Sasai, Y. Cytosystems dynamics in self-organization of tissue architecture. Nature 493, 318-326 (2013).

[0185] 23. Lancaster, M. A. & Knoblich, J. A. Organogenesis in a dish: Modeling development and disease using organoid technologies. Science 345, 1247125 (2014).

[0186] 24. Shi, Y., Inoue, EL, Wu, J. C. & Yamanaka, S. Induced pluripotent stem cell technology: a decade of progress. Nat. Rev. Drug Discov. 16, 115-130 (2017).

[0187] 25. Abel, A. & Sozen, B. Shifting early embryology paradigms: Applications of stem cell-based embryo models in bioengineering. Curr. Opin. Genet. Dev. 81, 102069 (2023).

[0188] 26. Wimmer, R. A. et al. Human blood vessel organoids as a model of diabetic vasculopathy. Nature 565, 505-510 (2019).

[0189] 27. Kanton, S. & Pa§ca, S. P. Human assembloids. Development 149, dev201120 (2022).

[0190] 28. Faustino Martins, J.-M. etal. Self-Organizing 3D Human Trunk Neuromuscular Organoids. Cell Stem Cell 26, 172-186.e6 (2020).

[0191] 29. Comai, G. & Tajbakhsh, S. Molecular and Cellular Regulation of Skeletal Myogenesis, in Current Topics in Developmental Biology vol. 110 1-73 (Elsevier, 2014).

[0192] 30. Lazaro, J. et al. A stem cell zoo uncovers intracellular scaling of developmental tempo across mammals. Cell Stem Cell 30, 938-949. e7 (2023).

[0193] 31. Sanaki-Matsumiya, M. et al. Periodic formation of epithelial somites from human pluripotent stem cells. Nat. Commun. 13, 2325 (2022). 32. Matsuda, M. et al. Recapitulating the human segmentation clock with pluripotent stem cells. Nature 580, 124-129 (2020).

[0194] 33. Matsuda, M. et al. Species-specific segmentation clock periods are due to differential biochemical reaction speeds. Science 369, 1450-1455 (2020).

[0195] 34. Bessho, Y., Hirata, H., Masamizu, Y. & Kageyama, R. Periodic repression by the bHLH factor Hes7 is an essential mechanism for the somite segmentation clock. Genes Dev. 17, 1451— 1456 (2003).

[0196] 35. Lewis, J. Autoinhibition with Transcriptional Delay. Curr. Biol. 13, 1398-1408 (2003).

[0197] 36. Chai, J. et al. Differentiation of pluripotent stem cells to muscle fiber to model Duchenne muscular dystrophy. Nat. BiotechnoL 33, 962-969 (2015).

[0198] 37. Chai, J. et al. Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro. Nat. Protoc. 11, 1833-1850 (2016).

[0199] 38. Chai, J. etal. Recapitulating early development of mouse musculoskeletal precursors of the paraxial mesoderm in vitro. Development 145, devl57339 (2018).

[0200] 39. Davis, G. W ., Smith, G. C., Carpenter, Z. L., Dutson, T. R. & Cross, H. R. Tenderness Variations among Beef Steaks from Carcasses of the Same USDA Quality Grade. J. Anim. Sci. 49, 103-114 (1979).

[0201] 40. Ertbjerg, P. & Puolanne, E. Muscle structure, sarcomere length and influences on meat quality: A review. Meat Sci. 132, 139-152 (2017).

[0202] 41. Urzi, A. et al. Efficient generation of a self-organizing neuromuscular junction model from human pluripotent stem cells. Nat. Commun. 14, 8043 (2023).

[0203] 42. Pereira, J. D. et al. Human sensorimotor organoids derived from healthy and amyotrophic lateral sclerosis stem cells form neuromuscular junctions. Nat. Commun. 12, 4744 (2021).

[0204] 43. Potente, M. & Makinen, T. Vascular heterogeneity and specialization in development and disease. Nat. Rev. Mol. Cell Biol. 18, 477-494 (2017).

[0205] 44. Loh, K. M. & Ang, L. T. Building human artery and vein endothelial cells from pluripotent stem cells, and enduring mysteries surrounding arteriovenous development. Semin. Cell Dev. Biol. 155, 62-75 (2024).

[0206] 45. Pardanaud, L. et al. Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis. Development 122, 1363-1371 (1996).

[0207] 46. Esner, M. et al. Smooth muscle of the dorsal aorta shares a common clonal origin with skeletal muscle of the myotome. Development 133, 737-749 (2006). 47. Pouget, C., Gautier, R., Teillet, M.-A. & Jaffredo, T. Somite-derived cells replace ventral aortic hemangioblasts and provide aortic smooth muscle cells of the trunk. Development 133, 1013-1022 (2006).

[0208] 48. Yvemogeau, L., Auda-Boucher, G. & Fontaine-Perus, J. Limb bud colonization by somite- derived angioblasts is a crucial step for myoblast emigration. Development 139, 277-287 (2012).

[0209] 49. Nguyen, P. D. etal. Haematopoietic stem cell induction by somite-derived endothelial cells controlled by meoxl. Nature 512, 314-318 (2014).

[0210] 50. Olmer, R. el al. Differentiation of Human Pluripotent Stem Cells into Functional Endothelial Cells in Scalable Suspension Culture. Stem Cell Rep. 10, 1657-1672 (2018).

[0211] 51. Wimmer, R. A., Leopoldi, A., Aichinger, M., Kerjaschki, D. & Penninger, J. M. Generation of blood vessel organoids from human pluripotent stem cells. Nat. Protoc. 14, 3082-3100 (2019).

[0212] 52. Ang, L. T. et al. Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses. Cell 185, 2523-2541. e30 (2022).

[0213] 53. Yoshii, Y. & Sugiyama, K. Intercapillary distance in the proliferating area of human glioma. Cancer Res. 48, 2938-2941 (1988).

[0214] 54. Rao, L., Qian, Y., Khodabukus, A., Ribar, T. & Bursae, N. Engineering human pluripotent stem cells into a functional skeletal muscle tissue. Nat. Commun. 9, 126 (2018).

[0215] 55. Mavrommatis, L. et al. Human skeletal muscle organoids model fetal myogenesis and sustain uncommitted PAX7 myogenic progenitors. eLife 12, RP87081 (2023).

[0216] 56. Maffioletti, S. M. et al. Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering. Cell Rep. 23, 899-908 (2018).

[0217] 57. Levenberg, S. et al. Engineering vascularized skeletal muscle tissue. Nat. BiotechnoL 23, 879-884 (2005).

[0218] 58. Gholobova, D. et al. Endothelial Network Formation Within Human Tissue-Engineered Skeletal Muscle. Tissue Eng. Part A 21, 2548-2558 (2015).

[0219] 59. Dailamy, A. et al. Programmatic introduction of parenchymal cell types into blood vessel organoids. Stem Cell Rep. 16, 2432-2441 (2021).

[0220] 60. Haase, K. et al. Physiologic flow-conditioning limits vascular dysfunction in engineered human capillaries. Biomaterials 280, 121248 (2022).

[0221] 61. Offeddu, G. S. etal. An on-chip model of protein paracellular and transcellular permeability in the microcirculation. Biomaterials 212, 115-125 (2019). 62. Nashimoto, Y. et al. Integrating perfusable vascular networks with a three-dimensional tissue in a microfluidic device. Integr. Biol. 9, 506-518 (2017).

[0222] 63. Homan, K. A. et al. Flow-enhanced vascularization and maturation of kidney organoids in vitro. Nat. Methods 16, 255-262 (2019).

[0223] 64. Kosyakova, N. et al. Differential functional roles of fibroblasts and pericytes in the formation of tissue-engineered microvascular networks in vitro. Npj Regen. Med. 5, 1 (2020).

[0224] 65. Curtis, M. B., Kelly, N., Hughes, C. C. W. & George, S. C. Organotypic stromal cells impact endothelial cell transcriptome in 3D microvessel networks. Sci. Rep. 12, 20434 (2022).

[0225] 66. Wataya, T. et al. Minimization of exogenous signals in ES cell culture induces rostral hypothalamic differentiation. Proc. Natl. Acad. Sci. 105, 11796-11801 (2008).

[0226] 67. Monke, G., Sorgenfrei, F. A., Schmal, C. & Granada, A. E. Optimal time frequency analysis for biological data - pyBOAT. bioRxiv 2020.04.29.067744 (2020) doi: 10.1101 / 2020.04.29.067744

[0227] 68. Rappez, L., Akinbote, A., Cherubini, M., Uhlmann, V. & Haase, K. Label -Free Phenotyping of Human Microvessel Networks, http: / / biorxiv.org / lookup / doi / 10.1101 / 2024.02.20.581133 (2024) doi: 10.1101 / 2024.02.20.581133.

Claims

Claims1. An in vitro method for the production of ungulate myocytes and muscle fibers, comprising the steps of i) providing a suitable ungulate stable-ESC line in a suitable culture medium free from fetal bovine serum; ii) contacting the uESC line with suitable concentrations of at least one WNT inducer / activator, bFGF, at least one TGFb inhibitor, and at least one BMP inhibitor for about 2 days to induce presomitic mesoderm cells (PSMs); iii) contacting the PSMs with suitable concentrations of HGF, IGF, and bFGF for about 4 days to induce myoblasts; and iv) contacting the myoblasts with suitable concentrations of HGF and IGF for about 9 days to induce myocytes and muscle fibers, wherein preferably the WNT inducer / activator is selected from a GSK-3 inhibitor, such as CHIR, e.g. CHIR 90221, the TGFb inhibitor is selected from SB431542, and the BMP inhibitor is selected from DMH1.

2. The method according to claim 1, wherein the myocytes and muscle fibers form a tissue furthermore comprising neuronal cells derived from neuromesodermal progenitors (NMPs), such as neurons.

3. The method according to claim 1 or 2, further comprising the co-production of endothelial cells, comprising further contacting the PSMs in step iii) with suitable concentrations of forskolin and VEGF to induce endothelial cells, and further contacting the endothelial cells in step iv) with suitable concentrations of VEGF to further induce endothelial cells.

4. The method according to any one of claims 1 to 3, wherein the production provides a 3D culture, further comprising adding suitable low concentrations of Matrigel, TrueGel3D™ and / or Cultrex to the PSMs in step iii) after aggregate formation, preferably at the end of step ii), andshaking the culture at about 50 to 100 rpm, preferably about 75 rpm after ECM addition in step iii) and in step iv), thereby inducing a 3D culture, wherein preferably the low concentration of Matrigel, TrueGel3D™ or Cultrex is between about 0.5 and 5%, preferably at between about 1 and 3%, more preferably at about 2%.

5. The method according to any one of claims 1 to 4, wherein the ungulate stable-ESC line is selected from CTFR-uESC and NBFR-uESC, or CTFR-bESC and NBFR-bESC.

6. The method according to any one of claims 1 to 5, wherein the ungulate is selected from pigs, peccaries, hippopotamuses, antelopes, deer, giraffes, camels, llamas, alpacas, sheep, goats, and cattle, and in particular bovines.

7. The method according to any one of claims 1 to 6, wherein the method comprises the selforganization of myocytes and muscle fibers with innervation, such as neurons.

8. The method according to any one of claims 1 to 7, wherein the method comprises forming of regular endothelial cellular networks inside muscle tissues, such as a vascular-like network, and / or the self-organization of myocytes and muscle fibers with innervation, such as neurons, and / or vascularization.

9. The method according to any one of claims 1 to 8, wherein the method is devoid of genetic manipulation or genetic modification of the cells.

10. The method according to any one of claims 1 to 9, wherein the suitable concentration of SB431542 is at about 10 pM, of DMH1 is at about 2 pM, of CHIR is at about 10 pM, bFGF is at about 20 ng / ml, HGF is at about 10 ng / ml, IGF (e.g. IGF-1) is at about 2 ng / ml, forskolin is at about 2 pM, and VEGF, such as rhVEGFies, is at about 100 to about 200 ng / ml.

11. The method according to any one of claims 1 to 10, wherein the method is performed in mTeSRl medium, StemFit medium, CTFR or NBFR medium at about 37 °C, and at about 5% CO2.

12. The method according to any one of claims 1 to 11, further comprising the detection of at least one cell specific marker selected from the group consisting of NANOG, OCT4, S0X2, BRACHYURY, TBX6, HES7, MYF5, MYODI, MYOG, MYH4, MYF6, MYH7, SAA, MHC, SMI- 32, and FUJI.

13. A method for producing a comestible meat product, comprising the method according to any one of claims 4 to 12 to produce a 3D skeletal muscle tissue, in particular a self-organized 3D tissue, and / or further comprising the step of suitably combining at least two or more of the 3D tissues as produced to obtain a comestible meat product.

14. A 3D culture, such as a 3D skeletal muscle tissue, in particular a self-organized 3D tissue produced according to any one of claims 4 to 12, or a comestible meat product produced according to claim 13, preferably comprising regular endothelial cellular networks inside muscle tissues, such as a vascular-like network and / or vascularization, and / or self-organized myocytes and muscle fibers with innervation, such as neurons.

15. Use of a 3D culture, such as a 3D skeletal muscle tissue, in particular a self-organized 3D tissue produced according to any one of claims 4 to 12 to make a comestible meat product.

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