Method for producing skeletal muscle cells and skeletal muscle tissue from pluripotent stem cells

Abstract

This application describes methods for producing artificial skeletal muscle tissue from pluripotent stem cells. Methods for producing skeletal myoblasts, skeletal myotubes, and satellite cells from pluripotent stem cells are also disclosed. In the described methods, differentiation and maturation of pluripotent stem cells are directed toward skeletal myotubes and satellite cells. This application also describes artificial skeletal muscle tissue having multinucleated skeletal muscle fibers with satellite cells. Furthermore, the present invention relates to mesodermally differentiated skeletal myoblast progenitor cells, myogenically specialized skeletal myoblast progenitor cells, skeletal myoblasts, satellite cells, and skeletal myotubes that can be produced by means of the disclosed methods. This application also describes the use of skeletal muscle tissue or the disclosed cells in drug testing or medicine. Finally, this application relates to in vitro methods in which skeletal muscle tissue or the disclosed cells are used.

Description

Background Art

[0001] Background of the Invention The human body is composed of 35 - 40% skeletal muscle that enables breathing, posture, and movement. Healthy skeletal muscle can fully regenerate from small injuries, such as lacerations or cuts, because muscle stem cells, also called satellite cells (SC), can fully regenerate the damaged tissue. However, large injuries do not heal and leave permanent damage.

[0002] The current theory of "tissue engineering" consists of generating the required cell types and differentiating them in a manipulated environment to produce in vivo - like tissues. It is important to note that in tissue engineering, the extracellular environment is lost during dissociation of differentiated cells, and thus information related to development can be lost. For example, dissociation breaks cell - cell interconnectivity, geometric cell arrangement, and cell - extracellular matrix connectivity. This environment must be reconstructed during tissue engineering (Zimmermann et al. 2004, Tiburcy et al. 2017). Furthermore, differentiated skeletal muscle tissue consists not only of skeletal muscle fibers but also of stromal / connective tissue cells, particularly satellite cells, formed in accordance with environmental and chemical stimuli.

[0003] Different tissue engineering methods for skeletal muscle cells using 2D cell cultures, small animal models, or muscle tissue extracted from small animals are known to those skilled in the art (Beldjilali - Labro et al. (2018) and Khodabukus et al. (2018)). For example, Chal et al. (2016) describe the production of muscle fibers in a 2D method.

[0004] In the past, small animal models have often been used to study biological processes. However, animal models generally have some limitations. When using animal models, there is fundamentally a question as to whether the results can be translated to humans, especially with regard to disease / cure processes and drug efficacy.

[0005] To overcome the limitations of animal models, the production of engineered skeletal muscle cells and / or skeletal muscle tissue is expected to be extremely beneficial.

[0006] To support the differentiation of stem cells into skeletal muscle cells, stem cells have often been transfected with muscle-specific transcription factors in the past. For example, Rao et al. (2018) describe the production of engineered skeletal muscle tissue in which the Pax7 transgene is transiently overexpressed. However, the transfection rate varies for different cells and can fluctuate with each experiment. In addition, many researchers use serum during the differentiation protocol. However, it is often unclear which factors are present in mammalian-derived serum and how they affect differentiation. Therefore, the reproducibility of these methods is severely limited, and differentiation protocols using transgenes or serum have weaknesses. Therefore, it is essential to develop a method by which human pluripotent stem cells can be differentiated and matured into skeletal muscle cells and satellite cells or skeletal muscle tissue by defined factors without the need for transgenes or serum.

[0007] Furthermore, there is increasing evidence suggesting that not only chemical stimuli but also physical stimuli play a role in skeletal muscle tissue development. Therefore, in addition to surface topography and structural composition, a combination of chemical and physical stimuli seems promising for the reliable production of functional muscle tissue in vitro (Liao et al. (2008), Pavesi et al. (2015)). However, the chronology, duration, and nature of these different stimuli during cell differentiation and maturation remain unclear.

[0008] The development of robust differentiation and maturation protocols is a very important step to enable the production of skeletal muscle cells and satellite cells, as well as engineered skeletal muscle tissue.

[0009] In patent application WO 2017 / 100498A1, the inventors disclosed a serum-free differentiation protocol for differentiating human pluripotent stem cells into skeletal myoblasts in a 2D method. However, this procedure requires a step of enriching skeletal myoblasts via flow cytometry to remove undifferentiated cell populations from the cell pool. Purification via flow cytometry does not allow for scale-up, is associated with a risk of infection and very high cell loss, and thus represents a significant barrier to the commercial application of cell products.

[0010] An efficient differentiation method for pluripotent stem cells that does not require an additional enrichment step for specific cell types, is transgene-free and serum-free, has not yet been successfully described. In particular, an efficient differentiation method for pluripotent stem cells into skeletal muscle tissue that describes chemical and physical stimuli while avoiding transgenes and serum has not yet been successfully described.

[0011] Shahriyari et al. (2018) only reported the preliminary production of engineered skeletal muscle tissue. However, Shahriyari et al. lack information on the essential features required for the production of the engineered skeletal muscle tissue of the present invention. Kramer et al. (2014) described the production of engineered skeletal muscle from rat myoblasts rather than from pluripotent stem cells. Summary of the Invention

[0012] The present invention describes a method for preparing engineered skeletal muscle tissue, as well as skeletal myoblasts, skeletal myotube cells, and satellite cells, wherein the medium used is serum-free and different chemicals and their concentrations, as well as physical stimuli, are defined. Additionally, the methods described herein are performed without transfection of transgenes into human cells. The engineered skeletal muscle cells exhibit myoblast-specific, myotube-specific, or satellite cell-specific gene markers, demonstrating efficient differentiation of these cell types. The skeletal muscle tissue, despite its engineered production, has very good stimulus-dependent contractility and exhibits contractions in response to different stimulation frequencies.

[0013] The present invention includes a method by which pluripotent stem cells are differentiated and matured into skeletal myoblasts, skeletal myotube cells, and satellite cells or skeletal muscle tissue. The skeletal muscle tissue is dispersed / embedded in the extracellular matrix.

[0014] The present invention is a method for producing engineered skeletal muscle tissue from pluripotent stem cells, (i) inducing mesodermal differentiation of pluripotent stem cells by culturing the pluripotent stem cells in a basal medium containing a serum-free additive comprising an effective amount of (a) FGF2, (b) a GSK3 inhibitor, (c) a SMAD inhibitor, and (d) transferrin, insulin, progesterone, putrescine, and selenium or a bioavailable salt thereof; (ii) culturing the cells obtained in step (i) in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive as in (i), followed by adding an effective amount of (d) HGF and continuing the culture in the medium, followed by culturing the cells in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive as in (i), and (d) KnockOut Serum Replacement (KSR) to induce myogenic specification; (iii) culturing the cells obtained in step (ii) in a basal medium containing an effective amount of (a) HGF, (b) a serum-free additive similar to that in (i), and (c) KnockOut Serum Replacement (KSR) to expand, proliferate, and mature the cells into skeletal myoblasts and satellite cells; (iv) culturing the cells obtained in step (iii) and dispersed in the extracellular matrix under mechanical stimulation in a basal medium containing an effective amount of (a) a serum-free additive similar to that in step (i), and (b) an additional serum-free additive containing albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3) to mature the cells into skeletal myotube cells and satellite cells comprising relating to a method for producing an engineered skeletal muscle tissue thereby.

[0015] Additionally, the present invention relates to a method for producing skeletal myoblasts, skeletal myotube cells, and satellite cells from pluripotent stem cells, comprising (i) inducing mesodermal differentiation of pluripotent stem cells by culturing the pluripotent stem cells in a basal medium containing an effective amount of (a) FGF2, (b) a GSK3 inhibitor, (c) a SMAD inhibitor, and (d) a serum-free additive containing transferrin, insulin, progesterone, putrescine, and selenium or a bioavailable salt thereof; (ii) culturing the cells obtained in step (i) in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive similar to that in (i), followed by adding an effective amount of (d) HGF and continuing the culturing in the medium, followed by culturing the cells in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive similar to that in (i), and (d) KnockOut Serum Replacement (KSR) a step of inducing myogenic specialization; (iii) maturing the cells obtained in step (ii) into skeletal myoblasts and satellite cells by culturing the cells in a basal medium containing an effective amount of (a) HGF, (b) a serum-free additive similar to that in (i), and (c) KnockOut Serum Replacement (KSR), followed by (iv) maturing the cells obtained in step (iii) into skeletal myotube cells and satellite cells by culturing the cells in a basal medium containing an effective amount of (a) a serum-free additive similar to that in step (i), and (b) an additional serum-free additive containing albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3) comprising a method for producing skeletal myoblasts, skeletal myotube cells, and satellite cells thereby.

[0016] Furthermore, the present invention relates to an engineered skeletal muscle tissue having multinucleated mature skeletal muscle fibers with satellite cells and having no blood supply and / or central nervous system control. In this regard, the presence of skeletal muscle fibers can be detected by staining with actinin and using DAPI.

[0017] Additionally, the present invention is directed to mesoderm-differentiated skeletal myoblast progenitor cells that are prepared and obtained according to step (i) and are characterized by the expression of the genes MSGN1 and / or TBX6, wherein the expression of MSGN1 and / or TBX6 can be determined by flow cytometry and / or immunostaining. These cells express mRNA SP5, and the expression of SP5 can be determined by RNA sequencing.

[0018] Additionally, the present invention relates to myogenic-specialized skeletal myoblast progenitor cells that are produced and obtained according to steps (i) to (ii), and are characterized by the expression of the gene PAX3, wherein the expression of PAX3 can be determined by flow cytometry and / or immunostaining. These cells express mRNA SIM1, and the expression of SIM1 can be determined by RNA sequencing.

[0019] Furthermore, the present invention relates to skeletal myoblasts that are produced and obtained according to steps (i) to (iii), and are characterized by the expression of actinin, wherein the expression of actinin can be determined by flow cytometry and / or immunostaining in skeletal myoblasts.

[0020] The present invention further relates to satellite cells that are prepared and obtained according to steps (i) to (iii), are characterized by the expression of the gene Pax7, wherein the expression of Pax7 can be determined by flow cytometry and / or immunostaining, and more preferably the satellite cells further express Ki67. Furthermore, a mixture of skeletal myoblasts and satellite cells is according to the present invention, and the proportion of satellite cells among all available cell amounts determined by the expression of Pax7 by flow cytometry is at least 10%, preferably at least 15%, more preferably at least 20%, even more preferably at least 30%; and / or the proportion of skeletal myoblasts among all available cell amounts determined by the expression of actinin by flow cytometry is at least 40%, preferably at least 50%, more preferably at least 60%, and most preferably at least 70%.

[0021] Furthermore, the present invention relates to skeletal myotube cells that are prepared and obtained according to steps (i) to (iv), and are characterized by the anisotropic orientation of the actinin protein-containing sarcomere structure.

[0022] The use of skeletal muscle tissue according to the invention, and / or cells according to the invention, and / or skeletal muscle tube cells according to the invention in in vitro drug assays is further disclosed. The drug test may be a toxicity assay or an assay of skeletal muscle tissue function under the influence of pharmacological drug candidates and gene therapy drug candidates.

[0023] Additionally, the present invention relates to skeletal muscle tissue and / or cells according to the invention, and / or skeletal muscle tube cells for use in medicine.

[0024] More particularly, the present invention relates to satellite cells according to the invention for use in the treatment of damaged skeletal muscle and / or skeletal muscle diseases, preferably genetic skeletal muscle defects, in particular Duchenne muscular dystrophy and / or Becker-Kiener muscular dystrophy, and / or lysosomal storage diseases, in particular Pompe disease, preferably wherein the skeletal muscle disease is Duchenne muscular dystrophy.

[0025] Finally, the present invention relates to the following in vitro methods: An in vitro method for testing the efficacy of a drug candidate on skeletal muscle tissue, comprising: (a) providing skeletal muscle tissue according to the invention; (b) optionally damaging the skeletal muscle tissue; and (c) contacting the skeletal muscle tissue of step (a) or (b) with a drug candidate. Preferably, the method further comprises determining the contractile force and / or structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters of the skeletal muscle tissue before and / or after step (c). A method.

[0026] An in vitro method for testing the toxicity of a substance on skeletal muscle tissue, comprising: (a) providing skeletal muscle tissue according to the invention; (b) Step of contacting the skeletal muscle tissue from step (a) with the substance to be tested comprising preferably, the method further comprises, before and / or after step (b), a step of determining the contractile force and / or structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters of the skeletal muscle tissue method.

[0027] An in vitro method for testing the effects of nutrients and dietary supplements on skeletal muscle tissue performance, comprising (a) Step of providing skeletal muscle tissue according to the present invention (b) Step of contacting the skeletal muscle tissue from step (a) with the nutrient or dietary supplement to be tested comprising preferably, the method further comprises, before and / or after step (b), a step of determining the contractile force and / or structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters of the skeletal muscle tissue method.

[0028] An in vitro method for testing the efficacy of drug candidates on mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells, comprising (a) Step of providing mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells according to the present invention (b) Optionally, a step of damaging the cells from step (a), and (c) Step of contacting the cells from step (a) or (b) with the drug candidate comprising; preferably, the method further comprises, before and / or after step (c), a step of determining the expression of actinin and / or Pax7, and the expression can be determined by flow cytometry and / or immunostaining Method

[0029] An in vitro method for testing the toxicity of a substance to mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells, comprising: (a) providing mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells according to the present invention; (b) contacting the cells of step (a) with the substance to be tested and preferably, the method further comprises determining the expression of actinin and / or Pax7 before and / or after step (b), and the expression can be determined by flow cytometry and / or immunostaining. Method Method

[0030] An in vitro method for testing the effect of nutrients and dietary supplements on mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells, comprising: (a) providing mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells according to the present invention; (b) contacting the cells of step (a) with the nutrient or dietary supplement to be tested and preferably, the method further comprises determining the expression of actinin and / or Pax7 before and / or after step (b), and the expression can be determined by flow cytometry and / or immunostaining. Method Method

[0031] Detailed Description of the Invention The present disclosure relates to a method for producing engineered skeletal muscle tissue from pluripotent stem cells, comprising: (i) inducing mesodermal differentiation of pluripotent stem cells by culturing the pluripotent stem cells in a basal medium containing a serum-free additive comprising an effective amount of (a) FGF2, (b) a GSK3 inhibitor, (c) a SMAD inhibitor, and (d) transferrin, insulin, progesterone, putrescine, and selenium or a bioavailable salt thereof; (ii) culturing the cells obtained in step (i) in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive as in (i), followed by adding an effective amount of (d) HGF and continuing the culture in the medium, followed by culturing the cells in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive as in (i), and (d) KnockOut Serum Replacement (KSR) to induce myogenic specification; (iii) expanding and maturing the cells into skeletal myoblasts and satellite cells by culturing the cells obtained in step (ii) in a basal medium containing an effective amount of (a) HGF, (b) a serum-free additive as in (i), and (c) KnockOut Serum Replacement (KSR); (iv) maturing the cells into skeletal myotube cells and satellite cells by culturing the cells obtained in step (iii) and dispersed in an extracellular matrix under mechanical stimulation in a basal medium containing an effective amount of (a) a serum-free additive as in step (i) and (b) an additional serum-free additive comprising albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3); comprising thereby producing engineered skeletal muscle tissue.

[0032] In a preferred embodiment, the pluripotent stem cells are cells of primate origin, particularly human pluripotent stem cells. In a particularly preferred embodiment, the pluripotent stem cells are selected from induced pluripotent stem cells, embryonic stem cells, parthenogenetic stem cells, pluripotent stem cells produced via nuclear transfer, and pluripotent cells produced via chemical reprogramming, and particularly the pluripotent stem cells are induced pluripotent stem cells.

[0033] "Pluripotent stem cells" can differentiate into any cell type in the body. Therefore, human pluripotent stem cells offer considerable potential for obtaining, for example, skeletal myoblasts, skeletal myotube cells, and satellite cells. Currently, the most commonly used pluripotent cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). The human ESC line was first produced by Thomson et al (Thomson et al, Science 282:1145-1147 (1998)). Today, human ESC research enables the development of new technologies for reprogramming somatic cells into ES-like cells. This technology was developed by Yamanaka et al. in 2006 and is applicable to human cells (Takahashi & Yamanaka Cell 126:663-676 (2006) and Takahashi, Kazutoshi et al. Cell, 131:(5)861-872 (2007)). The resulting induced pluripotent cells (iPSCs) exhibit behavior very similar to ESCs and can also differentiate into any cell in the body. Furthermore, in another aspect, parthenogenetic stem cells can be used. Parthenogenetic stem cells can be derived from blastocysts that develop after in vitro activation of unfertilized oocytes in mammals, preferably humans in addition to mice. These cells exhibit the key characteristics of pluripotent stem cells and as a result, can differentiate into any cell type in vitro (Espejel S et al. (2014)). Thus, the pluripotent stem cells can be selected from induced pluripotent stem cells, embryonic stem cells, and parthenogenetic stem cells. However, in the context of the present invention, the pluripotent stem cells are not produced by a method in which the genetic identity of a human is altered in the germ line or in which a human embryo is used for industrial or commercial purposes. In a particularly preferred aspect, induced pluripotent stem cells are selected.

[0034] To achieve directed cell differentiation of pluripotent stem cells into skeletal muscle tissue, differentiation is achieved using specific factors or additives. Generally, the differentiation process according to the present invention is carried out in the presence of a "basal medium". Any suitable basal medium can be used for the method. Preferably, the basal medium used in steps (i)-(iv) is selected from DMEM, DMEM / F12, RPMI, IMDM, alphaMEM, Medium 199, Hams F-10 and Hams F-12. Preferably, the basal medium used in steps (i)-(iv) is DMEM supplemented with pyruvate. Even more preferably, the basal medium used in steps (i)-(iv) is DMEM supplemented with pyruvate containing 1 g / l glucose. The basal medium can be commercially available or can be prepared according to published formulations, for example from the ATCC catalog. In a highly preferred embodiment, the basal medium is DMEM containing 1 g / l glucose + glutamine preparation (e.g., L-alanyl-L-glutamine or GlutaMAX™) and consisting of the substances listed in Table 3. If considered appropriate, the basal medium may be supplemented with an effective concentration of non-essential amino acids. In a preferred embodiment, the basal medium is supplemented with 1 effective concentration of the non-essential amino acids listed in Table 2. The basal media in steps (ii), (iii) and (iv) may be selected independently of the basal medium used in step (i). However, in a preferred embodiment, the basal media in steps (i)-(iv) are the same.

[0035] Generally, the different differentiation stages of steps (i)-(iv) can be detected using genes expressed that are characteristic of a particular stage. One method by which gene expression can be measured is RNA sequencing (RNA-Seq). RNA sequencing is also referred to as transcriptome analysis. RNA sequencing is the determination of the nucleotide sequence of RNA based on a high-throughput method. For this purpose, RNA is converted (transcribed) into cDNA so that DNA sequencing methods can be applied. Thus, RNA sequencing provides information about which mRNAs are expressed and is characterized by low background noise, higher resolution, and high replication rate. Those skilled in the art are proficient in and can perform the method of mRNA sequencing. Example 1 of the present invention shows exemplary data measured using RNA sequencing. In particular, FIG. 4 shows the time course of mRNA expression of different genes during a 0- to 60-day time frame during the differentiation protocol of the present invention.

[0036] The mRNA expression of NANOG, POU5F1 (OCT4), and ZFP42 is characteristic of pluripotent stem cells. This means that cells expressing these markers are pluripotent.

[0037] During the differentiation of pluripotent stem cells according to the present invention, "mesoderm differentiation" is induced by specific factors / additives in step (i). In addition to all bilaterian animals, in humans, the mesoderm is one of the three main germ layers in a very early embryo. In bilaterian animals, there are three main components of the mesoderm: axial mesoderm, intermediate mesoderm, and lateral plate mesoderm. The axial mesoderm in bilaterian animals gives rise to skeletal muscle in particular. The induction of mesoderm differentiation is characterized by the gene expression of specific genes, such as the mRNAs of MSGN1, TBX6, and MEOX1. The mRNA expression of these or other genes specific for axial mesoderm expression can be measured by RNA sequencing as described herein.

[0038] As described above, the basal medium for step (i) contains a serum-free additive containing an effective amount of (a) FGF2, (b) a GSK3 inhibitor, (c) a SMAD inhibitor, and (d) transferrin, insulin, progesterone, putrescine, and selenium or a bioavailable salt thereof. It is known to those skilled in the art that the effective concentration or amount of a receptor / enzyme agonist or inhibitor varies with the availability and biological activity of each substance.

[0039] In one embodiment, the effective amount of FGF2 is 1 to 15 ng / ml, preferably 2.5 to 14 ng / ml, more preferably 5 to 13 ng / ml, even more preferably 7.5 to 12.5 ng / ml, even more preferably 8 to 12 ng / ml, even more preferably 9 to 11 ng / ml, and most preferably about 10 ng / ml.

[0040] Glycogen synthase kinase 3 (GSK-3) is a serine / threonine protein kinase that selectively adds phosphate residues to serine and threonine residues of other proteins. Inhibition of glycogen synthase kinase 3 (GSK3) helps activate the Wnt signaling pathway for the differentiation of pluripotent stem cells. For example, the GSK3 inhibitor in the basal medium is selected from the group consisting of CHIR99021, CHIR98014, SB216763, TWS119, tideglusib, SB415286, 6-bromoindirubin-3-oxime, and valproate, and the GSK3 inhibitor CHIR99021 is preferred. However, any GSK3 inhibitor suitable for the method of the present invention can be used. When the GSK3 inhibitor is CHIR99021, the effective amount is 1 to 20 μM, preferably 2 to 19 μM, more preferably 3 to 18 μM, even more preferably 4 to 17 μM, even more preferably 5 to 16 μM, even more preferably 6 to 15 μM, even more preferably 7 to 14 μM, even more preferably 7.5 to 13 μM, even more preferably 8 to 12 μM, even more preferably 9 to 11 μM, and most preferably about 10 μM.

[0041] SMAD inhibitors inhibit proteins that are essential for regulating cell development and proliferation. For example, the SMAD inhibitor in the basal medium is selected from the group consisting of LDN193189, K02288, LDN214117, ML347, LDN212854, DMH1, and preferably the SMAD inhibitor is LDN193189. However, any SMAD inhibitor suitable for the method of the present invention can be used. When the SMAD inhibitor is LDN193189, the effective amount is 0.05 - 5 μM, preferably 0.1 - 2.5 μM, more preferably 0.2 - 1 μM, even more preferably 0.25 - 0.8 μM, even more preferably 0.3 - 0.75 μM, even more preferably 0.35 - 0.7 μM, even more preferably 0.4 - 0.6 μM, even more preferably 0.45 - 0.55 μM, and most preferably about 0.5 μM. The effective concentration or amount of the inhibitor varies with the availability and biological activity of each substance, and this is known to those skilled in the art to apply to all substances, such as proteins / peptides, nucleotides, or chemical compounds.

[0042] In one embodiment, the serum-free additive is 50 - 500 μg / ml of transferrin (preferably 70 - 300 μg / ml of transferrin, more preferably 80 - 200 μg / ml of transferrin, even more preferably 90 - 150 μg / ml of transferrin, most preferably about 100 μg / ml of transferrin) in steps (i), (ii), (iii), and (iv) of the method, 1 - 25 μg / ml of insulin (more preferably 2 - 13 μg / ml of insulin, more preferably 3 - 10 μg / ml of insulin, more preferably 4 - 6 μg / ml of insulin, most preferably about 5 μg / ml of insulin), 0.001 - 0.1 μg / ml of progesterone (preferably 0.002 - 0.05 μg / ml of progesterone, more preferably 0.004 - 0.01 μg / ml of progesterone, even more preferably 0.005 - 0.008 μg / ml of progesterone, most preferably about 0.0063 μg / ml of progesterone), Putrescine at a concentration of 5 to 50 μg / ml (preferably 10 to 35 μg / ml of putrescine, more preferably 12 to 25 μg / ml of putrescine, even more preferably 14 to 18 μg / ml of putrescine, and most preferably about 16 μg / ml of putrescine); and selenium at a concentration of 6 to 600 nM (preferably 12 to 300 nM of selenium, more preferably 20 to 150 nM of selenium, even more preferably 25 to 50 nM of selenium, and most preferably about 30 nM of selenium) or a bioavailable salt thereof is provided at a final concentration in the medium. In a preferred embodiment, selenium is present as selenite, and its effective concentration is 1 to 30 μg / l of selenite in the medium (preferably 2 to 20 μg / l of selenite, more preferably 3 to 10 μg / l of selenite, even more preferably 4 to 6 μg / l of selenite, and most preferably about 5 μg / l of selenite).

[0043] Serum-free additives that meet the above requirements can be commercially purchased. For example, N2 additive can be used. In a preferred embodiment, the serum-free additive is N2 additive at a concentration of 0.1 to 10% (v / v) of N2 additive, preferably 0.3 to 7.5% (v / v) of N2 additive, more preferably 0.5 to 5% (v / v) of N2 additive, more preferably 0.75% to 2% (v / v) of N2 additive, more preferably 0.9% to 1.2% (v / v) of N2 additive, and most preferably about 1% (v / v) of N2 additive. The N2 additive is commercially available at 100-fold effective concentration, and the composition is listed in Table 1. This means that 1% (v / v) of the N2 additive corresponds to 1 effective concentration.

[0044] In a preferred embodiment, step (i) of the method is carried out for 24 to 132 hours, preferably 48 to 120 hours, more preferably 60 to 114 hours, even more preferably 72 to 108 hours, more preferably 84 to 102 hours, and most preferably about 96 hours. The duration of step (i) as well as the concentrations of the substances (a) FGF2, (b) GSK3 inhibitor, (c) SMAD inhibitor, and (d) serum-free additive may be optimized by monitoring the efficiency of induction of mesoderm differentiation. As described above, the efficiency of mesoderm differentiation can be tracked by RNA sequencing. For example, when one or more of the gene markers MSGN1, TBX6, and MEOX1 have an expression value that is at least 5-fold higher (preferably at least 10-fold higher, more preferably 20-fold higher, even more preferably at least 30-fold higher, and most preferably at least 50-fold higher) compared to pluripotent stem cells as measured by "reads per million kilobases" by RNA sequencing, induction of mesoderm differentiation is indicated.

[0045] In step (ii) of the method according to the invention, "myogenic specification" is induced. This differentiation stage is characterized by the expression of specific factors. For example, mRNA Pax3 is expressed in myogenic specification, and its expression can be determined by RNA sequencing (see FIGS. 1 and 2 for a schematic overview; see FIG. 4 for experimental data regarding the expression of PAX3). For example, when the gene marker Pax3 has an expression value that is at least 5-fold higher (preferably at least 10-fold higher, more preferably 20-fold higher, even more preferably 30-fold higher) compared to pluripotent stem cells as measured by "reads per million kilobases" by RNA sequencing, myogenic specification is indicated.

[0046] As described above, step (ii) includes three culture steps. In particular, step (ii) involves culturing the cells obtained from step (i) in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive similar to that in (i), followed by adding an effective amount of (d) HGF and continuing the culture in the medium, and then culturing the cells in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive similar to that in (i), and (d) KnockOut Serum Replacement (KSR).

[0047] Similar to step (i), the basal medium in step (ii) may be selected from DMEM, DMEM / F12, RPMI, IMDM, alphaMEM, Medium 199, Hams F-10, and Hams F-12. Additionally, the basal medium may be supplemented with non-essential amino acids and / or pyruvate. Exemplary and preferred embodiments for the basal medium in step (ii) may be selected similarly to the exemplary and preferred embodiments in step (i). The basal medium in step (ii) may be selected independently of the basal medium used in step (i). However, in a preferred embodiment, the basal medium in steps (i) and (ii) is the same.

[0048] For example, the gamma-secretase / NOTCH inhibitor is selected from the group consisting of DAPT, RO4929097, semagacestat (LY450139), avagacestat (BMS-708163), dibenzazepine (YO-01027), LY411575, IMR-1, and L685458, and preferably the gamma-secretase / NOTCH inhibitor is DAPT. However, any gamma-secretase / NOTCH inhibitor suitable for the method of the present invention can be used. When the gamma-secretase / NOTCH inhibitor is DAPT, its effective amount is 1 to 20 μM, preferably 2 to 19 μM, more preferably 3 to 18 μM, even more preferably 4 to 17 μM, even more preferably 5 to 16 μM, even more preferably 6 to 15 μM, even more preferably 7 to 14 μM, even more preferably 7.5 to 13 μM, even more preferably 8 to 12 μM, even more preferably 9 to 11 μM, and most preferably about 10 μM.

[0049] In step (ii), the effective amount of FGF2 is, for example, 15 to 30 ng / ml, preferably 17.5 to 25 ng / ml, more preferably 18 to 22 ng / ml, even more preferably 19 to 21 ng / ml, and most preferably about 20 ng / ml.

[0050] For example, the effective amount of HGF is 1 to 15 ng / ml, preferably 2.5 to 14 ng / ml, more preferably 5 to 13 ng / ml, even more preferably 7.5 to 12.5 ng / ml, even more preferably 8 to 12 ng / ml, even more preferably 9 to 11 ng / ml, and most preferably about 10 ng / ml.

[0051] It is known to those skilled in the art that the effective concentration or amount of a receptor / enzyme agonist or inhibitor varies with the availability and biological activity of each substance.

[0052] As used herein, the term "KnockOut Serum Replacement" (KSR) means effective concentrations of ascorbic acid, insulin, transferrin, and albumin. In a preferred embodiment, KSR additionally contains effective concentrations of selenium or a bioavailable salt thereof, glutathione, and trace elements. In a more preferred embodiment, KSR contains the substances listed in Table 5 at effective concentrations. In the most preferred embodiment, KSR contains the substances of Table 5 at the indicated concentrations. "KnockOut Serum Replacement" (KSR) is known in the prior art and can be prepared according to the formulation on pages 27 - 29 of patent application WO 98 / 30679. Alternatively, KSR is commercially available, for example, from Gibco.

[0053] In a preferred embodiment, KSR is used in an amount of 5 - 20% (v / v), preferably 6 - 17.5% (v / v), more preferably 7 - 15% (v / v), more preferably 8% - 12% (v / v), more preferably 9% - 11% (v / v), and most preferably about 10% (v / v) of KSR. In a highly preferred embodiment, KSR is used in the presence of a reducing agent. Any suitable reducing agent can be used, and examples of reducing agents are beta - mercaptoethanol and / or alpha - thioglycerol. Beta - mercaptoethanol is typically used at a concentration of 0.02 - 0.5 mM, more preferably 0.05 - 0.2 mM, and most preferably about 0.1 mM. Alternatively, alpha - thioglycerol may be used, for example, at a concentration of 0.02 - 0.5 mM, more preferably 0.05 - 0.2 mM, and most preferably about 0.1 mM.

[0054] In one embodiment, the culture in step (ii) is carried out for 36 - 60 hours, preferably 42 - 54 hours, and most preferably about 48 hours in the presence of (a) a gamma - secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum - free additive; and / or The culture is carried out in the presence of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, (c) a serum-free additive, and (d) HGF for 36 to 60 hours, preferably 42 to 54 hours, most preferably about 48 hours; and / or The culture is carried out in the presence of (a) a gamma-secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive, and (d) KnockOut Serum Replacement (KSR) for 72 to 120 hours, preferably 76 to 114 hours, more preferably 84 to 108 hours, even more preferably 90 to 102 hours, most preferably about 96 hours.

[0055] In step (iii) of the method of the present invention, the cells are preferably matured and expanded in skeletal myoblasts and satellite cells. Skeletal myoblasts are characterized by their ability to be fusion competent and thus to fuse in a further step to become skeletal myotube cells. Satellite cells, also called muscle stem cells, are small multipotent cells. Satellite cells can give rise to (i) satellite cells or (ii) differentiated skeletal myoblasts. More particularly, after activation, satellite cells can re-enter the cell cycle and are capable of proliferation and differentiation into myoblasts. The differentiation stage of the method is characterized by the expression of specific factors. For example, the expression of Pax7 is characteristic of the presence of satellite cells. At the same time, the expression of MyoD is characteristic of skeletal myoblasts, and the expression of each of them can be determined by RNA sequencing (see FIGS. 1 and 2 for a schematic overview; see FIG. 4 for experimental data on the expression of MyoD1 and PAX7). For example, when the gene marker MyoD is measured by "reads / million kilobases" using RNA sequencing, a skeletal myoblast is present when it has an expression value that is at least 5-fold higher (preferably at least 10-fold higher, more preferably 15-fold higher, even more preferably 20-fold higher) compared to pluripotent stem cells. For example, when the gene marker PAX7 is measured by "reads / million kilobases" by RNA sequencing, a satellite cell is present when it has an expression value that is at least 5-fold higher (preferably at least 10-fold higher, more preferably 15-fold higher, even more preferably 20-fold higher) compared to pluripotent stem cells.

[0056] As described above, the basal medium in step (iii) contains an effective amount of (a) HGF, (b) a serum-free additive similar to that in (i), and (c) KnockOut Serum Replacement (KSR). Similar to steps (i) and (ii), the basal medium in step (iii) may be selected from DMEM, DMEM / F12, RPMI, IMDM, alphaMEM, Medium 199, Hams F-10, and Hams F-12, and the basal medium may be supplemented with non-essential amino acids and / or pyruvate. Exemplary and preferred embodiments for the basal medium in step (iii) may be selected similar to the exemplary and preferred embodiments in step (i). The basal medium in step (iii) may be selected independently of the basal media used in steps (i) and (ii). However, in a preferred embodiment, the basal media in steps (i), (ii), and (iii) are the same.

[0057] The KSR and any reducing agent in step (iii) include the same preferred embodiments as the KSR and any reducing agent in step (ii). Thus, the KSR can be prepared by those skilled in the art or purchased commercially.

[0058] In step (iii), the effective amount of HGF is, for example, 1 to 15 ng / ml, preferably 2.5 to 14 ng / ml, more preferably 5 to 13 ng / ml, even more preferably 7.5 to 12.5 ng / ml, even more preferably 8 to 12 ng / ml, even more preferably 9 to 11 ng / ml, and most preferably about 10 ng / ml; and / or The KSR is used in an amount of 5 to 20% (v / v), preferably 6 to 17.5% (v / v), more preferably 7 to 15% (v / v), more preferably 8% to 12% (v / v), more preferably 9% to 11% (v / v), and most preferably about 10% (v / v) of KSR; in particular, the KSR is used in the presence of a reducing agent such as beta-mercaptoethanol and / or alpha-thioglycerol.

[0059] It is known to those skilled in the art that the effective amount or concentration of a receptor / enzyme agonist or inhibitor varies with the availability and biological activity of each substance.

[0060] In step (iv) of the method according to the invention, the cells are preferably matured into skeletal myotube cells and satellite cells. Skeletal myotube cells are formed by the fusion of skeletal myoblasts. Thus, skeletal myotube cells are multinucleated cell structures formed by the fusion of mature myoblasts into long, thin myotube cells. Skeletal myotube cells are also called muscle cells or muscle fibers. Figure 2 provides a schematic overview of the developmental stages experienced by the engineered skeletal muscle tissue, and the formation of the engineered skeletal muscle tissue is known as myogenesis in the prior art. Skeletal myotube cells (muscle fibers) are characterized by the anisotropic orientation of the actinin-containing sarcomere structure. A regenerative competent satellite cell niche is formed adjacent to the fused skeletal myotube cells. The satellite cell niche is outside the skeletal myotube cells but in close contact with them. The satellite cell niche is anatomically characteristic and is also formed in natural skeletal muscle tissue. As a result, together with the generated satellite cells, it becomes another desirable quality characteristic of the engineered skeletal muscle tissue. This differentiation stage is also characterized by the expression of specific factors. For example, the expression of Pax7 is characteristic of the presence of satellite cells. At the same time, the expression of myogenin and actinin is characteristic of skeletal myotube cells, and the expression of each of them can be determined by RNA sequencing (see Figures 1 and 2 for a schematic overview; see Figure 4 for experimental data on the expression of PAX7 (paired box 7), ACTN2 (actinin alpha 2), DMD (dystrophin), and MYH3 (myosin heavy chain 3). For example, when the mRNA of PAX7 is measured by "reads / million kilobases" by RNA sequencing, satellite cells are present when it has an expression value at least 5 times higher (preferably at least 10 times higher, more preferably 15 times higher, even more preferably 20 times higher) compared to pluripotent stem cells. For example, when the gene marker ACTN2 is measured by "reads / million kilobases" by RNA sequencing, skeletal myotube cells are present when it has an expression value at least 5 times higher (preferably at least 50 times higher, more preferably 100 times higher, even more preferably 150 times higher) compared to pluripotent stem cells.In fact, the DMD and MYH3 gene markers typically have an expression value that is at least 200-fold higher (preferably at least 500-fold higher, more preferably 1000-fold higher) compared to pluripotent stem cells.

[0061] As described above, the cells obtained in step (iii) are dispersed in the extracellular matrix and matured under mechanical mimicry. Mechanical mimicry can be performed, for example, with the assistance of a stretching device, as is generally known and used in the art. Preferably, the stretching device exerts static, phasic, or dynamic strain forces. Therefore, the mechanical strain force can be (a) static, (b) phasic, or (c) dynamic. An example of a static strain force is isometric muscle contraction, in which case the muscle experiences only a change in tension and no change in length. Therefore, shortening does not occur in the muscle during isometric muscle contraction. A phasic strain force can be a quasi-isotonic muscle contraction, in which case the muscle shortens during contraction and the tension applied to the muscle remains the same. A dynamic strain force can occur, for example, when the muscle is suspended on a flexible support and overload contraction is promoted. In overload contraction, both the length and the tension of the muscle change. Preferably, the mechanical stimulation in step (iv) is a static mechanical stimulation, i.e., a static tension (static strain force). This means that the cells and extracellular matrix from step (iii) are under a force and an opposing force (opposing force).

[0062] As described above, the basal medium in step (iv) contains an effective amount of (a) a serum-free additive similar to that in step (i), and (b) an additional serum-free additive containing albumin, transferrin, ethanolamine, selenium or its bioavailable salt, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3).

[0063] Exemplary and preferred embodiments for the basal medium in step (iv) may be selected similar to the exemplary and preferred embodiments in step (i).

[0064] The additional serum-free additive in step (iv) of the method is prepared such that the additional serum-free additive provides the following final concentrations of substances: 0.5 to 50 mg / ml of albumin (preferably 1 to 40 mg / ml, more preferably 2 to 30 mg / ml, even more preferably 3 to 20 mg / ml, even more preferably 4 to 10 mg / ml, most preferably 4.5 to 7.5 mg / ml, for example about 5 mg / ml); 1 to 100 μg / ml of transferrin (preferably 2 to 90 μg / ml, more preferably 3 to 80 μg / ml, even more preferably 4 to 70 μg / ml, even more preferably 5 to 60 μg / ml, more preferably 6 to 50 μg / ml, more preferably 7 to 40 μg / ml, more preferably 8 to 30 μg / ml, more preferably 9 to 20 μg / ml, for example about 10 μg / ml); 0.1 to 10 μg / ml of ethanolamine (preferably 0.2 to 9 μg / ml, more preferably 0.3 to 8 μg / ml, even more preferably 0.4 to 7 μg / ml, even more preferably 0.5 to 6 μg / ml, more preferably 0.6 to 5 μg / ml, more preferably 0.7 to 4 μg / ml, more preferably 0.8 to 3 μg / ml, most preferably 1 to 2.5 μg / ml, for example about 2 μg / ml); 17.4 to 1744 nM of selenium or a bioavailable salt thereof (preferably 35 to 850 nM, more preferably 70 to 420 nM, even more preferably 120 to 220 μg / ml, most preferably about 174 nM); 0.4 to 40 μg / ml of L-carnitine HCl (preferably 0.5 to 30 μg / ml, more preferably 1 to 20 μg / ml, even more preferably 2 to 10 μg / ml, more preferably 3 to 5 μg / ml, most preferably about 4 μg / ml); 0.05 to 5 μl / ml of a fatty acid additive (preferably 0.1 to 4 μl / ml, more preferably 0.2 to 3 μl / ml, even more preferably 0.3 to 3 μl / ml, more preferably 0.4 to 2 μl / ml, most preferably 0.45 to 1 μl / ml, for example about 0.5 μl / ml); and 0.0001 to 0.1 μg / ml of triiodo-L-thyronine (T3) (preferably 0.001 to 0.01 μg / ml, more preferably 0.002 to 0.0075 μg / ml, even more preferably 0.003 to 0.005 μg / ml, most preferably about 0.004 μg / ml).

[0065] The fatty acid additive may contain, for example, linoleic acid and / or linolenic acid.

[0066] In a preferred embodiment, the additional serum-free additive further contains 0.1 to 10 μg / ml of hydrocortisone (preferably 0.2 to 9 μg / ml, more preferably 0.3 to 8 μg / ml, even more preferably 0.4 to 7 μg / ml, even more preferably 0.5 to 6 μg / ml, even more preferably 0.6 to 5 μg / ml, even more preferably 0.7 to 4 μg / ml, even more preferably 0.8 to 3 μg / ml, most preferably 0.9 to 2 μg / ml, for example about 1 μg / ml). In a similarly preferred embodiment, the additional serum-free additive further contains 0.3 to 30 μg / ml of insulin (preferably 0.5 to 25 μg / ml, more preferably 1 to 20 μg / ml, even more preferably 1.5 to 15 μg / ml, even more preferably 2 to 10 μg / ml, most preferably 2.5 to 5 μg / ml, for example about 3 μg / ml). For example, a bioavailable salt of selenium is sodium selenite such that a final concentration of 0.003 to 0.3 μg / ml (preferably 0.005 to 0.2 μg / ml, more preferably 0.01 to 0.1 μg / ml, even more preferably 0.02 to 0.05 μg / ml, most preferably 0.03 μg / ml, for example about 0.032 μg / ml) is provided in the basal medium.

[0067] Furthermore, the additional serum-free additive may further contain one or more components selected from the group consisting of hydrocortisone, ascorbic acid, vitamin A, D-galactose, linolenic acid, progesterone, and putrescine. These components are beneficial for cell survival. Suitable concentrations of each component are known to those skilled in the art or can be readily determined by conventional means.

[0068] Examples of additional serum-free additives referred to in step (iv) can be prepared according to published protocols (see also Brewer et al. 1993) or are commercially available. For example, B27 (Table 4) may be used. In a preferred embodiment, the B27 additive is used in an amount of 0.1-10% B27, preferably 0.5-8%, more preferably 1-6%, more preferably 1.5-4%, even more preferably 1.5-4%, most preferably about 2% B27.

[0069] In the present invention, a seeding step may be performed before step (i), and the obtained engineered skeletal muscle tissue is called bioengineered skeletal muscle (BSM). In the seeding step, pluripotent stem cells are seeded into a stem cell medium in the presence of a ROCK inhibitor, preferably, the seeding step is performed 18-30 hours before step (i). For example, the ROCK inhibitor is selected from the group consisting of Y27632, H-1152P, thiazovivin, fasudil, hydroxyfasudil, GSK429286A and RKI1447, preferably the ROCK inhibitor is selected from the group consisting of Y27632, H-1152P, thiazovivin, fasudil, and hydroxyfasudil, more preferably the ROCK inhibitor is selected from the group consisting of Y27632 and H-1152P, and particularly preferably the ROCK inhibitor is Y27632. However, any ROCK inhibitor suitable for the method of the present invention can be used. It will be understood by those skilled in the art that the concentration of the effective amount of the ROCK inhibitor will vary with the availability and inhibition constant of the inhibitor in question. For example, in the case of Y27632, the medium used in the seeding step may be used at a concentration of 0.5-10 μM, preferably 1-9 μM, more preferably 2-8 μM, more preferably 3-7 μM, more preferably 4-6 μM, most preferably about 5 μM. A stem cell medium may be used in the seeding step, and in principle, any stem cell medium suitable for the method can be used. Suitable stem cell media are known to those skilled in the art, and iPS-Brew XF stem cell medium is particularly preferred.

[0070] Furthermore, in the seeding step, the pluripotent stem cells may first be seeded in an engineered form in the presence of one or more components of the extracellular matrix in the master mix before the stem cell medium is added. In the seeding step, the pluripotent stem cells are dispersed in the extracellular matrix prior to step (i), such that the cells are embedded in the extracellular matrix and differentiated and matured into the engineered skeletal muscle tissue in a three-dimensional structure.

[0071] The "extracellular matrix" acts as a scaffold and provides a structural and functional microenvironment for cell growth and differentiation. The composition of the extracellular matrix is unique to each natural tissue, but the main components of the extracellular matrix are collagen, fibronectin, laminin, as well as various types of glycosaminoglycans and proteoglycans. Proteoglycans form a class of particularly strongly glycosylated glycoproteins that achieve stabilization between the cells of an organism. Here, they form large complexes with proteins, such as collagen, which is a main component of the extracellular matrix, in addition to both other proteoglycans and hyaluronic acid. Laminin is a glycoprotein similar to collagen. Fibronectin is also an important glycoprotein for the polymerization of extracellular collagen and can play an important role particularly in tissue repair. The components of the extracellular matrix in the master mix are preferably collagen, preferably type I collagen, more preferably of bovine origin, human origin, or mouse origin, particularly bovine origin collagen. Optionally, the extracellular matrix additionally contains laminin and / or fibronectin.

[0072] Pluripotent stem cells typically range from 1 to 6×10 6They are seeded in the medium at a ratio of cells / ml and 0.7 - 1.4 mg / ml of collagen. In one embodiment, the master mix contains, as an extracellular matrix component, the exudate of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells at 5 - 15% (v / v), preferably 7.5% - 12.5% (v / v), more preferably 9 - 11% (v / v), and most preferably about 10% (v / v). In a particularly preferred embodiment, the exudate is Matrigel. The pH of the master mix is typically pH 7.2 - pH 7.8. Matrigel is known to those skilled in the art and is further described in the prior art (Hughes et al. 2010).

[0073] As an alternative to the exudate of EHS mouse sarcoma cells, the master mix may contain stromal cells, which produce extracellular matrix components collagen, laminin, fibronectin, and / or proteoglycan. The pH of the master mix is typically pH 7.2 - pH 7.8.

[0074] In a preferred embodiment, the stem cell medium is added to the master mix in the engineered form about 1 hour later, and the stem cell medium preferably contains effective concentrations of KSR and FGF2. For example, the stem cell medium may contain KSR at 5 - 20% (v / v), preferably 6 - 17.5% (v / v), more preferably 7 - 15% (v / v), more preferably 8% - 12% (v / v), more preferably 9% - 11% (v / v), and most preferably about 10% (v / v).

[0075] An effective amount of FGF2 is typically 1 - 15 ng / ml, preferably 2.5 - 14 ng / ml, more preferably 5 - 13 ng / ml, even more preferably 7.5 - 12.5 ng / ml, even more preferably 8 - 12 ng / ml, even more preferably 9 - 11 ng / ml, and most preferably about 10 ng / ml of FGF2.

[0076] When manufacturing BSM, step (iii) is carried out for 7 to 11 days, preferably 8 to 10 days, and most preferably about 9 days.

[0077] Alternatively, skeletal myoblasts and satellite cells can be seeded in an additional step after step (iii) and before step (iv), and the obtained engineered skeletal muscle tissue is called engineered skeletal muscle (ESM). Here, the skeletal myoblasts and satellite cells are seeded in an engineered form in the presence of one or more components of the extracellular matrix in the master mix. Preferably, the components of the extracellular matrix in the master mix are collagen, preferably type I collagen, more preferably of bovine, human or mouse origin, particularly bovine origin collagen, and optionally the extracellular matrix additionally contains laminin and / or fibronectin. In the seeding step after step (iii) and before step (iv), the skeletal myoblasts and satellite cells are seeded, for example, at a ratio of 1 to 10×10 6 cells / ml and 0.7 to 1.4 mg / ml of collagen.

[0078] In one embodiment, the master mix contains 5 to 15% (v / v), preferably 7.5% to 12.5% (v / v), more preferably 9 to 11% (v / v), most preferably about 10% (v / v) of the exudate of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells as an extracellular matrix component. In a particularly preferred embodiment, the exudate is Matrigel. The pH of the master mix is typically pH 7.2 to pH 7.8.

[0079] As an alternative to the exudate of EHS mouse sarcoma cells, the master mix may contain stromal cells, and the stromal cells produce extracellular matrix components collagen, laminin, fibronectin, and / or proteoglycan. The pH of the master mix is typically pH 7.2 to pH 7.8.

[0080] In a preferred embodiment, after about 1 hour, a basal medium similar to that used in step (iii) is added to the master mix in the engineered form, and the medium additionally contains an effective amount of a ROCK inhibitor. For example, the ROCK inhibitor is selected from the group consisting of Y27632, H-1152P, thiazovivin, fasudil, hydroxyfasudil, GSK429286A, and RKI1447. Preferably, the ROCK inhibitor is selected from the group consisting of Y27632, H-1152P, thiazovivin, fasudil, and hydroxyfasudil. More preferably, the ROCK inhibitor is selected from the group consisting of Y27632 and H-1152P, and particularly preferably the ROCK inhibitor is Y27632. However, any ROCK inhibitor suitable for the method of the present invention can be used. The concentration of the effective amount of the ROCK inhibitor is known to those skilled in the art to vary with the availability and inhibition constant of the inhibitor in question. For example, in the case of Y27632, the medium used in the seeding step may be used at a concentration of 0.5 to 10 μM, preferably 1 to 9 μM, more preferably 2 to 8 μM, more preferably 3 to 7 μM, more preferably 4 to 6 μM, and most preferably about 5 μM.

[0081] When preparing the ESM, about 1 day after the seeding step performed between steps (iii) and (iv), the medium is replaced with a medium similar to that used in step (iii), and the cells are then further cultured in this medium for 5 to 9 days, preferably 6 to 8 days, and most preferably about 7 days.

[0082] In the preparation of BSM or ESM, the manipulated form may be, for example, in the form of a ring, ribbon, strand, patch, pouch, or cylinder, and optionally individual skeletal muscle tissues may be fused. It means that individual and / or different geometries can be fused to form skeletal muscle tissue and thus different muscle shapes can be achieved. In particular, the form of a ring, strand or ribbon is useful for applications in in vitro methods, for example for toxicity tests. Typically, since the manipulated form is obtained by pouring in the master mix, generally any desired pourable manipulated form can be manufactured.

[0083] Step (iv) can be carried out for at least 19 days, preferably at least 28 days, more preferably at least 56 days, even more preferably at least 120 days, particularly at least 240 days, and longer cultures are possible. The inventors have already been able to carry out a 240-day (8-month) culture, but do not exclude longer culture periods.

[0084] In contrast to many methods disclosed in the prior art, the method according to the present invention does not include a transfection step using a differentiation- or maturation-related transgene. Preferably, the method does not include a myogenic transgene, and more preferably, the method does not include either a Pax7 transgene or a MyoD transgene. A "transgene" refers to a gene introduced into a cell. Such a transgene may be transfected into a cell in the form of DNA (e.g., in the form of a plasmid) or RNA. The transgene is then expressed in the cell, thereby altering the characteristics of the cell. For example, a transcription factor can be introduced into a cell as a transgene, which then affects the expression of other genes. Thus, a myogenic transgene can increase the proportion of skeletal myoblasts in a cell population. However, transfection experiments using transgenes, such as Pax7 or MyoD, have different transfection efficiencies depending on the experiment and cell type. As a result, methods that require a transfection step have lower controllability and thus lower reproducibility. Therefore, a transgene-free method is advantageous over methods that require transfection with a transgene. However, it cannot be excluded that pluripotent stem cells are genetically modified in another form, for example, to simulate a disease pattern. Furthermore, genetic engineering of cell type and / or cell function (e.g., calcium or voltage signals) for labeling or control of cell function (e.g., contraction frequency) via, for example, optogenetic mechanisms is not excluded.

[0085] Another advantage of the method according to the invention is that no further selection step of specific cell types, such as skeletal myoblasts, etc., is required. Preferably, the method does not include a concentration step by cell selection, and more preferably, does not include a concentration step by antibody-based cell selection. This is advantageous because the cells do not need to be extracted from their environment in an additional step. One possible method of antibody-based cell selection is flow cytometry, which is known to those skilled in the art. Such cell selection by flow cytometry is associated with significant cell loss. Therefore, purification via flow cytometry does not allow for scale-up, is associated with an infection risk, and thus becomes a key barrier to the commercial application of cell products. Since the method of the present invention does not require cell selection, the production of the engineered skeletal muscle tissue and cells of the present invention is scalable and suitable for commercial or medical applications.

[0086] Additionally, the method is serum-free, and thus there is no variation regarding different batches of serum. This results in a robust and reproducible protocol for the production of engineered skeletal muscle tissue where all the necessary chemical and physical stimuli are defined.

[0087] The present invention is a method for producing skeletal myoblasts, skeletal myotube cells, and satellite cells from pluripotent stem cells, comprising: (i) inducing mesodermal differentiation of pluripotent stem cells by culturing the pluripotent stem cells in a basal medium containing a serum-free additive comprising an effective amount of (a) FGF2, (b) a GSK3 inhibitor, (c) a SMAD inhibitor, and (d) transferrin, insulin, progesterone, putrescine, and selenium or a bioavailable salt thereof; (ii) culturing the cells obtained in step (i) in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive similar to that in (i), followed by adding an effective amount of (d) HGF and continuing the culture in the medium, followed by Culturing cells in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive similar to that in (i), and (d) KnockOut Serum Replacement (KSR) to induce myogenic specification; (iii) maturing the cells obtained in step (ii) into skeletal myoblasts and satellite cells by culturing the cells in a basal medium containing an effective amount of (a) HGF, (b) a serum-free additive similar to that in (i), and (c) KnockOut Serum Replacement (KSR), followed by (iv) maturing the cells obtained in step (iii) into skeletal myotube cells and satellite cells by culturing the cells in a basal medium containing an effective amount of (a) a serum-free additive similar to that in step (i), and (b) an additional serum-free additive containing albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3) and thereby generating skeletal myoblasts, skeletal myotube cells, and satellite cells.

[0088] For example, in this method, the proportion of skeletal myoblasts in the amount of all available cells, determined by the expression of actin by flow cytometry, is at least 40%, preferably at least 50%, more preferably at least 60%, and most preferably at least 70%.

[0089] Preferably, the method achieves a proportion of satellite cells in the amount of all available cells of at least 10%, preferably at least 15%, more preferably at least 20%, and most preferably at least 30%, determined by the expression of Pax7 by flow cytometry.

[0090] The method of "flow cytometry" is known to those skilled in the art. In flow cytometry, the physical and / or chemical characteristics of a cell population are detected. For the invention described herein, the presence of skeletal muscle-specific proteins characteristic of differentiation into skeletal myoblasts, skeletal myotube cells, or satellite cells can be detected using fluorescence staining. In particular, the proteins sarcomeric α-actinin, myogenin, Pax7, and MyoD are incubated with a primary antibody and thereby labeled. With the aid of a fluorescently labeled secondary antibody, skeletal muscle-specific cells can be detected.

[0091] The main advantage of this method over the prior art is that it does not require a step of concentrating cells, such as skeletal myoblasts. Preferably, the method does not include a concentration step by cell selection, and more preferably, does not contain a concentration step by antibody-based cell selection, such as flow cytometry. This means that the method according to the invention does not require cell selection to achieve high-purity skeletal myoblasts, skeletal myotube cells and / or satellite cells. The method of cell selection is only disclosed herein for analytical purposes to demonstrate the high-purity skeletal myoblasts, skeletal myotube cells and satellite cells produced (see Figure 5).

[0092] As described above, the basal medium for step (i) contains a serum-free additive containing an effective amount of (a) FGF2, (b) a GSK3 inhibitor, (c) a SMAD inhibitor, and (d) transferrin, insulin, progesterone, putrescine, and selenium or a bioavailable salt thereof.

[0093] For example, the GSK3 inhibitor in the basal medium is selected from the group consisting of CHIR99021, CHIR98014, SB216763, TWS119, tidoglucib, SB415286, 6-bromoindirubin-3-oxime, and valproate, and the GSK3 inhibitor CHIR99021 is preferred. However, any GSK3 inhibitor suitable for the method of the present invention can be used. When the GSK3 inhibitor is CHIR99021, the effective amount is 4 to 18 μM, preferably 5 to 16 μM, more preferably 6 to 15 μM, even more preferably 7 to 14 μM, even more preferably 8 to 13 μM, even more preferably 9 to 12 μM, even more preferably 9.5 to 11 μM, and most preferably about 10 μM.

[0094] Preferred exemplary embodiments of steps (i)-(iii) are described in the method of preparing engineered skeletal muscle tissue and can be similarly applied to the method of preparing skeletal myoblasts, skeletal myotube cells, and satellite cells.

[0095] Each stage of differentiation can be determined by simple experimental evidence known to those skilled in the art. For example, the inventors analyzed the cells using fluorescence microscopy. This involves immunostaining of skeletal muscle-specific transcription factors (Pax7, MyoD, and myogenin). After step (iii) of the method, the fluorescence images show a high percentage of cells expressing Pax7, MyoD, and myogenin (Figure 3). This method shows that in addition to satellite cells (Pax7), skeletal myoblasts (MyoD and myogenin) are generated by the method.

[0096] In step (iv) of the method of the present invention, the cells are matured into skeletal myotube cells and satellite cells. Skeletal myotube cells are formed by the fusion of skeletal myoblasts. Thus, skeletal myotube cells are multinucleated cell structures formed by the fusion of mature myoblasts into long myotube cells. As described in step (iv) of the method for producing skeletal muscle tissue, this differentiation stage is also characterized by the expression of specific factors. For example, the expression of Pax7 is characteristic of the presence of satellite cells. At the same time, the expression of myogenin and actinin is characteristic of skeletal myotube cells, and the expression of each of them can be determined by RNA sequencing (see FIGS. 1 and 2 for a schematic overview; see FIG. 4 for experimental data on the expression of PAX7, ACTN2, DMD, and MYH3). For example, when the gene marker PAX7 has an expression value that is at least 5-fold higher (preferably at least 10-fold higher, more preferably 20-fold higher) compared to pluripotent stem cells when measured by "reads per million base pairs" using RNA sequencing, satellite cells are indicated. For example, when the gene marker ACTN2 has an expression value that is at least 5-fold higher (preferably at least 50-fold higher, more preferably 100-fold higher, even more preferably 150-fold higher) compared to pluripotent stem cells when measured by "reads per million base pairs" by RNA sequencing, skeletal myotube cells are indicated. The gene markers DMD and MYH3 exhibit expression values that are at least 200-fold higher (preferably at least 500-fold higher, more preferably 1000-fold higher) compared to pluripotent stem cells.

[0097] As described above, the basal medium for step (iv) contains an effective amount of (a) a serum-free additive similar to that in step (i), and (b) an additional serum-free additive containing albumin, transferrin, ethanolamine, selenium or its bioavailable salt, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3).

[0098] The additional serum-free additive in step (iv) of the method is prepared such that the additional serum-free additive provides the following final concentrations of substances: 0.5 to 50 mg / ml of albumin (preferably 1 to 40 mg / ml, more preferably 2 to 30 mg / ml, even more preferably 3 to 20 mg / ml, even more preferably 4 to 10 mg / ml, most preferably 4.5 to 7.5 mg / ml, for example about 5 mg / ml); 1 to 100 μg / ml of transferrin (preferably 2 to 90 μg / ml, more preferably 3 to 80 μg / ml, even more preferably 4 to 70 μg / ml, even more preferably 5 to 60 μg / ml, more preferably 6 to 50 μg / ml, more preferably 7 to 40 μg / ml, more preferably 8 to 30 μg / ml, more preferably 9 to 20 μg / ml, for example about 10 μg / ml); 0.1 to 10 μg / ml of ethanolamine (preferably 0.2 to 9 μg / ml, more preferably 0.3 to 8 μg / ml, even more preferably 0.4 to 7 μg / ml, even more preferably 0.5 to 6 μg / ml, more preferably 0.6 to 5 μg / ml, more preferably 0.7 to 4 μg / ml, more preferably 0.8 to 3 μg / ml, most preferably 1 to 2.5 μg / ml, for example about 2 μg / ml); 17.4 to 1744 nM of selenium or a bioavailable salt thereof (preferably 35 to 850 nM, more preferably 70 to 420 nM, even more preferably 120 to 220 μg / ml, most preferably about 174 nM); 0.4 to 40 μg / ml of L-carnitine HCl (preferably 0.5 to 30 μg / ml, more preferably 1 to 20 μg / ml, even more preferably 2 to 10 μg / ml, more preferably 3 to 5 μg / ml, most preferably about 4 μg / ml); 0.05 to 5 μl / ml of a fatty acid additive (preferably 0.1 to 4 μl / ml, more preferably 0.2 to 3 μl / ml, even more preferably 0.3 to 3 μl / ml, more preferably 0.4 to 2 μl / ml, most preferably 0.45 to 1 μl / ml, for example about 0.5 μl / ml); and 0.0001 to 0.1 μg / ml of triiodo-L-thyronine (T3) (preferably 0.001 to 0.01 μg / ml, more preferably 0.002 to 0.0075 μg / ml, even more preferably 0.003 to 0.005 μg / ml, most preferably about 0.004 μg / ml).

[0099] In a preferred embodiment, the additional serum-free additive further comprises 0.1 to 10 μg / ml of hydrocortisone (preferably 0.2 to 9 μg / ml, more preferably 0.3 to 8 μg / ml, even more preferably 0.4 to 7 μg / ml, even more preferably 0.5 to 6 μg / ml, even more preferably 0.6 to 5 μg / ml, even more preferably 0.7 to 4 μg / ml, even more preferably 0.8 to 3 μg / ml, most preferably 0.9 to 2 μg / ml, for example about 1 μg / ml). In a similarly preferred embodiment, the additional serum-free additive further comprises 0.3 to 30 μg / ml of insulin (preferably 0.5 to 25 μg / ml, more preferably 1 to 20 μg / ml, even more preferably 1.5 to 15 μg / ml, even more preferably 2 to 10 μg / ml, most preferably 2.5 to 5 μg / ml, for example about 3 μg / ml). For example, the bioavailable salt of selenium is sodium selenite such that a final concentration of 0.003 to 0.3 μg / ml (preferably 0.005 to 0.2 μg / ml, more preferably 0.01 to 0.1 μg / ml, even more preferably 0.02 to 0.05 μg / ml, most preferably 0.03 μg / ml, for example about 0.032 μg / ml) is provided in the basal medium.

[0100] Furthermore, the additional serum-free additive may further comprise one or more components selected from the group consisting of vitamin A, hydrocortisone, D-galactose, linolenic acid, progesterone, and putrescine. These components are beneficial for cell survival. Suitable concentrations of each component are known to those skilled in the art or can be readily determined by conventional means.

[0101] The additional serum-free additives referred to in step (iv) are also commercially available. For example, B27 may be used. In a preferred embodiment, the B27 additive is used in an amount of 0.1 to 10% B27, preferably 0.5 to 8%, preferably 1 to 6%, more preferably 1.5 to 4%, even more preferably 1.5 to 4%, and most preferably about 2% B27.

[0102] Additionally, step (iv) of this method may be carried out for at least 30 days, preferably at least 35 days, more preferably at least 40 days, even more preferably at least 50 days.

[0103] The seeding step may precede step (i) of this method. In the seeding step, pluripotent stem cells are seeded into a stem cell medium in the presence of a ROCK inhibitor. Preferably, the seeding step is carried out 18 - 30 hours before step (i), preferably 20 - 28 hours before, more preferably 22 - 26 hours before, even more preferably 23 - 25 hours before, and most preferably about 24 hours before. For example, the ROCK inhibitor is selected from the group consisting of Y27632, H-1152P, thiazovivin, fasudil, hydroxyfasudil, GSK429286A, and RKI1447. More preferably, the ROCK inhibitor is selected from the group consisting of Y27632, H-1152P, thiazovivin, fasudil, and hydroxyfasudil. Even more preferably, the ROCK inhibitor is selected from the group consisting of Y27632 and H-1152P. Particularly preferably, the ROCK inhibitor is Y27632. However, any ROCK inhibitor suitable for the method of the present invention can be used. Those skilled in the art understand that the concentration of the effective amount of the ROCK inhibitor will vary with the availability of the inhibitor in question and its inhibition constant. For example, in the case of Y27632, the medium used in the seeding step may be used at a concentration of 0.5 - 10 μM, preferably 1 - 9 μM, more preferably 2 - 8 μM, more preferably 3 - 7 μM, more preferably 4 - 6 μM, and most preferably about 5 μM. A stem cell medium may be used in the seeding step. In principle, any stem cell medium suitable for the method can be used. Suitable stem cell media are known to those skilled in the art, and iPS-Brew XF stem cell medium is particularly preferred. Preferably, the stem cell medium contains effective concentrations of KSR and FGF2. In particular, the stem cell medium contains, for example, 5 - 20% (v / v), preferably 6 - 17.5% (v / v), more preferably 7 - 15% (v / v), more preferably 8% - 12% (v / v), more preferably 9% - 11% (v / v), and most preferably about 10% (v / v) of KSR; and / or 1 - 15 ng / ml of FGF2, preferably 2.5 - 14 ng / ml, more preferably 5 - 13 ng / ml, even more preferably 7.5 - 12.5 ng / ml, even more preferably 8 - 12 ng / ml, even more preferably 9 - 11 ng / ml, and most preferably about 10 ng / ml of FGF2.

[0104] Similar to the method for producing skeletal muscle tissue, the differentiation stages of steps (i) to (iv) of the method for producing skeletal myoblasts, skeletal muscle tube cells, and satellite cells can be detected using genes expressed characteristic of specific stages. The method of RNA sequencing is used in this method similar to the method for producing skeletal muscle tissue. Thus, the same genes expressed (i.e., MSGN1, TBX6, MEOX1, PAX3, PAX7, MYOD1, ACTN2, DMD, MYH3) can be detected depending on the differentiation stage.

[0105] Traditional methods disclosed in the prior art for obtaining skeletal muscle cells often require large-scale digestion protocols and / or cell selection steps by flow cytometry. The digestion protocol transfers the cells to a different environment, whereby they lose cell-matrix connectivity in addition to cell-cell connectivity. This disrupts the extracellular environment and the spatial distribution of cell types formed during development and can have an inhibitory effect on the skeletal muscle differentiation process that is difficult to control. The present invention minimizes the number of digestion steps and, for example, does not require cell selection for concentrating skeletal myoblasts. The refined protocol produces highly pure skeletal myoblasts, skeletal muscle tube cells, and satellite cells (an exemplary cell population that is at least 70% actin positive and at least 30% PAX7 positive is shown in the examples. See also Figure 5).

[0106] By the method according to the present invention, skeletal muscle tissue can be obtained that is produced in an engineered manner with advantageous properties. In skeletal muscle tissue produced in an engineered manner, the presence of skeletal muscle tube cells can be detected by staining with actinin (see Figure 8). In particular, the skeletal muscle tissue does not contain either a differentiation-related transgene or a maturation-related transgene, preferably the skeletal muscle tissue does not contain a myogenic transgene, and more preferably the skeletal muscle tissue does not contain either the transgene Pax7 or MyoD. Compared to native skeletal muscle tissue, engineered skeletal muscle tissue, such as BSM or ESM, does not have a blood supply or central nervous system control. The central nervous system is known to those skilled in the art and consists of the brain and spinal cord in vertebrates. For example, engineered skeletal muscle tissue also does not have innervation by nerve cells. Blood supply means angiogenesis of the muscle, which supplies blood to the muscle. Another difference from native skeletal muscle tissue is that engineered skeletal muscle tissue does not have tendon parts or skeletal muscle attachments via bone and is generated completely ex vivo in the engineered skeletal muscle. Therefore, engineered skeletal muscle tissue is clearly distinguishable from native skeletal muscle tissue. Despite its engineered production, the skeletal muscle tissue according to the present invention exhibits many characteristics of native skeletal muscle. These include the morphological characteristics of the syncytium of fused muscle cells (muscle fibers, multinucleated skeletal muscle tube cells) as well as the contractile performance (positive force-frequency ratio and tetanus). Different from the tissue of the prior art, the satellite cell niche is formed in direct contact with the skeletal muscle fiber. Additionally, the skeletal muscle tissue produced according to the present invention exhibits typical fibers of striated skeletal muscle, and the skeletal muscle tissue consists of many muscle fibers (syncytium). It is known to those skilled in the art that native skeletal muscle tissue has multinucleated skeletal muscle fibers, and each skeletal muscle fiber consists of sarcomeres that are strung together. Therefore, multinucleated skeletal muscle fibers can be recognized by their characteristic striated skeletal muscle pattern in actin staining or actinin staining, because actin / actinin is stained within the sarcomere. Sarcomeres have a strict, regular structure, and they are arranged in rows and together form multinucleated muscle fibers. This means that the characteristic striated skeletal muscle pattern proves that multinucleated skeletal muscle fibers have formed.The characteristic skeletal muscle tissue structure (skeletal muscle fibers having a satellite cell niche) may be visualized by fluorescence microscopy after staining with actinin or actin and Pax7. In the present invention, the inventors have stained the structural protein actin in the engineered skeletal muscle tissue, and the fluorescence image of FIG. 8 exemplifies a characteristic striated pattern.

[0107] The key functional feature of the engineered skeletal muscle tissue is that the tissue contracts in response to electrical stimulation and as a result generates force. This force generation feature can be determined, for example, by measuring the contraction output. These contraction experiments measure the contraction frequency and contraction force of the engineered skeletal muscle tissue in response to electrical stimulation. Ring-shaped skeletal muscle tissue is tested in an organ bath (Fohr Medical Instruments) containing Tyrode's solution (e.g., in mmol / L, 120 NaCl, 1 MgCl2, 1.8 CaCl2, 5.4 KCl, 22.6 NaHCO3, 4.2 NaH2PO4, 5.6 glucose, and 0.56 ascorbate) at 37 °C with continuous gas treatment of 5% CO2 and 95% O2. The engineered skeletal muscle tissue is mechanically stretched and the maximum force amplitude (contraction force = FOC) is typically measured at an electric field stimulation frequency (4 ms rectangular pulse; 200 mA) within the range of 1 - 100 Hz. Exemplary measurement methods for the tissue according to the present invention are shown in FIGS. 6B, 6C, 7B, and 7C. These contraction experiments show that the engineered skeletal muscle tissue exhibits particularly advantageous properties when generating force in response to electrical stimulation. The engineered skeletal muscle tissue shows reproducible contraction frequency and contraction force in response to stimulation frequencies from 1 Hz to 100 Hz. Typically, for a single stimulus at 1 Hz, it takes about 0.5 seconds for contraction and complete relaxation. Since the contraction and relaxation times take about 0.5 seconds, initial or complete tetanus is formed at higher stimulation frequencies. Tetanus is also formed in native skeletal muscle tissue at increased stimulation frequencies, so the engineered skeletal muscle tissue behaves similarly to native skeletal muscle tissue even in this regard. Furthermore, the inventors can show that the contraction force of the muscle tissue increases with an increase in contraction frequency (positive force-frequency relationship). These properties are consistent with native skeletal muscle tissue, which also exhibits a positive force-frequency relationship in response to electrical stimulation in addition to single contractions and tetanus. Unlike the engineered skeletal muscle tissue, the electrical impulses in native muscle tissue originate from the action potential of neurons, while the engineered skeletal muscle tissue can contract spontaneously in response to electrical stimulation.

[0108] As illustrated in FIGS. 6B, 6C, 7B, 7C, and 8, the engineered skeletal muscle tissue produced by the method of the present invention has a characteristic formation of multinucleated muscle fibers (skeletal muscle tube cells) and generates force in response to electrical stimulation. Typically, the engineered skeletal muscle tissue can generate a contractile force of at least 0.3 millinewtons (mN), preferably at least 0.4 mN, more preferably at least 0.5 mN, more preferably at least 0.6 mN, more preferably at least 0.7 mN, more preferably at least 0.8 mN, more preferably at least 0.9 mN, more preferably at least 1 mN, more preferably at least 1.2 mN, more preferably at least 1.3 mN, more preferably at least 1.4 mN, more preferably at least 1.5 mN, more preferably at least 1.6 mN, more preferably at least 1.7 mN, more preferably at least 1.8 mN, more preferably at least 1.9 mN, and most preferably at least 2 mN at a stimulation of 100 Hz at 200 mA.

[0109] In principle, the engineered skeletal muscle tissue can have any desired form. For example, it may have an engineered form in the shape of a ring, ribbon, strand, patch, pouch, or cylinder, optionally with individual skeletal muscle tissues fused. For example, in the examples herein, the skeletal muscle tissue has the form of a ring. However, individual and / or different geometries may also be fused to the skeletal muscle tissue as desired, whereby many other different muscle forms can be achieved. In particular, forms in the shape of a ring, strand, patch or band are useful for applications in in vitro methods, for example for testing toxicity or for therapeutic applications for muscle repair in vivo. Usually, since the engineered form is already obtained by pouring in the master mix, generally any pourable engineered form can be produced.

[0110] Furthermore, the present invention relates to mesoderm-differentiated skeletal myoblast progenitor cells that are obtained according to step (i) of the present invention, characterized by the expression of the MSGN1 and / or TBX6 genes, and wherein the expression of MSGN1 and / or TBX6 can be determined by flow cytometry and / or immunostaining. These cells are also characterized by expressing the mRNA of SP5, and the expression of SP5 can be determined by RNA sequencing.

[0111] Additionally, the present invention relates to myogenic-specialized skeletal myoblast progenitor cells that are obtained according to step (ii) of the present invention, produced by steps (i) and (ii) of the present invention, characterized by the expression of the gene PAX3, and wherein the expression of PAX3 can be determined by flow cytometry and / or immunostaining. These cells are characterized by expressing the mRNA of SIM1, and the expression of SIM1 can be determined by RNA sequencing.

[0112] Furthermore, the present invention relates to skeletal myoblasts that are obtained according to step (iii) of the present invention, produced by steps (i) to (iii) of the present invention, characterized by the expression of actinin, and wherein the expression of actinin can be determined by flow cytometry and / or immunostaining in skeletal myoblasts.

[0113] Satellite cells are further provided by the present disclosure, which are obtainable according to step (iii) of the method disclosed herein and manufacturable by steps (i) to (iii) of the method disclosed herein, and are characterized by the expression of the gene Pax7. In this regard, the expression of Pax7 can be determined by flow cytometry and / or immunostaining. The satellite cells are characterized by an active or activatable cell cycle and express Pax7 and Ki67. In a particularly preferred embodiment, the satellite cells thus further express Ki67. Cell cycle activation in the engineered skeletal muscle tissue is more frequently observed after tissue injury (e.g., by pressure injury, cardiotoxic treatment, irradiation, or frostbite), which leads to the repair of tissue injury in the sense of endogenous regeneration.

[0114] A mixture of skeletal myoblasts and satellite cells is also disclosed, wherein the proportion of satellite cells in the total amount of all available cells, determined by the expression of Pax7 by flow cytometry, reaches at least 10%, preferably at least 15%, more preferably at least 20%, even more preferably at least 30%; and / or the proportion of skeletal myoblasts in the total amount of all available cells, determined by the expression of actinin by flow cytometry, reaches at least 40%, preferably at least 50%, more preferably at least 60%, most preferably at least 70%.

[0115] Furthermore, the present invention relates to skeletal myotube cells, which are obtained according to step (iv) of the present invention, manufactured by steps (i) to (iv) of the present invention, and are characterized by the anisotropic orientation of the actinin protein-containing sarcomere structure.

[0116] Advantageously, the engineered skeletal muscle tissue, mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, skeletal myoblasts, satellite cells, and / or skeletal muscle tube cells may be used in in vitro drug assays. The drug assays are preferably toxicity assays or assays of skeletal muscle tissue function under the influence of pharmacological drug candidates and gene therapy drug candidates. Pharmacological drug candidates are typically drug candidates that include protein-based molecules in addition to small molecule compounds. Gene therapy drug candidates typically alter the genome of skeletal muscle tissue by introducing the corresponding nucleic acid.

[0117] Furthermore, the engineered skeletal muscle tissue, mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, skeletal myoblasts, satellite cells, and / or skeletal muscle tube cells can be used in medicine.

[0118] Particularly important here are satellite cells. They are contemplated for use in the treatment of damaged skeletal muscle as well as / or skeletal muscle diseases, preferably genetic skeletal muscle defects, in particular Duchenne muscular dystrophy and / or Becker - Kearner muscular dystrophy, and / or lysosomal storage diseases, in particular Pompe disease, and preferably the skeletal muscle disease is Duchenne muscular dystrophy. Those skilled in the art recognize from the prior art that satellite cells have already been applied in clinical studies for the treatment of muscular dystrophy (Tedesco FS et al.2010). Additionally, satellite cells are being considered for use in the treatment of skeletal muscle diseases such as amyotrophic lateral sclerosis, myasthenia gravis, or myotonia. Myotonia encompasses various muscle diseases presenting with delayed relaxation and consequently pathological prolongation of tonic muscle contractions. Satellite cells are particularly suitable for the treatment of damaged skeletal muscle and / or for the treatment of skeletal muscle diseases because they continuously regenerate skeletal muscle tissue. The term "damaged skeletal muscle tissue" refers to tissue injury and trauma caused by external forces. Human satellite cells obtained according to step (iii) or (iv) of the method of the present invention exhibit the characteristic marker Pax7. Thus, since satellite cells lead to an increase in the regeneration of skeletal muscle tissue, the satellite cells according to the present invention are promising candidates for cell - based therapy in damaged skeletal muscle tissue (Yin et al.(2013)). Similarly, the engineered skeletal muscle tissue according to the present invention is a promising candidate for cell - based therapy in damaged skeletal muscle tissue, particularly for the treatment of large muscle defects. Direct transplantation of replacement tissue, such as engineered skeletal muscle tissue, is a promising approach, especially in cases involving trauma or large muscle destruction. Skeletal muscle transplantation can integrate functionally to restore or therapeutically support muscle function and can be controllable via electrical stimulation or optogenetic activation. The proportion of satellite cells in the engineered skeletal muscle tissue ensures the long - term endogenous regenerative capacity of skeletal muscle.

[0119] In addition to the engineered skeletal muscle tissue, mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells are also a suitable model system for studying the important cellular mechanisms for differentiation and maturation. Therefore, they are an important scientific tool for basic research. Therefore, for example, chemicals and optionally physical stimuli, such as stretching or injury, can be tested in human cells outside the human body or in engineered skeletal muscle tissue. The cells, skeletal myotube cells, and engineered skeletal muscle tissue according to the present invention enable pharmacological safety and efficacy experiments, whereby the effects on cells and tissues can be tested. This is a distinct advantage over animal experiments (e.g., mouse or rat tissue / cells), and the pharmacological effects can be tested, for example, in human tissue, in patient-specific tissue in certain embodiments. Due to the high similarity to native skeletal tissue, the skeletal tissue produced according to the methods disclosed above can advantageously be used in various in vitro procedures.

[0120] One such possible application is an in vitro method for testing the efficacy of a drug candidate on skeletal muscle tissue, comprising: (a) providing a skeletal muscle tissue according to the invention described herein; (b) optionally damaging the skeletal muscle tissue; and (c) contacting the skeletal muscle tissue of step (a) or (b) with the drug candidate. Preferably, the method further comprises determining the contractile force and / or structure and / or metabolic function and / or molecular and / or protein biochemical parameters of the skeletal muscle tissue before and / or after step (c).

[0121] ​​The contractile force and / or structure of skeletal muscle tissue may be measured by the contraction experiments described herein and the fluorescence microscopy experiments described herein. For example, the metabolic function can be measured by using a Seahorse Metabolic Flux Analyzer known to those skilled in the art. For example, the Seahorse Metabolic Flux Analyzer can measure the oxygen consumption of living cells and the rate of extracellular acid production, and can further measure important cellular functions such as mitochondrial respiration and glycolysis. Molecular parameters (markers) can be measured, for example, by transcriptome analysis (PCR or RNA sequencing). Protein biochemical parameters (markers) can be measured, for example, via mass spectrometry or common clinical chemistry measurement methods (such as ELISA or other antibody and / or chromatography and / or electrophoresis and / or affinity-based methods). These molecular and protein biochemical parameters are also called markers or biomarkers, and common biomarkers for skeletal muscle are known to those skilled in the art. For example, creatine kinase (also known as creatine kinase CK, CPK, or creatine phosphokinase) and L-lactate dehydrogenase (LDH) are such biomarkers.

[0122] Drug candidates include pharmacological drug candidates, such as small molecule compounds and drug candidates including protein-based or nucleic acid-based molecules. Furthermore, drug candidates include gene therapy drug candidates, which typically modify the genome of the cells of the present invention by introducing the corresponding nucleic acid. Additionally, drug candidates can also be substances of the body itself, and as a result, for example, the effects of hormones or hormone-like signaling substances can be tested. Examples of hormone-like signaling substances are myokines, such as myostatin, follistatin, irisin, visfatin, and myonectin.

[0123] A further in vitro method for testing the toxicity of a substance to skeletal muscle tissue, (a) providing skeletal muscle tissue according to the invention described herein, (b) Step of contacting the skeletal muscle tissue from step (a) with the substance to be tested comprising preferably, the method further comprises, before and / or after step (b), a step of determining the contractile force and / or structure and / or metabolic function and / or molecular and / or protein biochemical parameters of the skeletal muscle tissue a method is contemplated.

[0124] The contractile force and / or skeletal muscle tissue structure may be measured by the contraction experiments described herein and the fluorescence microscopy experiments described herein. The metabolic function may be measured, for example, by using a Seahorse Metabolic Flux Analyzer known to those skilled in the art.

[0125] The substances used in toxicity tests can be, for example, but not limited to, drug candidates. Rather, any substance whose toxicity is to be evaluated can be tested.

[0126] Another possible application is an in vitro method for testing the effects of nutrients and dietary supplements on skeletal muscle tissue performance, comprising (a) a step of providing skeletal muscle tissue according to the invention described herein, (b) a step of contacting the skeletal muscle tissue from step (a) with the nutrients and dietary supplements to be tested comprising preferably, the method further comprises, before and / or after step (b), a step of determining the contractile force and / or structure and / or metabolic function and / or molecular and / or protein biochemical parameters of the skeletal muscle tissue relating to a method.

[0127] This in vitro method provides an opportunity to measure the effects of nutrients and nutraceuticals on skeletal muscle tissue at clinically relevant concentrations. This method is of particular interest when measuring the effects of these substances in muscle growth, cachexia, or diabetes. Cachexia is understood to be a pathological, very severe wasting. Many patients with chronic diseases, such as cancer or autoimmune diseases, suffer from the additional condition of cachexia. The in vitro method gives the possibility to measure the effects of substances on skeletal muscle tissue outside the body.

[0128] However, similarly, various cells prepared according to the methods disclosed herein can also be used in such in vitro methods. For example, an in vitro method for testing the effectiveness of a drug candidate on mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells, (a) providing mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells according to the invention described herein; (b) optionally, damaging the cells from step (a); and (c) contacting the cells of step (a) or (b) with a drug candidate comprising; preferably, the method further comprises determining the expression of actinin and / or Pax7 before and / or after step (c), and the expression can be determined by flow cytometry and / or immunostaining; The method is described herein.

[0129] Another possible application is an in vitro method for testing the toxicity of a substance on mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells, (a) A step of providing mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells according to the invention described herein; (b) A step of contacting the cells of step (a) with the substance to be tested; comprising; Preferably, the method further comprises a step of determining the expression of actinin and / or Pax7 before and / or after step (b), and the expression can be determined by flow cytometry and / or immunostaining; relating to a method.

[0130] An additional possible application is an in vitro method for testing the effects of nutrients and dietary supplements on mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells, comprising: (a) A step of providing a mixture of mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells according to the invention described herein; (b) A step of contacting the cells of step (a) with the nutrient or dietary supplement to be tested; comprising; Preferably, the method further comprises a step of determining the expression of actinin and / or Pax7 before and / or after step (b), and the expression can be determined by flow cytometry and / or immunostaining; relating to a method.

[0131] In another preferred embodiment, the skeletal muscle tissue can generate a contractile force of at least 0.6 millinewtons (mN) with a 100 Hz stimulation, preferably at least 0.7 mN, more preferably at least 0.8 mN, more preferably at least 0.9 mN, more preferably at least 1 mN, more preferably at least 1.2 mN, more preferably at least 1.3 mN, more preferably at least 1.4 mN, more preferably at least 1.5 mN, more preferably at least 1.6 mN, more preferably at least 1.7 mN, more preferably at least 1.8 mN, more preferably at least 1.9 mN, and most preferably at least 2 mN. The contractile force is typically measured above the stimulation threshold. Suitable methods for determining the stimulation threshold are known to those skilled in the art. For example, the contractile force can be recorded in an electric field stimulation using 200 mA (see FIGS. 6, 7, 9, and 10). In a more preferred embodiment, the skeletal muscle tissue can generate a contractile force of at least 2 millinewtons (mN), preferably at least 2.3 mN, more preferably at least 2.6 mN, even more preferably at least 3 mM, even more preferably at least 3.3 mN, even more preferably at least 3.6 mN, and most preferably at least 4 mN with a 100 Hz stimulation. This typically occurs when step (iv) of the method is performed for at least 50 days, for example, 56 days. A typical characteristic of the engineered skeletal muscle tissue described herein is that the contractile force increases with the duration of maturation.

[0132] In another preferred embodiment, the skeletal muscle tissue has a contraction rate of at least 3 mN / sec, preferably at least 4 mN / sec, more preferably at least 5 mN, more preferably at least 6 mN / sec, even more preferably at least 6.5 mN / sec, even more preferably at least 7 mN / sec with a 100 Hz stimulation. For example, the contraction rate can be recorded with a 100 Hz stimulation (5 ms, single or biphasic) using 200 mA. The contraction rate, also called the force generation rate, is the time required for the engineered skeletal muscle tissue to accumulate a respective amount of tension, or the rate of increase of tension. The contraction rate is determined as the point of maximum increase in the contraction force (+dFOC / dt) in the context of an isometric contraction experiment.

[0133] In another preferred embodiment, the skeletal muscle tissue has a relaxation rate of at least 0.5 mN / sec, preferably at least 0.7 mN / sec, more preferably at least 0.9 mN / sec, more preferably at least 1 mN / sec, even more preferably at least 1.2 mN / sec, even more preferably at least 1.5 mN / sec at the end of a 100 Hz stimulation. The relaxation rate is determined during the relaxation phase of the skeletal muscle as the point of maximum decrease in the contraction force (-dFOC / dt) in the context of an isometric contraction experiment.

[0134] In a particularly preferred embodiment, the basal medium in step (iv) may contain an effective amount of creatine and / or triiodo-L-thyronine (T3). For example, if creatine is present in an effective amount in the basal medium of the maturation medium, the contractile force of the engineered skeletal muscle may be increased compared to maturation in step (iv) without using an effective amount of creatine. Such an increase in contractile force is shown with experimental data in Example 4 and FIG. 9. For example, an effective amount of creatine as the final concentration in the basal medium of step (iv) is 0.1 to 10 mM of creatine. More preferred concentrations are, for example, 0.2 to 6 mM of creatine, more preferably 0.4 to 4 mM of creatine, even more preferably 0.6 to 3 mM of creatine, even more preferably 0.7 to 2.5 mM of creatine, even more preferably 0.8 to 2 mM of creatine, even more preferably 0.85 to 1.5 mM of creatine, even more preferably 0.9 to 1.2 mM of creatine, and most preferably about 1 mM of creatine.

[0135] Furthermore, the maturation medium in step (iv) may also have an increased amount of T3. Such an increased amount of T3 may reduce the contraction rate and / or relaxation rate of the engineered skeletal muscle compared to the engineered skeletal muscle tissue prepared without the increased amount of T3 in step (iv). An exemplary increased amount of T3 in the basal medium of step (iv) is 0.001 to 1 μM of triiodo-L-thyronine (T3), preferably 0.005 to 0.7 μM of T3, more preferably 0.01 to 0.35 μM of T3, even more preferably 0.04 to 0.2 μM of T3, even more preferably 0.05 to 0.18 μM of T3, even more preferably 0.06 to 0.15 μM of T3, even more preferably 0.08 to 0.12 μM of T3, and even more preferably about 0.1 μM of T3. Furthermore, in addition to Example 4, FIG. 10 shows the beneficial effects with experimental data of increased concentrations of T3.

[0136] In a particularly highly preferred embodiment, the basal medium in step (iv) may contain an effective amount of creatine and / or an increased amount of triiodo-L-thyronine (T3) over a given maturation period. As shown in Example 4, such a period may be 4 weeks, for example, weeks 1 to 5 in step (iv), or weeks 5 to 9 in step (iv). However, other periods, such as 1 to 9 weeks, may be selected during any maturation period. For example, this period may be at least 1 week, preferably at least 2 weeks, more preferably at least 3 weeks, more preferably at least 4 weeks, even more preferably at least 5 weeks, even more preferably at least 6 weeks, even more preferably at least 7 weeks, even more preferably at least 8 weeks. Further, this period may be, for example, up to 9 weeks, more preferably up to 8 weeks, more preferably up to 7 weeks, even more preferably up to 6 weeks, even more preferably up to 5 weeks, even more preferably up to 4 weeks. In light of the present disclosure, one of ordinary skill in the art can freely combine the exemplary period endpoints.

[0137] In another preferred embodiment, the skeletal muscle tissue produced by the method described herein has regenerative properties. The regenerative properties are characterized by bringing about a natural restoration of a previously existing state. For example, the contractility of the manipulated skeletal muscle tissue can be restored. Thus, the contractility can be restored and / or the muscle can be rebuilt. In a highly preferred embodiment, the regenerative properties are characterized by restored contractility and / or muscle reconstruction, preferably, the ability to restore contractility and / or muscle reconstruction is measured 24 hours after exposure to cardiotoxin and / or muscle reconstruction, more preferably, the restored contractility and / or muscle reconstruction is measured 10 to 30 days after cardiotoxin exposure. The cardiotoxin is a polypeptide toxin that destroys skeletal muscle cells by inducing permanent depolarization. Functionally, incubation with cardiotoxin results in a loss of contractility of the manipulated skeletal muscle. Structurally, irreversible destruction of the formed myotube cells in the manipulated skeletal muscle is observed. For example, even after 2 days, no contraction could be recorded in Example 5 described herein. As shown in FIG. 11, the manipulated skeletal muscle tissue with regenerative properties can restore this contractility. For example, as described in Example 5, the muscle can contract again 21 days after cardiotoxin treatment. However, the manipulated skeletal muscle treated with gamma radiation (X-rays) does not exhibit regenerative properties and cannot contract even 21 days after cardiotoxin incubation. In the manipulated skeletal muscle tissue, when the skeletal muscle cells are irreversibly destroyed, skeletal muscle cell progenitor cells with regenerative capacity are preserved, and this example shows that the skeletal muscle structure with contractile function in the manipulated skeletal muscle tissue can be regenerated or rebuilt by cell division and differentiation into newly formed skeletal muscle cells. As shown in FIG. 11, the manipulated skeletal muscle tissue with regenerative properties can achieve this muscle reconstruction. FIG. 11B shows the regeneration of contractile force, and FIG. 11C (top) shows the structural regeneration of skeletal muscle. The failure of regeneration after gamma irradiation demonstrates that the cell division competent skeletal muscle progenitor cells (e.g., satellite cells) for regeneration survived the cardiotoxin treatment.

[0138] As shown in Example 4, step (iv) of the procedure can be carried out over several weeks. In a highly preferred embodiment, step (iv) is carried out for at least 50 days, more preferably at least 60 days, even more preferably at least 70 days, even more preferably at least 80 days. In the present knowledge of the inventors, there is no upper limit to the duration of step (iv). For example, the maximum duration of step (iv) can be 365 days, preferably 300 days, more preferably 250 days. A person skilled in the art can freely combine the exemplary period limits of step (iv) in light of this disclosure.

[0139] Furthermore, the present invention includes an engineered skeletal muscle tissue produced by a method as described herein. In a preferred embodiment, the skeletal muscle tissue generates a contractile force of at least 0.6 millinewtons (mN), preferably at least 0.7 mN, more preferably at least 0.8 mN, more preferably at least 0.9 mN, more preferably at least 1 mN, more preferably at least 1.2 mN, more preferably at least 1.3 mN, more preferably at least 1.4 mN, more preferably at least 1.5 mN, more preferably at least 1.6 mN, more preferably at least 1.7 mN, more preferably at least 1.8 mN, more preferably at least 1.9 mN, more preferably at least 2 mN, more preferably at least 2.3 mN, more preferably at least 2.6 mN, even more preferably at least 3 mN, even more preferably at least 3.3 mN, even more preferably at least 3.6 mN, and most preferably at least 4 mN in response to a stimulus of 100 Hz. The contractile force can be recorded, for example, at a stimulus of 200 mA.

[0140] In a particularly preferred embodiment, the skeletal muscle tissue has a contraction rate of at least 3 mN / sec, preferably at least 4 mN / sec, more preferably at least 5 mN / sec, more preferably at least 6 mN / sec, even more preferably at least 6.5 mN / sec, and even more preferably at least 7 mN / sec with a 100 Hz stimulation. In another preferred embodiment, the skeletal muscle tissue has a relaxation rate of at least 0.5 mN / sec, preferably at least 0.7 mN / sec, more preferably at least 0.9 mN / sec, more preferably at least 1 mN / sec, even more preferably at least 1.2 mN / sec, and even more preferably at least 1.5 mN / sec when the 100 mN / sec stimulation is terminated.

[0141] The present invention is further described by the following embodiments. 1. A method for producing engineered skeletal muscle tissue from pluripotent stem cells, comprising: (i) inducing mesodermal differentiation of pluripotent stem cells by culturing the pluripotent stem cells in a basal medium containing an effective amount of a serum-free additive comprising (a) FGF2, (b) a GSK3 inhibitor, (c) a SMAD inhibitor, and (d) transferrin, insulin, progesterone, putrescine, and selenium or a bioavailable salt thereof; (ii) culturing the cells obtained in step (i) in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive similar to that in (i), followed by adding an effective amount of (d) HGF and continuing the culture in the medium, followed by culturing the cells in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive similar to that in (i), and (d) KnockOut Serum Replacement (KSR) to induce myogenic specification; (iii) culturing the cells obtained in step (ii) in a basal medium containing an effective amount of (a) HGF, (b) a serum-free additive similar to that in (i), and (c) KnockOut Serum Replacement (KSR) to expand, proliferate, and mature the cells into skeletal myoblasts and satellite cells; (iv) culturing the cells obtained in step (iii) and dispersed in the extracellular matrix under mechanical stimulation in a basal medium containing an effective amount of (a) a serum-free additive similar to that in step (i), and (b) an additional serum-free additive containing albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3) to mature the cells into skeletal myotube cells and satellite cells comprising a method for producing an engineered skeletal muscle tissue thereby. 2. The method according to aspect 1, wherein the pluripotent stem cells are cells of primate origin, particularly human pluripotent stem cells; and / or the pluripotent stem cells are selected from induced pluripotent stem cells, embryonic stem cells, parthenogenetic stem cells, pluripotent stem cells produced via nuclear transfer, and pluripotent cells produced via chemical reprogramming, particularly the pluripotent stem cells are induced pluripotent stem cells. 3. The method according to aspect 1 or 2, wherein step (i) is carried out for 24 to 132 hours, preferably 48 to 120 hours, more preferably 60 to 114 hours, even more preferably 72 to 108 hours, more preferably 84 to 102 hours, and most preferably about 96 hours. 4. In step (i), the GSK3 inhibitor is selected from the group consisting of CHIR99021, CHIR98014, SB216763, TWS119, tideglusib, SB415286, 6-bromoindirubin-3-oxime, and valproate, preferably the GSK3 inhibitor is CHIR99021; and / or in step (i), the SMAD inhibitor is selected from the group consisting of LDN193189, K02288, LDN214117, ML347, LDN212854, and DMH1, preferably the SMAD inhibitor is LDN193189. The method according to any one of Aspects 1 to 3. 5. In step (i), the effective amount of FGF2 is 1 to 15 ng / ml, preferably 2.5 to 14 ng / ml, more preferably 5 to 13 ng / ml, even more preferably 7.5 to 12.5 ng / ml, even more preferably 8 to 12 ng / ml, even more preferably 9 to 11 ng / ml, and most preferably about 10 ng / ml; and / or The serum-free additive provides a final concentration in the medium of 50 to 500 μg / ml of transferrin, 1 to 20 μg / ml of insulin, 0.001 to 0.1 μg / ml of progesterone, 5 to 50 μg / ml of putrescine, and 6 to 600 nM of selenium or a bioavailable salt thereof, particularly sodium selenite; and / or The GSK3 inhibitor is CHIR99021 and the effective amount is 1 to 20 μM, preferably 2 to 19 μM, more preferably 3 to 18 μM, even more preferably 4 to 17 μM, even more preferably 5 to 16 μM, even more preferably 6 to 15 μM, even more preferably 7 to 14 μM, even more preferably 7.5 to 13 μM, even more preferably 8 to 12 μM, even more preferably 9 to 11 μM, and most preferably about 10 μM; and / or The SMAD inhibitor is LDN193189 and the effective amount is 0.05 to 5 μM, preferably 0.1 to 2.5 μM, more preferably 0.2 to 1 μM, even more preferably 0.25 to 0.8 μM, even more preferably 0.3 to 0.75 μM, even more preferably 0.35 to 0.7 μM, even more preferably 0.4 to 0.6 μM, even more preferably 0.45 to 0.55 μM, and most preferably about 0.5 μM. The method according to any one of Aspects 1 to 4. 6. The method according to any one of aspects 1 to 5, wherein the serum-free additive in step (i) is an N2 additive of 0.1 to 10% (v / v), more preferably an N2 additive of 0.3 to 7.5% (v / v), more preferably an N2 additive of 0.5 to 5% (v / v), more preferably an N2 additive of 0.75% to 2% (v / v), more preferably an N2 additive of 0.9% to 1.2% (v / v), and most preferably an N2 additive of about 1% (v / v). 7. The method according to any one of aspects 1 to 6, wherein the basal medium in step (i), step (ii), step (iii), and / or step (iv) is selected from DMEM, DMEM / F12, RPMI, IMDM, alphaMEM, Medium 199, Hams F-10, Hams F-12, the basal medium is preferably DMEM, and in particular the basal medium is supplemented with pyruvate and / or non-essential amino acids and / or contains 1 g / l of glucose. 8. In step (ii), the culture is carried out for 36 to 60 hours, preferably 42 to 54 hours, most preferably about 48 hours, in the presence of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive; and / or the culture is carried out for 36 to 60 hours, preferably 42 to 54 hours, most preferably about 48 hours, in the presence of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, (c) a serum-free additive, and (d) HGF; and / or the culture is carried out for 72 to 120 hours, preferably 76 to 114 hours, more preferably 84 to 108 hours, even more preferably 90 to 102 hours, most preferably about 96 hours, in the presence of (a) a gamma-secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive, and (d) KnockOut Serum Replacement (KSR). The method according to any one of aspects 1 to 7. 9. In step (ii), the gamma-secretase / NOTCH inhibitor is selected from the group consisting of DAPT, RO4929097, semagacestat (LY450139), avagacestat (BMS-708163), dibenzazepine (YO-01027), LY411575, IMR-1, L685458, preferably the gamma-secretase / NOTCH inhibitor is DAPT, the method according to any one of aspects 1 to 8. 10. In step (ii), the effective amount of FGF2 is 15 - 30 ng / ml, preferably 17.5 - 25 ng / ml, more preferably 18 - 22 ng / ml, even more preferably 19 - 21 ng / ml, most preferably about 20 ng / ml; and / or the effective amount of HGF is 1 - 15 ng / ml, preferably 2.5 - 14 ng / ml, more preferably 5 - 13 ng / ml, even more preferably 7.5 - 12.5 ng / ml, even more preferably 8 - 12 ng / ml, even more preferably 9 - 11 ng / ml, most preferably about 10 ng / ml; and / or the gamma-secretase / NOTCH inhibitor is DAPT and the effective amount is 1 - 20 μM, preferably 2 - 19 μM, more preferably 3 - 18 μM, even more preferably 4 - 17 μM, even more preferably 5 - 16 μM, even more preferably 6 - 15 μM, even more preferably 7 - 14 μM, even more preferably 7.5 - 13 μM, even more preferably 8 - 12 μM, even more preferably 9 - 11 μM, most preferably about 10 μM; KSR is used in an amount of 5 - 20% (v / v), preferably 6 - 17.5% (v / v), more preferably 7 - 15% (v / v), more preferably 8% - 12% (v / v), more preferably 9% - 11% (v / v), most preferably about 10% (v / v); in particular, KSR is used in the presence of a reducing agent such as beta-mercaptoethanol and / or alpha-thioglycerol, the method according to any one of aspects 1 to 9. 11. In step (iii), The effective amount of HGF is 1 to 15 ng / ml, preferably 2.5 to 14 ng / ml, more preferably 5 to 13 ng / ml, even more preferably 7.5 to 12.5 ng / ml, even more preferably 8 to 12 ng / ml, even more preferably 9 to 11 ng / ml, and most preferably about 10 ng / ml; and / or KSR is used in an amount of 5 to 20% (v / v), preferably 6 to 17.5% (v / v), more preferably 7 to 15% (v / v), more preferably 8% to 12% (v / v), more preferably 9% to 11% (v / v), and most preferably about 10% (v / v) of KSR; in particular, KSR is used in the presence of a reducing agent such as beta-mercaptoethanol and / or alpha-thioglycerol, The method of any one of Aspects 1 to 10. 12. In step (iv), an additional serum-free additive provides in the medium a final concentration of 0.5 to 50 mg / ml of albumin, 1 to 100 μg / ml of transferrin, 0.1 to 10 μg / ml of ethanolamine, 17.4 to 1744 nM of selenium or a bioavailable salt thereof, in particular sodium selenite, 0.4 to 40 μg / ml of L-carnitine, 0.05 to 5 μl / ml of a fatty acid additive, 0.0001 to 0.1 μg / ml of triiodo-L-thyronine (T3), the method of any one of Aspects 1 to 11. 13. The additional serum-free additive in step (iv) is 0.1 to 10% (v / v) of B27, preferably 0.5 to 8% (v / v), more preferably 1 to 6% (v / v), even more preferably 1.5 to 4% (v / v), and most preferably about 2% (v / v) of B27, the method of any one of Aspects 1 to 12. 14. In step (iv), the mechanical stimulation is static tension, or dynamic stimulation, or overload stimulation, and preferably the mechanical stimulation is static tension, the method of any one of Aspects 1 to 13. 15. The method includes a seeding step before step (i), in which the pluripotent stem cells are seeded into the stem cell medium in the presence of a ROCK inhibitor, and preferably, the seeding step is performed 18 to 30 hours before step (i), the method of any one of Aspects 1 to 14. 16. The ROCK inhibitor is selected from the group consisting of Y27632, H-1152P, thiazovivin, fasudil, hydroxyfasudil, GSK429286A, and RKI1447; preferably, the ROCK inhibitor is selected from the group consisting of Y27632, H-1152P, thiazovivin, fasudil, and hydroxyfasudil; more preferably, the ROCK inhibitor is selected from the group consisting of Y27632 and H-1152P; particularly preferably, the ROCK inhibitor is Y27632, the method of embodiment 15. 17. The ROCK inhibitor is Y27632 and is used at a concentration of 0.5 to 10 μM, preferably 1 to 9 μM, more preferably 2 to 8 μM, more preferably 3 to 7 μM, more preferably 4 to 6 μM, and most preferably about 5 μM; and / or the stem cell medium is iPS-Brew XF. The method of embodiment 15 or 16. 18. The pluripotent stem cells in the seeding step are first seeded in an engineered form in the presence of one or more components of the extracellular matrix in the master mix before the stem cell medium is added, the method of any one of embodiments 15 to 17. 19. The component of the extracellular matrix in the master mix is collagen, preferably type I collagen, more preferably of bovine, human, or murine origin, particularly bovine origin collagen, and optionally the extracellular matrix additionally contains laminin and / or fibronectin, the method of embodiment 18. 20. The pluripotent stem cells are seeded in the medium at a ratio of 1 to 6×10 6 cells / ml and 0.7 to 1.4 mg / ml of collagen, the method of embodiment 19. 21. The master mix contains 5 to 15% (v / v), preferably 7.5% to 12.5% (v / v), more preferably 9 to 11% (v / v), most preferably about 10% (v / v) of the exudate of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells as the extracellular matrix component, particularly the exudate is Matrigel; and / or the pH of the master mix is from pH 7.2 to pH 7.8. The method according to any one of aspects 18 to 20. 22. The master mix contains stromal cells, and the stromal cells produce extracellular matrix components collagen, laminin, fibronectin, and / or proteoglycan; and / or The pH of the master mix is from pH 7.2 to pH 7.8, The method according to any one of aspects 18 to 20. 23. The stem cell medium is added to the master mix in the engineered form after about 1 hour, and the stem cell medium contains KSR and FGF2. The method according to any one of aspects 18 to 22. 24. The stem cell medium contains 5-20% (v / v), preferably 6-17.5% (v / v), more preferably 7-15% (v / v), more preferably 8%-12% (v / v), more preferably 9%-11% (v / v), most preferably about 10% (v / v) of KSR; and / or The stem cell medium contains 1-15 ng / ml of FGF2, preferably 2.5-14 ng / ml, more preferably 5-13 ng / ml, even more preferably 7.5-12.5 ng / ml, even more preferably 8-12 ng / ml, even more preferably 9-11 ng / ml, most preferably about 10 ng / ml of FGF2. The method according to aspect 23. 25. Step (iii) is carried out for 7-11 days, preferably 8-10 days, most preferably about 9 days. The method according to any one of aspects 18 to 24. 26. After step (iii), skeletal myoblasts and satellite cells are seeded in the engineered form in the presence of one or more components of the extracellular matrix in the master mix in an additional step before step (iv). The method according to any one of aspects 1 to 17. 27. The component of the extracellular matrix in the master mix is collagen, preferably type I collagen, more preferably of bovine origin, human origin or mouse origin, especially bovine origin collagen, and optionally the extracellular matrix additionally contains laminin and / or fibronectin. The method according to aspect 26. 28. The skeletal myoblasts and satellite cells are 1-6×10 6The method of embodiment 27, seeded in a medium at a ratio of cells / ml and 0.7 to 1.4 mg / ml of collagen. 29. The master mix contains an exudate of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells at 5 to 15% (v / v), preferably 7.5% to 12.5% (v / v), more preferably 9 to 11% (v / v), most preferably about 10% (v / v) as an extracellular matrix component, in particular the exudate is Matrigel; and / or The pH of the master mix is from pH 7.2 to pH 7.8, The method of any one of embodiments 26 to 28. 30. The master mix contains stromal cells, and the stromal cells produce extracellular matrix components collagen, laminin, fibronectin, and / or proteoglycan; and / or The pH of the master mix is from pH 7.2 to pH 7.8, The method of any one of embodiments 26 to 28. 31. After about 1 hour, a medium similar to that used in step (iii) is added to the master mix in the manipulated form, and the medium additionally contains an effective amount of a ROCK inhibitor; In particular, the ROCK inhibitor is selected from the group consisting of Y27632, H-1152P, thiazovivin, fasudil, hydroxyfasudil, GSK429286A, and RKI1447, preferably the ROCK inhibitor is selected from the group consisting of Y27632, H-1152P, thiazovivin, fasudil, and hydroxyfasudil, more preferably the ROCK inhibitor is selected from the group consisting of Y27632 and H-1152P, particularly preferably the ROCK inhibitor is Y27632, The method of any one of embodiments 26 to 30. 32. The ROCK inhibitor is Y27632 and is used at a concentration of 0.5 to 10 μM, preferably 1 to 9 μM, more preferably 2 to 8 μM, more preferably 3 to 7 μM, more preferably 4 to 6 μM, most preferably about 5 μM, in the method of embodiment 31. 33. After about 1 day, the medium is replaced with a medium similar to that used in step (iii), and the cells are then further cultured in this medium for an additional 5 to 9 days, preferably 6 to 8 days, most preferably about 7 days, in any one of methods of embodiments 26 to 32. 34. Any method of embodiments 18 to 33, wherein the engineered form has the form of a ring, ribbon, strand, patch, pouch, or cylinder, and optionally individual skeletal muscle tissues are fused. 35. Any method of embodiments 1 to 34, wherein step (iv) is carried out for at least 19 days, preferably at least 28 days, more preferably at least 56 days, even more preferably at least 120 days, particularly at least 240 days. 36. Any method of embodiments 1 to 35, which does not contain a differentiation-related transgene or a maturation-related transgene, preferably does not contain a myogenic transgene, and more preferably does not contain the transgenes Pax7 or MyoD. 37. Any method of embodiments 1 to 36, which does not include a skeletal myoblast enrichment step, preferably does not include a concentration step by cell selection, and more preferably does not include a concentration step by antibody-based cell selection. 38. A method for producing skeletal myoblasts, skeletal myotube cells, and satellite cells from pluripotent stem cells, (i) Inducing mesodermal differentiation of pluripotent stem cells by culturing the pluripotent stem cells in a basal medium containing a serum-free additive comprising an effective amount of (a) FGF2, (b) a GSK3 inhibitor, (c) a SMAD inhibitor, and (d) transferrin, insulin, progesterone, putrescine, and selenium or a bioavailable salt thereof; (ii) Culturing the cells obtained in step (i) in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive similar to that in (i), followed by adding an effective amount of (d) HGF and continuing the culture in the medium, followed by Culturing cells in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive as in (i), and (d) KnockOut Serum Replacement (KSR) to induce myogenic specification; (iii) culturing the cells obtained in step (ii) in a basal medium containing an effective amount of (a) HGF, (b) a serum-free additive as in (i), and (c) KnockOut Serum Replacement (KSR) to mature the cells into skeletal myoblasts and satellite cells, followed by (iv) culturing the cells obtained in step (iii) in a basal medium containing an effective amount of (a) a serum-free additive as in step (i), and (b) an additional serum-free additive containing albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3) to mature the cells into skeletal myotube cells and satellite cells comprising A method for producing skeletal myoblasts, skeletal myotube cells, and satellite cells thereby. 39. The method of embodiment 38, wherein the percentage of skeletal myoblasts in the amount of all available cells, determined by the expression of actinin by flow cytometry, is at least 40%, preferably at least 50%, more preferably at least 60%, and most preferably at least 70%, achieved by said method. 40. The method of embodiment 38 or 39, wherein the percentage of satellite cells in the amount of all available cells, determined by the expression of Pax7 by flow cytometry, is at least 10%, preferably at least 15%, more preferably at least 20%, and most preferably at least 30%, achieved by said method. 41. The method according to any one of embodiments 38 to 40, not including a skeletal myoblast enrichment step, preferably not including a concentration step by cell selection, more preferably not including a concentration step by antibody-based cell selection. 42. The method according to any one of aspects 38 to 41, wherein the pluripotent stem cells are cells of primate origin, particularly human pluripotent stem cells; and / or the pluripotent stem cells are selected from induced pluripotent stem cells, embryonic stem cells, parthenogenetic stem cells, pluripotent stem cells produced via nuclear transfer, and pluripotent cells produced via chemical reprogramming, particularly wherein the pluripotent stem cells are induced pluripotent stem cells. 43. The method according to any one of aspects 38 to 42, wherein step (i) is carried out for 48 to 132 hours, preferably 48 to 120 hours, more preferably 60 to 114 hours, even more preferably 72 to 108 hours, more preferably 84 to 102 hours, and most preferably about 96 hours. 44. In step (i), the GSK3 inhibitor is selected from the group consisting of CHIR99021, CHIR98014, SB216763, TWS119, tideglusib, SB415286, 6-bromoindirubin-3-oxime, and valproate, preferably the GSK3 inhibitor is CHIR99021; and / or In step (i), the SMAD inhibitor is selected from the group consisting of LDN193189, K02288, LDN214117, ML347, LDN212854, and DMH1, preferably the SMAD inhibitor is LDN193189. The method according to any one of aspects 38 to 43. 45. In step (i), the effective amount of FGF2 is 1 to 15 ng / ml, preferably 2.5 to 14 ng / ml, more preferably 5 to 13 ng / ml, even more preferably 7.5 to 12.5 ng / ml, even more preferably 8 to 12 ng / ml, even more preferably 9 to 11 ng / ml, and most preferably about 10 ng / ml; and / or The serum-free additive provides in the medium a final concentration of 50 to 500 mg / l of transferrin, 1 to 20 mg / l of insulin, 1 to 30 μg / l of progesterone, 5 to 50 μg / ml of putrescine, and 6 to 600 nM of selenium or a bioavailable salt thereof, particularly sodium selenite; and / or The GSK3 inhibitor is CHIR99021, and the effective amount is 4 - 18 μM, preferably 5 - 16 μM, more preferably 6 - 15 μM, even more preferably 7 - 14 μM, even more preferably 8 - 13 μM, even more preferably 9 - 12 μM, even more preferably 9.5 - 11 μM, and most preferably about 10 μM; and / or The SMAD inhibitor is LDN193189, and the effective amount is 0.05 - 5 μM, preferably 0.1 - 2.5 μM, more preferably 0.2 - 1 μM, even more preferably 0.25 - 0.8 μM, even more preferably 0.3 - 0.75 μM, even more preferably 0.35 - 0.7 μM, even more preferably 0.4 - 0.6 μM, even more preferably 0.45 - 0.55 μM, and most preferably about 0.5 μM. The method of any one of Aspects 38 - 44. 46. The serum-free additive in step (i) is 0.1 - 10% (v / v) N2 additive, preferably 0.3 - 7.5% (v / v) N2 additive, more preferably 0.5 - 5% (v / v) N2 additive, more preferably 0.75% - 2% (v / v) N2 additive, more preferably 0.9% - 1.2% (v / v) N2 additive, and most preferably about 1% (v / v) N2 additive, the method of any one of Aspects 38 - 45. 47. The basal medium in step (i), step (ii), step (iii), and / or step (iv) is selected from DMEM, DMEM / F12, RPMI, IMDM, alphaMEM, Medium 199, Hams F-10, Hams F-12, the basal medium is preferably DMEM, and in particular the basal medium is supplemented with pyruvate and / or non-essential amino acids, and / or contains 1 g / l glucose, the method of any one of Aspects 38 - 46. 48. In step (ii), the culture is carried out for 36 - 60 hours, preferably 42 - 54 hours, and most preferably about 48 hours in the presence of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive; and / or The culturing is carried out for 36 to 60 hours, preferably 42 to 54 hours, most preferably about 48 hours, in the presence of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, (c) a serum-free additive, and (d) HGF; and / or The culturing is carried out for 72 to 120 hours, preferably 76 to 114 hours, more preferably 84 to 108 hours, even more preferably 90 to 102 hours, most preferably about 96 hours, in the presence of (a) a gamma-secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive, and (d) KnockOut Serum Replacement (KSR). The method according to any one of aspects 38 to 47. 50. In step (ii), the gamma-secretase / NOTCH inhibitor is selected from the group consisting of DAPT, RO4929097, semagacestat (LY450139), avagacestat (BMS-708163), dibenzazepine (YO-01027), LY411575, IMR-1, L685458, and the gamma-secretase / NOTCH inhibitor is preferably DAPT. The method according to any one of aspects 38 to 48. 50. In step (ii), the effective amount of FGF2 is 15 to 30 ng / ml, preferably 17.5 to 25 ng / ml, more preferably 18 to 22 ng / ml, even more preferably 19 to 21 ng / ml, most preferably about 20 ng / ml; and / or The effective amount of HGF is 1 to 15 ng / ml, preferably 2.5 to 14 ng / ml, more preferably 5 to 13 ng / ml, even more preferably 7.5 to 12.5 ng / ml, even more preferably 8 to 12 ng / ml, even more preferably 9 to 11 ng / ml, most preferably about 10 ng / ml; and / or The gamma-secretase / NOTCH inhibitor is DAPT, and the effective amount is 1 to 20 μM, preferably 2 to 19 μM, more preferably 3 to 18 μM, even more preferably 4 to 17 μM, even more preferably 5 to 16 μM, even more preferably 6 to 15 μM, even more preferably 7 to 14 μM, even more preferably 7.5 to 13 μM, even more preferably 8 to 12 μM, even more preferably 9 to 11 μM, and most preferably about 10 μM; KSR is used in an amount of 6 to 14% (v / v), preferably 7 to 13% (v / v), more preferably 8% to 12% (v / v), more preferably 9% to 11% (v / v), and most preferably about 10% (v / v) of KSR; in particular, KSR is used in the presence of a reducing agent such as beta-mercaptoethanol and / or alpha-thioglycerol, Any method of embodiments 38 to 49. 51. In step (iii), the effective amount of HGF is 1 to 15 ng / ml, preferably 2.5 to 14 ng / ml, more preferably 5 to 13 ng / ml, even more preferably 7.5 to 12.5 ng / ml, even more preferably 8 to 12 ng / ml, even more preferably 9 to 11 ng / ml, and most preferably about 10 ng / ml; KSR is used in an amount of 5 to 20% (v / v), preferably 6 to 17.5% (v / v), more preferably 7 to 15% (v / v), more preferably 8% to 12% (v / v), more preferably 9% to 11% (v / v), and most preferably about 10% (v / v) of KSR; in particular, KSR is used in the presence of a reducing agent such as beta-mercaptoethanol and / or alpha-thioglycerol, Any method of embodiments 38 to 50. 52. Any method of embodiments 38 to 51, wherein step (iii) is carried out for 7 to 11 days, preferably 8 to 10 days, and most preferably about 9 days. 53. In step (iv), an additional serum-free additive provides a final concentration in the medium of 0.5 to 50 mg / ml of albumin, 1 to 100 μg / ml of transferrin, 0.1 to 10 μg / ml of ethanolamine, 17.4 to 1744 nM of selenium or a bioavailable salt thereof, particularly sodium selenite, 0.4 to 40 μg / ml of L-carnitine, 0.05 to 5 μl / ml of a fatty acid additive, 0.0001 to 0.1 μg / ml of triiodo-L-thyronine (T3), according to any of the methods of embodiments 38 to 52. 54. The additional serum-free additive in step (iv) is 0.1 to 10% (v / v) of B27, preferably 0.5 to 8% (v / v), more preferably 1 to 6% (v / v), even more preferably 1.5 to 4% (v / v), most preferably about 2% (v / v) of B27, according to any of the methods of embodiments 38 to 53. 55. Step (iv) is carried out for at least 30 days, preferably at least 35 days, more preferably at least 40 days, even more preferably at least 50 days, according to any of the methods of embodiments 38 to 54. 56. A seeding step is included before step (i), in which the pluripotent stem cells are seeded in a stem cell medium in the presence of a ROCK inhibitor, preferably the seeding step is carried out 18 to 30 hours before step (i), according to any of the methods of embodiments 38 to 55. 57. The ROCK inhibitor is selected from the group consisting of Y27632, H-1152P, thiazovivin, fasudil, hydroxyfasudil, GSK429286A and RKI1447, preferably the ROCK inhibitor is selected from the group consisting of Y27632, H-1152P, thiazovivin, fasudil, and hydroxyfasudil, more preferably the ROCK inhibitor is selected from the group consisting of Y27632 and H-1152P, particularly preferably the ROCK inhibitor is Y27632, according to the method of embodiment 56. 58. The ROCK inhibitor is Y27632 and is used at a concentration of 0.5 to 10 μM, preferably 1 to 9 μM, more preferably 2 to 8 μM, more preferably 3 to 7 μM, more preferably 4 to 6 μM, most preferably about 5 μM; and / or The method according to any one of aspects 56 to 58, wherein the stem cell medium is iPS-Brew XF. The method of aspect 56 or 57. 59. The stem cell medium contains 5 to 20% (v / v), preferably 6 to 17.5% (v / v), more preferably 7 to 15% (v / v), still more preferably 8% to 12% (v / v), still more preferably 9% to 11% (v / v), and most preferably about 10% (v / v) of KSR; and / or The stem cell medium contains 1 to 15 ng / ml of FGF2, preferably 2.5 to 14 ng / ml, more preferably 5 to 13 ng / ml, even more preferably 7.5 to 12.5 ng / ml, even more preferably 8 to 12 ng / ml, even more preferably 9 to 11 ng / ml, and most preferably about 10 ng / ml of FGF2. The method of aspect 58. 60. An engineered skeletal muscle tissue having multinucleated mature skeletal muscle fibers with satellite cells and without blood supply and / or central nervous system control; in particular, the engineered skeletal muscle tissue in which the presence of skeletal muscle fibers is determined by staining with actinin and using DAPI. 61. The engineered skeletal muscle tissue of aspect 60, wherein the skeletal muscle tissue is serum-free and / or does not contain differentiation-related transgenes or maturation-related transgenes, preferably the skeletal muscle tissue does not contain myogenic transgenes, and more preferably the skeletal muscle tissue does not contain the transgenes Pax7 or MyoD. 62. The skeletal muscle tissue generates a contractile force of at least 0.3 millinewtons (mN), preferably at least 0.4 mN, more preferably at least 0.5 mN, more preferably at least 0.6 mN, more preferably at least 0.7 mN, more preferably at least 0.8 mN, more preferably at least 0.9 mN, more preferably at least 1 mN, more preferably at least 1.2 mN, more preferably at least 1.3 mN, more preferably at least 1.4 mN, more preferably at least 1.5 mN, more preferably at least 1.6 mN, more preferably at least 1.7 mN, more preferably at least 1.8 mN, more preferably at least 1.9 mN, and most preferably at least 2 mN in response to a stimulus of 100 Hz at 200 mA, and is the operated skeletal muscle tissue of embodiment 60 or embodiment 61. 63. The skeletal muscle tissue is formed by operation, preferably has an operated form in the form of a ring, ribbon, strand, patch, pouch, or cylinder, optionally the individual skeletal muscle tissues are fused, and in particular the skeletal muscle tissue has the form of a ring, and is the operated skeletal muscle tissue of any of embodiments 60 to 62. 64. Mesoderm-differentiated skeletal myoblast progenitor cells, which are obtained according to step (i) of embodiment 1 or embodiment 38, prepared by the method of embodiment 1(i) or embodiment 38(i), characterized by the expression of the genes MSGN1 and / or TBX6, and the expression of MSGN1 and / or TBX6 can be determined by flow cytometry and / or immunostaining; and / or mRNA SP5 is expressed, and the expression of SP5 can be determined by RNA sequencing. 65. Myogenic-specialized skeletal myoblast progenitor cells, which are obtained according to step (ii) of embodiment 1 or embodiment 38, manufactured by the method of embodiment 1(i)-(ii) or embodiment 38(i)-(ii), characterized by the expression of the gene PAX3, and the expression of PAX3 can be determined by flow cytometry and / or immunostaining; and / or mRNA SIM1 is expressed, and the expression of SIM1 can be determined by RNA sequencing. 66. A skeletal myoblast that is obtained according to step (iii) of embodiment 1 or embodiment 38, manufactured by the method of embodiment 1 (i)-(iii) or embodiment 38 (i)-(iii), characterized by the expression of actinin, and preferably the expression of actinin can be determined by flow cytometry and / or immunostaining. 67. A satellite cell that is obtained according to step (iii) of embodiment 1 or embodiment 38, manufactured by the method of embodiment 1 (i)-(iii) or embodiment 38 (i)-(iii), characterized by the expression of the gene Pax7, the expression of Pax7 can be determined by flow cytometry and / or immunostaining, and more preferably the satellite cell further expresses Ki67. 68. A mixture of the skeletal myoblast of embodiment 66 and the satellite cell of embodiment 67, wherein the proportion of satellite cells in the amount of all available cells, determined by the expression of Pax7 by flow cytometry, is at least 10%, preferably at least 15%, more preferably at least 20%, even more preferably at least 30%; and / or the proportion of skeletal myoblasts in the amount of all existing cells, determined by the expression of actinin by flow cytometry, is at least 40%, preferably at least 50%, more preferably at least 60%, and most preferably at least 70%. 69. A skeletal myotube cell that is obtained according to step (iv) of embodiment 1 or embodiment 38, prepared by the method of embodiment 1 (i)-(iv) or embodiment 38 (i)-(iv), and characterized by the anisotropic orientation of the actinin protein-containing sarcomere structure. 70. Use of any of the skeletal muscle tissues of embodiments 60-63, and / or any of the cells of embodiments 64-68, and / or the skeletal myotube cells of embodiment 69 in an in vitro drug assay; in particular, the drug assay is a toxicity assay or an assay of skeletal muscle tissue function under the influence of a pharmacological drug candidate and a gene therapy drug candidate. 71. For use in medicine, skeletal muscle tissue of any of aspects 60 - 63 and / or cells of any of aspects 64 - 68, and / or skeletal muscle tube cells of aspect 69. 72. Satellite cells for use in the treatment of damaged skeletal muscle and / or skeletal muscle diseases, preferably genetic skeletal muscle defects, particularly Duchenne muscular dystrophy and / or Becker - Keiner muscular dystrophy, and / or lysosomal storage diseases, particularly Pompe disease, preferably wherein the skeletal muscle disease is Duchenne muscular dystrophy, satellite cells of aspect 67. 73. An in vitro method for testing the effectiveness of a drug candidate on skeletal muscle tissue, (a) providing skeletal muscle tissue of any of aspects 60 - 63, (b) optionally damaging the skeletal muscle tissue, and (c) contacting the skeletal muscle tissue from step (a) or (b) with the drug candidate comprising; preferably, the method further comprises, before and / or after step (c), determining the contractile force and / or skeletal muscle tissue structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters, method. 74. An in vitro method for testing the toxicity of a substance on skeletal muscle tissue, (a) providing skeletal muscle tissue of any of aspects 60 - 63, (b) contacting the skeletal muscle tissue from step (a) with the substance to be tested comprising, preferably, the method further comprises, before and / or after step (b), determining the contractile force and / or skeletal muscle tissue structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters, method. 75. An in vitro method for testing the effect of nutrients and dietary supplements on skeletal muscle tissue performance, (a) providing skeletal muscle tissue of any of aspects 60 - 63, (b) Step of contacting the skeletal muscle tissue from step (a) with the nutrient or dietary supplement to be tested comprising preferably, the method further comprises, before and / or after step (b), a step of determining contractile force and / or skeletal muscle tissue structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters Method 76. An in vitro method for testing the effectiveness of a drug candidate on mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells, comprising (a) Step of providing mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells according to any one of aspects 64-69 (b) Optionally, a step of damaging the cells from step (a), and (c) Step of contacting the cells of step (a) or (b) with the drug candidate comprising preferably, the method further comprises, before and / or after step (c), a step of determining the expression of actinin and / or Pax7, which expression can be determined by flow cytometry and / or immunostaining Method 77. An in vitro method for testing the toxicity of a substance on mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells, comprising (a) Step of providing mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells according to any one of aspects 64-69 (b) Step of contacting the cells of step (a) with the substance to be tested comprising Preferably, the method further comprises a step of determining the expression of actin and / or Pax7 before and / or after step (b), and the expression can be determined by flow cytometry and / or immunostaining. Method. 78. An in vitro method for testing the effects of nutrients and dietary supplements on mesoderm-differentiated skeletal myoblast progenitor cells, myogenic-specialized skeletal myoblast progenitor cells, satellite cells, skeletal myoblasts, skeletal myotube cells, or a mixture of skeletal myoblasts and satellite cells, comprising: (a) providing a mesoderm-differentiated skeletal myoblast progenitor cell, myogenic-specialized skeletal myoblast progenitor cell, satellite cell, skeletal myoblast, skeletal myotube cell, or a mixture of skeletal myoblasts and satellite cells according to any one of aspects 64-69; (b) contacting the cells of step (a) with the nutrient or dietary supplement to be tested. comprising: Preferably, the method further comprises a step of determining the expression of actin and / or Pax7 before and / or after step (b), and the expression can be determined by flow cytometry and / or immunostaining. Method. 79. The method according to any one of aspects 1-59, wherein the skeletal muscle tissue generates a contractile force of at least 0.6 millinewtons (mN), preferably at least 0.7 mN, more preferably at least 0.8 mN, more preferably at least 0.9 mN, more preferably at least 1 mN, more preferably at least 1.2 mN, more preferably at least 1.3 mN, more preferably at least 1.4 mN, more preferably at least 1.5 mN, more preferably at least 1.6 mN, more preferably at least 1.7 mN, more preferably at least 1.8 mN, more preferably at least 1.9 mN, and most preferably at least 2 mN in response to a 100 Hz stimulus. 80. The method according to any one of aspects 1 to 59 or 79, wherein the skeletal muscle tissue generates a contractile force of at least 2 millinewtons (mN), preferably at least 2.3 mN, more preferably at least 2.6 mN, even more preferably at least 3 mN, even more preferably at least 3.3 mN, even more preferably at least 3.6 mN, and most preferably at least 4 mN in response to a 100 Hz stimulus. 81. The method according to any one of aspects 1 to 59, 79, or 80, wherein the skeletal muscle tissue has a contraction rate of at least 3 mN / second, preferably at least 4 mN / second, more preferably at least 5 mN / second, more preferably at least 6 mN / second, even more preferably at least 6.5 mN / second, and even more preferably at least 7 mN / second in response to a 100 Hz stimulus. 82. The method according to any one of aspects 1 to 59 or 79 to 81, wherein the skeletal muscle tissue has a relaxation rate of at least 0.5 mN / second, preferably at least 0.7 mN / second, more preferably at least 0.9 mN / second, more preferably at least 1 mN / second, even more preferably at least 1.2 mN / second, and even more preferably at least 1.5 mN / second at the end of a 100 Hz stimulus. 83. The method according to any one of aspects 1 to 59 or 79 to 82, wherein the basal medium in step (iv) contains an effective amount of creatine and / or triiodo-L-thyronine (T3). 84. The method of aspect 83, wherein the effective amount of creatine in the basal medium increases the contractile force of the engineered skeletal muscle as compared to maturation in step (iv) without the use of an effective amount of creatine. 85. The method of aspect 83 or 84, wherein the effective amount of T3 in the basal medium reduces the contraction rate and / or relaxation rate of the engineered skeletal muscle as compared to maturation in step (iv) without the use of an effective amount of T3. 86. The method according to any one of aspects 1 to 59 or 79 to 85, wherein the basal medium in step (iv) provides a final concentration of creatine of 0.1 to 10 mM, preferably 0.2 to 6 mM, more preferably 0.4 to 4 mM, even more preferably 0.6 to 3 mM, even more preferably 0.7 to 2.5 mM, even more preferably 0.8 to 2 mM, even more preferably 0.85 to 1.5 mM, even more preferably 0.9 to 1.2 mM, and most preferably about 1 mM. 87. The method according to any one of aspects 1 to 59 or 79 to 86, wherein the basal medium in step (iv) provides a final concentration of triiodo-L-thyronine (T3) of 0.001 to 1 μM, preferably 0.005 to 0.7 μM, more preferably 0.01 to 0.35 μM, even more preferably 0.04 to 0.2 μM, even more preferably 0.05 to 0.18 μM, even more preferably 0.06 to 0.15 μM, even more preferably 0.08 to 0.12 μM, and even more preferably about 0.1 μM. 88. The method according to any one of aspects 1 to 59 or 79 to 87, wherein the skeletal muscle tissue has the property of self-regeneration. 89. The method of aspect 88, wherein the regenerative property is characterized by restored contractility and / or muscle recovery, preferably, the restored contractility and / or muscle recovery is measured after irreversible muscle damage using a cardiotoxin, and more preferably, the restored contractility and / or muscle recovery is measured 10 to 30 days after incubation with the cardiotoxin. 90. The method according to any one of aspects 1 to 59 or 79 to 89, wherein step (iv) is carried out for at least 50 days, more preferably at least 60 days, even more preferably at least 70 days, and even more preferably at least 80 days. 91. An engineered skeletal muscle tissue produced by the method according to any one of aspects 1 to 59 or 79 to 90. The engineered skeletal muscle tissue of any of aspects 60-63 or 91 that generates a contractile force of at least 0.6 millinewtons (mN) at a 92.100 Hz stimulus, preferably at least 0.7 mN, more preferably at least 0.8 mN, more preferably at least 0.9 mN, more preferably at least 1 mN, more preferably at least 1.2 mN, more preferably at least 1.3 mN, more preferably at least 1.4 mN, more preferably at least 1.5 mN, more preferably at least 1.6 mN, more preferably at least 1.7 mN, more preferably at least 1.8 mN, more preferably at least 1.9 mN, more preferably at least 2 mN, more preferably at least 2.3 mN, more preferably at least 2.6 mN, even more preferably at least 3 mN, even more preferably at least 3.3 mN, even more preferably at least 3.6 mN, and most preferably at least 4 mN. The engineered skeletal muscle tissue of any of aspects 60-63 or 91-92 that has a contraction rate of at least 3 mN / second at a 93.100 Hz stimulus, preferably at least 4 mN / second, more preferably at least 5 mN / second, more preferably at least 6 mN / second, even more preferably at least 6.5 mN / second, and even more preferably at least 7 mN / second. The engineered skeletal muscle tissue of any of aspects 60-63 or 91-93 that has a relaxation rate of at least 0.5 mN / second at the end of a 94.100 Hz stimulus, preferably at least 0.7 mN / second, more preferably at least 0.9 mN / second, more preferably at least 1 mN / second, even more preferably at least 1.2 mN / second, and even more preferably at least 1.5 mN / second. Use of the skeletal muscle tissue of any of aspects 60-63 or 91-94, and / or the cells of any of aspects 64-68, and / or the skeletal muscle tube cells of aspect 69 in an in vitro drug assay; in particular, where the drug assay is a toxicity assay or an assay of skeletal muscle tissue function under the influence of a pharmacological drug candidate and a gene therapy drug candidate. For use in medicine, skeletal muscle tissue of any of aspects 60 - 63 and 91 - 94, and / or cells of any of aspects 64 - 68, and / or skeletal muscle tube cells of aspect 69.

Brief Description of the Drawings

[0142]

Figure 1

Figure 2

Figure 3

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Figure 5

Figure 6

Figure 7A

Figure 7B

Figure 7C

Figure 8

Figure 9

Figure 10A

Figure 10B

Figure 10C

Figure 11A

Figure 11B

Figure 11C

Figure 11D

Figure 11E

Example

[0143] The following examples are intended to further illustrate examples of the present invention and are not intended to limit the present invention. The examples describe technical features, and the present invention also relates to combinations of the technical features presented in this section. The methods and materials used in all examples are described after the examples.

[0144] Example 1 : Directed differentiation of human pluripotent stem cells (hPSCs) into skeletal muscle cells and satellite cells in 2D cell culture. A method for the directed differentiation of induced pluripotent stem cells into skeletal muscle cells and satellite cells in 2D cell culture was developed. The method described herein is transgene-free and serum-free. Human skeletal myoblasts, skeletal muscle tube cells, and satellite cells can be generated in high purity by this method. In this method, a specific temporal sequence of agents (small molecules and inhibitors and stimulators) was used to induce the differentiation of human pluripotent stem cells. Different genes were expressed at different differentiation stages of pluripotent stem cells. Typical gene expression during differentiation is also called a gene expression pattern. These gene expression patterns are also experienced during embryonic skeletal muscle development in the human body. A schematic of the differentiation protocol is shown in FIG. 1, which shows a series of additions of different agents to the medium. Additionally, FIG. 1 shows the differentiation stages experienced during differentiation into skeletal myoblasts / muscle tube cells and satellite cells, namely, induction of mesoderm differentiation, induction of myogenic specification, (myogenic) expansion and maturation into skeletal myoblasts and satellite cells, and maturation into skeletal muscle tube cells and satellite cells.

[0145] To perform the method, human pluripotent stem cells were seeded at 1.7×10 4 cells / cm 2Plated the day before on plates coated with Matrigel at a density of [[ID=]], and cultured in the presence of 12 ml of StemMACS™ iPS-Brew XF medium containing 5 μM of a Rock inhibitor (Stemolecule Y27632) (the method of coating Matrigel on cell culture plates is at the end of Example 1), and the cell culture was made approximately 30% confluent on the next day (day 0). However, the optimal cell number for plating must be determined individually for each cell line.

[0146] Culture in N2-FCL medium induced mesodermal differentiation of pluripotent stem cells. On each of days 0, 1, 2, and 3, the medium was replaced with 15 ml of N2-FCL medium and changed daily. N2-FCL medium: DMEM containing 1 g / l glucose and L-alanyl-L-glutamine (GlutaMAX™) (Gibco) supplemented with pyruvate, 1% Pen / Strep (Invitrogen), 1% serum-free additive N-2 (100x) (Thermo Scientific), 1% non-essential amino acids (100x) (MEM-NEAA, Invitrogen), 10 ng / ml recombinant bFGF (Peprotech), 10 μM CHIR-99021 (Stemgent), 0.5 μM LDN193189 (Stemgent).

[0147] Myogenic specification was induced by culturing in N2-FD, N2-FHD, and N2-HKD media. On days 4 and 5, the medium was replaced with N2-FD medium and changed daily. N2-FD medium: DMEM containing 1 g / l glucose and L-alanyl-L-glutamine (GlutaMAX™) (Gibco) supplemented with pyruvate, 1% Pen / Strep (Invitrogen), 1% serum-free additive N-2 (100x) (Thermo Scientific), 1% non-essential amino acids (100x) (MEM-NEAA, Invitrogen), 20 ng / ml recombinant bFGF (Peprotech), 10 uM DAPT (TOCRIS).

[0148] On days 6 and 7, the medium was replaced with N2-FHD medium and exchanged daily. N2-FHD medium: 1 g / l glucose supplemented with pyruvate and L-alanyl-L-glutamine (GlutaMAX™) (Gibco), 1% Pen / Strep (Invitrogen), 1% serum-free supplement N-2 (100x) (Thermo Scientific), 1% non-essential amino acids (100x) (MEM-NEAA, Invitrogen), 20 ng / ml recombinant bFGF (Peprotech), 10 μM DAPT (TOCRIS), 10 ng / ml recombinant HGF (Peprotech) in DMEM.

[0149] On days 8, 9, 10 and 11, the medium was replaced with N2-HKD medium and exchanged daily. N2-HKD medium: 1 g / l glucose supplemented with pyruvate and L-alanyl-L-glutamine (GlutaMAX™) (Gibco), 1% Pen / Strep (Invitrogen), 1% serum-free supplement N-2 (100x) (Thermo Scientific), 1% non-essential amino acids (100x) (MEM-NEAA, Invitrogen), 0.1 mM 2-mercaptoethanol (Invitrogen), 10 μM DAPT (TOCRIS), 10 ng / ml recombinant HGF (Peprotech), 10% KnockOut Serum Replacement (Life Technologies) in DMEM.

[0150] By culturing in N2-HK medium, the cells were myogenically expanded and matured into skeletal myoblasts and satellite cells. On days 12 to 20, the medium was replaced with N2-HK medium (expansion medium) and changed every other day. N2-HK medium: DMEM supplemented with 1 g / l glucose and L-alanyl-L-glutamine (GlutaMAX™) (Gibco), 1% Pen / Strep (Invitrogen), 1% serum-free supplement N-2 (100x) (Thermo Scientific), 1% non-essential amino acids (100x) (MEM-NEAA, Invitrogen), 0.1 mM 2-mercaptoethanol (Invitrogen), 10 ng / ml recombinant HGF (Peprotech), 10% KnockOut Serum Replacement (Life Technologies).

[0151] From day 21, the cells were either further cultured on cell culture plates, frozen, or used in the method of Example 2. When the cells were further cultured, the medium was replaced with differentiation medium (maturation medium). Maturation medium: DMEM supplemented with 1 g / l glucose and L-alanyl-L-glutamine (GlutaMAX™) (Gibco), 1% Pen / Strep (Invitrogen), 1% serum-free supplement N-2 (Thermo Scientific), 1% B27 serum-free supplement (Invitrogen). Skeletal myoblasts, skeletal myotube cells, and satellite cells were generated by further culturing on cell culture plates.

[0152] To track the directed differentiation during the described culture process, the gene expression pattern of the cells was determined using RNA sequencing over a 60-day period. RNA sequencing was used to determine the increases and decreases in the expression of specific genes, i.e., to analyze the onset and end of specific differentiation or maturation stages.

[0153] In particular, the expression of genes specific for pluripotency, axial mesoderm, skeletal muscle-specific transcription factors, and sarcomeres was measured. Genes typical of pluripotency, such as NANOG, POU5F1, and ZFP42, showed high expression on days 0 and 1 (the day after seeding and the following day) (Figure 4a). NANOG and POU5F1 showed the highest expression on day 0, and ZFP42 showed the highest expression on day 1 (Figure 4a). Genes typical of axial mesoderm, such as MSGN1, TBX6, and MEOX1, showed high expression from days 1 to 8 (Figure 4b). MSGN1 showed the highest expression on day 1, TBX6 showed the highest expression on day 4, and MEOX showed the highest expression on day 8 (Figure 4b). Skeletal muscle-specific transcription factors, such as PAX3, PAX7, and MYOD1, showed the highest expression on days 8, 29, and 60, respectively (Figure 4c). Genes typical of sarcomeres, such as ACTN2, DMD, and MYH3, showed the highest expression on day 60 (Figure 4d). Furthermore, the gene expression patterns show sharp increases or decreases of different markers, especially during the first 21 days. For example, TBX6 and MEOX1 are strongly expressed only on days 4 and 8, respectively, while the expression is at least four-fold weaker on other days (Figure 4d). This time course indicates a homogeneous progression of the differentiation process.

[0154] To determine differentiation using a second independent method, the inventors analyzed the cells using fluorescence microscopy after a 21-day differentiation method. This involved staining the DNA of the cells with Hoechst, as well as immunostaining for actin and skeletal muscle-specific transcription factors (Pax7, MyoD, and myogenin). After 21 days, the fluorescence images showed a high percentage of cells expressing Pax7, MyoD, and myogenin (Figure 3). Thus, using another method, it was shown that a cell population of myogenic cells was generated by the differentiation protocol.

[0155] To determine differentiation by a third independent method, cells were analyzed by flow cytometry. The flow cytometry used here measures the expression of skeletal muscle-specific factors using immunostaining. In particular, the percentages of skeletal myoblasts and skeletal muscle tube cells (expression of markers actinin, myogenin, MyoD) and satellite cells (expression of marker PAX7) were determined in four independent pluripotent stem cell lines (iPSC(WT 1), iPSC(WT 2), DMD iPSC, corrected DMD iPSC) (Figure 5). The percentage of actinin-positive cells was 71 - 77.6% in the four cell lines, the percentage of myogenin-positive cells was 41.4% - 60.4% in the four cell lines, the percentage of MyoD-positive cells was 40% - 54.1% in the four cell lines, and the percentage of PAX7-positive cells was 33.4% - 43.8% in the four cell lines (Figure 5).

[0156] Flow cytometry also showed that the cells analyzed produced satellite cell-specific markers in addition to skeletal myoblast and skeletal muscle tube cell-specific markers at high purity (>70% actinin-positive and >30% PAX7-positive muscle cells).

[0157] Therefore, using three different methods, it was measured that pluripotent stem cells differentiated into a skeletal myoblast-containing cell pool and thus underwent mesoderm induction, myogenic specification, and myogenic maturation.

[0158] Materials and Methods The following pluripotent stem cell lines were used: TC1133 (iPSC WT1; Baghbaderani et al. Stem Cell Reports 2015), iPSC WT2, DMD iPSC (DMD Del; Long et al. Sci Adv 2018), corrected DMD iPSC (Long et al. Sci Adv 2018). In the DMD iPSC stem cell line, the X-linked dystrophin gene (DMD) is mutated, which is also mutated in Duchenne muscular dystrophy (DMD) disease and causes the disease.

[0159] To prepare Matrigel-coated cell culture plates, BD Matrigel (Basement Membrane Matrix Growth Factor Reduced) was diluted at a ratio of 1:30 in ice-cold PBS and immediately stored at 4°C. To prepare Matrigel-coated plates, a 1:120 Matrigel dilution was made using ice-cold PBS. 0.1 ml / cm 2 of the dilution was added to the cell culture flask. The flask was stored at 4°C for at least overnight and up to 2 weeks. Before use, the plates were placed in a 37°C incubator for at least 30 minutes.

[0160] For passage (e.g., to detach cells for cryopreservation), the cells were washed once with 3 ml of TrypLE (Invitrogen) and subsequently incubated in 5 ml of TrypLE at room temperature for approximately 7 minutes. The TrypLE was then washed away and the digestion was stopped with 10 ml of N2-HK medium containing 5 μM Rock inhibitor. To induce aggregation, the cell suspension was pipetted using a 10 ml pipette. The separation of the cells had to be gentle enough not to reduce cell viability. The cells were counted using a CASY counter (by adding 20 μl of the cell suspension to 10 ml of CASY buffer). The cells were pelleted at 100×g for 10 minutes at room temperature. The supernatant was removed and the pellet was gently resuspended in N2-HK medium containing 5 μM Rock inhibitor. The cells were plated onto Matrigel-coated plates at a density of 60 - 70000 cells / cm 2 in N2-HK medium containing 5 μM Rock inhibitor. Starting the next day, the N2-HK medium was replaced every other day for 9 days.

[0161] For cell freezing (e.g., on day 21, cryopreservation), the cells were washed once with 3 ml of TrypLE (Invitrogen) and then incubated in 5 ml of TrypLE at room temperature for approximately 7 minutes. Subsequently, the TrypLE was removed by washing and digestion was stopped with 10 ml of N2-HK medium containing 5 μM of a Rock inhibitor. To induce aggregation, the cell suspension was pipetted using a 10 ml pipette. The separation of the cells must be gentle enough not to reduce cell viability. The cells were counted using a CASY counter (by adding 20 μl of the cell suspension to 10 ml of CASY buffer). The cells were pelleted at 100×g for 10 minutes at room temperature. The supernatant was removed and the pellet was gently resuspended in N2-HK medium containing 5 μM of a Rock inhibitor and 10% DMSO (Sigma) at 4 °C. 10×10 6 cells per cryovial were frozen overnight at -80 °C at 2 ml per cryovial using Mr Frosty (Thermo). The cells were then transferred to -150 °C.

[0162] For RNA extraction, cell lysates embedded in Trizol reagent (Thermo Fisher) were homogenized by vortexing. For every 1 ml of Trizol reagent, 200 μl of chloroform (AppliChem) was added. The reagent tube was tightly closed, inverted 5 times, and then incubated at room temperature for 5 minutes. The sample was then centrifuged at 10,000 - 12,000 × g for 15 minutes. The aqueous phase containing RNA was transferred to a new reagent tube, and subsequently 500 μl of isopropanol (Roth) was added to precipitate the RNA. The reagent tube was vortexed and left standing at room temperature for 10 minutes, and then centrifuged at 12,000 × g for an additional 10 minutes. The supernatant was removed, and 1 ml of 70% EtOH / diethyl pyrocarbonate (DEPC) H2O was added to wash the pellet. After gently tapping the reagent tube to dissolve and wash the pellet, the sample was centrifuged one more time at 12,000 × g for 5 minutes, and the supernatant was removed. The pellet was left open for 5 - 10 minutes until the residual liquid evaporated, and the RNA was resuspended in DEPC H2O. The RNA concentration and quality were determined using a Nanodrop ND-1000. Before sequencing, the quality and RNA integrity were further analyzed using the Advanced Analytical Fragment Analyzer (Standard Sensitivity RNA Analysis Kit (DNF-471)). A modified strand-specific ultraparallel cDNA sequencing (RNA-Seq) protocol (Illumina: TruSeq Stranded Total RNA (Cat. No. RS-122-2301)) was used to generate RNA-Seq libraries. The protocol was optimized to keep the rRNA content in the dataset below 5% (RiboMinus™ technology). Residual total transcriptome RiboMinus™ RNA is suitable for direct sequencing. The ligation step was optimized to increase the ligation efficiency (>94%), and the PCR protocol was adjusted for an optimal final product of the library.For accurate quantification of the cDNA library, a fluorescence-based system, Promega's quantiFluor™ dsDNA system, was used. The size of the final cDNA library was determined using the dsDNA 905 Reagent Kit (Fragment Analyzer from Advanced Bioanalytical), and the average size was 300 bp.

[0163] The libraries were pooled and sequenced on an Illumina HiSeq 4000 (Illumina) to generate 50-bp single-end reads (30 - 40×10^6 reads / sample). The sequence images were converted to BCL files using the Illumina software BaseCaller and then demultiplexed to fastq files using bcl2fastq v2.17.1.14. Quality was assessed using FastQC version 0.11.5 (Andrews, 2014). The sequence reads were mapped to the human genome reference library (UCSC version hg19 with Bowtie 2.0 (Langmead and Salzberg, 2012)). Next, the number of reads mapped to each identified gene was counted, and differential gene expression was evaluated using the DESeq2 software (Anders and Huber, 2010). Reads per kilobase transcript per million (RPKM) was calculated based on the length of Ensembl transcripts extracted from biomaRt (v2.24).

[0164] For flow cytometry, single cell suspensions were prepared by digesting the cells with TrypLE Select (Thermo Fisher). The cells were resuspended in culture medium, centrifuged at 300 g for 5 minutes, and fixed in 4% formalin (Histofix, Roth). After fixation, the cells were centrifuged again and resuspended in blocking buffer (PBS containing 1 mg / ml BSA (Sigma-Aldrich), 5% FCS (Thermo Fisher), and 0.1% Triton 100X (Sigma)). After 10 minutes of blocking, the cells were pelleted by centrifugation and resuspended in blocking buffer with primary antibodies (sarcomeric α-actinin 1:4,000 (Sigma-Aldrich); Pax7 1:50 (DSHB); MyoD 1:100 (DAKO); myogenin 1:50 (DSHB)) or appropriate IgG1 isotype control for 45 minutes at 4 °C.

[0165] The cells were washed twice with PBS, followed by a washing step in blocking buffer, and then incubated with secondary antibodies (1:1000 anti-mouse 488 [A-11001] or 633 [A-21052], Thermo Fisher) and Hoechst (10 ng / ml; Thermo Fisher) for 30 minutes at 4 °C. The cells were washed with PBS and finally resuspended in PBS for analysis. 10,000 live cell events were analyzed per sample. Measurements were performed on an LSRII SORP cytometer and analyzed using DIVA software (BD Biosciences).

[0166] Example 2: Production of engineered skeletal muscle (ESM) tissue from skeletal myoblasts and satellite cells (cells from Example 1) derived from pluripotent stem cells For the construction of engineered skeletal muscle tissue, the cells obtained in Example 1 (cells from day 21) were used as starting material and mixed with the extracellular matrix. By mixing with the extracellular matrix, the cells were dispersed in the matrix to generate a three-dimensional skeletal muscle tissue. This method is also serum-free and transgene-free. Therefore, the reproducibility of manufacturing skeletal muscle tissue is increased because all the required substances and their concentrations are defined. By this method, a force-generating skeletal muscle tissue that contracts in a controlled manner in response to electrical stimulation can be generated. A specific temporal sequence of agents and physical stimuli is used, which is schematically shown in FIG. 6A and described in detail below.

[0167] To construct engineered skeletal muscle tissue, cells from Example 1 (cells from day 21) were mixed with the extracellular matrix and poured into a ring mold to support the self-assembly of the cells into contractile skeletal muscle. This means either (a) dissociating the cells from the differentiated cell culture according to Example 1 or (b) using the frozen cells from Example 1 (see below for a detailed description of the method of thawing the cells).

[0168] To mix the cells from Example 1 with the extracellular matrix, the master mix was mixed in a 50 ml reaction tube on ice. Collagen was added using a 2 ml pipette. The following exact pipetting order was followed: TIFF0007702739000001.tif60149

[0169] Alternatively, the master mix was pipetted in according to the following volumes: TIFF0007702739000002.tif60149

[0170] The master mix was poured into a ring mold, and the ring mold was carefully transferred to an incubator, and the mixture was left to stand at 37 °C for 1 hour. After the incubation period, 8 ml of expansion growth medium containing 5 μM of a Rock inhibitor was carefully added per mold (Figure 6A, left panel). Expansion growth medium (N2-HK medium): 1 g / l glucose and L-alanyl-L-glutamine (GlutaMAX™) (Gibco) supplemented with pyruvate, 1% Pen / Strep (Invitrogen), 1% serum-free additive N-2 (Thermo Scientific), 1% non-essential amino acids (MEM-NEAA, Invitrogen), 0.1 mM 2-mercaptoethanol (Invitrogen), 10 ng / ml recombinant HGF (Peprotech), 10% KnockOut Serum Replacement (Life Technologies) in DMEM.

[0171] The cells were thus cultured in the expansion growth medium for 7 days. On days 1, 3, and 5, the medium was replaced with fresh expansion growth medium (N2-HK medium; without Rock inhibitor). After pouring, the mixture was compressed in the ring mold, and as a result, the mixture was sufficiently compressed after 24 hours.

[0172] After 7 days, the mold ring was transferred to a 6-well plate of an expansion growth tray (Figure 6A, middle panel). There, the cells were further cultured under physical stimulation, i.e., mechanical stretching. Additionally, cell maturation was induced by the maturation medium by adding 5 ml of the maturation medium per well. Maturation medium: 1 g / l glucose and L-alanyl-L-glutamine (GlutaMAX™) (Gibco) supplemented with pyruvate, 1% Pen / Strep (Invitrogen), 1% N serum-free additive N-2 (Thermo Scientific), 2% B27 serum-free additive (Invitrogen) in DMEM.

[0173] To mature the cells into skeletal muscle tube cells and satellite cells, the maturation medium was changed every other day during the subsequent 6-week maturation.

[0174] To experimentally test the production of engineered skeletal muscle tissue, the generated skeletal muscle tissue was analyzed using fluorescence microscopy. The characteristic striated pattern demonstrated that multinucleated skeletal muscle fibers were formed and force-generating skeletal muscle was produced.

[0175] The inventors visualized the structural protein actin of the eukaryotic cytoskeleton using immunostaining and stained the DNA in the nucleus with the dye DAPI. The fluorescence images showed a characteristic striated pattern, demonstrating that multinucleated mature skeletal muscle fibers were formed by this method (Figure 8A). Immunostaining demonstrated that the engineered skeletal muscle tissue exhibited the structure of mature multinucleated muscle fibers.

[0176] Furthermore, to functionally test the artificially generated muscle tissue, the inventors performed contraction experiments (Figure 6A, right panel). These contraction experiments in an organ bath measure the contraction frequency and contraction force of the produced skeletal muscle tissue in response to electrical stimulation.

[0177] For this purpose, ring-shaped skeletal muscle tissue was isometrically transferred to an organ bath (Fohr Medical Instruments) containing Tyrode's solution (in mmol / L: 120 NaCl, 1 MgCl2, 1.8 CaCl2, 5.4 KCl, 22.6 NaHCO3, 4.2 NaH2PO4, 5.6 glucose, and 0.56 ascorbate) with continuous gas treatment at 37°C, 5% CO2, and 95% O2. The ESM was mechanically stretched at 125-μm intervals until the maximum force amplitude (contraction force = FOC) was observed. FOC measurements were performed at an electric field stimulation frequency (4-ms rectangular pulse; 200 mA) in the range of 1-100 Hz.

[0178] The results of the contraction experiment are shown in FIGS. 6B and 6C. FIG. 6B shows representative contraction force curves of the engineered skeletal muscle tissue at different stimulation frequencies. At a stimulation of 1 Hz (dashed line), eight single contractions with a single duration of about 0.5 seconds were recorded. At a stimulation of 10 Hz (solid line), the initial twitch was measured. At a stimulation of 100 Hz (dotted-dashed line), a fully developed twitch was detected. FIG. 6C shows the contraction force of the engineered skeletal muscle tissue as a function of the stimulation frequency. The contraction force ("FOC") was measured in millinewtons (mN) of the skeletal muscle tissue depending on the electrical stimulation frequency (n = 3). At a stimulation of 1 Hz, the average contraction force was 0.5 millinewtons; at a stimulation of 10 Hz, the average contraction force was 0.9 millinewtons; at a stimulation of 20 Hz, the average contraction force was 1.1 millinewtons; at a stimulation of 40 Hz, the average contraction force was 1.4 millinewtons; at a stimulation of 60 Hz, the average contraction force was 1.55 millinewtons; at a stimulation of 80 Hz, the average contraction force was 1.6 millinewtons; at a stimulation of 100 Hz, the average contraction force was 2.1 millinewtons.

[0179] The tested skeletal muscle tissue showed reproducible contraction frequency and contraction force in response to stimulation frequencies from 1 Hz to 100 Hz. At a single stimulation of 1 Hz, it took about 0.5 seconds for contraction and complete relaxation. Since the contraction and relaxation times were about 0.5 seconds, the onset or full twitch was recorded at higher stimulation frequencies. Since twitches are also formed in native skeletal muscle tissue at increased stimulation frequencies, the engineered skeletal muscle tissue behaves similarly to native skeletal muscle tissue in this regard. Furthermore, the inventors were able to show that the contraction force of the muscle tissue increases with an increase in the contraction frequency. These properties are consistent with native skeletal muscle tissue, which also exhibits a positive force-frequency relationship in response to electrical stimulation, in addition to single contractions and twitches. In contrast to the engineered skeletal muscle tissue, in native muscle tissue, electrical impulses are triggered by neurotransmitter stimulation (acetylcholine) from the motor endplate.

[0180] Thus, the described method produced engineered muscle tissue that exhibited characteristic formation of multinucleated muscle fibers (myotubes) and generated force in response to electrical stimulation.

[0181] Materials and Methods For dissociation of cells from cell cultures (at the volume described for T75 cell culture flasks), cells were washed once with 3 ml of TrypLE (Invitrogen) and then incubated in 5 ml of TrypLE for approximately 7 minutes at room temperature. The TrypLE was removed by washing and digestion was stopped with 10 ml of expansion growth medium containing 5 μM Rock inhibitor. The cell suspension was triturated using a 10 ml pipette to induce aggregation. The cell dissociation had to be gentle enough not to reduce cell viability. Cells were counted using a CASY counter (by adding 20 μl of cell suspension to 10 ml of CASY buffer). Cells were pelleted at 100×g for 10 minutes at room temperature. The supernatant was removed and the pellet was gently resuspended in an appropriate volume of expansion growth medium (see master mix) containing 5 μM Rock inhibitor, depending on the number of ESMs. The cell suspension was placed on ice.

[0182] To thaw the cells, the vial was removed from the -152 °C freezer. The cells were rapidly thawed in a 37 °C water bath for 2 minutes. The vial was sprayed with alcohol and transferred under a cell culture hood. The contents of the cryovial were transferred to a 15 ml reaction tube using a 2 ml serological pipette. The cryovial was washed with 1 ml of expansion growth medium at room temperature containing 5 μM Rock inhibitor, and the expansion growth medium was added dropwise to the cells to avoid osmotic shock. Another 8 ml of expansion growth medium containing 5 μM Rock inhibitor was added slowly. To avoid cell damage, the suspension was pipetted up and down no more than 2 times before cell counting. The cells were counted using a CASY counter (by adding 20 μl of cell suspension to 10 ml of CASY buffer). The cells were pelleted at 100 × g for 10 minutes at room temperature. The supernatant was removed, and the pellet was gently resuspended in an appropriate volume of expansion growth medium containing 5 μM Rock inhibitor, and a defined volume of cell suspension was prepared depending on the number of ESMs (see master mix). The cell suspension was placed on ice.

[0183] Example 3: Production of engineered skeletal muscle tissue (biologically engineered skeletal muscle, BSM) from pluripotent stem cells In this example, pluripotent stem cells and extracellular matrix were used to construct engineered skeletal muscle tissue (BSM). In contrast to Examples 1 and 2, the transfer from Matrigel-coated cell culture plates to extracellular matrix was not performed in the production of BSM. Instead, human induced pluripotent stem cells were directly dispersed / embedded in a defined extracellular matrix. The self-assembly of pluripotent stem cells into skeletal muscle tissue was supported in the extracellular matrix in the presence of chemical and physical stimuli. Since this method is also serum-free and transgene-free, all required substances and their concentrations are defined. Therefore, the differentiation and maturation of human pluripotent stem cells into skeletal muscle tube cells and satellite cells (skeletal muscle fibers) were controlled.

[0184] A schematic of the differentiation protocol is shown in Fig. 7A, which depicts a series of different agents added to the medium and physical stimuli with a stretching device. During the method depicted in Fig. 7A, mesoderm differentiation was induced (days 0 - 4), myogenic specification was induced (days 4 - 12), cells were matured into skeletal myoblasts and satellite cells (days 12 - 21), and finally into skeletal myotube cells and satellite cells (days 21 - 50).

[0185] To perform the method, induced pluripotent stem cells were dissociated from the cell culture the previous day, counted, and the pellet was gently resuspended in an appropriate volume of medium (iPS - Brew XF containing 10% KO serum replacement (Life Technologies), 5 μM Rock inhibitor, and 10 ng / ml bFGF (Peptrotech)). The stem cells were placed on ice as a cell suspension.

[0186] Human pluripotent stem cells were mixed with collagen / Matrigel, and the master mix was mixed in a 50 - ml reaction tube on ice for pouring into the ring mold. Collagen was added using a 2 - ml pipette, following the exact pipetting order below. TIFF0007702739000003.tif60128

[0187] The master mix was poured into the ring mold. The ring mold was carefully transferred to the incubator and the mixture was left to stand at 37 °C for 1 hour. After the incubation period, 8 ml of medium per mold (iPS - Brew XF containing 10% KO serum replacement (Life Technologies), 5 μM Rock inhibitor, and 10 ng / ml bFGF (Peptrotech)) was carefully added.

[0188] Culturing in N2 - FCL medium induced mesoderm differentiation of pluripotent stem cells. The medium was replaced with N2 - FCL medium 24 hours after pouring. On days 1, 2, and 3, the medium was replaced daily with fresh N2 - FCL medium (see Example 1 for composition).

[0189] Myogenic specialization was induced by culturing in N2-FD, N2-FHD, and N2-HKD media. On days 4 and 5, the medium was replaced with N2-FD medium and changed daily (see Example 1 for composition). On days 6 and 7, the medium was replaced with N2-FHD medium and changed daily (see Example 1 for composition). On days 8, 9, 10, and 11, the medium was replaced with N2-HKD medium and changed daily (see Example 1 for composition).

[0190] From days 12 to 20, the medium was replaced with N2-HK medium (expansion growth medium) and changed every other day (see Example 1 for composition). By culturing in the expansion growth medium, the cells were matured into skeletal myoblasts.

[0191] On day 21, the formed rings were transferred to a 6-well plate of a stretching device and further cultured under maturation conditions. Thus, the cells were further cultured under physical stimulation, i.e., mechanical stretching. Additionally, by adding 5 ml of maturation medium per well, cell maturation was induced by the maturation medium (see Example 2 for the composition of the maturation medium). The maturation medium was changed every other day during the subsequent 4-week maturation to mature the cells into skeletal myotube cells and satellite cells.

[0192] To experimentally test the production of engineered skeletal muscle tissue from induced pluripotent stem cells, the generated skeletal muscle tissue was analyzed using fluorescence microscopy as in Example 2. As in Example 2, immunostaining was used to visualize the structural protein actin of the eukaryotic cytoskeleton, and the DNA in the nucleus was stained with the dye DAPI. As in Example 2, the fluorescence images showed a characteristic striated pattern, demonstrating the formation of multinucleated mature skeletal muscle fibers (Figure 8b). Thus, BSM also exhibits multinucleated mature skeletal muscle fibers formed by this method.

[0193] Furthermore, to functionally test the artificially generated muscle tissue, as in Example 2, the inventors conducted a contraction experiment. These contraction experiments in an organ bath measure the contraction frequency and contraction force of the fabricated skeletal muscle tissue in response to electrical stimulation.

[0194] The results of the contraction experiment are shown in FIGS. 7B and 7C.

[0195] FIG. 7B shows representative contraction force curves of the engineered skeletal muscle tissue at different stimulation frequencies. At a stimulation of 1 Hz (dashed line), eight single contractions with a single duration of about 0.5 seconds were recorded, and at a stimulation of 100 Hz (solid line), a well-developed tetanus was detected. FIG. 7C shows the contraction force of the engineered skeletal muscle tissue depending on the stimulation frequency. The contraction force (“FOC”) was measured in millinewtons (mN) of the skeletal muscle tissue (n = 3). At a stimulation of 1 Hz, the average contraction force was 0.3 millinewtons; at a stimulation of 10 Hz, the average contraction force was 0.5 millinewtons; at a stimulation of 20 Hz, the average contraction force was 0.55 millinewtons; at a stimulation of 40 Hz, the average contraction force was 0.6 millinewtons; at a stimulation of 60 Hz, the average contraction force was 0.65 millinewtons; at a stimulation of 80 Hz, the average contraction force was 0.72 millinewtons; at a stimulation of 100 Hz, the average contraction force was 0.9 millinewtons.

[0196] These contraction experiments also demonstrate that BSM can also generate force in response to electrical stimulation. The tested skeletal muscle tissue showed reproducible contraction frequencies and contraction forces in response to stimulation frequencies from 1 Hz to 100 Hz, and the contraction and relaxation times after a single stimulation were about 0.6 seconds. Additionally, ESM and BSM show the same characteristics in terms of tetanus formation and increase in contraction force. Similar to the ESM described in Example 2, BSM generates a tetanus at increased stimulation frequencies, such as 100 Hz. Also similar to Example 2, the contraction force of BSM increases with an increase in the stimulation frequency.

[0197] Both of these characteristics are similar to the contraction behavior in natural muscle tissue because, in natural skeletal muscle, strong contractions are also formed and the contractile force increases with the increase in the frequency of stimulation. Similar to natural skeletal muscle, the engineered skeletal muscle tissue showed a positive force-frequency relationship in response to electrical stimulation, in addition to single contractions and tetanic contractions.

[0198] Therefore, the engineered skeletal muscle tissues (ESM and BSM) of Examples 2 and 3 behave similarly to natural skeletal muscle tissue in response to electrical stimulation.

[0199] Example 4 - Increased Function of Engineered Skeletal Muscle Tissue To further increase the function of the engineered skeletal muscle, for example, the contractile force can be increased by adding specific molecules. In this example, we specifically tested the enhancement of contraction and relaxation times in addition to the contractile force in response to the addition of creatine and the increase in the concentration of thyroid hormone T3 (triiodo-L-thyronine (T3); from 3 nmol / L to 100 nmol / L in the maturation medium of step iv). Here, the procedures according to Examples 1 and 2 were first carried out. In contrast to Example 2, the maturation medium was supplemented with creatine or increased concentrations of T3 on either days 28 - 56 or days 56 - 84 of the method.

[0200] Creatine supplementation: When the maturation medium was supplemented with 1 mM creatine from day 28 to day 56 of the procedure, the contractile force (FOC) increased from 1.8 mN to 2.5 mN during tetanic contraction at 100 Hz stimulation (Figure 9B, top). Thus, this medium addition increased the contractile force by 39%. Furthermore, the possible increase in contractile force in the long-term of this method was tested. For this purpose, the method was extended for an additional 4 weeks as described in Example 2, and during this time, the medium was supplemented with 1 mM creatine. By supplementing the maturation medium with 1 mM creatine from day 56 to day 84 of the procedure, the contractile force (single twitch tension) increased from 4.0 mN to 5.2 mN during tetanic contraction at 100 Hz stimulation (Figure 9B, bottom). This medium addition thus increased the contractile force by 30%.

[0201] From this, the addition of creatine to the maturation medium significantly increases the contractile force in both experiments.

[0202] Supplementation with T3: By supplementing the maturation medium with 0.1 μM of T3 from day 28 to day 56 of the method, the contraction and relaxation rates were significantly decreased as determined by the Student's t-test (Figure 10B). Furthermore, when the method was extended as described in Example 2 and the medium was supplemented with 0.1 μM of T3 from day 56 to day 84, the contraction and relaxation rates decreased (Figure 10B). Thus, the engineered skeletal muscle responds more rapidly to strong contraction stimuli and relaxes more rapidly after the end of the stimulus.

[0203] Generally, a maturation medium with an increased T3 concentration can be expected to lead to an improvement in skeletal muscle contractility in terms of accelerating the contraction and relaxation times.

[0204] To investigate the molecular cause of this improved muscle function, the expression of different proteins was analyzed by Western blot. MYH2 is the heavy chain of fast myosin (MYH2; fast myosin heavy chain); MYH7 is the heavy chain of slow myosin (MYH7; slow myosin heavy chain); MYH3 is the heavy chain of embryonic myosin (MYH3; embryonic myosin heavy chain). Protein expression was analyzed on day 84. As shown in Figure 10C, the protein expression of MYH2 is significantly increased by the addition of 0.1 μM of T3 for 4 weeks. Based on three independent experiments, the expression is increased by at least 5-fold. The expression of MYH7 remained unchanged by the addition of 0.1 μM of T3. The expression of MYH3 was reduced by approximately half on average. These protein expression data support the functional data from Figure 10B, and the reduction in the response time of the engineered skeletal muscle can be explained by an increase in the expression of the fast myosin (MYH2) isoform by T3.

[0205] In conclusion, the addition of creatine and / or T3 during maturation was shown to increase the function of engineered skeletal muscle. In particular, the addition of creatine was shown to greatly increase contractile force. Additionally, the addition of T3 was shown to increase the reaction rate of engineered skeletal muscle. This increase in function is supported by an increase in the expression of MYH2.

[0206] It can also be envisioned that the increase in the function of engineered skeletal muscle occurs in the same manner when engineered skeletal muscle tissue is prepared according to Example 3 (BSM) and then creatine and / or T3 are added to the maturation medium.

[0207] Example 5 - Regenerative Capacity of Engineered Skeletal Muscle Tissue Engineered skeletal muscle tissue ideally has regenerative properties so that it can be used, for example, as an implant or as a model for testing regenerative or muscle growth-inducing drugs. This regenerative property is characterized by the fact that damage to the engineered skeletal muscle tissue can be repaired. For this repair process, engineered skeletal muscle tissue requires cells with regenerative properties, such as satellite cells (skeletal muscle progenitor cells). In Figure 11A, the protein expression of markers expressed in skeletal muscle cell progenitors (PAX7, PAX3, MYF5, and BARX2) was analyzed. In contrast to 2D cultures, all four markers were clearly expressed in the ESM on day 60 of the method culture. Furthermore, PAX3, MYF5, and BARX2 were expressed higher in engineered skeletal muscle than in skeletal muscle cells cultured in 2D plates. This indicates that in engineered skeletal muscle tissue, skeletal muscle cell progenitors are maintained and additionally proliferate, in contrast to parallel 2D cultures. Figure 11B also shows a well-differentiated satellite cell niche in the ESM, and a scattered and less differentiated satellite cell niche was also seen in 2D cultures similar to the method described herein.

[0208] To test the regenerative properties, the engineered skeletal muscle tissue (60 days old) was incubated with myotoxin cardiotoxin (25 μg / ml) for 24 hours. Contractile force was measured 2 and 21 days after incubation (Figure 11C). As shown in Figure 11D, the engineered skeletal muscle tissue did not show contraction 2 days after incubation with CTX, and 21 days after incubation, the engineered skeletal muscle contracted again with a contractile force of 1 mM. Thus, the engineered skeletal muscle is capable of regeneration. As a comparison, skeletal muscle additionally treated with gamma radiation (30 Gy) did not recover from incubation with CTX. This indicates that the regeneration of the engineered skeletal muscle is dependent on the activation of the skeletal muscle cell progenitor cells contained therein. Irradiation inhibits these and all other cells with cell division activity. This experiment further demonstrates that the molecularly and microscopically detectable skeletal muscle cell progenitor cells (Figures 11A - B) are functional. As a comparison, Figure 11E also shows muscle remodeling in non - irradiated ESM compared to irradiated ESM by fluorescence microscopy. Detection of cells with sarcomeric actinin 21 days after CTX - induced muscle cell destruction demonstrates muscle remodeling in ESM via activation involving cell division and differentiation of skeletal muscle cell progenitor cells. No regenerative activity was detectable in irradiated ESM. These morphological observations are consistent with the functional observations in Figure 11D. This indicates that the engineered skeletal muscle contracts again at approximately 1 mN 21 days after CTX - mediated muscle cell destruction.

[0209] Methods of Examples 4 and 5 Maturation Conditions The maturation medium was changed every other day and cultured under mechanical stretching for up to 9 weeks. The maturation medium consisted of DMEM containing low glucose, GlutaMAX™ Supplement, pyruvate (Thermo Fisher Scientific), 1% N-2 Supplement (Thermo Fisher Scientific), 2% B-27 Supplement (Thermo Fisher Scientific), and optional antibiotics (e.g., 1% Pen / Strep - Thermo Fisher Scientific). At the indicated time points (e.g., days 28 - 56 or 56 - 84), 0.1 μM T3 (Sigma - Aldrich) or 1 mM creatine monohydrate (Sigma - Aldrich) was added to the maturation medium for a 4 - week period.

[0210] Isometric Force Measurement The contractile function of the engineered skeletal muscle tissue was measured under isometric conditions at 37 °C in an organ bath filled with Tyrode's solution (containing, in mmol / L, 120 NaCl, 1 MgCl2, 0.2 CaCl2, 5.4 KCl, 22.6 NaHCO3, 4.2 NaH2PO4, 5.6 glucose, and 0.56 ascorbate) and gassed with 5% CO2 / 95% O2. To verify the force - length relationship, the muscle length was increased by mechanical stretching at 125 - μm intervals until maximum contractile force was observed while electrically stimulating the ESM at 1 Hz with 200 - mA 5 - ms rectangular pulses. At the length of maximum force generation, tetanic force was evaluated under defined stimulation frequencies (4 - s stimulation at 10, 20, 40, 60, 80, and 100 Hz).

[0211] Cardiotoxic Injury Model The control, manipulated skeletal muscle was subjected to cardiotoxic injury (CTX) in parallel with irradiated ESM. To induce injury, the tissue was maintained in maturation medium (Latoxan) containing 25 μg / ml CTX for 24 h (Tiburcy et al., 2019). The injured tissue was then rinsed and placed in expansion growth medium consisting of DMEM, low glucose, GlutaMAX™ Supplement, pyruvate (Thermo Fisher Scientific), 1% N-2 Supplement (Thermo Fisher Scientific), 1% MEM non-essential amino acid solution (Thermo Fisher Scientific), 10 ng / ml HGF (Peprotech), and 10% KnockOut Serum Replacement (Thermo Fisher Scientific) for 1 week, and then cultured for a further 2-week regeneration period in maturation medium consisting of DMEM, low glucose, GlutaMAX™ Supplement, pyruvate (Thermo Fisher Scientific), 1% N-2 Supplement (Thermo Fisher Scientific), 2% B-27 Supplement (Thermo Fisher Scientific), and 1 mM creatine monohydrate (Sigma-Aldrich). The medium was changed every other day. Optionally, antibiotics (e.g., 1% Pen / Strep - Thermo Fisher Scientific) can be added.

[0212] Irradiation of ESM Twenty-four hours prior to CTX treatment, ESM was placed in culture dishes in the STS Biobeam 8000 gamma irradiator and exposed to a single dose of 30 Gy irradiation for 10 min (Tiburcy et al., 2019).

[0213] Immunostaining and Confocal Imaging The 2D cell cultures were fixed in 4% formaldehyde (Carl Roth) in phosphate-buffered saline (PBS) for 15 min at room temperature. The manipulated skeletal muscle was fixed overnight at 4 °C in 4% paraformaldehyde in PBS. After fixation, the manipulated skeletal muscle was immersed in 70% ethanol (Carl Roth) for 1 min and then embedded in 2% agarose (peqGOLD) in 1X Tris-acetate-EDTA (TAE) buffer. Sections were cut to 400 μm using a Leica Vibrotome (LEICA VT1000S) and stored in cold 1X PBS. Before staining, both the 2D cell cultures and ESM sections were washed with 1X PBS. To induce blocking and permeabilization, samples were incubated in blocking buffer (1X PBS containing 5% fetal bovine serum, 1% bovine serum albumin (BSA), and 0.5% Triton-X). All primary and secondary antibody stainings were performed in the same blocking solution. The following antibodies were used for primary staining at RT for 4 h or at 4 °C for 24 - 72 h: Pax3 (1:100, DSHB), Pax7 (1:100, DSHB), MyoD (1:100, Dako), and myogenin (1:10, DSHB). Sarcomeric α-actinin (1:500, Sigma-Aldrich), laminin (1:50, Sigma-Aldrich). After washing 3 times with PBS, appropriate Alexa fluorochrome-conjugated secondary antibodies (1:1000, Thermo Fisher Scientific) were applied for 2 h at room temperature. In parallel with the secondary antibodies, Alexa 633-conjugated phalloidin (1:100, Thermo Fisher Scientific) and Hoechst 33342 (1:1000, Molecular Probes) were used for f-actin and nuclear staining, respectively. After washing 3 times with PBS, samples were stained with Fluoromount-G (Southern Biotech). All images were acquired using a Zeiss LSM 710 / NLO confocal microscope. To quantify the labeled cells, three random focal planes per sample from three different experiments were selected for analysis using the ImageJ Cell Counter Tool.

[0214] Western Blot Analysis For protein isolation, the manipulated skeletal muscle was placed into an Eppendorf tube and snap-frozen in liquid nitrogen. To the manipulated skeletal muscle, 150 μl of ice-cold protein lysis buffer (2.38 g of HEPES, 10.20 g of NaCl, 100 ml of glycerol, 102 mg of MgCl2, 93 mg of EDTA, 19 mg of EGTA, 5 ml of NP-40 in a total volume of 500 ml of ddH2O) containing 1 / 10 phosphatase inhibitor (Roche) and 1 / 7 protease inhibitor (Roche) was added. A 7 mm stainless steel ball (Qiagen) was added to the Eppendorf tube, and the sample was homogenized using a TissueLyser II (Qiagen) at 30 Hz and 4 °C for 30 seconds, followed by incubation on ice for 2 hours and then centrifugation at 12,000 rpm and 4 °C for 30 minutes. The supernatant was collected as the protein sample, and the protein concentration was measured by Bradford protein assay. 30 μg of the protein sample was loaded onto a 4–15% sodium dodecyl sulfate (SDS)-polyacrylamide gel (Bio-Rad) and electrophoretically separated at 100 V for approximately 2.5 hours, and then transferred onto a polyvinylidene fluoride (PVDF) membrane at 30 V into a box filled with ice from a cold storage overnight. To visualize total proteins, the PVDF membrane was stained with Ponceau Red. Staining with the primary antibody (4 hours at room temperature) and secondary antibody (1 hour at room temperature) was performed in a blocking solution containing 5% milk and 0.1% Tween 20 in 1x Tris-buffered saline (TBS). Protein expression in ESM was analyzed by Western blot using the following primary antibodies: monoclonal embryonic myosin heavy chain 3 (1:500, F1.652, DSHB), slow myosin heavy chain type 7 (1:500, A4.951, DSHB), and fast myosin heavy chain type 2 (1:100, A4.74, DSHB). Protein loading was controlled by vinculin (VCL) antibody (1:5000, V3131, Sigma-Aldrich). The membrane was washed with 1x Tris-buffered saline (TBS) and 0.1% Tween 20 for 5 minutes. A horseradish peroxidase-conjugated goat anti-mouse IgG antibody (1:10,000, P0260, Dako) was used for secondary staining.The membrane was washed with 1x Tris Buffered Saline (TBS) and 0.1% Tween 20 for 5 minutes, after which the blot was covered with Femto LUCENT™ Luminol reagent (Gbiosciences) and protein bands were imaged using a BIO-RAD ChemiDoc™ MP system. Protein quantification from the Western blot was performed using ImageJ.

[0215] Quantitative Real-Time PCR Total RNA was isolated from 2D cell cultures and engineered skeletal muscle using Trizol reagent (Thermo Fisher Scientific). Trizol was added to the 2D cells in the culture plate, the cells were scraped off, and the cell lysate was homogenized by vortexing. Engineered skeletal muscle was placed into polypropylene tubes (Eppendorf) and snap frozen in liquid nitrogen. 1 ml of Trizol was added to the engineered skeletal muscle in the presence of a 7 mm stainless steel ball (Qiagen), and the samples were lysed at 30 Hz and 4 °C for 2 minutes using a TissueLyser II (Qiagen). RNA isolation was performed according to the manufacturer's protocol. RNA concentration was quantified using a Nanodrop spectrophotometer (Thermo Fisher Scientific). 1 μg of RNA sample was treated with DNase I (Roche) and then reverse transcribed into complementary DNA (cDNA) using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's instructions. Quantitative PCR was performed using Fast SYBR Green Master Mix (Thermo Fisher Scientific) and an AB7900 HT Fast Real-Time PCR System (Applied Biosystems). Alternatively, transcriptome analysis was performed by RNA sequencing using an Illumina platform.

[0216] Materials Used in All Examples The materials used in this specification are commercially available unless otherwise stated. For example, penicillin / streptomycin, B27 serum-free additive, non-essential amino acids (MEM-NEAA), and 2-mercaptoethanol are available from Invitrogen. The company name is indicated with each of the materials used.

[0217] Stock solutions of N2 and B27 serum-free additive solutions were stored at -20 °C. Once thawed, they were added to the medium and stored at 4 °C for a maximum of 1 week. KnockOut Serum Replacement stock solutions were also stored at -20 °C. Once thawed, they were stored at 4 °C for a maximum of 2 weeks. The LDN193189 stock solution had a concentration of 10 mM in DMSO and was stored at -20 °C. The DAPT stock solution had a concentration of 20 mM in DMSO and was stored at -20 °C. The bFGF stock solution had a concentration of 10 μg / ml in PBS containing 0.1% human recombinant albumin and was stored at -20 °C. The HGF stock solution had a concentration of 10 μg / ml in PBS containing 0.1% human recombinant albumin and was stored at -20 °C. The Rock inhibitor had a concentration of 10 mM in DMSO and was stored at -20 °C.

[0218] Once the stock solutions of growth factors and small molecules were thawed, they were stored at 4 °C for a maximum of 1 week.

[0219] (Table 1) Composition of serum-free additive N-2 at 100x effective concentration (liquid form), i.e., 1% (v / v) corresponds to 1 (1x) effective concentration TIFF0007702739000004.tif35161

[0220] (Table 2) Composition of non-essential amino acids at 100x effective concentration (100x) TIFF0007702739000005.tif41169

[0221] (Table 3) DMEM, low glucose, 1 g / l, supplemented with GlutaMAX™ (Gibco, catalog number: 10567014) TIFF0007702739000006.tif211167

[0222] (Table 4) Composition of additional serum-free B27 supplement at 50X concentration (liquid form) 10 ml of 50X B27 supplement per 500 ml of medium corresponds to 2% (v / v) TIFF0007702739000007.tif121128

[0223] (Table 5) Composition of Knockout Serum Replacement (KSR) TIFF0007702739000008.tif210166

[0224] References TIFF0007702739000009.tif232161TIFF0007702739000010.tif144161

Claims

**Claim 1** A serum-free method for producing engineered skeletal muscle tissue from pluripotent stem cells, comprising: (i) inducing mesodermal differentiation of pluripotent stem cells by culturing the pluripotent stem cells in a basal medium containing a serum-free additive comprising an effective amount of (a) FGF2, (b) a GSK3 inhibitor, (c) a SMAD inhibitor, and (d) transferrin, insulin, progesterone, putrescine, and selenium or a bioavailable salt thereof; (ii) culturing the cells obtained in step (i) in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive similar to that in (i), followed by adding an effective amount of (d) HGF and continuing culturing in the medium, followed by culturing the cells in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) HGF, (c) a serum-free additive similar to that in (i), and (d) KnockOut Serum Replacement (KSR) to induce myogenic specification; (iii) expanding, proliferating, and maturing the cells into skeletal myoblasts and satellite cells by culturing the cells obtained in step (ii) in a basal medium containing an effective amount of (a) HGF, (b) a serum-free additive similar to that in (i), and (c) KnockOut Serum Replacement (KSR); (iv) maturing the cells into skeletal myotube cells and satellite cells by culturing the cells obtained in step (iii), which are dispersed in the extracellular matrix, under mechanical stimulation in a basal medium containing an effective amount of (a) a serum-free additive similar to that in step (i) and (b) an additional serum-free additive containing albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3). The method produces engineered skeletal muscle tissue. **Claim 2** The method of claim 1, wherein the skeletal muscle tissue generates a contractile force of at least 0.6 millinewtons (mN) at a stimulation of 100 Hz. **Claim 3** The method according to claim 1 or 2, wherein in step (iv), the mechanical stimulation is static tension, dynamic stimulation, or overload stimulation. **Claim 4** [[ ​ The method according to any one of claims 1 to 3, comprising a seeding step before step (i), in which the pluripotent stem cells are seeded in a stem cell medium in the presence of a ROCK inhibitor.

5. After step (iii), skeletal myoblasts and satellite cells are seeded in an engineered form in the presence of one or more components of the extracellular matrix in the master mix in an additional step before step (iv), the method according to any one of claims 1 to 4.

6. The method according to claim 4 or 5, wherein the engineered form has the form of a ring, ribbon, strand, patch, pouch, or cylinder.

7. Not containing a differentiation-related transgene nor a maturation-related transgene; and / or Not including a skeletal myoblast enrichment step, The method according to any one of claims 1 to 6.

8. The method according to any one of claims 1 to 7, wherein the basal medium in step (iv) contains an effective amount of creatine and / or triiodo-L-thyronine (T3).

9. The skeletal muscle tissue has a contraction rate of at least 3 mN / sec with a 100 Hz stimulus; and / or The skeletal muscle tissue has a relaxation rate of at least 0.5 mN / sec at the end of a 100 Hz stimulus, The method according to any one of claims 1 to 8.

Images