A method for culturing pluripotent cells to differentiate them into progenitor cells or mature muscle cells, and a composition containing said cells

Abstract

Provided herein are artificial cultured somites containing mature muscle progenitor cells and / or mature muscle cells, methods for obtaining same, and methods for rapid and large-scale production of cultivated meat containing mature muscle cells.

Description

Technical Field

[0001] Provided herein are artificial cultured somites containing mature muscle progenitor cells and / or mature muscle cells, a method for obtaining the same, and a method for rapidly and massively producing cultured meat containing mature muscle cells.

Background Art

[0002] The meat industry is one of the main causes of environmental and climate deterioration. Due to the growth of the world population and the increasing demand, meat consumption is expected to double between 1999 and 2050. This industry is also highly related to the emergence of antibiotic resistance and zoonotic diseases. Although a completely plant-based diet may be a good option for reducing environmental impact, there are many reasons to believe that most of the world's population will not voluntarily adapt to such a diet while meat is still available. Cultured meat is produced in vitro using cell culture and provides an alternative production method for meat that is inexpensive, ethical, and environmentally friendly. At the same time, however, it has characteristic taste, texture, and nutritional value, and further maintains the same biological origin as animal-derived products. Since skeletal muscle is the main edible tissue used in the meat of most animals, it is necessary to generate a large amount of skeletal muscle in vitro in order to efficiently produce cultured meat.

[0003] Schmidt et al. (Cellular and Molecular Life Sciences, 76:2559-2570, 2019) disclose skeletal muscle regeneration from satellite cells, which are skeletal muscle stem cells.

[0004] Suzuki et al. (Differentiation, 96:70-81, 2017) disclose a protocol for directly (without genetic recombination) inducing myogenic precursors from human pluripotent cells using free-floating spherical culture through a long differentiation period of more than 6 weeks in a medium supplemented with 100 ng / ml FGF-2 and 100 ng / ml EGF.

[0005] Nachman et al. (bioRxiv, 728642, 2019) have disclosed that the basic rules governing the generation of embryonic endoderm cells from within the mesoderm progenitor cell population involve self-selection of Sox17+ cells preceding upregulation of E-cadherin, and that a minor subpopulation of highly expressed Bra cells is involved in the life and death of Sox17+ independently of external Wnt signals.

[0006] There is a need for an efficient, rapid, and cost-effective process for producing in vitro muscle cells, preferably within a tissue structure, but this need has not been met. SUMMARY OF THE INVENTION

[0007] Provided herein is a serum-free and genome-modification-free protocol based on the developmental pathway for generating somites, myogenic precursors, muscle cells, and muscle tissue in culture. This protocol includes inducing muscle differentiation in a three-dimensional suspension cell culture starting from embryonic stem cells. Additionally, cultured somites and somite-like structures containing muscle cells are provided.

[0008] Advantageously, the protocol disclosed herein for the preparation of cultured meat products combines the self-organizing properties of a microscale three-dimensional embryonic development system with biotechnological solutions and is highly suitable for the mass production of meat products and the maturation of muscle tissue in organoid culture. This protocol resolves the large dissociation between existing established muscle differentiation protocols and the scale-up and safety requirements of food production.

[0009] Surprisingly, the protocol disclosed herein generates somites and somitoid structures, which are closed structures that give rise to vertebrae and skeletal muscle in the embryo. The generation of somites has several advantages. First, the generation of somites indicates that the protocol (method / process) disclosed herein closely mimics the developmental stages that cells undergo during embryonic development from pluripotent (ES) cells to fully differentiated skeletal muscle cells in a three-dimensional suspension organoid format. As a result, the cultured tissue produced herein contains all the characteristics of "real" meat. That is, it is mainly composed of muscle tissue but also contains adipocytes, connective tissue, and blood, which contribute to the structure and flavor of meat produced from livestock. Second, the presence of somites in the production process allows for in-organoid signaling and promotes the necessary differentiation independent of external addition. Thus, the amount of growth factors used for addition to the medium is minimal (e.g., on the order of about 10 ng / ml). Therefore, the protocol disclosed herein is cost-effective as it does not require large amounts of expensive growth factors, which are typically a major expense in the production of cultured tissue.

[0010] Surprisingly, when the protocol disclosed herein is applied to pluripotent cells, myogenic precursors are produced in a short period, such as just 2 weeks, using FGF-2 and EGF growth factors at about 20 ng / ml or less.

[0011] Furthermore, since the developmental processes, including somitogenesis and myogenesis, are highly conserved among vertebrates, the protocol disclosed herein that follows embryonic development guidelines can be successfully implemented to produce cultured meat from precursor cells of any livestock species.

[0012] Finally, this protocol allows for differentiation in suspension (e.g., even in large-scale bioreactors or conventional stirrers) from stem cells until myogenic precursors are obtained.

[0013] According to some embodiments, from the above perspective, the protocol disclosed herein is rapid, cost-effective, reproducible, suitable for large-scale bioreactors, and capable of producing cultured meat that includes the characteristics of livestock meat, and is very suitable for the industrial production of cultured myogenic precursors and cultured meat.

[0014] The disclosed protocol consists of a series of steps, and each step attempts to efficiently mimic the in-vivo signaling environment sensed by the myogenic lineage at the relevant developmental stage, such as mesoderm, presomitic mesoderm, myogenic precursor, and mature muscle cells. The first step of the protocol involves aggregating mES cells under a first culture medium following the differentiation of aggregates in suspension, and the medium may be changed during several consecutive steps as further detailed and exemplified below.

[0015] According to some embodiments, a method for generating somites is provided, the method comprising: (a) suspending embryonic stem cells in a medium to obtain aggregates that are substantially spherical; (b) adding at least one Wnt activator to the medium to obtain aggregates containing mesoderm cells and endoderm cells; (c) removing the Wnt activator; (d) observing the morphology of the aggregates containing the mesoderm cells and endoderm cells, and when the aggregates have an egg-like morphology, adding an extracellular matrix or a component thereof to obtain aggregates containing paraxial mesoderm; (e) incubating the aggregates of step (d) for 10 to 48 hours to thereby obtain aggregates containing a plurality of somites.

[0016] According to some embodiments, the method further comprises adding one or more of insulin, knockout serum replacement, transferrin-selenium, and BMP4 to the medium of step (a).

[0017] According to some embodiments, the Wnt activator is selected from the group consisting of Chir99021, Wnt3a, and Rspo3.

[0018] According to some embodiments, adding at least one Wnt activator to the medium is performed within 12 to 36 hours after obtaining the substantially spherical aggregates.

[0019] According to some embodiments, removing the Wnt activator is performed within 8 to 36 hours after adding the Wnt activator. According to some embodiments, removing the Wnt activator is performed within 12 to 36 hours after adding the Wnt activator.

[0020] According to some embodiments, the method may further include adding at least one Nodal inhibitor to the medium after the appearance of the aggregates containing the mesoderm cells and endoderm cells.

[0021] According to some embodiments, the amount of the extracellular matrix or its components is in the range of about 1 to 15% vol / vol.

[0022] According to some embodiments, the method may further include adding at least one compound selected from the group consisting of a Wnt activator and a BMP inhibitor in step (d).

[0023] According to some embodiments, the method may further include removing the extracellular matrix or its components after obtaining the plurality of somites.

[0024] According to some embodiments, a method for generating myogenic precursors is provided, the method comprising: (a) propagating embryonic stem cells in a medium to obtain spherical aggregates; (b) adding at least one Wnt activator to the medium to obtain aggregates containing mesoderm cells and endoderm cells; (c) The step of removing the Wnt activator; (d) Observing the morphology of the aggregate containing the mesodermal cells and endodermal cells, and when the aggregate has an egg-like morphology, adding an extracellular matrix or a component thereof to obtain an aggregate containing paraxial mesoderm; (e) Incubating the aggregate of step (d) for 10 to 48 hours to thereby obtain an aggregate containing a plurality of somites; (f) Adding one or more growth factors to the medium of step (e) such that the concentration is less than 30 ng / ml, thereby obtaining myogenic precursors.

[0025] According to some embodiments, the one or more growth factors may include HGF, IGF, and FGF2.

[0026] According to some embodiments, step (a) may be performed in a suspended state.

[0027] According to some embodiments, the method may further include adding at least one compound selected from a BMP inhibitor and a Wnt activator to the medium of step (d). According to some embodiments, the Wnt activator is selected from the group consisting of Chir99021, Rspo3, and Wnt3a.

[0028] According to some embodiments, adding at least one Wnt activator to the medium is performed within 12 to 36 hours after obtaining the spherical aggregate.

[0029] According to some embodiments, the method may further include removing the extracellular matrix (ECM) or a component thereof before step (e). According to some embodiments, the method may further include removing the extracellular matrix or a component thereof before step (f).

[0030] According to some embodiments, the concentration of each of the growth factors is in the range of about 1 to 25 ng / ml.

[0031] According to some embodiments, the one or more growth factors are IGFs, and the concentration of the IGFs is in the range of about 1 to 5 ng / ml.

[0032] According to some embodiments, the myogenic precursors include myoblasts and muscle cells.

[0033] According to some embodiments, the method may further include adding one or more of insulin, knockout serum replacement, transferrin-selenium, and BMP4 to the medium of step (a).

[0034] According to some embodiments, removing the Wnt activator may be performed within about 8 to 36 hours. According to some embodiments, removing the Wnt activator may be performed within about 12 to 36 hours after the addition of the Wnt activator.

[0035] According to some embodiments, the method may further include adding at least one Nodal inhibitor to the medium after the appearance of the aggregate including the mesoderm cells and endoderm cells.

[0036] According to some embodiments, the amount of the extracellular matrix or its components may be in the range of about 1 to 15% vol / vol.

[0037] According to some embodiments, provided is a cultured somite containing mature muscle precursor cells and / or mature muscle cells prepared by the method disclosed herein.

[0038] Other objects, features, and advantages of the present invention will become apparent from the following description, examples, and drawings.

[0039] Certain embodiments of the present disclosure may include some or all of the above advantages, or may include none of them. One or more other technical advantages will be readily apparent to those skilled in the art from the figures, descriptions, and claims included herein. Further, although specific advantages are listed above, various embodiments may include all, some, or none of the listed advantages.

Brief Description of the Drawings

[0040] Some embodiments of the present disclosure are described herein with reference to the accompanying drawings. This description, together with the figures, will make it apparent to those skilled in the art how some embodiments may be implemented. The figures are for illustrative purposes only and are not intended to show the structural details of the embodiments in more detail than is necessary for a basic understanding of the present disclosure. For clarity, some of the objects depicted in the figures are not to scale. The figures are as follows.

[0041] Figure 1A shows aggregates containing mesodermal cells (Brachyury-positive cells (Bra-GFP), upper panel) and endodermal cells (Sox17-positive cells (Sox-RFP), middle panel) obtained from ESC cells seeded on a plate after step 2 of the disclosed protocol.

[0042] Figure 1B shows aggregates containing mesodermal cells (Brachyury-positive cells, thin arrows) and endodermal cells (Sox17-positive cells, thick white arrows) obtained from ESC cells in suspension after step 2 of the disclosed protocol.

[0043] Figure 2A represents the spatiotemporal pattern of posterior presomitic mesoderm (pPSM) in a mouse three-dimensional organoid formed during step 3 of the disclosed protocol. The organoid was aggregated from Msgn1-YFP mES cells and cultured in suspension for 6 days. The scale bar is 100 μm.

[0044] Figure 2B plots the fluorescence levels against the absolute organoid length (left panel) and the normalized organoid length (right panel) measured for 20 organoids formed during step 3 of the disclosed protocol. Msgn1-YFP labels pPSM.

[0045] Figure 2C represents an elongated organoid consisting of two clusters. Msgn1+ cells have migrated to form two clusters.

[0046] Figure 2D represents an organoid consisting of three clusters. Msgn1+ cells have migrated to form three clusters, all of which extend from the center of the organoid.

[0047] Figure 2E represents a histogram of the number of Msgn1+ clusters per organoid.

[0048] Figure 3A represents a 5-day-old organoid formed in step 3 that was fixed and immunostained for brachyury (located at the left / rear pole, indicated by the gray arrow), and Msgn1-YFP labels pPSM (bright region, indicated by the dashed arrow).

[0049] Figure 3B is a schematic diagram of the organoid shown in Figure 3A, including posterior pole brachyury-positive cells, Msgn1-positive pPSM cells, and anterior presomitic mesoderm (aPSM) cells at the anterior pole.

[0050] Figure 3C represents the average delta CT values of pPSM-related genes on day 4 (black columns) and day 6 (gray columns) of the differentiation of mESC-derived organoids into PSM.

[0051] Figure 3D represents an exemplary image of a Msgn1-YFP mESC organoid immunostained for Pax3 (right panel) on day 6 of differentiation (central panel). The scale bar is 100 μm.

[0052] Figure 3E represents an exemplary image of a Msgn1-YFP mESC organoid immunostained for N-cadherin (Cdh2, right panel) on day 6 of differentiation (central panel). The scale bar is 100 μm.

[0053] Figures 4A-4C represent images of the aggregates obtained in step 3. Msgn1-YFP (bright regions) indicates the presence of pPSM. The scale bar is 100 μm.

[0054] Figures 5A-5C are images of the elongated aggregates obtained at the end of step 4, stained with the somite marker (Pax3) and imaged in two z-sections (left, center) and bright field (right). The somites are numbered. The thick white arrow points to the Sox17-RFP positive region, indicating the endoderm. The scale bar is 100 μm.

[0055] Figure 5D represents an image of the elongated aggregates obtained at the end of step 4, with positive regions for Brachyury GFP and Sox17-RFP, labeling the mesoderm and endoderm, respectively. The somites are indicated by small white arrows pointing to the circular structures in the anterior region. The scale bar is 100 μm.

[0056] Figure 6A represents an image of the aggregates obtained at the end of step 5, which aggregates are stained with a myogenic precursor marker. There are Pax7 (bright regions in the upper half of the aggregates) and a Sox17-RFP positive endoderm region (lower left, marked with a white arrow). The scale bar is 100 μm.

[0057] Figure 6B represents an image of the aggregates obtained at the end of step 5, which aggregates are stained with the myoblast marker MyoD (bright regions). The scale bar is 100 μm.

[0058] Figure 6C represents an image of the aggregates obtained at the end of step 5, which aggregates are stained with the myocyte marker MyoG (bright regions). The scale bar is 100 μm.

[0059] Figure 6D shows an image of mature somites, and the black arrows indicate representative somites. The scale bar is 100 μm.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The principles, usage, and implementation of the teachings herein will be better understood by referring to the accompanying description and drawings. By carefully reading the description and drawings described herein, those skilled in the art will be able to implement the teachings herein without undue effort or experimentation. In the figures, the same reference numerals indicate the same parts throughout. In the figures, the same reference numerals indicate the same parts throughout.

[0061] Cell-based meat science has evolved in recent years, but obtaining large amounts of skeletal muscle tissue is a major challenge. For this, a large-scale feasible and efficient proliferation and muscle differentiation process is required. The suspension culture method is the most promising method for scale-up, but currently, it is a hindrance to muscle differentiation because a physical anchor point is required.

[0062] In recent years, three-dimensional suspension culture of small organ-like three-dimensional structures (called organoids) that mimic various aspects of native organs containing cell compositions has advanced by leaps and bounds. Many of the organoid systems start from embryonic stem cells, provide a complete differentiation process from start to finish, mimic the embryonic process to some extent, and do not require repeated collection of starting cell populations from animals.

[0063] Briefly, there are mainly two approaches to the production of cultured meat. (1) Starting from satellite cells. The relative advantage of cultured satellite cells is that their differentiation into muscle cells is relatively fast and easy. However, since satellite cells are post-mitotic (post-terminal differentiation) cells, they do not proliferate, and using them as starting cells is not suitable for mass production. (2) Starting from pluripotent cells (embryonic stem cells). The advantages of using pluripotent cells are their excellent long-term proliferation ability and the ability to differentiate when exposed to appropriate conditions. However, the process of these cells differentiating into mature muscle cells takes a very long time (a period exceeding one month, usually a period exceeding six weeks).

[0064] The main challenge is to efficiently grow a large number of muscle cells, preferably in a tissue similar to cultured meat, which mainly consists of muscle tissue (about 90%) and further contains a combination of adipocytes, blood, and connective tissue, similar to the content of meat products. Current solutions mainly start from adult progenitor cells (such as satellite cells) and rely on differentiation protocols for single cell types, so there are limitations in both proliferation ability and similarity to a complete tissue structure. Therefore, a large-scale feasible and efficient proliferation and muscle differentiation process is required.

[0065] According to some embodiments, by utilizing the protocol disclosed herein, mainly because this protocol produces somatic cells, cultured meat containing various components of meat products can be produced.

[0066] As used herein, the term "protocol" is interchangeable with the term "method" or "process" in the context of the protocol for obtaining somites and three-dimensional edible organoids.

[0067] Known myogenic differentiation protocols are either based on two-dimensional adherent culture or are inefficient. Many protocols use serum and / or genetic modification to activate specific important genes. Some use partially differentiated cells (such as satellite cells) as the starting cell type, and the proliferation rate and capacity of these starting cells may be limited. The protocol disclosed herein combines a differentiation protocol based only on signaling with a suspension 3D culture system (starting from ES cells), resulting in a large-scale realizable and efficient myogenic differentiation method that does not contain serum and other animal-derived factors. By using pluripotent stem cells, it is not necessary to repeatedly collect starting cell populations from animals.

[0068] According to some embodiments, a method of generating somites is provided, the method comprising: (a) suspending embryonic stem cells in a medium to obtain aggregates that are substantially or sufficiently spherical; (b) adding at least one Wnt activator to the medium to obtain aggregates containing mesodermal cells and endodermal cells; (c) removing the Wnt activator; (d) observing the morphology of the aggregates containing the mesodermal cells and endodermal cells, and when the aggregates have an egg-like morphology, adding an extracellular matrix (ECM) or a component thereof to obtain aggregates containing paraxial mesoderm; (e) incubating the aggregates of step (d) for 10 to 48 hours to thereby obtain aggregates containing a plurality of somites.

[0069] According to some embodiments, as used herein, the terms "propagation" and "suspension" refer to culturing in two dimensions or in suspension (three dimensions) under conditions suitable for the proliferation and maintenance of pluripotent cells. Cell proliferation generally increases the size of the aggregates to form larger aggregates, but by constantly dissociating these aggregates mechanically or enzymatically into smaller aggregates, cell proliferation can be maintained in the culture and the number of cells can be increased. Usually, cells cultured in aggregates in maintenance culture maintain pluripotency markers. Pluripotency can be determined in part by evaluating the pluripotent characteristics of the cells. Characteristics of pluripotency include (i) the morphology of pluripotent stem cells, (ii) the potential for unlimited self-renewal, (iii) the expression of pluripotent stem cell markers, examples of pluripotent stem cell markers including, but not limited to, SSEA1 (mouse only), SSEA3 / 4, SSEA5, TRA1-60 / 81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133 / prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30, and / or CD50, (iv) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm, endoderm), (v) the formation of teratomas consisting of the three somatic cell lineages, and (vi) the formation of embryoid bodies consisting of cells from the three somatic cell lineages, but are not limited thereto. The aggregates of pluripotent stem cells require further cues for differentiation to induce differentiation.

[0070] According to some embodiments, at the end of step (a) of the method, a plurality of spherical aggregates are obtained. According to some embodiments, the plurality of spherical aggregates obtained at the end of step (a) includes at least 50 spherical aggregates, at least 100 spherical aggregates, at least 150 spherical aggregates, at least 200 spherical aggregates, at least 250 spherical aggregates, at least 300 spherical aggregates, about 300 spherical aggregates. Each possible configuration represents a separate embodiment.

[0071] As used herein, the term "about" may be used to specify a value of a quantity or parameter (e.g., amount, percentage, length) within a continuous range of values near (and including) a given (stated) value. According to some embodiments, "about" may specify that the value of the parameter is between 80% and 120% of the given value. According to some embodiments, "about" may specify that the value of the parameter is between 90% and 110% of the given value. According to some embodiments, "about" may specify that the value of the parameter is between 95% and 105% of the given value.

[0072] The terms "pluripotent stem cell", "embryonic stem cell", and ESC as used herein are interchangeable.

[0073] According to some embodiments, the embryonic stem cells are derived from livestock. According to some embodiments, the embryonic stem cells are derived from an animal selected from the group consisting of cows, sheep, goats, fish, pigs, and poultry.

[0074] It will be understood that the embryonic stem cells (ESCs) used herein have not been genetically engineered.

[0075] The morphology of the pluripotent stem cells has the classical morphological characteristics of embryonic stem cells. The morphology of normal embryonic stem cells is characterized by being round (spherical) and small, having a high ratio of nucleus to cytoplasm, the presence of prominent nucleoli, and typical intercellular spaces. In contrast, the aggregates containing mesoderm cells and endoderm cells obtained by step (b) of the method disclosed herein have, for example, an egg-like morphology as shown in Figure 2A on day 4.5.

[0076] According to some embodiments, step (a) is performed on a culture plate (e.g., a petri dish and / or a multi-well plate). Thus, according to some embodiments, embryonic stem cells form an adherent cell culture within the culture plate. According to some embodiments, the method may further include adding one or more of insulin, transferrin-selenium, knockout serum replacement, and BMP4 to the medium of step (a). Each possible configuration represents a separate embodiment.

[0077] To increase the fraction of PSM cells in the organoid / aggregate, the step of adding one or more of insulin, transferrin-selenium, knockout serum replacement, and BMP4 to the medium of step (a) is performed. Thus, adding insulin, transferrin-selenium, knockout serum replacement, and / or BMP4 to the medium of step (a) improves the quality of the protocol disclosed herein.

[0078] According to some embodiments, insulin may be added to the medium. According to some embodiments, the concentration of insulin added to the medium is in the range of 5-15 μl / ml, 5-13 μl / ml, 7-13 μl / ml, 7-12 μl / ml, 5-12 μl / ml, about 7 μl / ml, or about 10 μl / ml. Each possible configuration represents a separate embodiment.

[0079] According to some embodiments, transferrin-selenium may be added to the medium. According to some embodiments, the concentration of transferrin-selenium added to the medium is in the range of 0.1%-5%, 0.2%-4%, 0.2%-3%, 0.5%-3%, 0.5%-2%, 0.8%-3%, 0.8%-2%, about 2%, about 1.5%, or about 1%. Each possible configuration represents a separate embodiment.

[0080] According to some embodiments, BMP4 may be added to the medium. According to some embodiments, the concentration of BMP4 added to the medium is in the range of 5 - 15 μl / ml, 5 - 13 μl / ml, 7 - 13 μl / ml, 7 - 12 μl / ml, 5 - 12 μl / ml, about 7 μl / ml, or about 10 μl / ml. Each possible configuration represents a separate embodiment.

[0081] According to some embodiments, knockout serum replacement (KSR) may be added to the medium.

[0082] According to some embodiments, step (a) may be performed in N2B27 medium. According to some embodiments, step (a) may be performed in a medium supplemented with at least one of knockout serum replacement (KSR), bovine serum albumin (BSA), insulin, and BMP4. According to some embodiments, step (a) may be performed in a medium supplemented with low - concentration insulin. According to some embodiments, the low - concentration insulin is in the range of about 1 - 25 μg / ml of insulin, about 1 - 20 μg / ml of insulin, about 1 - 15 μg / ml of insulin, about 5 - 25 μg / ml of insulin, or about 5 - 20 μg / ml of insulin. Each possible configuration represents a separate embodiment.

[0083] According to some embodiments, the cells are maintained in a suspended state by rotating the plate.

[0084] According to some embodiments, the Wnt activator added in step (b) is selected from the group consisting of Chir99021, Rspo3, and Wnt3a. Each possible configuration represents a separate embodiment.

[0085] According to some embodiments, the concentration of the Wnt activator added in step (b) is in the range of about 0.5 - 10 μM, 0.5 - 7 μM, 0.7 - 10 μM, 0.7 - 7 μM, 1 - 7 μM, or 1 - 5 μM. Each possible configuration represents a separate embodiment.

[0086] According to some embodiments, the Wnt activator is added in step (b) about 24 hours after a plurality of aggregates are formed in the culture. According to some embodiments, the Wnt activator is added in step (b) about 24 hours after a plurality of aggregates are formed in a suspended state.

[0087] According to some embodiments, the Wnt activator is removed from the medium within about 1 day after being added to the medium, within 8 to 36 hours after being added, within 12 to 36 hours after being added, within 18 to 30 hours after being added, or within about 24 hours after being added. Each possible configuration represents a separate embodiment. In some embodiments, the removal of the Wnt activator may be achieved by replacing the medium with a similar medium lacking the Wnt activator.

[0088] According to some embodiments, the method may further comprise adding at least one Nodal inhibitor to the medium after the appearance of the aggregates comprising the mesodermal cells and endodermal cells. Adding at least one Nodal inhibitor to the medium helps to suppress the expansion of non-somatic cell populations such as embryonic endoderm.

[0089] According to some embodiments, the inhibitor is selected from the group consisting of SB-431542 and SB-505124. Each possible configuration represents a separate embodiment. According to some embodiments, the concentration of the Nodal inhibitor added in step (b) is in the range of 200 nM, 50 - 700 nM, 5 - 15 μM, 5 - 12 μM, 7 - 15 μM, 7 - 12 μM, or is about 7 μM, or about 10 μM. Each possible configuration represents a separate embodiment.

[0090] According to some embodiments, the scaffold matrix (extracellular matrix), or a component thereof, is incorporated into the aggregates in the suspension. According to some embodiments, the medium contains extracellular matrix (ECM) or a component thereof at a low concentration, specifically less than about 20% vol / vol, less than 15% vol / vol, less than 10% vol / vol, less than 8% vol / vol, less than 5% vol / vol. According to some embodiments, the amount of ECM in the medium is in the range of about 1-15% vol / vol. According to some embodiments, the amount of ECM in the medium is in the range of about 1-12% vol / vol. According to some embodiments, the amount of ECM in the medium is in the range of about 2-12% vol / vol. According to some embodiments, the amount of ECM in the medium is in the range of about 3-13% vol / vol. According to some embodiments, the amount of ECM in the medium is in the range of about 2-12% vol / vol. According to some embodiments, the amount of ECM in the medium is in the range of about 3-11% vol / vol. According to some embodiments, the amount of ECM in the medium is in the range of about 3-10% vol / vol. According to some embodiments, the amount of ECM in the medium is in the range of about 4-10% vol / vol. According to some embodiments, the amount of ECM in the medium is in the range of about 5-10% vol / vol.

[0091] According to some embodiments, the ECM includes Matrigel (trademark). According to some embodiments, the medium contains a component of the ECM. According to some embodiments, the components of the ECM include, but are not limited to, fibronectin, collagen, and laminin.

[0092] According to some embodiments, the addition of the ECM or its components is performed after observing aggregates having an egg-like morphology. Thus, the protocol is not based on a random / fixed timing for adding the ECM or its components. The appropriate timing has been found to vary from batch to batch, and surprisingly, it is best to add the ECM or its components based on the observation of the aggregates. The ideal timing is when the aggregates exhibit an egg-like form.

[0093] When the ECM or its components are added, paraxial mesoderm cells are formed. According to some embodiments, the ECM or its components are removed from the medium after the formation of paraxial mesoderm cells within the aggregates. The removal of the ECM or its components from the medium provides an additional advantage to the disclosed protocol. That advantage is to reduce the overall cost of the entire protocol without compromising the desired results.

[0094] According to some embodiments, there is also an option to maintain the ECM or its components in the medium.

[0095] According to some embodiments, the method may further comprise adding at least one Wnt activator and / or at least one BMP inhibitor to the medium in step (d). Thus, according to some embodiments, the medium in step (d) may further comprise at least one Wnt activator and / or at least one BMP inhibitor. Adding a Wnt activator and / or a BMP inhibitor to the medium in step (d), i.e., the medium containing the ECM or its components, is beneficial because it can improve the yield of somitogenesis. Further, adding the Wnt activator(s) and / or BMP inhibitor(s) to the medium at this stage reduces the overall viscosity of the medium, thereby promoting growth under mixed suspension conditions, which is particularly suitable for industrial-scale bioreactors.

[0096] According to some embodiments, the at least one Wnt activator is selected from Chir99201 and Rspo3. Each possible configuration represents a separate embodiment.

[0097] According to some embodiments, the at least one BMP inhibitor is selected from LDN193189 and Noggin. Each possible configuration represents a separate embodiment.

[0098] According to some embodiments, a plurality of organoids are embedded in a scaffold matrix at the end of the differentiation process.

[0099] As used herein, the terms "aggregate", "three-dimensional organoid" and "organoid" are interchangeable.

[0100] According to some embodiments, a method for generating myogenic precursors is provided, the method comprising the above steps for obtaining somites, (e) incubating the aggregates of step (d) for 10 to 48 hours, thereby obtaining aggregates comprising a plurality of somites, and (f) adding one or more growth factors such that their concentration is less than about 30 ng / ml, thereby obtaining myogenic precursors.

[0101] According to some embodiments, the one or more growth factors include, but are not limited to, HGF, IGF, and FGF2, and the concentration of the one or more growth factors is in the range of about 1 to 25 ng / ml. According to some embodiments, at least one compound selected from a BMP inhibitor and a Wnt activator is further added to the medium of step (f).

[0102] According to some embodiments, the one or more growth factors may include HGF. According to some embodiments, the concentration of HGF added to the medium in step (f) is in the range of 5-15 μl / ml, 5-13 μl / ml, 7-13 μl / ml, 7-12 μl / ml, 5-12 μl / ml, about 7 μl / ml, or about 10 μl / ml. Each possible configuration represents a separate embodiment.

[0103] According to some embodiments, the one or more growth factors may include IGF. According to some embodiments, the concentration of IGF added to the medium in step (f) is in the range of about 0.5-5 μl / ml, 0.5-3 μl / ml, 0.7-3 μl / ml, 0.7-2.5 μl / ml, 1-2.5 μl / ml, about 2.5 μl / ml, or about 2 μl / ml. Each possible configuration represents a separate embodiment.

[0104] According to some embodiments, the one or more growth factors may include FGF2. According to some embodiments, the concentration of FGF2 added to the medium in step (f) is in the range of about 10-25 μl / ml, 12-25 μl / ml, 12-23 μl / ml, 15-25 μl / ml, 15-23 μl / ml, about 25 μl / ml, or about 20 μl / ml. Each possible configuration represents a separate embodiment.

[0105] According to some embodiments, the obtained myogenic precursors include myoblasts, muscle cells, and satellite cells.

[0106] It has been demonstrated that the maturation of muscle cells depends on the fixation of cells to a (semi) rigid surface, seemingly mimicking the fixation to bone structures. Thus, the method disclosed herein may further include the step of embedding the late-stage organoids in an edible matrix for the purpose of generating an anchor point for the maturation of muscle cells. Examples of any matrix include, but are not limited to, mycelium, alginate, and cellulose (e.g., derived from decellularized apples).

[0107] According to some embodiments, the method further includes monitoring differentiation in the suspended three-dimensional organoids.

[0108] According to some embodiments, the monitoring may be performed using at least one technique selected from the group consisting of high-throughput two-photon 3D imaging, live imaging using GFP-tagged developmental markers (such as Msgn1, Pax3, Pax7, etc.), and immunostaining against marker proteins.

[0109] Imaging is also used to monitor cell compositions at various stages of the process for the purpose of identifying efficiency bottlenecks with respect to the fraction of myogenic cells within the organoids.

[0110] According to some embodiments, the method may further include characterizing cell populations within the three-dimensional organoids at different time points.

[0111] According to some embodiments, the method may further include characterizing the spatial configuration of cell populations within the three-dimensional organoids at different time points.

[0112] According to some embodiments, characterizing cell populations in the three-dimensional organoids may be performed in various ways, examples of which include RT-PCR and FISH, but are not limited thereto.

[0113] According to some embodiments, the method may further include nutritional profiling of the three-dimensional organoids. Various approaches can be applied to nutritional profiling, such as GC, HPLC, GC-MS using cold EI, etc.

[0114] According to some embodiments, the three-dimensional organoids are in a format selected from the group consisting of hanging drop, rotary suspension, free suspension, patterned microwells, and high-throughput 96-well.

[0115] According to some embodiments, provided are cultured somites and cultured tissues comprising mature muscle progenitor cells or mature muscle cells obtained by the protocols disclosed herein.

[0116] In the specification and claims of this application, the words "include" and "have" and their forms are not limited to the members of the list with which the word may be associated

[0117] Those skilled in the art will readily understand that the present invention is well adapted to carry out its purpose and obtain the mentioned objectives and advantages, as well as those inherent in the present invention. The examples provided herein represent preferred embodiments and are illustrative and are not intended to limit the scope of the present invention.

Examples

[0118] Example 1 Protocol for Somite and Myogenic Progenitor Generation

[0119] The 3D differentiation model disclosed herein forms the basis for industry-level scale-up processes and adaptation to other food species. A complete suspension, cell cluster-based protocol as disclosed herein can be incorporated into bioreactors without size limitations. Furthermore, the protocol does not use serum and does not involve genetic recombination, making it suitable for industrial processing.

[0120] The following protocol provides myogenic progenitors in a few days. The protocol includes the following steps.

[0121] Step 1 - Formation of ESC aggregates. ESCs were seeded in N2B27 or NDIFF medium (for example, in a ULA u-bottom plate, Figure 1A), and aggregates of 50 - 300 ESCs were obtained. One or more of insulin (for example, 10 μg / ml), transferrin - selenium (for example, 1%), and BMP4 (for example, 10 ng / ml) may be added to the medium. Also, in N2B27 medium supplemented with 1 - 5% knockout serum replacement (KSR), 10 - 30 μg / ml bovine serum albumin (BSA), 5 - 15 μg / ml low - concentration insulin, and 5 - 15 ng / ml BMP4, ESCs were cultured at a density of 10K - 30K cells / ml to obtain aggregates of 20 - 300 ESCs (Figure 1B). Preferably, the cells should be kept in a suspended state, for example, by rotating the plate at a speed of 100 - 300 rpm.

[0122] Step 2 - Germ layer formation. This step was performed approximately 1 day after multiple aggregates were formed, or after multiple floating cell aggregates with a diameter of 50 - 200 μm were generated in each well. In Step 2, the Wnt pathway was activated by adding a Wnt activator, Chir99021 (1 - 5 mM), or Rspo3 (10 - 30 ng / ml), or Wnt3a (50 - 300 ng / ml) to the medium for about 12 - 24 hours. Then, the medium containing the Wnt activator was removed and replaced with a new medium without the Wnt activator.

[0123] The addition of the Wnt activator promoted differentiation into the mesoderm lineage. As a result, at the end of this step, mesoderm cells (marked by Brachyury GFP, indicated by thin arrows in Figure 1B for example) and endoderm cells (marked by Sox17 RFP, indicated by thick arrows in Figure 1B) were seen in the aggregates, as shown in Figures 1A and 1B. It should be noted that the morphology of the aggregates obtained at the end of Step 2 and germ layer formation is typically an egg - like shape, as seen in Figure 1B.

[0124] Step 3A - Formation of the paraxial mesoderm (also referred to as PSM, presomitic mesoderm). PSM cells were observed within 1 - 2 days after the appearance of the first mesodermal cells (Brachyury - positive cells) and could be identified using the marker Mesogenin1 (Msgn1) (Figs. 2A - 2E and 4A - 4C). As shown in Fig. 2A, the cells initially spread randomly within the aggregate (Fig. 2A, left panel, day 4) and gradually concentrated in one location of the aggregate.

[0125] One to two days after the appearance of the first PSM cells, in parallel with the maturation of the paraxial mesoderm, the morphology of the aggregate changed from circular, spherical to ovoid (e.g., Fig. 2A central panel, Fig. 2C, and Fig. 2F). PSM cells at this stage migrated and aggregated at one (or several) poles within the aggregate (also called "PSM polarization") (e.g., Fig. 2A and 2D, bright regions).

[0126] Once the morphology of the aggregate has changed from spherical to ovoid - like, add a low - percentage extracellular matrix (ECM) such as 5 - 10% Matrigel, or one of the ECM components of fibronectin, collagen, and / or laminin. Optionally, 12 - 24 hours after the onset of mesodermal cell development, add one or more of the following to the medium. (a) A Nodal inhibitor (e.g., 5 - 15 μM SB - 431542). (b) A high - concentration Wnt activator (e.g., Chir99201, or Rspo3), usually in the range of 3 μM - 9 μM and 20 - 60 ng / ml respectively. (c) A BMP inhibitor (e.g., LDN193189, or Noggin).

[0127] Step 3B - Maturation of the PSM and formation of presomitic mesoderm (aPSM) cells that are Pax3 - positive occur in parallel with the aforementioned (Step 3A) PSM polarization. The aPSM cells are generated at the anterior pole of the PSM domain within the aggregate (e.g., Figs. 3A and 3B). These cells can give rise to somites and then myogenic precursors.

[0128] Step 4 - Somite formation and axis elongation After the formed paraxial mesoderm aggregates were fully elongated and contained somite-like structures (each numbered and indicated in FIGS. 5A - 5B and 5D), the ECM (or ECM components) was removed from the medium after about 1 or 2 days. However, the removal of the ECM is optional. Further, it has been morphologically changed from spherical (e.g., FIG. 1A), through oval (e.g., FIG. 2C), to the elongated shape shown in FIGS. 5A - 5D, which should be noted that the protocol disclosed herein is similar to embryonic development.

[0129] Step 5 - Formation of myogenic precursors About 1 - 2 days after the formation of somite-like structures, low concentrations of one or more of the following growth factors (GF) were added to the medium: 10 ng / ml HGF, 2 ng / ml IGF, 20 ng / ml FGF2 If necessary, the GF may be added alone or in combination with one or more of a BMP inhibitor, a Wnt activator, and ECM as described in the previous step.

[0130] At the end of this step, about 1 - 2 days after the addition of GF, myogenic precursors such as myoblasts and muscle cells were observed in the mature somites (FIGS. 6A - 6D).

[0131] Throughout the protocol, the medium was changed every other day.

[0132] Example 2 Spatial expression of stage-specific markers of paraxial mesoderm differentiation

[0133] The molecular and cellular properties of the in vitro model were compared with those of the embryo. This comparison is very important because adjacent cell layers provide signal transduction and mechanical support essential for the formation of muscle tissue.

[0134] For this purpose, the spatial expression of stage-specific markers of paraxial mesoderm differentiation (Brachyury / T, Msgn1, Pax3, Mesp2, Meox1), somites (Uncx, Tbx18), muscle precursors (Pax7), muscle cells (MyoG), and mature muscle cells (fast MHC) was characterized. This was done using existing fluorescent live marker cell lines such as Brachyury, Msgn1, Pax3, Pax7, MyoG, or immunostaining with marker-specific antibodies.

[0135] Using fluorescence in-situ hybridization (FISH), spatial mRNA expression maps at various stages were also obtained for an extended set of genes preselected at each stage. This method is essential for genes for which validated antibodies do not exist. This becomes important in non-model organisms such as cows and chickens where antibody availability is particularly limited.

[0136] Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and modifications of the present invention can be devised by those skilled in the art without departing from the true spirit and scope of the present invention. The appended claims are intended to be construed to include all such embodiments and equivalent modifications.

[0137] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. In case of conflict, the patent specification, including definitions, will prevail. As used herein, the indefinite articles "a" and "an" mean "at least one" or "one or more" unless the context clearly dictates otherwise.

Claims

1. (a) suspending embryonic stem cells in a medium to obtain spherical aggregates; (b) adding at least one Wnt activator to the medium within 12 to 36 hours after obtaining the spherical aggregates to obtain aggregates containing mesodermal cells and endodermal cells; (c) removing the Wnt activator within the range of 8 to 36 hours after the addition; (d) observing the morphology of the aggregates containing the mesodermal cells and endodermal cells, and after the aggregates show an egg-like morphology, adding an extracellular matrix or a component thereof containing fibronectin, collagen, and / or laminin within the range of 1 to 15% vol / vol to obtain aggregates containing paraxial mesoderm; (e) incubating the aggregates of step (d) for 10 to 48 hours to thereby obtain aggregates containing a plurality of somites. A method for generating somites.

2. The method according to claim 1, further comprising adding one or more of insulin, transferrin-selenium, and BMP4 to the medium in step (a).

3. The Wnt activator is selected from the group consisting of Chir99021, Wnt3a, and Rspo3. The method according to any one of claims 1 to 2.

4. The method according to any one of claims 1 to 3, further comprising adding at least one Nodal inhibitor to the medium after the appearance of the aggregates containing the mesodermal cells and endodermal cells.

5. The method according to any one of claims 1 to 4, further comprising adding at least one compound selected from the group consisting of a Wnt activator and a BMP inhibitor in step (d).

6. The method according to any one of claims 1 to 5, further comprising removing the extracellular matrix or a component thereof after obtaining the plurality of somites.

7. (a) suspending embryonic stem cells in a medium to obtain spherical aggregates; (b) within 12 to 36 hours after obtaining the spherical aggregates, adding at least one Wnt activator to the medium to obtain aggregates containing mesodermal cells and endodermal cells; (c) removing the Wnt activator within the range of 8 to 36 hours after the addition; (d) observing the morphology of the aggregates containing the mesodermal cells and endodermal cells, and after the aggregates show an egg-like morphology, adding an extracellular matrix or a component thereof containing fibronectin, collagen, and / or laminin within the range of 1 to 15% vol / vol to obtain aggregates containing paraxial mesoderm; (e) incubating the aggregates of step (d) for 10 to 48 hours to thereby obtain aggregates containing a plurality of somites; (f) adding one or more growth factors to the medium of step (e) such that the concentration is less than 30 ng / ml, thereby obtaining myogenic precursors, a method for generating myogenic precursors.

8. The method according to claim 7, wherein the one or more growth factors are selected from HGF, IGF, and FGF2.

9. The method according to claim 7 or 8, further comprising adding at least one compound selected from a BMP inhibitor and a Wnt activator to the medium of step (d).

10. The method according to any one of claims 7 to 9, further comprising removing the extracellular matrix or a component thereof after step (d).

11. The method according to any one of claims 7 to 10, wherein the concentration of each of the growth factors is within the range of 1 to 25 ng / ml.

12. The method according to claim 11, wherein the one or more growth factors are IGF, and the concentration of the IGF is within the range of 1 to 5 ng / ml.

13. The method according to any one of claims 7 to 12, wherein the myogenic precursor includes myoblasts and muscle cells.

14. The method according to any one of claims 7 to 13, further comprising adding one or more of insulin, transferrin-selenium, and BMP4 to the medium of step (a).

15. The method according to any one of claims 7 to 14, wherein the Wnt activator is selected from the group consisting of Chir99021, Rspo3, and Wnt3a.

16. The method according to any one of claims 7 to 15, further comprising adding at least one Nodal inhibitor to the medium after the appearance of the aggregate containing the mesoderm cells and endoderm cells.

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