Production method of skeletal muscle cells and skeletal muscle tissue from pluripotent stem cells

Abstract

To provide methods for producing an artificial skeletal muscle tissue from pluripotent stem cells.SOLUTION: The present invention describes a method for preparing an engineered skeletal muscle tissue, as well as skeletal myoblasts, skeletal myotubes, and satellite cells, a medium used being serum-free and different chemicals and their concentrations, as well as the physical stimuli, being defined. Additionally, the methods described herein are performed without transfection of human cells with transgenes. The engineered skeletal muscle cells exhibit myoblast-, myotube-, or satellite cell-specific genetic markers, demonstrating efficient differentiation of these cell types. Despite its engineered production, the skeletal muscle tissue possesses excellent stimulus-dependent contractility and exhibits contractions in response to different stimulation frequencies.SELECTED DRAWING: None

Description

[Background technology]

[0001] Background of the Invention The human body is composed of 35-40% skeletal muscle, which enables breathing, posture, and movement. Healthy skeletal muscle can fully regenerate from minor injuries, such as tears or cuts, because muscle stem cells, also known as satellite cells (SCs), can completely regenerate injured tissue. However, major injuries do not heal and leave permanent damage.

[0002] The current theory of "tissue engineering" consists of generating desired cell types and differentiating them in an engineered environment to produce in vivo-like tissues. It is important to note that during tissue engineering, the extracellular environment is lost during dissociation of differentiated cells, so developmentally relevant information may be lost. For example, dissociation disrupts cell-to-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 is not composed solely of skeletal muscle fibers but also of interstitial / connective tissue cells, particularly satellite cells, which form in response to environmental and chemical stimuli.

[0003] Different tissue engineering methods for skeletal muscle cells using 2D cell cultures, small animal models, or extracted muscle tissue 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 manner.

[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 a fundamental question of whether the results can be transferred to humans, especially regarding disease / healing 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 likely to be extremely beneficial.

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

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

[0008] The development of robust differentiation and maturation protocols is a critical 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 have disclosed a serum-free differentiation protocol for human pluripotent stem cells into skeletal myoblasts in a 2D manner. However, this procedure requires a flow cytometry-mediated enrichment step for skeletal myoblasts to remove non-differentiated cell types from the cell pool. Purification via flow cytometry is not scalable and is associated with infection risk and high cell loss, thus posing a significant barrier to commercial application of cell products.

[0010] A transgenic-free and serum-free method for efficient differentiation of pluripotent stem cells that does not require additional enrichment steps for specific cell types has not yet been successfully described. In particular, a method for efficient differentiation of pluripotent stem cells into skeletal muscle tissue that avoids transgenes and serum while using chemical and physical stimuli has not yet been successfully described.

[0011] Shahriyari et al. (2018) only reports preliminary production of engineered skeletal muscle tissue. However, Shahriyari et al. lacks information regarding the essential characteristics required for the production of engineered skeletal muscle tissue of the present invention. Kramer et al. (2014) describes 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 myotubes, and satellite cells, in which 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 human cells with transgenes. The engineered skeletal muscle cells exhibit myoblast-, myotube-, or satellite cell-specific genetic markers, demonstrating efficient differentiation of these cell types. Despite its engineered production, the skeletal muscle tissue possesses excellent stimulus-dependent contractility and exhibits contractions in response to different stimulation frequencies.

[0013] The present invention includes methods in which pluripotent stem cells are differentiated and matured into skeletal myoblasts, skeletal myotubes, and satellite cells or skeletal muscle tissue, which is dispersed / embedded in an extracellular matrix.

[0014] The present invention provides 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 effective amounts 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) the same serum-free additives as in (i); and adding an effective amount of (d) HGF and continuing the culture in the medium; Culturing the cells in a basal medium containing an effective amount of (a) a gamma secretase / NOTCH inhibitor, (b) HGF, (c) the same serum-free additives as in (i), and (d) KnockOut Serum Replacement (KSR). inducing myogenic specification by (iii) expanding and maturing the cells obtained in step (ii) into skeletal myoblasts and satellite cells by culturing the cells in a basal medium containing effective amounts of (a) HGF, (b) the same serum-free additive as in (i), and (c) KnockOut Serum Replacement (KSR); (iv) maturing 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) additional serum-free additives including albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3), thereby maturing the cells into skeletal myotubes and satellite cells. Including, This relates to a method for producing engineered skeletal muscle tissue.

[0015] Additionally, the present invention provides a method for producing skeletal myoblasts, skeletal myotubes, 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 effective amounts 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) the same serum-free additives as in (i); and adding an effective amount of (d) HGF and continuing the culture in the medium; Culturing the cells in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) HGF, (c) the same serum-free additives as in (i), and (d) KnockOut Serum Replacement (KSR). inducing myogenic specification by (iii) culturing the cells obtained in step (ii) in a basal medium containing an effective amount of (a) HGF, (b) the same serum-free additive as in (i), and (c) KnockOut Serum Replacement (KSR), thereby maturing the cells into skeletal myoblasts and satellite cells; (iv) maturing the cells obtained in step (iii) into skeletal myotubes and satellite cells by culturing the cells in a basal medium containing effective amounts of (a) the same serum-free additives as in step (i) and (b) additional serum-free additives including albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3); Including, This relates to a method for producing skeletal myoblasts, skeletal myotubes, and satellite cells.

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

[0017] Additionally, the present invention is directed to mesodermally differentiated skeletal myoblast precursor cells, which are prepared and obtained according to step (i) and characterized by the expression of genes MSGN1 and / or TBX6, where 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 myogenically committed skeletal myoblast progenitor cells, which are produced and obtained according to steps (i) to (ii) and are characterized by the expression of the gene PAX3, which can be determined by flow cytometry and / or immunostaining. These cells express mRNA SIM1, which can be determined by RNA sequencing.

[0019] Furthermore, the present invention relates to skeletal myoblasts, which 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 prepared and obtained according to steps (i) to (iii) and characterized by the expression of the gene Pax7, which can be determined by flow cytometry and / or immunostaining, more preferably the satellite cells further express Ki67. Furthermore, the present invention relates to a mixture of skeletal myoblasts and satellite cells, in which the proportion of satellite cells in the amount of all available cells, as 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 available cells, as determined by the expression of actinin by flow cytometry, is at least 40%, preferably at least 50%, more preferably at least 60%, most preferably at least 70%.

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

[0022] Further disclosed is the use of skeletal muscle tissue according to the invention, and / or cells according to the invention, and / or skeletal myotube cells according to the invention in in vitro drug assays, which may be toxicity assays or assays 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 and / or skeletal myotube cells according to the present invention for use in medicine.

[0024] More particularly, the present invention relates to satellite cells according to the invention for use in the therapy of damaged skeletal muscle and / or in the treatment of skeletal muscle diseases, preferably genetic skeletal muscle defects, in particular Duchenne muscular dystrophy and / or Becker-Keener 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: 1. An in vitro method for testing the effectiveness of a drug candidate on skeletal muscle tissue, comprising: (a) providing skeletal muscle tissue according to the present invention; (b) optionally damaging skeletal muscle tissue; and (c) contacting the skeletal muscle tissue of step (a) or (b) with a drug candidate. Includes; Preferably, the method further comprises a step of determining the contractile force and / or structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters of skeletal muscle tissue before and / or after step (c), method.

[0026] 1. An in vitro method for testing the toxicity of a substance to skeletal muscle tissue, comprising: (a) providing skeletal muscle tissue according to the present invention; (b) contacting the skeletal muscle tissue from step (a) with the substance to be tested. Including, Preferably, the method further comprises a step of determining the contractile force and / or structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters of skeletal muscle tissue before and / or after step (b), method.

[0027] 1. An in vitro method for testing the effects of nutrients and dietary supplements on skeletal muscle tissue performance, comprising: (a) providing skeletal muscle tissue according to the present invention; (b) contacting the skeletal muscle tissue from step (a) with the nutrient or dietary supplement to be tested. Including, Preferably, the method further comprises a step of determining the contractile force and / or structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters of skeletal muscle tissue before and / or after step (b), method.

[0028] 1. An in vitro method for testing the effectiveness of a drug candidate on mesodermally differentiated skeletal myoblast precursor cells, myogenically committed skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, or a mixture of skeletal myoblasts and satellite cells, comprising: (a) providing mesodermally differentiated skeletal myoblast precursor cells, myogenically committed skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, or a mixture of skeletal myoblasts and satellite cells according to the present invention; (b) optionally damaging the cells from step (a); and (c) contacting the cells of step (a) or (b) with a drug candidate. Includes; Preferably, the method further comprises determining the expression of actinin and / or Pax7 before and / or after step (c), wherein the expression may be determined by flow cytometry and / or immunostaining. method.

[0029] 1. An in vitro method for testing the toxicity of a substance to mesodermally differentiated skeletal myoblast precursor cells, myogenically committed skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, or a mixture of skeletal myoblasts and satellite cells, comprising: (a) providing mesodermally differentiated skeletal myoblast precursor cells, myogenically committed skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, 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. Including, Preferably, the method further comprises determining the expression of actinin and / or Pax7 before and / or after step (b), wherein the expression may be determined by flow cytometry and / or immunostaining. method.

[0030] 1. An in vitro method for testing the effects of nutrients and dietary supplements on mesodermally differentiated skeletal myoblast precursor cells, myogenically committed skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, or a mixture of skeletal myoblasts and satellite cells, comprising: (a) providing mesodermally differentiated skeletal myoblast precursor cells, myogenically committed skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, 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. Including, Preferably, the method further comprises determining the expression of actinin and / or Pax7 before and / or after step (b), wherein the expression may be determined by flow cytometry and / or immunostaining. method. DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed Description of the Invention The present disclosure provides 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 effective amounts 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) the same serum-free additives as in (i); and adding an effective amount of (d) HGF and continuing the culture in the medium; Culturing the cells in a basal medium containing an effective amount of (a) a gamma secretase / NOTCH inhibitor, (b) HGF, (c) the same serum-free additives as in (i), and (d) KnockOut Serum Replacement (KSR). inducing myogenic specification by (iii) expanding and maturing the cells obtained in step (ii) into skeletal myoblasts and satellite cells by culturing the cells in a basal medium containing effective amounts of (a) HGF, (b) the same serum-free additive as in (i), and (c) KnockOut Serum Replacement (KSR); (iv) maturing 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) additional serum-free additives including albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3), thereby maturing the cells into skeletal myotubes and satellite cells. Including, This relates to a method for 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 in particular, the pluripotent stem cells are induced pluripotent stem cells.

[0033] Pluripotent stem cells (PSCs) can differentiate into any cell type in the body. Therefore, human pluripotent stem cells offer considerable possibilities for obtaining, for example, skeletal myoblasts, skeletal myotubes, and satellite cells. Currently, the most commonly used pluripotent cells are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Human ESC systems were first produced by Thomson et al. (Thomson et al., Science 282:1145-1147 (1998)). Today, human ESC research allows for the development of new techniques for reprogramming somatic cells into ES-like cells. This technique, developed by Yamanaka et al. in 2006, is also 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) behave very similarly to ESCs and can differentiate into any cell type in the body. Furthermore, in another embodiment, 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 mice, as well as humans. These cells exhibit key characteristics of pluripotent stem cells and, as a result, can differentiate into any cell type in vitro (Espejel S et al. (2014)). Thus, 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, pluripotent stem cells are not produced by methods that alter the genetic identity of humans in the germline or that use human embryos for industrial or commercial purposes. In a particularly preferred embodiment, 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 step 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) to (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) to (iv) is DMEM supplemented with pyruvate. Even more preferably, the basal medium used in steps (i) to (iv) is DMEM supplemented with pyruvate containing 1 g / l glucose. Basal media are commercially available or can be prepared according to published recipes, for example, from the ATCC catalog. In a highly preferred embodiment, the basal medium is DMEM containing 1 g / L glucose plus a glutamine preparation (e.g., L-alanyl-L-glutamine or GlutaMAX™) and consists of the substances listed in Table 3. If deemed appropriate, the basal medium may be supplemented with an effective concentration of a non-essential amino acid. In a preferred embodiment, the basal medium is supplemented with an effective concentration of a non-essential amino acid listed in Table 2. The basal medium in steps (ii), (iii), and (iv) may be selected independently from the basal medium used in step (i). However, in a preferred embodiment, the basal medium in steps (i) to (iv) is the same.

[0035] Generally, the different differentiation stages in steps (i) to (iv) can be detected using expressed genes characteristic of a particular stage. One method for measuring gene expression is RNA sequencing (RNA-Seq). RNA sequencing is also called 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, and then DNA sequencing methods can be applied. Therefore, RNA sequencing provides information about which mRNAs are expressed and is characterized by low background noise, higher resolution, and a high replication rate. Those skilled in the art are familiar with and able to perform mRNA sequencing. Example 1 of the present invention shows exemplary data measured using RNA sequencing. In particular, Figure 4 shows the time course of mRNA expression of different genes during the differentiation protocol of the present invention from 0 to 60 days.

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

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

[0038] As described above, the basal medium in step (i) contains effective amounts 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. Those skilled in the art will know 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 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, with the GSK3 inhibitor CHIR99021 being preferred. However, any suitable GSK3 inhibitor can be used for the method of the present invention. 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 play an essential role in 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, and DMH1, with LDN193189 being preferred. However, any SMAD inhibitor suitable for the methods of the present invention may 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. It is known to those skilled in the art that the effective concentration or amount of an inhibitor varies with the availability and biological activity of each substance, and this applies to all substances, e.g., proteins / peptides, nucleotides or chemical compounds.

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

[0043] Serum-free additives that meet the above requirements can be purchased commercially. For example, N2 additive can be used. In a preferred embodiment, the serum-free additive 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. N2 additive is commercially available in 100x effective concentration, and the composition is listed in Table 1. This means that 1% (v / v) of 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) and 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 mesodermal differentiation. As described above, the efficiency of mesodermal differentiation can be tracked by RNA sequencing. For example, induction of mesoderm differentiation is indicated when one or more of the genetic markers MSGN1, TBX6 and MEOX1 have an expression value that is at least 5-fold higher (preferably at least 10-fold higher, more preferably at least 20-fold higher, even more preferably at least 30-fold higher, and most preferably at least 50-fold higher) compared to pluripotent stem cells when measured by "reads / million kilobases" by RNA sequencing.

[0045] In step (ii) of the method according to the present 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 Figures 1 and 2 for a schematic overview; see Figure 4 for experimental data on PAX3 expression). For example, myogenic specification is indicated when the gene marker Pax3 has an expression value at least 5-fold higher (preferably at least 10-fold higher, more preferably 20-fold higher, and even more preferably 30-fold higher) compared to pluripotent stem cells, as measured by "reads / million kilobases" by RNA sequencing.

[0046] As described above, step (ii) comprises three culture steps. Specifically, step (ii) comprises 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) the same 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) the same serum-free additive as in (i), and (d) KnockOut Serum Replacement (KSR).

[0047] As in 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 from 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 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) refers to effective concentrations of ascorbic acid, insulin, transferrin, and albumin. In a preferred embodiment, KSR additionally contains effective concentrations of selenium or its bioavailable salt, glutathione, and trace elements. In a more preferred embodiment, KSR contains effective concentrations of the substances listed in Table 5. In a most preferred embodiment, KSR contains the indicated concentrations of the substances in Table 5. "KnockOut Serum Replacement" (KSR) is known in the prior art and can be prepared according to the formulations 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). 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 0.05-0.02 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 0.05-0.02 mM, and most preferably about 0.1 mM.

[0054] In one embodiment, the culturing in step (ii) is carried out in the presence of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive for 36 to 60 hours, preferably 42 to 54 hours, and most preferably about 48 hours; and / or the culturing 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, and 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, and most preferably about 96 hours.

[0055] In step (iii) of the method of the present invention, the cells are advantageously matured and expanded into skeletal myoblasts and satellite cells. Skeletal myoblasts are characterized by their fusion-competence and thus their ability to fuse into skeletal myotubes in a further step. 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 proliferate and differentiate 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 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 MyoD1 and PAX7). For example, skeletal myoblasts are present when the genetic marker MyoD has an expression value at least 5 times higher (preferably at least 10 times higher, more preferably at least 15 times higher, and even more preferably at least 20 times higher) than pluripotent stem cells when measured by "reads / million kilobases" using RNA sequencing. For example, satellite cells are present when the genetic marker PAX7 has an expression value at least 5 times higher (preferably at least 10 times higher, more preferably at least 15 times higher, and even more preferably at least 20 times higher) than pluripotent stem cells when measured by "reads / million kilobases" using RNA sequencing.

[0056] As described above, the basal medium in step (iii) contains an effective amount of (a) HGF, (b) the same serum-free additive as in step (i), and (c) KnockOut Serum Replacement (KSR). As in 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 of the basal medium in step (iii) may be selected similarly to the exemplary and preferred embodiments in step (i). The basal medium in step (iii) may be selected independently from the basal medium used in steps (i) and (ii). However, in a preferred embodiment, the basal medium in steps (i), (ii), and (iii) is the same.

[0057] The KSR and optional reducing agent in step (iii) include the same preferred embodiments as the KSR and optional reducing agent in step (ii). Thus, KSR can be prepared by one 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 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) KSR; in particular, 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 will vary with the availability and biological activity of each substance.

[0060] In step (iv) of the method according to the present invention, the cells are advantageously matured into skeletal myotubes and satellite cells. Skeletal myotubes are formed by the fusion of skeletal myoblasts. Skeletal myotubes are therefore multinucleated cellular structures formed by the fusion of mature myoblasts into long, thin myotubes. Skeletal myotubes are also called muscle cells or muscle fibers. Figure 2 provides a schematic overview of the developmental stages undergone by engineered skeletal muscle tissue; the formation of engineered skeletal muscle tissue is known in the prior art as myogenesis. Skeletal myotubes (muscle fibers) are characterized by anisotropic orientation of actinin-containing sarcomere structures. A regeneration-competent satellite cell niche is formed adjacent to the fused skeletal myotubes. The satellite cell niche is external to, but in close contact with, the skeletal myotubes. The satellite cell niche is anatomically distinct and is also formed in native skeletal muscle tissue. As a result, along with the generated satellite cells, it is another desirable quality characteristic of 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 myotubes, and the expression of each can be determined by RNA sequencing (see Figures 1 and 2 for a schematic overview; PAX7 (paired box See Figure 4 for experimental data regarding the expression of PAX7 (actinin alpha 2), ACTN2 (actinin alpha 2), DMD (dystrophin), and MYH3 (myosin heavy chain 3). For example, satellite cells are present when PAX7 mRNA has an expression value at least 5 times higher (preferably at least 10 times higher, more preferably at least 15 times higher, and even more preferably at least 20 times higher) than pluripotent stem cells when measured by "reads / million kilobases" using RNA sequencing. For example, skeletal myotubes are present when the gene marker ACTN2 has an expression value at least 5 times higher (preferably at least 50 times higher, more preferably at least 100 times higher, and even more preferably at least 150 times higher) than pluripotent stem cells when measured by "reads / million kilobases" using RNA sequencing.Indeed, the DMD and MYH3 gene markers typically have 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.

[0061] As described above, the cells obtained in step (iii) are dispersed in an extracellular matrix and matured under mechanical mimicry. Mechanical mimicry can be performed, for example, with the aid of a stretching device, as is commonly known and used in the art. Preferably, the stretching device exerts static, phasic, or dynamic strain. Therefore, the mechanical strain can be (a) static, (b) phasic, or (c) dynamic. An example of a static strain is isometric muscle contraction, in which the muscle only experiences a change in tension, but not in length. Therefore, shortening does not occur in the muscle during isometric muscle contraction. A phasic strain can be quasi-isotonic muscle contraction, in which the muscle shortens during contraction, and the tension on the muscle remains the same. A dynamic strain can occur, for example, when the muscle is suspended on a flexible support to facilitate load-load contraction. During load-load contraction, both the muscle length and muscle tension change. Preferably, the mechanical stimulus in step (iv) is a static mechanical stimulus, i.e., a static strain (static strain force), which means that the cells and extracellular matrix from step (iii) are under a force and an opposing force (counterforce).

[0062] As described above, the basal medium of step (iv) contains effective amounts of (a) the same serum-free additives as in step (i), and (b) additional serum-free additives including albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, 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 similarly 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 a final concentration of the following substances: 0.5 to 50 mg / ml 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, e.g. 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 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 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); 0.4-40 μg / ml L-carnitine HCl (preferably 0.5-30 μg / ml, more preferably 1-20 μg / ml, even more preferably 2-10 μg / ml, more preferably 3-5 μg / ml, most preferably about 4 μg / ml); 0.05 to 5 μl / ml of 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, e.g., 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, and most preferably about 0.004 μg / ml).

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

[0066] 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, and most preferably 0.9 to 2 μg / ml, for example, about 1 μg / ml). Similarly, in a 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, and most preferably 2.5 to 5 μg / ml, for example, about 3 μg / ml). For example, a bioavailable salt of selenium is sodium selenite, provided in the basal medium at a final concentration of 0.003-0.3 μg / ml (preferably 0.005-0.2 μg / ml, more preferably 0.01-0.1 μg / ml, even more preferably 0.02-0.05 μg / ml, and most preferably 0.03 μg / ml, e.g., about 0.032 μg / ml).

[0067] Furthermore, the additional serum-free additive may further comprise 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. The appropriate concentration of each component is known to those skilled in the art or can be easily determined by routine means.

[0068] Examples of additional serum-free additives mentioned in step (iv) can be prepared according to published protocols (see also Brewer et al. 1993) or can be purchased commercially. 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%, and most preferably about 2% B27.

[0069] In the present invention, a seeding step may be performed prior to step (i), and the resulting engineered skeletal muscle tissue is referred to as bioengineered skeletal muscle (BSM). In the seeding step, pluripotent stem cells are seeded in stem cell medium in the presence of a ROCK inhibitor. Preferably, the seeding step is performed 18 to 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. Particularly preferably, the ROCK inhibitor is Y27632. However, any ROCK inhibitor suitable for the method of the present invention may be used. Those skilled in the art will understand that the effective concentration of a 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 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. Stem cell medium may be used in the seeding step, and in principle, any stem cell medium suitable for the method may be used. Suitable stem cell media are known to those skilled in the art, with iPS-Brew XF stem cell medium being particularly preferred.

[0070] Furthermore, in the seeding step, the pluripotent stem cells may be 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. In the seeding step, the pluripotent stem cells are dispersed in the extracellular matrix before step (i), so that the cells are embedded in the extracellular matrix and differentiate and mature into engineered skeletal muscle tissue in a three-dimensional structure.

[0071] The "extracellular matrix" acts as a scaffold, providing a structural and functional microenvironment for cell growth and differentiation. While the composition of the extracellular matrix is ​​unique to each natural tissue, its main components are collagen, fibronectin, laminin, and various types of glycoaminoglycans and proteoglycans. Proteoglycans form a class of particularly heavily glycosylated glycoproteins that achieve stabilization between living cells. Here, they form large complexes with proteins, such as collagen, the main component of the extracellular matrix, in addition to 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 may play an important role, particularly in tissue repair. The extracellular matrix component in the master mix is ​​preferably collagen, preferably type I collagen, more preferably of bovine, human, or murine origin, especially bovine collagen. Optionally, the extracellular matrix additionally contains laminin and / or fibronectin.

[0072] Pluripotent stem cells typically range from 1 to 6 x 10 6The cells are seeded in medium at a ratio of 0.7-1.4 mg / ml of collagen and 500 mg / ml of cellulose. In one embodiment, the master mix contains 5-15% (v / v), preferably 7.5-12.5% ​​(v / v), more preferably 9-11% (v / v), and most preferably about 10% (v / v) of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cell exudate as an extracellular matrix component. In a particularly preferred embodiment, the exudate is Matrigel. The pH of the master mix is ​​typically 7.2-7.8. Matrigel is known to those skilled in the art and has been described further in the prior art (Hughes et al. 2010).

[0073] As an alternative to EHS murine sarcoma cell exudates, the master mix may also contain stromal cells, which produce the extracellular matrix components collagen, laminin, fibronectin, and / or proteoglycans. The pH of the master mix is ​​typically between pH 7.2 and pH 7.8.

[0074] In a preferred embodiment, 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 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) KSR.

[0075] An effective amount of FGF2 is typically 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 of FGF2.

[0076] When producing 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 resulting 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 component of the extracellular matrix in the master mix is ​​collagen, preferably type I collagen, more preferably collagen of bovine, human, or mouse origin, particularly bovine 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 in a medium at, for example, 1 to 10 × 10 6 They are seeded at a ratio of cells / ml and collagen of 0.7-1.4 mg / ml.

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

[0079] As an alternative to EHS murine sarcoma cell exudates, the master mix may also contain stromal cells, which produce the extracellular matrix components collagen, laminin, fibronectin, and / or proteoglycans. The pH of the master mix is ​​typically between pH 7.2 and pH 7.8.

[0080] In a preferred embodiment, after about 1 hour, the same basal medium as 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.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 is known to those skilled in the art that the concentration of an effective amount of a ROCK inhibitor varies 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 ESM, about one day after the seeding step performed during step (iii) and step (iv), the medium is replaced with the same medium as 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, and most preferably about 7 days.

[0082] In preparing BSM or ESM, the engineered shape may be, for example, a ring, ribbon, strand, patch, pouch, or cylinder, and optionally, individual skeletal muscle tissues may be fused together. This means that individual and / or different geometric shapes can be fused together to form skeletal muscle tissue, and thus different muscle shapes can be achieved. In particular, ring, strand, or ribbon shapes are useful for applications in in vitro methods, such as toxicity testing. Typically, the engineered shape is obtained by casting a master mix, so generally, any desired castable engineered shape can be produced.

[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, and particularly at least 240 days, and longer culture periods are possible. The present inventors have already achieved culture for 240 days (8 months), but this does not preclude longer culture periods.

[0084] In contrast to many methods disclosed in the prior art, the method according to the present invention does not involve a transfection step using a differentiation- or maturation-related transgene. Preferably, the method does not involve a myogenic transgene, and more preferably, the method does not involve a Pax7 or MyoD transgene. "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., 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. This makes methods requiring a transfection step less controllable and therefore less reproducible. Therefore, transgene-free methods are advantageous over methods requiring transfection with a transgene. However, it cannot be excluded that pluripotent stem cells may be genetically modified in other ways, for example to simulate disease patterns, or even genetically engineered labeling of cell types and / or cell functions (e.g., calcium or voltage signals) or control of cell functions (e.g., contraction frequency) via, for example, optogenetic mechanisms.

[0085] Another advantage of the method according to the present invention is that no additional selection step is required for specific cell types, such as skeletal myoblasts. Preferably, the method does not include a cell selection enrichment step, and more preferably, does not include an antibody-based cell selection enrichment step. This is advantageous because 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 scalability and is associated with infection risks, thus becoming a key barrier to commercial application of cell products. Because the method of the present invention does not require cell selection, the production of engineered skeletal muscle tissues and cells of the present invention is scalable and suitable for commercial or medical applications.

[0086] Additionally, the method is serum-free, eliminating variability associated with different serum batches, resulting in a robust and reproducible protocol for the production of engineered skeletal muscle tissue, with all necessary chemical and physical stimuli defined.

[0087] The present invention provides a method for producing skeletal myoblasts, skeletal myotubes, 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 effective amounts 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) the same serum-free additives as in (i); and adding an effective amount of (d) HGF and continuing the culture in the medium; Culturing the cells in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) HGF, (c) the same serum-free additives as in (i), and (d) KnockOut Serum Replacement (KSR). inducing myogenic specification by (iii) culturing the cells obtained in step (ii) in a basal medium containing an effective amount of (a) HGF, (b) the same serum-free additive as in (i), and (c) KnockOut Serum Replacement (KSR), thereby maturing the cells into skeletal myoblasts and satellite cells; (iv) maturing the cells obtained in step (iii) into skeletal myotubes and satellite cells by culturing the cells in a basal medium containing effective amounts of (a) the same serum-free additives as in step (i) and (b) additional serum-free additives including albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3); thereby producing skeletal myoblasts, skeletal myotubes, and satellite cells.

[0088] For example, the cells produced by this method have a skeletal myoblast percentage of the total available cells of at least 40%, preferably at least 50%, more preferably at least 60%, and most preferably at least 70%, as determined by actinin expression by flow cytometry.

[0089] Preferably, the method achieves a proportion of satellite cells of the total available cell mass of at least 10%, preferably at least 15%, more preferably at least 20%, and most preferably at least 30%, as determined by Pax7 expression 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 properties 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 myotubes, or satellite cells can be detected using fluorescent staining. In particular, the proteins sarcomeric α-actinin, myogenin, Pax7, and MyoD are incubated with and thereby labeled by a primary antibody. With the aid of a fluorescently labeled secondary antibody, skeletal muscle-specific cells can be detected.

[0091] A major advantage of the present method over the prior art is that it does not require a step of enriching cells, such as skeletal myoblasts. Preferably, the method does not include an enrichment step by cell selection, and more preferably, does not contain an enrichment step by antibody-based cell selection, such as flow cytometry. This means that the method according to the present invention does not require cell selection to achieve highly pure skeletal myoblasts, skeletal myotubes, and / or satellite cells. The cell selection method is disclosed herein solely for analytical purposes to demonstrate the highly pure skeletal myoblasts, skeletal myotubes, and satellite cells produced (see FIG. 5).

[0092] As described above, the basal medium of step (i) comprises effective amounts of (a) FGF2, (b) a GSK3 inhibitor, (c) a SMAD inhibitor, and (d) a serum-free additive comprising 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, tideglusib, SB415286, 6-bromoindirubin-3-oxime, and valproate, with the GSK3 inhibitor CHIR99021 being preferred. However, any GSK3 inhibitor suitable for the method of the present invention may be used. When the GSK3 inhibitor is CHIR99021, 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.

[0094] Preferred exemplary embodiments of steps (i)-(iii) are described in the methods for preparing engineered skeletal muscle tissue and can be similarly applied to the methods for preparing skeletal myoblasts, skeletal myotubes, 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 present inventors analyzed cells using fluorescence microscopy, which involves immunostaining for skeletal muscle-specific transcription factors (Pax7, MyoD, and myogenin). After step (iii) of the method, fluorescence images show a high percentage of cells expressing Pax7, MyoD, and myogenin (Figure 3). This method demonstrates that the method generates skeletal myoblasts (MyoD and myogenin) in addition to satellite cells (Pax7).

[0096] In step (iv) of the method of the present invention, the cells are matured into skeletal myotubes and satellite cells. Skeletal myotubes arise from the fusion of skeletal myoblasts. Thus, skeletal myotubes are multinucleated cellular structures formed by the fusion of mature myoblasts into elongated myotubes. 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 myotubes, and the expression of each 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, ACTN2, DMD, and MYH3). For example, when the gene marker PAX7 is measured by "reads / million kilobases" using RNA sequencing, the expression value is at least 5 times higher (preferably at least 10 times higher, more preferably 20 times higher) than that of pluripotent stem cells, indicating satellite cells.For example, when the gene marker ACTN2 is measured by "reads / million kilobases" using RNA sequencing, the expression value is at least 5 times higher (preferably at least 50 times higher, more preferably 100 times higher, even more preferably 150 times higher) than that of pluripotent stem cells, indicating skeletal myotube cells.The gene markers DMD and MYH3 even exhibit at least 200 times higher expression values ​​(preferably at least 500 times higher, more preferably 1000 times higher) than that of pluripotent stem cells.

[0097] As described above, the basal medium of step (iv) contains effective amounts of (a) the same serum-free additives as in step (i), and (b) additional serum-free additives including albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, 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 a final concentration of the following substances: 0.5 to 50 mg / ml 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, e.g. 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 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 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); 0.4-40 μg / ml L-carnitine HCl (preferably 0.5-30 μg / ml, more preferably 1-20 μg / ml, even more preferably 2-10 μg / ml, more preferably 3-5 μg / ml, most preferably about 4 μg / ml); 0.05 to 5 μl / ml of 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, e.g., 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, and 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, and most preferably 0.9 to 2 μg / ml, for example, about 1 μg / ml). Similarly, in a 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, and most preferably 2.5 to 5 μg / ml, for example, about 3 μg / ml). For example, a bioavailable salt of selenium is sodium selenite, provided in the basal medium at a final concentration of 0.003-0.3 μg / ml (preferably 0.005-0.2 μg / ml, more preferably 0.01-0.1 μg / ml, even more preferably 0.02-0.05 μg / ml, and most preferably 0.03 μg / ml, e.g., about 0.032 μg / ml).

[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. The appropriate concentration of each component is known to those skilled in the art or can be easily determined by routine means.

[0101] The additional serum-free additives mentioned 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 0.1-10% B27, preferably 0.5-8%, preferably 1-6%, more preferably 1.5-4%, even more preferably 1.5-4%, and most preferably about 2% B27.

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

[0103] Step (i) of this method may be preceded by a seeding step, in which pluripotent stem cells are seeded in stem cell medium in the presence of a ROCK inhibitor. Preferably, the seeding step is performed 18 to 30 hours, preferably 20 to 28 hours, more preferably 22 to 26 hours, even more preferably 23 to 25 hours, and most preferably about 24 hours prior to step (i). 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. 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 may be used. Those skilled in the art will understand that the effective concentration of a 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 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. Stem cell medium may also be used in the seeding step, and in principle, any stem cell medium suitable for the method may be used. Suitable stem cell media are known to those skilled in the art, with iPS-Brew XF stem cell medium being particularly preferred. Preferably, the stem cell medium contains effective concentrations of KSR and FGF2. In particular, the stem cell medium contains, for example, 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) KSR; and / or It 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.

[0104] Similar to the method for producing skeletal muscle tissue, the differentiation stage of steps (i) to (iv) of the method for producing skeletal myoblasts, skeletal myotubes, and satellite cells can be detected using expressed genes characteristic of specific stages. RNA sequencing is used in this method similarly to the method for producing skeletal muscle tissue. Thus, the same expressed genes (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 extensive digestion protocols and / or flow cytometry cell selection steps. Digestion protocols transfer cells to a different environment, causing them to lose cell-matrix connectivity as well as cell-cell connectivity. This disrupts the extracellular environment and the spatial distribution of cell types formed during development, which can have an inhibitory effect on the skeletal muscle differentiation process, which is difficult to control. The present invention minimizes the number of digestion steps and does not require cell selection to enrich, for example, skeletal myoblasts. The refined protocol produces highly pure skeletal myoblasts, skeletal myotubes, and satellite cells (an exemplary cell population at least 70% actinin-positive and at least 30% PAX7-positive is shown in the Examples; see also Figure 5).

[0106] The method according to the present invention allows for the production of engineered skeletal muscle tissue with advantageous properties. In engineered skeletal muscle tissue, the presence of skeletal myotubes can be detected by actinin staining (see FIG. 8). In particular, the skeletal muscle tissue does not contain differentiation-related or maturation-related transgenes, preferably does not contain myogenic transgenes, and more preferably does not contain the Pax7 or MyoD transgenes. Compared to native skeletal muscle tissue, engineered skeletal muscle tissue, such as BSM or ESM, lacks a blood supply and 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 lacks innervation by nerve cells. Blood supply refers to muscle vascularization, which supplies blood to the muscle. Another difference from native skeletal muscle tissue is that engineered skeletal muscle tissue does not have musculoskeletal attachments via tendons or bones, but rather occurs entirely ex vivo. Therefore, engineered skeletal muscle tissue is clearly distinguishable from natural skeletal muscle tissue. Despite its engineered production, skeletal muscle tissue according to the present invention exhibits many characteristics of native skeletal muscle. These include the morphological characteristics of a syncytium of fused muscle cells (muscle fibers, multinucleated skeletal myotubes) and contractile performance (positive force-frequency ratio and tetanus). Unlike prior art tissue, satellite cell niches are formed in direct contact with skeletal muscle fibers. Additionally, skeletal muscle tissue produced according to the present invention exhibits the typical fibers of striated skeletal muscle, consisting of many muscle fibers (syncytium). It is known to those skilled in the art that natural skeletal muscle tissue has multinucleated skeletal muscle fibers, each consisting of sarcomeres strung together. Therefore, multinucleated skeletal muscle fibers can be recognized by their characteristic striated skeletal muscle pattern in actin or actinin staining, because actin / actinin stains within the sarcomere. Sarcomere structures are rigid and regular, and they are arranged in rows and join together to form multinucleated muscle fibers. This means that the characteristic striated skeletal muscle pattern demonstrates the formation of multinucleated skeletal muscle fibers.The characteristic skeletal muscle tissue structure (skeletal muscle fibers with satellite cell niches) may be visualized by fluorescence microscopy after staining for actinin or actin and Pax 7. In this study, we stained the structural protein actin in engineered skeletal muscle tissue, and the fluorescence image in Figure 8 illustrates the characteristic striped pattern.

[0107] A key functional characteristic of engineered skeletal muscle tissue is that it contracts in response to electrical stimulation, thereby generating force. This force-generation characteristic can be determined, for example, by measuring contractile output. These contraction experiments measure the contraction frequency and force of engineered skeletal muscle tissue in response to electrical stimulation. Skeletal muscle tissue in the form of rings was 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 gassing with 5% CO2 and 95% O2. The engineered skeletal muscle tissue was mechanically stretched, and the maximum force amplitude (force of contraction = FOC) was measured, typically at an electrical field stimulation frequency (4 ms rectangular pulse; 200 mA) within the range of 1-100 Hz. Exemplary measurement methods for tissues according to the present invention are shown in Figures 6B, 6C, 7B, and 7C. These contraction experiments demonstrate that engineered skeletal muscle tissue exhibits particularly advantageous properties when generating force in response to electrical stimulation. Engineered skeletal muscle tissue exhibits reproducible contraction frequency and contraction force in response to stimulation frequencies between 1 Hz and 100 Hz. Typically, contraction and full relaxation take approximately 0.5 seconds with a single 1 Hz stimulation. Because contraction and relaxation times require approximately 0.5 seconds, initial or full tetanus is formed at higher stimulation frequencies. Because tetanus also forms in native skeletal muscle tissue at increased stimulation frequencies, engineered skeletal muscle tissue behaves similarly to native skeletal muscle tissue even in this respect. Furthermore, we can show that the contraction force of muscle tissue increases with increasing contraction frequency (a 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 twitching and tetanus. Unlike engineered skeletal muscle tissue, where electrical impulses in natural muscle tissue arise from neuronal action potentials, engineered skeletal muscle tissue can spontaneously contract in response to electrical stimulation.

[0108] As illustrated in Figures 6B, 6C, 7B, 7C, and 8, the engineered skeletal muscle tissue produced by the methods of the invention has a characteristic formation of multinucleated muscle fibers (skeletal myotubes) 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, upon stimulation at 100 Hz at 200 mA.

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

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

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

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

[0113] The present disclosure further provides satellite cells, which can be obtained according to step (iii) of the method disclosed herein and can be produced by steps (i) to (iii) of the method disclosed herein, characterized by expression of the gene Pax7. In this regard, Pax7 expression 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 particularly preferred embodiments, the satellite cells therefore further express Ki67. Cell cycle activation in engineered skeletal muscle tissue is more frequently observed after tissue injury (e.g., due to pressure injury, cardiotoxin treatment, irradiation, or frostbite) and leads to repair of tissue damage in the sense of endogenous regeneration.

[0114] Also disclosed is a mixture of skeletal myoblasts and satellite cells, in which a proportion of satellite cells of the total available cell mass is achieved of at least 10%, preferably at least 15%, more preferably at least 20%, and even more preferably at least 30%, as determined by expression of Pax7 by flow cytometry; and / or a proportion of skeletal myoblasts of the total available cell mass is achieved of at least 40%, preferably at least 50%, more preferably at least 60%, and most preferably at least 70%, as determined by expression of actinin by flow cytometry.

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

[0116] Advantageously, engineered skeletal muscle tissue, mesodermally differentiated skeletal myoblast precursor cells, myogenically specialized skeletal myoblast precursor cells, skeletal myoblasts, satellite cells, and / or skeletal myotube 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 typically include protein-based molecules in addition to small molecule compounds. Gene therapy drug candidates typically modify the genome of skeletal muscle tissue by introducing the corresponding nucleic acid.

[0117] Furthermore, engineered skeletal muscle tissue, mesodermally differentiated skeletal myoblast precursor cells, myogenically committed skeletal myoblast precursor cells, skeletal myoblasts, satellite cells, and / or skeletal myotube cells may be used in medicine.

[0118] Of particular importance here are satellite cells. They are intended 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-Keener muscular dystrophy, and / or lysosomal storage diseases, particularly Pompe disease, preferably the skeletal muscle disease is Duchenne muscular dystrophy. Those skilled in the art will 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 considered for use in the treatment of skeletal muscle diseases, such as amyotrophic lateral sclerosis, myasthenia gravis, or myotonia. Myotonia includes various muscle diseases that exhibit delayed relaxation and consequently pathological prolongation of tonic muscle contractions. Satellite cells are particularly suitable for the treatment of damaged skeletal muscle and / or the treatment of skeletal muscle diseases because they continuously regenerate skeletal muscle tissue. The term "damaged skeletal muscle tissue" refers to tissue damage and injury 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. Therefore, because satellite cells lead to increased regeneration of skeletal muscle tissue, satellite cells according to the present invention are promising candidates for cell-based therapy in damaged skeletal muscle tissue (Yin et al. (2013)). Similarly, 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 transplants can be functionally integrated and controllable via electrical stimulation or optogenetic activation to restore or therapeutically support muscle function. The proportion of satellite cells in engineered skeletal muscle tissue ensures the endogenous regenerative capacity of skeletal muscle for a long period of time.

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

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

[0121] The contractile force and / or structure of skeletal muscle tissue may be measured by the contraction experiments and fluorescence microscopy experiments described herein. For example, metabolic function may be measured using a Seahorse Metabolic Flux Analyzer, known to those skilled in the art. For example, the Seahorse Metabolic Flux Analyzer measures the oxygen consumption and extracellular acid production rates of living cells, and can further measure important cellular functions such as mitochondrial respiration and glycolysis. Molecular parameters (markers) may be measured, for example, by transcriptome analysis (PCR or RNA sequencing). Protein biochemical parameters (markers) may be measured, for example, via mass spectrometry or common clinical chemistry measurement methods (e.g., 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 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 native to the body, so that the effects of, for example, 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, comprising: (a) providing skeletal muscle tissue according to the invention described herein; (b) contacting the skeletal muscle tissue from step (a) with the substance to be tested. Including, Preferably, the method further comprises a step of determining the contractile force and / or structure and / or metabolic function and / or molecular and / or protein biochemical parameters of skeletal muscle tissue before and / or after step (b), A method is envisaged.

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

[0125] The substance used in the toxicity test may be, for example, but is not limited to, a drug candidate. Rather, any substance whose toxicity is to be evaluated may 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, (a) providing skeletal muscle tissue according to the invention described herein; (b) contacting the skeletal muscle tissue from step (a) with the nutrients and dietary supplements to be tested. Including, Preferably, the method further comprises a step of determining the contractile force and / or structure and / or metabolic function and / or molecular and / or protein biochemical parameters of skeletal muscle tissue before and / or after step (b), Regarding the method.

[0127] This in vitro method provides the opportunity to measure the effect of nutrients and nutritional supplements on skeletal muscle tissue at clinically relevant concentrations. This method is particularly interesting when measuring the effect of these substances on muscle growth, cachexia, or diabetes. Cachexia is understood to be a pathological, very severe form of wasting. Many patients with chronic diseases, such as cancer or autoimmune diseases, suffer from the additional condition of cachexia. This in vitro method allows the possibility of measuring the effect 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, such as an in vitro method for testing the effectiveness of a drug candidate on mesodermally differentiated skeletal myoblast precursor cells, myogenically committed skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, or a mixture of skeletal myoblasts and satellite cells, comprising: (a) providing mesodermally differentiated skeletal myoblast precursor cells, myogenically committed skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, 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. Includes; Preferably, the method further comprises determining the expression of actinin and / or Pax7 before and / or after step (c), wherein the expression may be determined by flow cytometry and / or immunostaining. Methods are described herein.

[0129] Another possible application is an in vitro method for testing the toxicity of substances to mesodermally differentiated skeletal myoblast precursor cells, myogenically committed skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, or a mixture of skeletal myoblasts and satellite cells, comprising: (a) providing mesodermally differentiated skeletal myoblast precursor cells, myogenically committed skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, or a mixture of skeletal myoblasts and satellite cells according to the invention described herein; (b) contacting the cells of step (a) with the substance to be tested. Including, Preferably, the method further comprises determining the expression of actinin and / or Pax7 before and / or after step (b), wherein the expression may be determined by flow cytometry and / or immunostaining. Regarding the method.

[0130] An additional possible application is an in vitro method for testing the effects of nutrients and nutritional supplements on mesodermally differentiated skeletal myoblast precursor cells, myogenically committed skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, or a mixture of skeletal myoblasts and satellite cells, comprising: (a) providing a mixture of mesodermally differentiated skeletal myoblast precursor cells, myogenically committed skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, or a mixture of skeletal myoblasts and satellite cells according to the invention described herein; (b) contacting the cells of step (a) with the nutrient or dietary supplement to be tested. Including, Preferably, the method further comprises determining the expression of actinin and / or Pax7 before and / or after step (b), wherein the expression may be determined by flow cytometry and / or immunostaining. Regarding the method.

[0131] In another preferred embodiment, the skeletal muscle tissue can generate a contractile force of at least 0.6 milliNewtons (mN) at 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. 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, contractile force can be recorded during electrical field stimulation using 200 mA (see Figures 6, 7, 9, and 10). In more preferred embodiments, the skeletal muscle tissue is capable of generating 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, at 100 Hz stimulation. This typically occurs when step (iv) of the method is carried out for at least 50 days, e.g., 56 days. A typical characteristic of the engineered skeletal muscle tissue described herein is that contractile force increases with the duration of maturation.

[0132] In another preferred embodiment, the skeletal muscle tissue has a contraction velocity of at least 3 mN / s, preferably at least 4 mN / s, more preferably at least 5 mN, more preferably at least 6 mN / s, even more preferably at least 6.5 mN / s, and even more preferably at least 7 mN / s under 100 Hz stimulation. For example, contraction velocity can be recorded under 100 Hz stimulation (5 ms, monophasic or biphasic) using 200 mA. Contraction velocity, also referred to as force production rate, is the time required for the manipulated skeletal muscle tissue to accumulate the amount of tension or the rate of tension increase, respectively. Contraction velocity is determined in the context of isometric contraction experiments as the time of maximum increase in contractile force (+dFOC / dt).

[0133] In another preferred embodiment, the skeletal muscle tissue has a relaxation rate at the end of 100 Hz stimulation 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. The relaxation rate is determined during the relaxation phase of the skeletal muscle in the context of an isometric contraction experiment as the time of maximum decrease in contractile force (-dFOC / dt).

[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, when 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 an effective amount of creatine. Such an increase in contractile force is demonstrated with experimental data in Example 4 and Figure 9. For example, an effective amount of creatine as a final concentration in the basal medium in step (iv) is 0.1 to 10 mM creatine. More preferred concentrations are, for example, 0.2 to 6 mM creatine, more preferably 0.4 to 4 mM creatine, even more preferably 0.6 to 3 mM creatine, even more preferably 0.7 to 2.5 mM creatine, even more preferably 0.8 to 2 mM creatine, even more preferably 0.85 to 1.5 mM creatine, even more preferably 0.9 to 1.2 mM creatine, and most preferably about 1 mM creatine.

[0135] Additionally, the maturation medium in step (iv) may also have an increased amount of T3. Such increased amounts of T3 may reduce the contraction and / or relaxation rates of the engineered skeletal muscle compared to engineered skeletal muscle tissue prepared in step (iv) without the increased amount of T3. Exemplary increased amounts of T3 in the basal medium in step (iv) are 0.001 to 1 μM triiodo-L-thyronine (T3), preferably 0.005 to 0.7 μM T3, more preferably 0.01 to 0.35 μM T3, even more preferably 0.04 to 0.02 μM T3, even more preferably 0.05 to 0.18 μM T3, even more preferably 0.06 to 0.15 μM T3, even more preferably 0.08 to 0.12 μM T3, and even more preferably about 0.1 μM T3. Furthermore, Example 4 as well as Figure 10 show the beneficial effects with experimental data of increasing 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 increasing amount of triiodo-L-thyronine (T3) over a given maturation period. As shown in Example 4, such a period may be 4 weeks, e.g., from week 1 to week 5 in step (iv), or from week 5 to week 9 in step (iv). However, other periods, e.g., from 1 to 9 weeks, may be selected for any maturation period. For example, the 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, and even more preferably at least 8 weeks. Furthermore, the 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, and even more preferably up to 4 weeks. In light of the present disclosure, those skilled in the art may freely combine exemplary period end points.

[0137] In another preferred embodiment, the skeletal muscle tissue produced by the methods described herein possesses regenerative properties. Regenerative properties are characterized by a restoration of a naturally occurring state that previously existed. For example, the contractility of the engineered skeletal muscle tissue can be restored. Thus, contractility can be restored and / or muscle can be remodeled. In a highly preferred embodiment, the regenerative properties are characterized by restored contractility and / or muscle remodeling. Preferably, the ability to restore contractility and / or muscle remodeling is measured 24 hours after exposure to a cardiotoxin and / or muscle remodeling, and more preferably, the restored contractility and / or muscle remodeling is measured 10 to 30 days after cardiotoxin exposure. Cardiotoxins are polypeptide toxins that destroy skeletal muscle cells by inducing permanent depolarization. Functionally, incubation with cardiotoxin results in a loss of contractility in the engineered skeletal muscle. Structurally, irreversible destruction of myotube cells formed in the engineered skeletal muscle is observed. For example, no contraction was recorded even after two days in Example 5 described herein. As shown in Figure 11, engineered skeletal muscle tissue with regenerative properties can restore this contractility. For example, as described in Example 5, muscles can contract again 21 days after cardiotoxin treatment. However, engineered skeletal muscle treated with gamma radiation (X-rays) does not exhibit regenerative properties and is unable to contract, even after 21 days of cardiotoxin incubation. This example demonstrates that when skeletal muscle cells are irreversibly destroyed in engineered skeletal muscle tissue, skeletal muscle progenitor cells with regenerative capabilities are preserved and can regenerate or reconstruct skeletal muscle structure with contractile function in engineered skeletal muscle tissue through cell division and differentiation into newly formed skeletal muscle cells. As shown in Figure 11, engineered skeletal muscle tissue with regenerative properties can achieve this muscle reconstruction. Figure 11B shows the regeneration of contractile force, and Figure 11C (top) shows the structural regeneration of skeletal muscle. The failure of regeneration after gamma irradiation demonstrates that skeletal muscle progenitor cells (e.g., satellite cells) competent for cell division 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, and even more preferably at least 80 days. According to the present inventors' knowledge, 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, and more preferably 250 days. Those skilled in the art can freely combine exemplary duration limits for step (iv) in light of the present disclosure.

[0139] Furthermore, the present invention includes engineered skeletal muscle tissue produced by the methods described herein. In preferred embodiments, the skeletal muscle tissue generates a contractile force of at least 0.6 milliNewtons (mN) at 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, 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. Contractile force can be recorded, for example, at a 200 mA stimulation.

[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 at 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 aspects. 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 effective amounts 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) the same serum-free additives as in (i); and (d) adding an effective amount of HGF and continuing the culture in the medium; and Culturing the cells in a basal medium containing an effective amount of (a) a gamma secretase / NOTCH inhibitor, (b) HGF, (c) the same serum-free additives as in (i), and (d) KnockOut Serum Replacement (KSR). inducing myogenic specification by (iii) expanding and maturing the cells obtained in step (ii) into skeletal myoblasts and satellite cells by culturing the cells in a basal medium containing effective amounts of (a) HGF, (b) the same serum-free additive as in (i), and (c) KnockOut Serum Replacement (KSR); (iv) maturing 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) additional serum-free additives including albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3), thereby maturing the cells into skeletal myotubes and satellite cells. Including, thereby producing engineered skeletal muscle tissue. 2. The method of embodiment 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 wherein the pluripotent stem cells are induced pluripotent stem cells. 3. The method of embodiment 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, and preferably the SMAD inhibitor is LDN193189; The method of 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 50-500 μg / ml transferrin, 1-20 μg / ml insulin, 0.001-0.1 μg / ml progesterone, 5-50 μg / ml putrescine, and 6-600 nM 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 of any one of aspects 1 to 4. 6. The method of any of aspects 1 to 5, wherein the serum-free additive in step (i) is 0.1 to 10% (v / v) N2 additive, more preferably 0.3 to 7.5% (v / v) N2 additive, more preferably 0.5 to 5% (v / v) N2 additive, more preferably 0.75% to 2% (v / v) N2 additive, more preferably 0.9% to 1.2% (v / v) N2 additive, and most preferably about 1% (v / v) N2 additive. 7. The method of any 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, wherein the basal medium is preferably DMEM, in particular the basal medium is supplemented with pyruvate and / or a non-essential amino acid and / or comprises 1 g / l glucose. 8. In step (ii), the culturing is carried out in the presence of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive for 36 to 60 hours, preferably 42 to 54 hours, and most preferably about 48 hours; and / or the culturing 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, and 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, and most preferably about 96 hours; The method of any one of aspects 1 to 7. 9. The method of any of aspects 1 to 8, wherein 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. 10. 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, and 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, and 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 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); 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 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); 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. The method of any of aspects 1-11, wherein in step (iv), the additional serum-free additives provide final concentrations in the medium of 0.5-50 mg / ml albumin, 1-100 μg / ml transferrin, 0.1-10 μg / ml ethanolamine, 17.4-1744 nM selenium or a bioavailable salt thereof, particularly sodium selenite, 0.4-40 μg / ml L-carnitine, 0.05-5 μl / ml fatty acid additive, 0.0001-0.1 μg / ml triiodo-L-thyronine (T3). 13. The method of any of aspects 1 to 12, wherein the additional serum-free additive in step (iv) is 0.1 to 10% (v / v) 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) B27. 14. The method of any one of aspects 1 to 13, wherein in step (iv), the mechanical stimulus is static tension, or dynamic tension, or load-increasing tension, and preferably the mechanical stimulus is static tension. 15. The method of any of aspects 1 to 14, comprising a seeding step prior to step (i), in which the pluripotent stem cells are seeded in a stem cell medium in the presence of a ROCK inhibitor, and preferably, the seeding step is carried out 18 to 30 hours before step (i). 16. The method of embodiment 15, wherein 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. 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 method of any of aspects 15-17, wherein 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 a master mix before the stem cell medium is added. 19. The method of embodiment 18, wherein the component of the extracellular matrix in the master mix is ​​collagen, preferably type I collagen, more preferably of bovine, human or murine origin, in particular collagen of bovine origin, and optionally the extracellular matrix additionally comprises laminin and / or fibronectin. 20. Pluripotent stem cells are 1-6 x 10 6 20. The method of embodiment 19, wherein the cells are seeded in medium at a ratio of cells / ml and collagen of 0.7 to 1.4 mg / ml. 21. The master mix contains 5-15% (v / v), preferably 7.5%-12.5% ​​(v / v), more preferably 9-11% (v / v), and most preferably about 10% (v / v) of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cell exudate as an extracellular matrix component, in particular the exudate is Matrigel; and / or The pH of the master mix is ​​between pH 7.2 and pH 7.8. The method of any one of aspects 18 to 20. 22. The master mix comprises stromal cells, and the stromal cells produce the extracellular matrix components collagen, laminin, fibronectin, and / or proteoglycans; and / or The pH of the master mix is ​​between pH 7.2 and pH 7.8. The method of any one of aspects 18 to 20. 23. The method of any of embodiments 18-22, wherein stem cell medium is added to the master mix in the engineered form after about 1 hour, and the stem cell medium comprises KSR and FGF2. 24. The stem cell culture medium comprises 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) KSR; and / or 24. The method of embodiment 23, wherein the stem cell culture medium comprises 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. 25. The method of any of embodiments 18-24, wherein step (iii) is carried out for 7-11 days, preferably 8-10 days, and most preferably about 9 days. 26. The method of any of embodiments 1-17, wherein after step (iii), 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 in an additional step before step (iv). 27. The method of embodiment 26, wherein the component of the extracellular matrix in the master mix is ​​collagen, preferably type I collagen, more preferably of bovine, human or murine origin, in particular collagen of bovine origin, and optionally the extracellular matrix additionally comprises laminin and / or fibronectin. 28. Skeletal myoblasts and satellite cells are cultured at 1–6 × 10 628. The method of embodiment 27, wherein the cells are seeded in medium at a ratio of cells / ml and collagen of 0.7 to 1.4 mg / ml. 29. The master mix contains 5-15% (v / v), preferably 7.5%-12.5% ​​(v / v), more preferably 9-11% (v / v), and most preferably about 10% (v / v) of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cell exudate as an extracellular matrix component, in particular the exudate is Matrigel; and / or The pH of the master mix is ​​between pH 7.2 and pH 7.8. The method of any one of aspects 26 to 28. 30. The master mix comprises stromal cells, and the stromal cells produce the extracellular matrix components collagen, laminin, fibronectin, and / or proteoglycans; and / or The pH of the master mix is ​​between pH 7.2 and pH 7.8. The method of any one of aspects 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 engineered form, the medium additionally containing 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, and particularly preferably the ROCK inhibitor is Y27632. The method of any one of aspects 26 to 30. 32. The method of embodiment 31, wherein 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. 33. The method of any one of aspects 26-32, wherein after about 1 day, the medium is replaced with the same medium as 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, and most preferably about 7 days. 34. The method of any of aspects 18-33, wherein the engineered form has the form of a ring, ribbon, strand, patch, pouch, or cylinder, optionally with fused individual skeletal muscle tissues. 35. The method of any of embodiments 1-34, 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, and in particular at least 240 days. 36. The method of any of aspects 1-35, wherein the method comprises no differentiation-associated transgene or maturation-associated transgene, preferably no myogenic transgene, more preferably no transgene Pax7 or MyoD. 37. The method of any of aspects 1-36, which does not include a skeletal myoblast enrichment step, preferably does not include an enrichment step by cell selection, more preferably does not include an enrichment step by antibody-based cell selection. 38. A method for producing skeletal myoblasts, skeletal myotubes, 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 effective amounts 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) the same serum-free additives as in (i); and (d) adding an effective amount of HGF and continuing the culture in the medium; and Culturing the cells in a basal medium containing an effective amount of (a) a gamma-secretase / NOTCH inhibitor, (b) HGF, (c) the same serum-free additives as in (i), and (d) KnockOut Serum Replacement (KSR). inducing myogenic specification by (iii) culturing the cells obtained in step (ii) in a basal medium containing an effective amount of (a) HGF, (b) the same serum-free additive as in (i), and (c) KnockOut Serum Replacement (KSR), thereby maturing the cells into skeletal myoblasts and satellite cells; (iv) maturing the cells obtained in step (iii) into skeletal myotubes and satellite cells by culturing the cells in a basal medium containing effective amounts of (a) the same serum-free additives as in step (i) and (b) additional serum-free additives including albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3); Including, This method produces skeletal myoblasts, skeletal myotubes, and satellite cells. 39. The method of embodiment 38, wherein the method achieves a proportion of skeletal myoblasts out of the total amount of available cells of at least 40%, preferably at least 50%, more preferably at least 60%, and most preferably at least 70%, as determined by actinin expression by flow cytometry. 40. The method of embodiment 38 or 39, wherein the method achieves a proportion of satellite cells out of the total amount of available cells of at least 10%, preferably at least 15%, more preferably at least 20%, and most preferably at least 30%, as determined by the expression of Pax7 by flow cytometry. 41. The method of any of aspects 38 to 40, which does not include a skeletal myoblast enrichment step, preferably does not include an enrichment step by cell selection, more preferably does not include an enrichment step by antibody-based cell selection. 42. The method of any 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 of any of aspects 38-42, 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, and preferably the SMAD inhibitor is LDN193189; The method of 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 a final concentration in the medium of 50-500 mg / L transferrin, 1-20 mg / L insulin, 1-30 μg / L progesterone, 5-50 μg / mL putrescine, and 6-600 nM selenium or a bioavailable salt thereof, in particular sodium selenite; and / or the GSK3 inhibitor is CHIR99021 and 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; 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 of any one of embodiments 38 to 44. 46. ​​The method of any of aspects 38 to 45, wherein the serum-free additive in step (i) is 0.1 to 10% (v / v) N2 additive, preferably 0.3 to 7.5% (v / v) N2 additive, more preferably 0.5 to 5% (v / v) N2 additive, more preferably 0.75% to 2% (v / v) N2 additive, more preferably 0.9% to 1.2% (v / v) N2 additive, and most preferably about 1% (v / v) N2 additive. 47. The method of any of aspects 38 to 46, 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, wherein the basal medium is preferably DMEM, in particular the basal medium is supplemented with pyruvate and / or a non-essential amino acid and / or contains 1 g / l glucose. 48. In step (ii), the culturing is carried out in the presence of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive for 36 to 60 hours, preferably 42 to 54 hours, and most preferably about 48 hours; and / or the culturing 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, and 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, and most preferably about 96 hours; The method of any one of aspects 38 to 47. 49. The method of any of aspects 38 to 48, wherein 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. 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, and 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, and 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), 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 of any one of aspects 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); 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 38 to 50. 52. The method of any of embodiments 38-51, wherein step (iii) is carried out for 7-11 days, preferably 8-10 days, and most preferably about 9 days. 53. The method of any of aspects 38-52, wherein in step (iv), the additional serum-free additives provide final concentrations in the medium of 0.5-50 mg / ml albumin, 1-100 μg / ml transferrin, 0.1-10 μg / ml ethanolamine, 17.4-1744 nM selenium or a bioavailable salt thereof, particularly sodium selenite, 0.4-40 μg / ml L-carnitine, 0.05-5 μl / ml fatty acid additive, 0.0001-0.1 μg / ml triiodo-L-thyronine (T3). 54. The method of any of aspects 38 to 53, wherein the additional serum-free additive in step (iv) is 0.1 to 10% (v / v) 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) B27. 55. The method of any of embodiments 38-54, wherein step (iv) is carried out for at least 30 days, preferably at least 35 days, more preferably at least 40 days, and even more preferably at least 50 days. 56. The method of any of aspects 38 to 55, comprising a seeding step prior to step (i), in which the pluripotent stem cells are seeded in a stem cell medium in the presence of a ROCK inhibitor, and preferably, the seeding step is carried out 18 to 30 hours before step (i). 57. The method of embodiment 56, wherein 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. 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, and most preferably about 5 μM; and / or The stem cell medium is iPS-Brew XF. 58. The method of embodiment 56 or 57. 59. The stem cell culture medium comprises 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) KSR; and / or The stem cell culture 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 embodiment 58. 60. Engineered skeletal muscle tissue having multinucleated mature skeletal muscle fibers with satellite cells and no blood supply and / or central nervous system control; in particular, engineered skeletal muscle tissue wherein the presence of skeletal muscle fibers is determined by staining for actinin and using DAPI. 61. The engineered skeletal muscle tissue of embodiment 60, wherein the skeletal muscle tissue is serum-free and / or does not comprise a differentiation-associated transgene or a maturation-associated transgene, preferably wherein the skeletal muscle tissue does not comprise a myogenic transgene, more preferably wherein the skeletal muscle tissue does not comprise the transgene Pax7 or MyoD. 62. The engineered skeletal muscle tissue of embodiment 60 or embodiment 61, wherein 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, upon stimulation at 100 Hz at 200 mA. 63. The engineered skeletal muscle tissue of any of embodiments 60-62, wherein the skeletal muscle tissue has been engineered to have an engineered form, preferably in the form of a ring, ribbon, strand, patch, pouch, or cylinder, and optionally wherein individual skeletal muscle tissues are fused together, particularly wherein the skeletal muscle tissue has the form of a ring. 64. Mesodermally differentiated skeletal myoblast progenitor cells, obtained according to step (i) of embodiment 1 or embodiment 38, and prepared by a method of embodiment 1(i) or embodiment 38(i), and characterized by expression of genes MSGN1 and / or TBX6, wherein expression of MSGN1 and / or TBX6 can be determined by flow cytometry and / or immunostaining; and / or wherein mRNA SP5 is expressed, wherein expression of SP5 can be determined by RNA sequencing. 65. Myogenically committed skeletal myoblast progenitor cells, obtained according to step (ii) of embodiment 1 or embodiment 38, and produced by a method of embodiment 1(i)-(ii) or embodiment 38(i)-(ii), and characterized by expression of the gene PAX3, wherein the expression of PAX3 can be determined by flow cytometry and / or immunostaining; and / or in which mRNA SIM1 is expressed, wherein the expression of SIM1 can be determined by RNA sequencing. 66. A skeletal myoblast, obtained according to step (iii) of embodiment 1 or embodiment 38, or produced by a method of embodiment 1(i) to (iii) or embodiment 38(i) to (iii), characterized by expression of actinin, preferably wherein the expression of actinin can be determined by flow cytometry and / or immunostaining. 67. Satellite cells obtained according to step (iii) of embodiment 1 or embodiment 38, and produced by a method of embodiment 1(i) to (iii) or embodiment 38(i) to (iii), and characterized by expression of the gene Pax7, wherein the expression of Pax7 can be determined by flow cytometry and / or immunostaining, more preferably wherein the satellite cells further express Ki67. 68. A mixture of skeletal myoblasts of embodiment 66 and satellite cells of embodiment 67, wherein a proportion of satellite cells of the total amount of available cells is obtained that is at least 10%, preferably at least 15%, more preferably at least 20%, and even more preferably at least 30%, as determined by the expression of Pax7 by flow cytometry; and / or a proportion of skeletal myoblasts of the total amount of cells present that is at least 40%, preferably at least 50%, more preferably at least 60%, and most preferably at least 70%, as determined by the expression of actinin by flow cytometry. 69. A skeletal myotube cell obtained according to step (iv) of embodiment 1 or embodiment 38 and prepared by a method of embodiment 1(i) to (iv) or embodiment 38(i) to (iv), wherein the skeletal myotube cell is characterized by anisotropic orientation of actinin protein-containing sarcomere structures. 70. Use of skeletal muscle tissue of any of embodiments 60 to 63, and / or cells of any of embodiments 64 to 68, and / or 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 pharmacological drug candidates and gene therapy drug candidates. 71. Skeletal muscle tissue according to any of embodiments 60 to 63 and / or a cell according to any of embodiments 64 to 68, and / or a skeletal myotube cell according to embodiment 69, for use in medicine. 72. Satellite cells according to embodiment 67 for use in therapy of damaged skeletal muscle and / or in the treatment of a skeletal muscle disease, preferably a genetic skeletal muscle defect, in particular Duchenne muscular dystrophy and / or Becker-Keener muscular dystrophy, and / or a lysosomal storage disease, in particular Pompe disease, preferably wherein the skeletal muscle disease is Duchenne muscular dystrophy. 73. An in vitro method for testing the effectiveness of a drug candidate on skeletal muscle tissue, comprising: (a) providing skeletal muscle tissue according to any one of aspects 60 to 63; (b) optionally damaging skeletal muscle tissue; and (c) contacting the skeletal muscle tissue from step (a) or (b) with a drug candidate. Includes; Preferably, the method further comprises the step of determining contractile force and / or skeletal muscle tissue structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters before and / or after step (c), method. 74. An in vitro method for testing the toxicity of a substance to skeletal muscle tissue, comprising: (a) providing skeletal muscle tissue according to any one of aspects 60 to 63; (b) contacting the skeletal muscle tissue from step (a) with the substance to be tested. Including, Preferably, the method further comprises the step of determining contractile force and / or skeletal muscle tissue structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters before and / or after step (b), method. 75. An in vitro method for testing the effects of nutrients and dietary supplements on skeletal muscle tissue performance, comprising: (a) providing skeletal muscle tissue according to any one of aspects 60 to 63; (b) contacting the skeletal muscle tissue from step (a) with the nutrient or dietary supplement to be tested. Including, Preferably, the method further comprises the step of determining contractile force and / or skeletal muscle tissue structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters before and / or after step (b), method. 76. An in vitro method for testing the effectiveness of a drug candidate on mesodermally differentiated skeletal myoblast precursor cells, myogenically specialized skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, or a mixture of skeletal myoblasts and satellite cells, comprising: (a) providing a mesodermally differentiated skeletal myoblast precursor cell, a myogenically committed skeletal myoblast precursor cell, a satellite cell, a skeletal myoblast, a skeletal myotube cell, or a mixture of skeletal myoblasts and satellite cells according to any one of embodiments 64 to 69; (b) optionally damaging the cells from step (a); and (c) contacting the cells of step (a) or (b) with a drug candidate. Including, Preferably, the method further comprises determining the expression of actinin and / or Pax7 before and / or after step (c), wherein said expression may be determined by flow cytometry and / or immunostaining. method. 77. An in vitro method for testing the toxicity of a substance to mesodermally differentiated skeletal myoblast precursor cells, myogenically specialized skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, or a mixture of skeletal myoblasts and satellite cells, comprising: (a) providing a mesodermally differentiated skeletal myoblast precursor cell, a myogenically committed skeletal myoblast precursor cell, a satellite cell, a skeletal myoblast, a skeletal myotube cell, or a mixture of skeletal myoblasts and satellite cells according to any one of embodiments 64 to 69; (b) contacting the cells of step (a) with the substance to be tested. Including, Preferably, the method further comprises determining the expression of actinin and / or Pax7 before and / or after step (b), wherein said 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 mesodermally differentiated skeletal myoblast precursor cells, myogenically specialized skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, or a mixture of skeletal myoblasts and satellite cells, comprising: (a) providing a mesodermally differentiated skeletal myoblast precursor cell, a myogenically committed skeletal myoblast precursor cell, a satellite cell, a skeletal myoblast, a skeletal myotube cell, or a mixture of skeletal myoblasts and satellite cells according to any one of embodiments 64 to 69; (b) contacting the cells of step (a) with the nutrient or dietary supplement to be tested. Including, Preferably, the method further comprises determining the expression of actinin and / or Pax7 before and / or after step (b), wherein said expression can be determined by flow cytometry and / or immunostaining. method. 79. The method of any 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, upon stimulation at 100 Hz. 80. The method of any of embodiments 1-59 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, upon stimulation at 100 Hz. 81. The method of any of aspects 1-59, 79, or 80, wherein the skeletal muscle tissue has a contraction velocity 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 at 100 Hz stimulation. 82. The method of any of aspects 1-59 or 79-81, wherein the skeletal muscle tissue has a relaxation rate at the end of the 100 Hz stimulation 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. 83. The method of any of aspects 1-59 or 79-82, wherein the basal medium in step (iv) comprises an effective amount of creatine and / or triiodo-L-thyronine (T3). 84. The method of embodiment 83, wherein an effective amount of creatine in the basal medium increases the contractile force of the engineered skeletal muscle compared to maturation in step (iv) without the effective amount of creatine. 85. The method of embodiment 83 or 84, wherein an effective amount of T3 in the basal medium shortens the contraction rate and / or relaxation rate of the engineered skeletal muscle compared to maturation in step (iv) without the effective amount of T3. 86. The method of any of aspects 1-59 or 79-85, wherein the basal medium in step (iv) provides a final concentration of 0.1-10 mM creatine, preferably 0.2-6 mM creatine, more preferably 0.4-4 mM creatine, even more preferably 0.6-3 mM creatine, even more preferably 0.7-2.5 mM creatine, even more preferably 0.8-2 mM creatine, even more preferably 0.85-1.5 mM creatine, even more preferably 0.9-1.2 mM creatine, and most preferably about 1 mM creatine. 87. The method of any of aspects 1-59 or 79-86, wherein the basal medium in step (iv) provides a final concentration of 0.001-1 μM triiodo-L-thyronine (T3), preferably 0.005-0.7 μM T3, more preferably 0.01-0.35 μM T3, even more preferably 0.04-0.02 μM T3, even more preferably 0.05-0.18 μM T3, even more preferably 0.06-0.15 μM T3, even more preferably 0.08-0.12 μM T3, and even more preferably about 0.1 μM T3. 88. The method of any of aspects 1-59 or 79-87, wherein the skeletal muscle tissue has the property of self-regenerating. 89. The method of embodiment 88, wherein the regenerative properties are characterized by restored contractility and / or muscle recovery, preferably, said restored contractility and / or muscle recovery is measured after irreversible muscle damage using a cardiotoxin, more preferably, said restored contractility and / or muscle recovery is measured 10 to 30 days after incubation with the cardiotoxin. 90. The method of any of embodiments 1-59 or 79-89, 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. Engineered skeletal muscle tissue produced by the method of any of aspects 1-59 or 79-90. 92. The engineered skeletal muscle tissue of any of aspects 60-63 or 91, wherein at 100 Hz stimulation, the engineered skeletal muscle tissue produces 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. 93. The engineered skeletal muscle tissue of any of aspects 60-63 or 91-92, having a contraction velocity at 100 Hz stimulation 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. 94. The engineered skeletal muscle tissue of any of aspects 60-63 or 91-93, having a relaxation rate at the end of 100 Hz stimulation 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. 95. Use of skeletal muscle tissue of any of embodiments 60 to 63 or 91 to 94, and / or cells of any of embodiments 64 to 68, and / or 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 pharmacological drug candidates and gene therapy drug candidates. 96. Skeletal muscle tissue according to any of embodiments 60 to 63 and 91 to 94, and / or a cell according to any of embodiments 64 to 68, and / or a skeletal myotube cell according to embodiment 69, for use in medicine. [Brief explanation of the drawings]

[0142] [Figure 1] Schematic of differentiation protocol in 2D cell culture. Differentiation protocol for directed differentiation of human pluripotent stem cells (hPSCs) into skeletal myoblasts and satellite cells in 2D. hPSCs are seeded the day before. For mesoderm induction, cells are cultured from day 0 to day 4 in mesoderm induction medium (DMEM supplemented with pyruvate and 1 g / L glucose) containing CHIR-99021, LDN193189, and FGF-2. For myogenic specification, cells are cultured from day 4 to day 6 in medium containing DAPT and FGF-2, followed by culture from day 6 to day 8 in medium containing DAPT, FGF-2, and HGF, followed by culture from day 8 to day 12 in medium containing DAPT, HGF, and "KnockOut Serum Replacement" (KSR). For myogenic expansion and maturation, cells are cultured from day 12 to day 21 in medium containing HGF and KSR. For myogenic maturation, cells are cultured in a medium containing albumin, transferrin, ethanolamine, selenium, carnitine, fatty acids, and T3 starting on day 21. Additionally, the medium contains serum-free supplement N-2 from day 0 to day 21. [Figure 2]Schematic of cell differentiation from pluripotent stem cells to myotubes and satellite cells. Schematic representation of the differentiation of pluripotent stem cells into myotubes, including the following stages: (i) pluripotent stem cells, (ii) unsegmented mesoderm cells, (iii) myoblasts with satellite cells, (iv) myotubes with satellite cells, and (v) myotubes with a satellite cell niche. Additionally, the order of expression of marker genes during different stages of differentiation is shown. Oct4 expression is characteristic of pluripotent stem cells. MSGN1 and Tbx6 expression is characteristic of unsegmented mesoderm cells. Pax3 is primarily expressed during the transition from unsegmented mesoderm cells to myoblasts. Pax7 expression is characteristic of the presence of satellite cells and is first expressed at the end of the unsegmented mesoderm stage and the beginning of the myoblast stage. Pax7 expression is highest in the myoblast stage and flattens out by the myotube stage, remaining as a sign of satellite cell niche formation. MyoD expression is strongest in myoblasts and is also detectable in myotubes. Myogenin and actinin expression are characteristic of myotubes and are barely expressed in myoblasts. Myotubes form the satellite cell niche, the muscle stem cell niche. The satellite cell niche is Pax7-positive and quiescent. The cell cycle is activated upon muscle injury, at which point cells are also Ki67-positive. [Figure 3] Fluorescence microscopy of skeletal muscle cells and satellite cells. Immunostaining of myogenic cells in a representative cell culture after 21 days (Example 1). Fluorescence images show the expression of skeletal muscle-specific transcription factors PAX7 (top row left), MyoD (middle row left), and myogenin (bottom row left). PAX7 detects satellite cells, while MyoD and myogenin detect skeletal myoblasts and / or skeletal myotubes. Additionally, the fluorescence images show cell nuclei (nuclei, right column) and actin expression (middle column). Scale: 100 μm. [Figure 4A]Gene expression patterns during differentiation of human pluripotent stem cells (hPSCs) into skeletal muscle cells analyzed by RNA sequencing. Directed differentiation exhibits gene expression patterns similar to embryonic skeletal muscle development. Expression values ​​(reads per million kilobases, RPKM) of genes typical of pluripotent and paraxial mesoderm are shown graphically over time for differentiation and maturation (Figure 4A and Figure 4B). In RNA sequencing, genes typical of pluripotency, such as NANOG, POU5F1, and ZFP42, show high expression on days 0 and 1. NANOG and POU5F1 show highest expression on day 0, and ZFP42 shows highest expression on day 1. Genes typical of paraxial mesoderm, such as MSGN1, TBX6, and MEOX1, show high expression on days 1–8. MSGN1 shows highest expression on day 1, TBX6 shows highest expression on day 4, and MEOX shows highest expression on day 8. The expression values ​​(reads per million kilobases, RPKM) of skeletal muscle-specific transcription factors and sarcomere-typical genes over the time course of differentiation and maturation are shown graphically (Figures 4C and 4D). Skeletal muscle-specific transcription factors, such as PAX3, PAX7, and MYOD1, show highest expression at days 8, 29, and 60, respectively. Sarcomere-typical genes, such as ACTN2, DMD, and MYH3, show highest expression at day 60. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 5]Analysis of the efficiency of directed differentiation of human pluripotent stem cells (hPSCs) into skeletal muscle cells. Flow cytometry was used to determine the percentage of muscle cells (positive for myogenin or MyoD in addition to actinin) and satellite cells (positive for PAX7) from four independent pluripotent stem cell lines (iPSCs (WT 1), iPSCs (WT 2), DMD iPSCs, and corrected DMD iPSCs). The percentage of actinin-positive cells ranged from 71% to 77.6% in the four cell lines, the percentage of myogenin-positive cells ranged from 41.4% to 60.4% in the four cell lines, the percentage of MyoD-positive cells ranged from 40% to 54.1% in the four cell lines, and the percentage of PAX7-positive cells ranged from 33.4% to 43.8% in the four cell lines. [Figure 6]Production of engineered skeletal muscle tissue (ESM) from hPSC-derived skeletal myoblasts. (A) Schematic of the ESM culture protocol. 21-day-old cell pools from Example 1 are poured into extracellular matrix (collagen / Matrigel) and cultured in ring-shaped molds (left panel) under extended growth conditions for 7 days. The formed rings are then transferred to a stretching device (center image) and further cultured under maturation conditions. After a further 4 weeks, ESM function is measured in organ baths (right image); scale: 5 mm. (B) Representative contraction force curves of engineered skeletal muscle tissue at different stimulation frequencies: 1 Hz (dashed line: eight twitches with a single duration of approximately 500 ms each with a force of contraction ("FOC") of approximately 0.5 millinewtons), 10 Hz (solid line: initial tetanic contraction with a force of contraction ("FOC") of approximately 1 millinewton, individual contractions are graphically distinguishable), 100 Hz (dashed-dotted line: fully developed tetanic contraction with a force of contraction ("FOC") of approximately 2.2 millinewtons). (C) Contractile force ("FOC") in milliNewtons (mN) of skeletal muscle tissue depending on electrical stimulation frequency; n=3. For 1 Hz stimulation, the average contractile force was 0.5 milliNewtons; for 10 Hz stimulation, the average contractile force was 0.9 milliNewtons; for 20 Hz stimulation, the average contractile force was 1.1 milliNewtons; for 40 Hz stimulation, the average contractile force was 1.4 milliNewtons; for 60 Hz stimulation, the average contractile force was 1.55 milliNewtons; for 80 Hz stimulation, the average contractile force was 1.6 milliNewtons; and for 100 Hz stimulation, the average contractile force was 2.1 milliNewtons. [Figure 7A]Fabrication of bioengineered skeletal muscle (BSM) from hPSCs. (A) Schematic of the BSM culture protocol. Human induced pluripotent stem cells are cast into ring-shaped molds in collagen / Matrigel hydrogel and differentiated into skeletal muscle tissue in 3D. The formed rings are transferred to a stretching apparatus on day 21 and further cultured under maturation conditions. After an additional 4 weeks, BSM function is measured, typically in an organ bath. Specifically, for this purpose, human induced pluripotent stem cells are first dispersed in collagen / Matrigel hydrogel and conditioned for 24 h in Brew XF containing Y-27632 and KSR (day -1). The cells are then cultured from day 0 to day 4 in medium containing CHIR-99021, LDN193189, and FGF-2. Cells are cultured in medium containing DAPT and FGF-2 from day 4 to day 6, followed by culture in medium containing DAPT, FGF-2, and HGF from day 6 to day 8, followed by culture in medium containing DAPT, HGF, and "KnockOut Serum Replacement" (KSR) from day 8 to day 12. Cells are cultured in medium containing HGF and KSR from day 12 to day 21. From day 21 to day 50, cells are cultured in maturation medium on a static stretching apparatus, i.e., under mechanical stretching. Additionally, from day 0 to day 50, the medium contains serum-free additive N-2. (B) Representative contractile force curves of engineered skeletal muscle tissue at different stimulation frequencies: 1 Hz (dashed line: eight twitches with a single duration of approximately 600 ms, each with a force of contraction ("FOC") of approximately 0.7 millinewtons) and 100 Hz (solid line: fully developed tetanic contraction with a force of contraction ("FOC") of approximately 1.1 millinewtons). (C) Force of contraction ("FOC") in millinewtons (mN) of skeletal muscle tissue depending on electrical stimulation frequency; n=3.For 1 Hz stimulation, the average contraction force is 0.3 millinewtons; for 10 Hz stimulation, the average contraction force is 0.5 millinewtons; for 20 Hz stimulation, the average contraction force is 0.55 millinewtons; for 40 Hz stimulation, the average contraction force is 0.6 millinewtons; for 60 Hz stimulation, the average contraction force is 0.65 millinewtons; for 80 Hz stimulation, the average contraction force is 0.72 millinewtons; and for 100 Hz stimulation, the average contraction force is 0.9 millinewtons. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 8] Fluorescence microscopy of skeletal muscle tissue produced by ESM and BSM methods. Immunostaining of actin and DNA in representative skeletal muscle tissue prepared by ESM (Examples 1 and 2) and BSM (Example 3) methods. Fluorescent images show multinucleated mature skeletal muscle fibers characterized by a characteristic striped pattern (actin staining). Scale: 50 μm (ESM) and 10 μm (BSM). [Figure 9] Enhancement of ESM function by creatine treatment. A) Experimental procedure for ESM maturation over 5 or 9 weeks with the addition of 1 mM creatine over 4 weeks. B) Force of contraction (FOC) in response to 100 Hz field stimulation in ESM after 5 and 9 weeks of culture; cultures were as shown in A with (right bar) or without (left bar) the addition of creatine; n=3 / group; *p<0.05 Student's t-test. [Figure 10A]Enhancement of ESM function by thyroid hormone treatment. A) Experimental scheme of ESM maturation over 5 or 9 weeks with additional administration of 0.1 μmol / L triiodo-L-thyronine (T3) over 4 weeks. (B) Representative curves showing the maximum velocity of contraction (+dFOC / dt) and relaxation (-dFOC / dt) during and after 100 Hz electrical field stimulation. Comparison of ESM treated with (gray) and without (black) T3 at 5 weeks (day 56) and 9 weeks (day 84); n = 4–11 / group; *p < 0.05 Student's t-test. C) Protein content of myosin heavy chain protein 2 (MYH2; fast isoform), myosin heavy chain protein 7 (MYH7; slow isoform), and myosin heavy chain protein 3 (MYH3; embryonic isoform) in 9-week ESM cultures with (gray bars) and without (black bars) T3; n = 3 / group; *p < 0.05 Student's t-test. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 11A]Regenerative capacity of engineered skeletal muscle. A) RNA detection by RNA sequencing (reads per million kilobases, RPKM) of skeletal muscle progenitor / stem cell markers in ESM (prepared from 2D cultures at day 22) as well as in 2D cultures at day 22 and day 60. *p<0.05 by one-way ANOVA and Tukey's multiple comparison test. B) Immunofluorescent staining of skeletal muscle progenitor cells (Pax7) in ESM (left) and 2D cultures (right) at day 60: Pax7 (bright nuclei), laminin, f-actin (elongated muscle cell bodies), and nuclei; bar: 20 μm. Enlarged views show satellite cell niches (skeletal muscle cell precursors) in ESM and 2D cultures. C) Experimental scheme of cardiotoxin (CTX) injury. ESM was incubated with 25 μg / ml CTX for 24 hours. The irradiated group was treated with 30 Gy (gamma irradiation) 24 hours after CTX injury to inhibit cell proliferation and associated regeneration. D) Force of contraction (FOC) at 100 Hz tetanic contraction of ESM without (left bar) or with (right bar) gamma irradiation at the indicated time points after CTX injury (25 μg / ml) or vehicle treatment (Vehicle); n = 3-4, *p < 0.05 vs. control + 2 days, *p < 0.05 by one-way ANOVA and Tukey's multiple comparison test. E) Immunostaining of sarcomeric actinin and nuclei in ESM 21 days after CTX injury in groups with and without gamma irradiation according to the scheme in A. Muscle growth in the non-irradiated control group is due to the proliferation and differentiation of new muscle cells from muscle cell precursors in the ESM. Bar: 50 μm. [Figure 11B] See legend to Figure 11A. [Figure 11C] See legend to Figure 11A. [Figure 11D] See legend to Figure 11A. [Figure 11E] See legend to Figure 11A. [Example]

[0143] The following examples are intended to further illustrate the present invention, but are not intended to limit the present invention. The examples describe technical features, and the present invention also relates to the combination of 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. We have developed a method for the directed differentiation of induced pluripotent stem cells into skeletal muscle cells and satellite cells in 2D cell culture. The method described here is transgene-free and serum-free. Human skeletal myoblasts, skeletal myotubes, and satellite cells can be produced with high purity using 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 gene expression patterns. 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 Figure 1, which shows the sequential addition of different agents to the culture medium. Additionally, FIG. 1 shows the differentiation stages experienced during differentiation into skeletal myoblasts / myotubes and satellite cells: induction of mesodermal differentiation, induction of myogenic specification, (myogenic) expansion and maturation into skeletal myoblasts and satellite cells, and maturation into skeletal myotubes and satellite cells.

[0145] To perform the method, 1.7 x 10 human pluripotent stem cells were cultured. 4 cells / cm 2Cells were plated the previous day onto Matrigel-coated plates at a density of 1000 kJ / ml and cultured in the presence of 12 ml of StemMACS™ iPS-Brew XF medium containing 5 μM Rock inhibitor (Stemolecule Y27632) (methods for coating Matrigel onto cell culture plates are provided at the end of Example 1) until the cell cultures were approximately 30% confluent the following 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 days 0, 1, 2, and 3, the medium was replaced with 15 ml of N2-FCL medium, which was changed daily. N2-FCL medium: DMEM supplemented with pyruvate containing 1 g / L glucose and L-alanyl-L-glutamine (GlutaMAX™) (Gibco), 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), and 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 media was replaced with N2-FD media and changed daily. N2-FD media: DMEM supplemented with pyruvate containing 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), 20 ng / ml recombinant bFGF (Peprotech), and 10 μM DAPT (TOCRIS).

[0148] On days 6 and 7, the medium was replaced with N2-FHD medium, which was changed daily. N2-FHD medium: DMEM supplemented with pyruvate, 1 g / L glucose and L-alanyl-L-glutamine (GlutaMAX™) (Gibco), 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 μM DAPT (TOCRIS), and 10 ng / ml recombinant HGF (Peprotech).

[0149] On days 8, 9, 10, and 11, the medium was replaced with N2-HKD medium, which was changed daily. N2-HKD medium: DMEM supplemented with pyruvate containing 1 g / L glucose and L-alanyl-L-glutamine (GlutaMAX™) (Gibco), 1% Pen / Strep (Invitrogen), 1% serum-free additive 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), and 10% KnockOut Serum Replacement (Life Technologies).

[0150] Cells were cultured in N2-HK medium to allow myogenic expansion and maturation into skeletal myoblasts and satellite cells. From 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 pyruvate, 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), and 10% KnockOut Serum Replacement (Life Technologies).

[0151] From day 21, the cells were 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 pyruvate containing 1 g / l glucose and L-alanyl-L-glutamine (GlutaMAX™) (Gibco), 1% Pen / Strep (Invitrogen), 1% serum-free additive N-2 (Thermo Scientific), and 1% B27 serum-free additive (Invitrogen). Skeletal myoblasts, skeletal myotubes, and satellite cells were generated by further culturing on cell culture plates.

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

[0153] In particular, we measured the expression of genes specific to pluripotency, paraxial mesoderm, skeletal muscle-specific transcription factors, and sarcomeres. Genes typical of pluripotency, such as NANOG, POU5F1, and ZFP42, showed high expression on days 0 and 1 (the day after the seeding process and the day after) (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 paraxial mesoderm, such as MSGN1, TBX6, and MEOX1, showed high expression on days 1–8 (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, show the highest expression at day 60 (Figure 4d). Furthermore, the gene expression pattern shows a steep increase or decrease in different markers, especially during the first 21 days. For example, TBX6 and MEOX1 are strongly expressed only on days 4 and 8, respectively, while expression is at least fourfold 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, we analyzed cells using fluorescence microscopy after 21 days of differentiation. This involved staining cellular DNA with Hoechst and immunostaining for actin and skeletal muscle-specific transcription factors (Pax7, MyoD, and myogenin). After 21 days, fluorescence images showed a high percentage of cells expressing Pax7, MyoD, and myogenin (Figure 3). Therefore, using another method, we demonstrated that a 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. Flow cytometry, used here, uses immunostaining to measure the expression of skeletal muscle-specific factors. Specifically, the percentages of skeletal myoblasts and skeletal myotubes (expression of markers actinin, myogenin, and MyoD) as well as satellite cells (expression of marker PAX7) were determined in four independent pluripotent stem cell lines (iPSC(WT 1), iPSC(WT 2), DMD iPSC, and corrected DMD iPSC) (Figure 5). The percentages of actinin-positive cells ranged from 71% to 77.6% in the four cell lines, the percentages of myogenin-positive cells ranged from 41.4% to 60.4% in the four cell lines, the percentages of MyoD-positive cells ranged from 40% to 54.1% in the four cell lines, and the percentages of PAX7-positive cells ranged from 33.4% to 43.8% in the four cell lines (Figure 5).

[0156] Flow cytometry also showed that the analyzed cells produced skeletal myoblast- and myotube-specific markers as well as satellite cell-specific markers with high purity (>70% actinin-positive and >30% PAX7-positive myocytes).

[0157] Therefore, using three different methods, pluripotent stem cells were measured to differentiate into a skeletal myoblast-containing cell pool that therefore undergoes mesoderm induction, myogenic specification, and myogenic maturation.

[0158] material and method 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), and 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) and causes the disease.

[0159] To prepare Matrigel-coated cell culture plates, BD Matrigel (Basement Membrane Matrix Growth Factor Reduced) was diluted 1:30 in ice-cold PBS and immediately stored at 4°C. To prepare Matrigel-coated plates, a 1:120 dilution of Matrigel was made with ice-cold PBS. 0.1 ml / cm 2 Dilutions of 1000 mg ...

[0160] For passaging (e.g., to detach cells for cryopreservation), 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. The TrypLE was then washed away, and digestion was stopped with 10 ml of N2-HK medium containing 5 μM Rock inhibitor. To induce clumping, the cell suspension was pipetted using a 10 ml pipette. Cell detachment must 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 N2-HK medium containing 5 μM Rock inhibitor. Cells were plated onto Matrigel-coated plates at 60–70,000 cells / cm. 2 The cells were plated in N2-HK medium containing 5 μM Rock inhibitor at a density of 100 μg / ml. Starting the next day, the N2-HK medium was replaced every other day for 9 days.

[0161] For cell freezing (e.g., day 21, cryopreservation), 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. The TrypLE was then washed away, and digestion was stopped with 10 ml of N2-HK medium containing 5 μM Rock inhibitor. To induce clumping, the cell suspension was pipetted using a 10 ml pipette. Cell detachment must 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 N2-HK medium containing 5 μM Rock inhibitor and 10% DMSO (Sigma) at 4 °C. 10 × 10 6 Cells were frozen overnight at -80°C in 2 ml per cryovial using Mr Frosty (Thermo). Cells were then transferred to -150°C.

[0162] For RNA extraction, cell lysates embedded in Trizol reagent (Thermo Fisher) were homogenized by vortexing. 200 μl of chloroform (AppliChem) was added for every 1 ml of Trizol reagent. The reagent tube was tightly closed, inverted five 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 the RNA was transferred to a new reagent tube, followed by the addition of 500 μl of isopropanol (Roth) to precipitate the RNA. The reagent tube was vortexed, allowed to stand at room temperature for 10 minutes, and then centrifuged at 12,000 × g for an additional 10 minutes. The supernatant was removed, and the pellet was washed by adding 1 ml of 70% EtOH / diethylpyrocarbonate (DEPC) H2O. After gently tapping the reagent tube to dissolve and wash the pellet, the sample was centrifuged once more at 12,000 × g for 5 minutes, and the supernatant was removed. The pellet was left open for 5–10 min until the residual liquid evaporated, and the RNA was resuspended in DEPC HO. RNA concentration and quality were determined using a Nanodrop ND-1000. Prior to sequencing, quality and RNA integrity were further analyzed using an Advanced Analytical Fragment Analyzer (Standard Sensitivity RNA Analysis Kit (DNF-471)). RNA-Seq libraries were generated using a modified strand-specific massively parallel cDNA sequencing (RNA-Seq) protocol (Illumina: TruSeq Stranded Total RNA (Cat. No. RS-122-2301)). The protocol was optimized to keep rRNA content in the dataset below 5% (RiboMinus™ technology). The remaining whole-transcriptome RiboMinus™ RNA was suitable for direct sequencing. The ligation step was optimized to increase ligation efficiency (>94%), and the PCR protocol was adjusted for optimal final production of the library.For accurate quantification of the cDNA library, a fluorometric-based system, Promega's quantiFluor™ dsDNA system, was used. The size of the final cDNA library was determined using the dsDNA 905 Reagent Kit (Advanced Bioanalytical's Fragment Analyzer), with an average size of 300 bp.

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

[0164] For flow cytometry, single-cell suspensions were prepared by digesting cells with TrypLE Select (Thermo Fisher). Cells were resuspended in culture medium, centrifuged at 300 g for 5 minutes, and fixed in 4% formalin (Histofix, Roth). After fixation, 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, 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 the appropriate IgG1 isotype control for 45 minutes at 4°C.

[0165] Cells were washed twice with PBS, followed by a wash step in blocking buffer, and then incubated with secondary antibodies (anti-mouse 488 [A-11001] or 633 [A-21052] at 1:1000, Thermo Fisher) and Hoechst (10 ng / ml; Thermo Fisher) for 30 minutes at 4°C. 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 pluripotent stem cell-derived skeletal myoblasts and satellite cells (cells from Example 1) To construct engineered skeletal muscle tissue, the cells obtained in Example 1 (cells from day 21) were used as starting materials and mixed with an extracellular matrix. By mixing with the extracellular matrix, the cells were dispersed in the matrix to generate three-dimensional skeletal muscle tissue. This method is also serum-free and transgene-free. This increases the reproducibility of skeletal muscle tissue production because all required substances and their concentrations are defined. This method can generate force-generating skeletal muscle tissue that contracts in a controlled manner in response to electrical stimulation. A specific temporal sequence of agents and physical stimuli was used, which is shown diagrammatically in Figure 6A and described in detail below.

[0167] To construct engineered skeletal muscle tissue, cells from Example 1 (cells from day 21) were mixed with extracellular matrix and cast into ring molds to support self-assembly of the cells into contractile skeletal muscle. This means that either (a) cells were dissociated from differentiated cell cultures according to Example 1, or (b) frozen cells from Example 1 were used (see below for a detailed description of how to thaw 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 pipette addition order was followed: TIFF2025134825000001.tif60149

[0169] Alternatively, the master mix was pipetted according to the following volumes: TIFF2025134825000002.tif60149

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

[0171] The cells were cultured in this expansion medium for 7 days. On days 1, 3, and 5, the medium was replaced with fresh expansion medium (N2-HK medium; without Rock inhibitors). After casting, the mixture was compressed in a ring mold, so that the mixture was fully compressed after 24 hours.

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

[0173] The maturation medium was changed every other day for the following 6 weeks of maturation to allow the cells to mature into skeletal myotubes and satellite cells.

[0174] To experimentally test the production of engineered skeletal muscle tissue, the generated skeletal muscle tissue was analyzed using fluorescence microscopy. The characteristic striped pattern demonstrates the formation of multinucleated skeletal muscle fibers to produce force-generating skeletal muscle.

[0175] We used immunostaining to visualize the structural protein actin of the eukaryotic cytoskeleton and stained DNA in the nucleus with the dye DAPI. Fluorescent images showed a characteristic striped 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, we performed contraction experiments (Figure 6A, right panel). These contraction experiments in organ baths measure the contraction frequency and force of the engineered skeletal muscle tissue in response to electrical stimulation.

[0177] For this purpose, skeletal muscle tissue in the form of rings was isometrically transferred to an organ bath (Fohr Medical Instruments) containing Tyrode's solution (in mmol / L: 120 NaCl, 1 MgCl, 1.8 CaCl, 5.4 KCl, 22.6 NaHCO, 4.2 NaHPO, 5.6 glucose, and 0.56 ascorbate) at 37 °C with continuous gassing at 5% CO and 95% O. The ESM was mechanically stretched at 125 µm intervals until a maximum force amplitude (force of contraction = FOC) was observed. FOC measurements were performed at electrical field stimulation frequencies (4 ms rectangular pulses; 200 mA) ranging from 1 to 100 Hz.

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

[0179] The tested skeletal muscle tissues exhibited reproducible contraction frequencies and forces in response to stimulation frequencies ranging from 1 Hz to 100 Hz. With a single 1 Hz stimulation, contraction and complete relaxation took approximately 0.5 seconds. Because the contraction and relaxation time was approximately 0.5 seconds, onset or full tetanus was recorded at higher stimulation frequencies. Because tetanus also forms in native skeletal muscle tissue with increased stimulation frequency, engineered skeletal muscle tissue behaves similarly to native skeletal muscle tissue in this respect. Furthermore, we were able to demonstrate that the contractile force of muscle tissue increases with increasing 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 twitching and tetanus. In contrast to engineered skeletal muscle tissue, electrical impulses in native muscle tissue are triggered by neurotransmitter stimulation (acetylcholine) from the motor endplate.

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

[0181] material and method To dissociate cells from the cell culture (volume listed for a T75 cell culture flask), 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 washed away, and digestion was stopped with 10 ml of Expansion Growth Medium containing 5 μM Rock inhibitor. To induce clumping, the cell suspension was triturated using a 10 ml pipette. Cell detachment must be gentle enough not to reduce cell viability. Cells were counted using a CASY counter (by adding 20 μl of the 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 the appropriate volume of Expansion Growth Medium containing 5 μM Rock inhibitor (see Master Mix), 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 placed 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 room-temperature expansion medium containing 5 μM Rock inhibitor, and the expansion medium was added dropwise to the cells to avoid osmotic shock. Another 8 ml of expansion medium containing 5 μM Rock inhibitor was slowly added. To avoid cell damage, the suspension was pipetted up and down no more than twice 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. Remove the supernatant and gently resuspend the pellet in an appropriate volume of Expansion Medium containing 5 µM Rock inhibitor to prepare a defined volume of cell suspension, depending on the number of ESMs (see Master Mix). Place the cell suspension on ice.

[0183] Example 3: Production of engineered skeletal muscle tissue (bioengineered skeletal muscle, BSM) from pluripotent stem cells In this example, engineered skeletal muscle tissue (BSM) was constructed using pluripotent stem cells and an extracellular matrix. In contrast to Examples 1 and 2, no transfer from a Matrigel-coated cell culture plate to the extracellular matrix was performed in the preparation of BSM. Instead, human induced pluripotent stem cells were directly dispersed / embedded in a defined extracellular matrix. Self-assembly of pluripotent stem cells into skeletal muscle tissue was supported in the extracellular matrix in the presence of chemical and physical stimuli. This method is also serum-free and transgene-free, so all required substances and their concentrations were defined. Therefore, differentiation and maturation of human pluripotent stem cells into skeletal myotubes and satellite cells (skeletal muscle fibers) was controlled.

[0184] A schematic of the differentiation protocol is shown in Figure 7A, which shows a series of different agents added to the culture medium and physical stimulation with a stretching device. During the process depicted in Figure 7A, mesodermal differentiation was induced (days 0-4), myogenic specification was induced (days 4-12), cells matured into skeletal myoblasts and satellite cells (days 12-21), and finally, cells matured into skeletal myotubes and satellite cells (days 21-50).

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

[0186] To mix the human pluripotent stem cells with collagen / Matrigel and pour into the ring mold, the master mix was mixed in a 50 ml reaction tube on ice. The collagen was added using a 2 ml pipette, following the exact pipette addition order: TIFF2025134825000003.tif60128

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

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

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

[0190] Between days 12 and 20, the medium was replaced with N2-HK medium (Expansion Medium) and changed every other day (see Example 1 for composition). Culturing in Expansion Medium allowed the cells to mature into skeletal myoblasts.

[0191] On day 21, the formed rings were transferred to a 6-well plate in a stretching apparatus and further cultured under maturation conditions. There, the cells were further cultured under physical stimulation, i.e., mechanical stretching. Additionally, 5 ml of maturation medium was added per well to induce cell maturation with maturation medium (see Example 2 for the composition of maturation medium). The maturation medium was replaced every other day during the following 4 weeks of maturation to allow the cells to mature into skeletal myotubes 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 DNA in the nucleus was stained with the dye DAPI. As in Example 2, the fluorescence images showed a characteristic striped pattern, demonstrating the formation of multinucleated mature skeletal muscle fibers ( FIG. 8 b). Thus, BSM also exhibits multinucleated mature skeletal muscle fibers formed by this method.

[0193] Furthermore, to functionally test the artificially generated muscle tissue, we performed contraction experiments as in Example 2. These contraction experiments in organ baths measure the contraction frequency and force of the engineered skeletal muscle tissue in response to electrical stimulation.

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

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

[0196] These contraction experiments demonstrate that BSM also generates force in response to electrical stimulation. The skeletal muscle tissue tested exhibited reproducible contraction frequency and contraction force in response to stimulation frequencies between 1 Hz and 100 Hz, with contraction and relaxation times of approximately 0.6 seconds after a single stimulation. Additionally, ESM and BSM exhibit similar characteristics in terms of tetanus formation and increased contraction force. Similar to the ESM described in Example 2, BSM generates tetanus at increased stimulation frequencies, e.g., 100 Hz. Also similar to Example 2, the contraction force of BSM increases with increasing stimulation frequency.

[0197] Both of these properties are similar to the contractile behavior of native muscle tissue, since in native skeletal muscle, tetanus is formed and contractile force increases with increasing frequency of stimulation. Similar to native skeletal muscle, the engineered skeletal muscle tissue exhibited a positive force-frequency relationship in response to electrical stimulation, in addition to twitches and tetanus.

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

[0199] Example 4 - Increased function of engineered skeletal muscle tissue To further increase the function of engineered skeletal muscles, for example, contractile force can be increased by adding specific molecules. In this example, we specifically examined the enhancement of contractile force as well as contraction and relaxation time in response to the addition of creatine and an increase in the concentration of the thyroid hormone T3 (triiodo-L-thyronine (T3); from 3 nmol / L to 100 nmol / L in the maturation medium in step iv). Here, the procedures according to Examples 1 and 2 were first performed. In contrast to Example 2, the maturation medium was supplemented with creatine or increasing concentrations of T3 either from days 28 to 56 or from days 56 to 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 force of contraction (FOC) increased from 1.8 mN to 2.5 mN during tetanus stimulation at 100 Hz (Figure 9B, top). Thus, this medium addition increased the force of contraction by 39%. Furthermore, we tested the possible increase in force of contraction over the long term with this method. To this end, the method was extended for an additional 4 weeks as described in Example 2, during which time the medium was supplemented with 1 mM creatine. Supplementing the maturation medium with 1 mM creatine from day 56 to day 84 of the procedure increased the force of contraction (twitch tension) from 4.0 mN to 5.2 mN during tetanus stimulation at 100 Hz (Figure 9B, bottom). This medium addition thus increased the force of contraction by 30%.

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

[0202] Supplementation with T3: Supplementing the maturation medium with 0.1 μM T3 from day 28 to day 56 of the procedure significantly reduced contraction and relaxation rates as determined by Student's T-test (FIG. 10B). Furthermore, when the procedure was prolonged as described in Example 2 and the medium was supplemented with 0.1 μM T3 from day 56 to day 84, contraction and relaxation rates were reduced (FIG. 10B). Thus, engineered skeletal muscle responds more rapidly to tetanic stimulation and relaxes more rapidly after the stimulation has ceased.

[0203] In general, it can be assumed that maturation media with increased T3 concentrations lead to improved skeletal muscle contractility in terms of accelerated contraction and relaxation times.

[0204] To investigate the molecular basis 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); and 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, MYH2 protein expression was significantly increased with the addition of 0.1 μM T3 for 4 weeks. Based on three independent experiments, expression increased at least 5-fold. MYH7 expression remained unchanged with the addition of 0.1 μM T3. MYH3 expression was reduced by approximately half on average. These protein expression data support the functional data from Figure 10B, suggesting that the reduced response time of engineered skeletal muscle may be explained by increased expression of the fast myosin (MYH2) isoform by T3.

[0205] In conclusion, the addition of creatine and / or T3 during maturation has been shown to increase the function of engineered skeletal muscle. In particular, the addition of creatine has been shown to significantly increase contractile force. Additionally, the addition of T3 has been shown to increase the kinetics 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 an 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 precursors was analyzed (PAX7, PAX3, MYF5, and BARX2). In contrast to 2D cultures, all four markers were clearly expressed in ESM at day 60 of culture. Furthermore, PAX3, MYF5, and BARX2 were more highly expressed in engineered skeletal muscle than in skeletal muscle cells cultured in 2D plates. This indicates that skeletal muscle cell precursors are maintained and expanded in engineered skeletal muscle tissue, in contrast to parallel 2D cultures. FIG. 11B also shows well-differentiated satellite cell niches in the ESM, with scattered and less differentiated satellite cell niches also seen in 2D cultures similar to the methods described herein.

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

[0209] Methods of Examples 4 and 5 maturation conditions 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 Contractile function of engineered skeletal muscle tissue was measured under isometric conditions at 37°C in organ baths filled with gassed (5% CO2 / 95% O2) 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). To examine the force-length relationship, muscle length was increased by mechanical stretching at 125 μm intervals while electrically stimulating the ESM at 1 Hz with 200 mA 5 ms rectangular pulses until maximal contractile force was observed. Tetanic force was assessed at the length of maximal force generation under defined stimulation frequencies (4 s stimulation at 10, 20, 40, 60, 80, and 100 Hz).

[0211] Cardiotoxic injury model Control and engineered skeletal muscle were subjected to cardiotoxin injury (CTX) in parallel with irradiated ESM. To induce injury, tissue was maintained in maturation medium (Latoxan) containing 25 μg / ml CTX for 24 hours (Tiburcy et al., 2019). The injured tissue was then rinsed and placed in Expansion 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 one week, and then cultured 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) for an additional two-week regeneration period. 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 before CTX treatment, ESM were placed in culture dishes in an STS Biobeam 8000 gamma irradiator and exposed to a single dose of 30 Gy irradiation for 10 minutes ( Tiburcy et al., 2019 ).

[0213] Immunostaining and confocal imaging 2D cell cultures were fixed in 4% formaldehyde (Carl Roth) in phosphate-buffered saline (PBS) for 15 minutes at room temperature. Engineered skeletal muscles were fixed overnight in 4% paraformaldehyde in PBS at 4°C. After fixation, the engineered skeletal muscles were immersed in 70% ethanol (Carl Roth) for 1 minute and then embedded in 2% agarose (peqGOLD) in 1X Tris-acetate-EDTA (TAE) buffer. Sections were cut at 400 μm using a Leica Vibrotome (LEICAVT1000S) and stored in cold 1X PBS. Both 2D cell cultures and ESM sections were washed with 1X PBS before staining. 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 staining was 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), and laminin (1:50, Sigma-Aldrich). After three PBS washes, the appropriate Alexa fluorochrome-conjugated secondary antibody (1:1000, Thermo Fisher Scientific) was 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 three washes with PBS, samples were stained with Fluoromount-G (Southern Biotech). All images were acquired using a Zeiss LSM 710 / NLO confocal microscope. To quantify 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, engineered skeletal muscle was placed in an Eppendorf tube and snap-frozen in liquid nitrogen. 150 μl of ice-cold protein lysis buffer (2.38 g HEPES, 10.20 g NaCl, 100 ml glycerol, 102 mg MgCl2, 93 mg EDTA, 19 mg EGTA, 5 ml NP-40 in a total volume of 500 ml ddH2O) containing 1 / 10 phosphatase inhibitor (Roche) and 1 / 7 protease inhibitor (Roche) was added to the engineered skeletal muscle. 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. The sample was then incubated on ice for 2 hours and then centrifuged at 12,000 rpm and 4°C for 30 minutes. The supernatant was collected as a protein sample, and protein concentration was measured by Bradford protein assay. Thirty micrograms of protein samples were loaded onto a 4-15% sodium dodecyl sulfate (SDS)-polyacrylamide gel (Bio-Rad) and electrophoretically separated at 100 V for approximately 2.5 hours. They were then transferred to a polyvinylidene difluoride (PVDF) membrane at 30 V in an ice-filled box placed in a cold storage chamber overnight. To visualize total protein, the PVDF membrane was stained with Ponceau Red. Staining with primary and secondary antibodies (4 hours 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 7 (1:500, A4.951, DSHB), and fast myosin heavy chain 2 (1:100, A4.74, DSHB). Protein loading was controlled with vinculin (VCL) antibody (1:5000, V3131, Sigma-Aldrich). Membranes were washed for 5 min with 1x Tris-buffered saline (TBS) and 0.1% Tween 20. Horseradish peroxidase-conjugated goat anti-mouse IgG antibody (1:10,000, P0260, Dako) was used for secondary staining.After washing the membrane with 1x Tris-buffered saline (TBS) and 0.1% Tween 20 for 5 min, 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 Western blots was performed using ImageJ.

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

[0216] Materials used in all examples Materials used herein are commercially available unless otherwise noted. For example, penicillin / streptomycin, B27 serum-free supplement, essential amino acids (MEM-NEAA), and 2-mercaptoethanol are available from Invitrogen. Company names are listed 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 culture medium and stored at 4°C for up to one week. KnockOut Serum Replacement stock solutions were also stored at -20°C. Once thawed, they were stored at 4°C for up to two weeks. LDN193189 stock solutions had a concentration of 10 mM in DMSO and were stored at -20°C. DAPT stock solutions had a concentration of 20 mM in DMSO and were stored at -20°C. bFGF stock solutions had a concentration of 10 μg / ml in PBS containing 0.1% human recombinant albumin and were stored at -20°C. HGF stock solutions had a concentration of 10 μg / ml in PBS containing 0.1% human recombinant albumin and were stored at -20°C. Rock inhibitors had a concentration of 10 mM in DMSO and were stored at -20°C.

[0218] Once the growth factor and small molecule stock solutions were thawed, they were stored at 4°C for up to 1 week.

[0219] Table 1. Composition of serum-free additive N-2 (liquid form) at 100x effective concentration, i.e., 1% (v / v) corresponds to 1 (1x) effective concentration. TIFF2025134825000004.tif35161

[0220] Table 2. Composition of non-essential amino acids at 100x effective concentration (100x) TIFF2025134825000005.tif41169

[0221] Table 3. DMEM, 1 g / L low glucose, supplemented with pyruvate GlutaMAX™ (Gibco, Catalog Number: 10567014) TIFF2025134825000006.tif211167

[0222] Table 4. Composition of additional serum-free B27 additive at 50X effective concentration (liquid form) 10 ml of 50X B27 additive per 500 ml of medium corresponds to 2% (v / v) TIFF2025134825000007.tif121128

[0223] Table 5. Composition of Knockout Serum Replacement (KSR) TIFF2025134825000008.tif210166

[0224] References TIFF2025134825000009.tif232161TIFF2025134825000010.tif144161

Claims

1. 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 effective amounts 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 effective amounts of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) the same serum-free additives as in (i); and (d) adding an effective amount of HGF and continuing the culture in the medium; Culturing the cells in a basal medium containing an effective amount of (a) a gamma secretase / NOTCH inhibitor, (b) HGF, (c) the same serum-free additives as in (i), and (d) KnockOut Serum Replacement (KSR). inducing myogenic specification by (iii) expanding and maturing the cells obtained in step (ii) into skeletal myoblasts and satellite cells by culturing the cells in a basal medium containing effective amounts of (a) HGF, (b) the same serum-free additive as in (i), and (c) KnockOut Serum Replacement (KSR); (iv) maturing the cells obtained in step (iii) and dispersed in the extracellular matrix under mechanical stimulation in a basal medium containing effective amounts of (a) the same serum-free additives as in step (i) and (b) additional serum-free additives including albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3), thereby maturing the cells into skeletal myotubes and satellite cells. Including, thereby producing engineered skeletal muscle tissue.

2. 2. The method of claim 1, 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 at 100 Hz stimulation.

3. 3. The method of claim 1 or 2, 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.

4. 4. The method of any one of claims 1 to 3, wherein step (i) is carried out for a period of 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.

5. 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, and preferably the SMAD inhibitor is LDN193189; 5. The method of any one of claims 1 to 4.

6. 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 50-500 μg / ml transferrin, 1-20 μg / ml insulin, 0.001-0.1 μg / ml progesterone, 5-50 μg / ml putrescine, and 6-600 nM 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; 6. The method of any one of claims 1 to 5.

7. 7. The method according to any one of claims 1 to 6, wherein the serum-free additive in step (i) is 0.1 to 10% (v / v) N2 additive, preferably 0.3 to 7.5% (v / v) N2 additive, more preferably 0.5 to 5% (v / v) N2 additive, more preferably 0.75% to 2% (v / v) N2 additive, more preferably 0.9% to 1.2% (v / v) N2 additive, and most preferably about 1% (v / v) N2 additive.

8. 8. The method according to any one of claims 1 to 7, 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, preferably DMEM, in particular the basal medium is supplemented with pyruvate and / or non-essential amino acids and / or contains 1 g / l glucose.

9. In step (ii), the culturing is carried out in the presence of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive for 36 to 60 hours, preferably 42 to 54 hours, and most preferably about 48 hours; and / or the culturing 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, and most preferably about 48 hours; and / or The culturing 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, and most preferably about 96 hours; 9. The method of any one of claims 1 to 8.

10. 10. The method according to any one of claims 1 to 9, wherein 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.

11. 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, and 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, and 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, and 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) of KSR; in particular, KSR is used in the presence of a reducing agent, such as beta-mercaptoethanol and / or alpha-thioglycerol; 11. The method of any one of claims 1 to 10.

12. In step (iii), 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, and most preferably about 10 ng / ml; and / or 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) of KSR; in particular, KSR is used in the presence of a reducing agent, such as beta-mercaptoethanol and / or alpha-thioglycerol; 12. The method of any one of claims 1 to 11.

13. 13. The method of any one of claims 1 to 12, wherein in step (iv) the additional serum-free additives provide final concentrations in the medium of 0.5 to 50 mg / ml albumin, 1 to 100 μg / ml transferrin, 0.1 to 10 μg / ml ethanolamine, 17.4 to 1744 nM selenium or a bioavailable salt thereof, in particular sodium selenite, 0.4 to 40 μg / ml L-carnitine, 0.05 to 5 μl / ml fatty acid additive, 0.0001 to 0.1 μg / ml triiodo-L-thyronine (T3).

14. 14. The method of any one of claims 1 to 13, wherein the additional serum-free additive in step (iv) is 0.1 to 10% (v / v) 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) B27.

15. The method according to any one of claims 1 to 14, wherein in step (iv), the mechanical stimulus is static tension, dynamic tension, or load-inducing tension, preferably the mechanical stimulus is static tension.

16. 16. The method according to any one of claims 1 to 15, comprising a seeding step prior to step (i), in which the pluripotent stem cells are seeded in a stem cell medium in the presence of a ROCK inhibitor, and preferably the seeding step is carried out 18 to 30 hours before step (i).

17. 16. The method of claim 15, wherein 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.

18. 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.

18. The method of claim 16 or 17.

19. 19. The method of any one of claims 15 to 18, wherein 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 a master mix before the stem cell medium is added.

20. 20. The method of claim 19, wherein the component of the extracellular matrix in the master mix is ​​collagen, preferably type I collagen, more preferably of bovine, human or murine origin, in particular collagen of bovine origin, and optionally the extracellular matrix additionally comprises laminin and / or fibronectin.

21. Pluripotent stem cells are 1-6 x 10 6 21. The method of claim 20, wherein the cells are seeded in a medium at a ratio of cells / ml and collagen of 0.7 to 1.4 mg / ml.

22. the master mix comprises 5-15% (v / v), preferably 7.5%-12.5% ​​(v / v), more preferably 9-11% (v / v), and most preferably about 10% (v / v) of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cell exudate as an extracellular matrix component, in particular the exudate is Matrigel; and / or The pH of the master mix is ​​between pH 7.2 and pH 7.

8.

22. The method of any one of claims 19 to 21.

23. the master mix comprises stromal cells, the stromal cells producing the extracellular matrix components collagen, laminin, fibronectin, and / or proteoglycans; and / or The pH of the master mix is ​​between pH 7.2 and pH 7.

8.

22. The method of any one of claims 19 to 21.

24. 24. The method of any one of claims 19 to 23, wherein stem cell medium is added to the master mix in the engineered form after about 1 hour, and the stem cell medium comprises KSR and FGF2.

25. the stem cell medium comprises 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) KSR; and / or 25. The method of claim 24, wherein the stem cell culture medium comprises 1 to 15 ng / ml 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 FGF2.

26. 26. The method of any one of claims 19 to 25, wherein step (iii) is carried out for 7 to 11 days, preferably 8 to 10 days, most preferably about 9 days.

27. 19. The method of any one of claims 1 to 18, wherein after step (iii), 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 a master mix in an additional step before step (iv).

28. 28. The method of claim 27, wherein the component of the extracellular matrix in the master mix is ​​collagen, preferably type I collagen, more preferably of bovine, human or murine origin, in particular collagen of bovine origin, and optionally the extracellular matrix additionally comprises laminin and / or fibronectin.

29. Skeletal myoblasts and satellite cells, 1–6 × 10 6 29. The method of claim 28, wherein the cells are seeded in a medium at a ratio of cells / ml and 0.7 to 1.4 mg / ml of collagen.

30. the master mix comprises 5-15% (v / v), preferably 7.5%-12.5% ​​(v / v), more preferably 9-11% (v / v), and most preferably about 10% (v / v) of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cell exudate as an extracellular matrix component, in particular the exudate is Matrigel; and / or The pH of the master mix is ​​between pH 7.2 and pH 7.

8.

30. The method of any one of claims 27 to 29.

31. the master mix comprises stromal cells, the stromal cells producing the extracellular matrix components collagen, laminin, fibronectin, and / or proteoglycans; and / or The pH of the master mix is ​​between pH 7.2 and pH 7.

8.

30. The method of any one of claims 27 to 29.

32. After about 1 hour, a medium similar to that used in step (iii) is added to the master mix in engineered form, the medium additionally containing 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, and particularly preferably the ROCK inhibitor is Y27632.

32. The method of any one of claims 27 to 31.

33. 33. The method of claim 32, wherein 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.

34. 34. The method of any one of claims 27 to 33, wherein after about 1 day the culture medium is replaced with the same medium as used in step (iii), and the cells are then further cultured in this medium for a further 5 to 9 days, preferably 6 to 8 days, most preferably about 7 days.

35. 35. The method of any one of claims 19 to 34, wherein the engineered form has the form of a ring, ribbon, strand, patch, pouch, or cylinder, optionally with fused individual skeletal muscle tissues.

36. 36. The method according to any one of claims 1 to 35, 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, in particular at least 240 days.

37. 37. The method of any one of claims 1 to 36, wherein the cell is free of differentiation-associated transgenes or maturation-associated transgenes, preferably free of myogenic transgenes, more preferably free of the transgenes Pax7 and MyoD.

38. 38. The method of any one of claims 1 to 37, which does not include a skeletal myoblast enrichment step, preferably does not include an enrichment step by cell selection, more preferably does not include an enrichment step by antibody-based cell selection.

39. 1. A method for producing skeletal myoblasts, skeletal myotubes, 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 effective amounts 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 effective amounts of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) the same serum-free additives as in (i); and (d) adding an effective amount of HGF and continuing the culture in the medium; 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). inducing myogenic specification by (iii) culturing the cells obtained in step (ii) in a basal medium containing effective amounts of (a) HGF, (b) the same serum-free additive as in (i), and (c) KnockOut Serum Replacement (KSR), thereby maturing the cells into skeletal myoblasts and satellite cells; and (iv) maturing the cells obtained in step (iii) into skeletal myotubes and satellite cells by culturing the cells in a basal medium containing effective amounts of (a) the same serum-free additives as in step (i) and (b) additional serum-free additives including albumin, transferrin, ethanolamine, selenium or a bioavailable salt thereof, L-carnitine, a fatty acid additive, and triiodo-L-thyronine (T3); Including, Thereby, skeletal myoblasts, skeletal myotubes, and satellite cells are generated.

40. 40. The method of claim 39, wherein the method achieves a proportion of skeletal myoblasts in the total amount of available cells of at least 40%, preferably at least 50%, more preferably at least 60%, and most preferably at least 70%, as determined by actinin expression by flow cytometry.

41. A method according to claim 39 or 40, wherein the method achieves a proportion of satellite cells of at least 10%, preferably at least 15%, more preferably at least 20%, and most preferably at least 30% of the total amount of available cells, as determined by the expression of Pax7 by flow cytometry.

42. 42. The method of any one of claims 39 to 41, wherein the method does not include a skeletal myoblast enrichment step, preferably does not include an enrichment step by cell selection, more preferably does not include an enrichment step by antibody-based cell selection.

43. 43. The method of any one of claims 39 to 42, 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.

44. 44. The method of any one of claims 39 to 43, 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.

45. 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, and preferably the SMAD inhibitor is LDN193189; 45. The method of any one of claims 39 to 44.

46. 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 50-500 mg / l transferrin, 1-20 mg / l insulin, 1-30 μg / l progesterone, 5-50 μg / ml putrescine, and 6-600 nM selenium or a bioavailable salt thereof, particularly sodium selenite; and / or the GSK3 inhibitor is CHIR99021 and 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; 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; 46. ​​The method of any one of claims 39 to 45.

47. 47. The method of any one of claims 39 to 46, wherein the serum-free additive in step (i) is 0.1 to 10% (v / v) N2 additive, preferably 0.3 to 7.5% (v / v) N2 additive, more preferably 0.5 to 5% (v / v) N2 additive, more preferably 0.75% to 2% (v / v) N2 additive, more preferably 0.9% to 1.2% (v / v) N2 additive, and most preferably about 1% (v / v) N2 additive.

48. 48. The method of any one of claims 39 to 47, 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, preferably DMEM, in particular the basal medium is supplemented with pyruvate and / or non-essential amino acids and / or contains 1 g / l glucose.

49. In step (ii), the culturing is carried out in the presence of (a) a gamma-secretase / NOTCH inhibitor, (b) FGF2, and (c) a serum-free additive for 36 to 60 hours, preferably 42 to 54 hours, and most preferably about 48 hours; and / or the culturing 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, and most preferably about 48 hours; and / or The culturing 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, and most preferably about 96 hours; 49. The method of any one of claims 39 to 48.

50. 50. The method of any one of claims 39 to 49, wherein 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.

51. 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, and 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, and 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, and most preferably about 10 μM; KSR is used in an amount of 6-14% (v / v), preferably 7-13% (v / v), more preferably 8%-12% (v / v), more preferably 9%-11% (v / v), 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; 51. The method of any one of claims 39 to 50.

52. 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 5-20% (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; in particular, KSR is used in the presence of a reducing agent, such as beta-mercaptoethanol and / or alpha-thioglycerol; 50. The method of any one of claims 39 to 49.

53. 53. The method of any one of claims 39 to 52, wherein step (iii) is carried out for 7 to 11 days, preferably 8 to 10 days, most preferably about 9 days.

54. 54. The method of any one of claims 39 to 53, wherein in step (iv) the additional serum-free additives provide final concentrations in the medium of 0.5 to 50 mg / ml albumin, 1 to 100 μg / ml transferrin, 0.1 to 10 μg / ml ethanolamine, 17.4 to 1744 nM selenium or a bioavailable salt thereof, in particular sodium selenite, 0.4 to 40 μg / ml L-carnitine, 0.05 to 5 μl / ml fatty acid additive, 0.0001 to 0.1 μg / ml triiodo-L-thyronine (T3).

55. 55. The method of any one of claims 39 to 54, wherein the additional serum-free additive in step (iv) is 0.1 to 10% (v / v) 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) B27.

56. 56. The method of any one of claims 39 to 55, wherein step (iv) is carried out for at least 30 days, preferably at least 35 days, more preferably at least 40 days, and even more preferably at least 50 days.

57. 57. The method of any one of claims 39 to 56, comprising a seeding step prior to step (i), in which the pluripotent stem cells are seeded in a stem cell medium in the presence of a ROCK inhibitor, and preferably the seeding step is carried out 18 to 30 hours before step (i).

58. 58. The method of claim 57, wherein 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.

59. 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.

59. The method of claim 57 or 58.

60. the stem cell medium comprises 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) KSR; and / or the stem cell medium comprises 1-15 ng / ml 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 FGF2; 60. The method of claim 59.

61. Engineered skeletal muscle tissue having multinucleated mature skeletal muscle fibers with satellite cells and no blood supply and / or central nervous system control; in particular, engineered skeletal muscle tissue in which the presence of skeletal muscle fibers is detected by staining for actinin and using DAPI.

62. 62. The engineered skeletal muscle tissue of claim 61, wherein the skeletal muscle tissue is serum-free and / or does not contain a differentiation-associated transgene or a maturation-associated transgene, preferably the skeletal muscle tissue does not contain a myogenic transgene, more preferably the skeletal muscle tissue does not contain a Pax7 transgene or a MyoD transgene.

63. 63. The engineered skeletal muscle tissue of claim 61 or claim 62, wherein the skeletal muscle tissue generates a contractile force of at least 0.3 milliNewtons (mN) at 100 Hz stimulation at 200 mA, 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.

64. 64. The engineered skeletal muscle tissue of any one of claims 61 to 63, wherein the skeletal muscle tissue has been engineered to have an engineered form, preferably in the form of a ring, ribbon, strand, patch, pouch, or cylinder, optionally with individual skeletal muscle tissues fused together, particularly wherein the skeletal muscle tissue has the form of a ring.

65. A mesodermally differentiated skeletal myoblast progenitor cell, obtained according to step (i) of claim 1 or claim 39 and prepared by the method of claim 1(i) or claim 39(i), characterized by the expression of genes MSGN1 and / or TBX6, wherein the expression of MSGN1 and / or TBX6 can be determined by flow cytometry and / or immunostaining; and / or wherein mRNA SP5 is expressed, wherein the expression of SP5 can be determined by RNA sequencing.

66. 1. A myogenically committed skeletal myoblast precursor cell, obtained according to step (ii) of claim 1 or claim 39 and produced by the method of claim 1(i)-(ii) or claim 39(i)-(ii), characterized by the expression of the gene PAX3, wherein the expression of PAX3 can be determined by flow cytometry and / or immunostaining; and / or wherein mRNA SIM1 is expressed, wherein the expression of SIM1 can be determined by RNA sequencing.

67. A skeletal myoblast obtained according to step (iii) of claim 1 or claim 39, produced by a method according to claim 1(i) to (iii) or claim 39(i) to (iii), characterized by expression of actinin, preferably wherein the expression of actinin can be determined by flow cytometry and / or immunostaining.

68. Satellite cells obtained according to step (iii) of claim 1 or claim 39 and produced by the method of claim 1(i) to (iii) or claim 39(i) to (iii), characterized by the expression of the gene Pax7, wherein the expression of Pax7 can be determined by flow cytometry and / or immunostaining, more preferably the satellite cells further express Ki67.

69. A mixture of skeletal myoblasts as described in claim 67 and satellite cells as described in claim 68, wherein a proportion of satellite cells of the total available cell amount is obtained that is at least 10%, preferably at least 15%, more preferably at least 20%, and even more preferably at least 30%, as determined by expression of Pax7 by flow cytometry; and / or a proportion of skeletal myoblasts of the total available cell amount is obtained that is at least 40%, preferably at least 50%, more preferably at least 60%, and most preferably at least 70%, as determined by expression of actinin by flow cytometry.

70. Skeletal myotubes obtained according to step (iv) of claim 1 or claim 39, and A skeletal myotube cell prepared by the method of claim 39(i) to (iv), characterized by anisotropic orientation of actinin protein-containing sarcomere structures.

71. Use of skeletal muscle tissue according to any one of claims 61 to 64, and / or cells according to any one of claims 64 to 68, and / or skeletal myotube cells according to claim 70 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 pharmacological drug candidates and gene therapy drug candidates.

72. A skeletal muscle tissue according to any one of claims 61 to 64 and / or a cell according to any one of claims 65 to 69 and / or a skeletal myotube cell according to claim 70 for use in medicine.

73. 69. The satellite cell of claim 68, for use in therapy of damaged skeletal muscle and / or in the treatment of skeletal muscle diseases, preferably genetic skeletal muscle defects, in particular Duchenne muscular dystrophy and / or Becker-Keener muscular dystrophy, and / or lysosomal storage diseases, in particular Pompe disease, wherein preferably the skeletal muscle disease is Duchenne muscular dystrophy.

74. 1. An in vitro method for testing the effectiveness of a drug candidate on skeletal muscle tissue, comprising: (a) providing a skeletal muscle tissue according to any one of claims 60 to 63; (b) optionally damaging skeletal muscle tissue; and (c) contacting the skeletal muscle tissue of step (a) or (b) with a drug candidate. Includes; Preferably, the method further comprises the step of determining contractile force and / or skeletal muscle tissue structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters before and / or after step (c), method.

75. 1. An in vitro method for testing the toxicity of a substance to skeletal muscle tissue, comprising: (a) providing a skeletal muscle tissue according to any one of claims 61 to 64; (b) contacting the skeletal muscle tissue from step (a) with the substance to be tested. Including, Preferably, the method further comprises the step of determining contractile force and / or skeletal muscle tissue structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters before and / or after step (b), method.

76. 1. An in vitro method for testing the effects of nutrients and dietary supplements on skeletal muscle tissue performance, comprising: (a) providing a skeletal muscle tissue according to any one of claims 61 to 64; (b) contacting the skeletal muscle tissue from step (a) with the nutrient or dietary supplement to be tested. Including, Preferably, the method further comprises the step of determining contractile force and / or skeletal muscle tissue structure and / or metabolic function and / or molecular parameters and / or protein biochemical parameters before and / or after step (b), method.

77. 1. An in vitro method for testing the effectiveness of a drug candidate on mesodermally differentiated skeletal myoblast precursor cells, myogenically committed skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, or a mixture of skeletal myoblasts and satellite cells, comprising: (a) providing a mesodermally differentiated skeletal myoblast precursor cell, a myogenically committed skeletal myoblast precursor cell, a satellite cell, a skeletal myoblast, a skeletal myotube cell, or a mixture of skeletal myoblasts and satellite cells according to any one of claims 65 to 70; (b) optionally damaging the cells from step (a); and (c) contacting the cells of step (a) or (b) with a drug candidate. Including, Preferably, the method further comprises determining the expression of actinin and / or Pax7 before and / or after step (c), wherein said expression may be determined by flow cytometry and / or immunostaining. method.

78. 1. An in vitro method for testing the toxicity of a substance to mesodermally differentiated skeletal myoblast precursor cells, myogenically committed skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, or a mixture of skeletal myoblasts and satellite cells, comprising: (a) providing a mesodermally differentiated skeletal myoblast precursor cell, a myogenically committed skeletal myoblast precursor cell, a satellite cell, a skeletal myoblast, a skeletal myotube cell, or a mixture of skeletal myoblasts and satellite cells according to any one of claims 65 to 70; (b) contacting the cells of step (a) with the substance to be tested Including, Preferably, the method further comprises determining the expression of actinin and / or Pax7 before and / or after step (b), wherein said expression can be determined by flow cytometry and / or immunostaining. method.

79. 1. An in vitro method for testing the effects of nutrients and dietary supplements on mesodermally differentiated skeletal myoblast precursor cells, myogenically committed skeletal myoblast precursor cells, satellite cells, skeletal myoblasts, skeletal myotubes, or a mixture of skeletal myoblasts and satellite cells, comprising: (a) providing a mesodermally differentiated skeletal myoblast precursor cell, a myogenically committed skeletal myoblast precursor cell, a satellite cell, a skeletal myoblast, a skeletal myotube cell, or a mixture of skeletal myoblasts and satellite cells according to any one of claims 65 to 70; (b) contacting the cells of step (a) with the nutrient or dietary supplement to be tested. Including, Preferably, the method further comprises determining the expression of actinin and / or Pax7 before and / or after step (b), wherein said expression can be determined by flow cytometry and / or immunostaining. method.

80. 61. The method of any one of claims 1 to 60, 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, at 100 Hz stimulation.

81. 81. The method of any one of claims 1 to 60 or 80, wherein the skeletal muscle tissue has a contraction velocity 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 at 100 Hz stimulation.

82. 82. The method of any one of claims 1 to 60, 80, or 81, wherein the skeletal muscle tissue has a relaxation rate at the end of the 100 Hz stimulation 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.

83. 83. The method of any one of claims 1 to 60 or 80 to 82, wherein the basal medium in step (iv) comprises an effective amount of creatine and / or triiodo-L-thyronine (T3).

84. 84. The method of claim 83, wherein the effective amount of creatine in the basal medium increases the contractile force of the engineered skeletal muscle compared to maturation in step (iv) without the effective amount of creatine.

85. 85. The method of claim 83 or 84, wherein an effective amount of T3 in the basal medium shortens the contraction and / or relaxation rate of the engineered skeletal muscle compared to maturation in step (iv) without the effective amount of T3.

86. 86. The method of any one of claims 1 to 60 or 80 to 85, wherein the basal medium in step (iv) provides a final concentration of 0.1 to 10 mM creatine, preferably 0.2 to 6 mM creatine, more preferably 0.4 to 4 mM creatine, even more preferably 0.6 to 3 mM creatine, even more preferably 0.7 to 2.5 mM creatine, even more preferably 0.8 to 2 mM creatine, even more preferably 0.85 to 1.5 mM creatine, even more preferably 0.9 to 1.2 mM creatine, and most preferably about 1 mM creatine.

87. 87. The method of any one of claims 1 to 60 or 80 to 86, wherein the basal medium in step (iv) provides a final concentration of 0.001 to 1 μM triiodo-L-thyronine (T3), preferably 0.005 to 0.7 μM T3, more preferably 0.01 to 0.35 μM T3, even more preferably 0.04 to 0.02 μM T3, even more preferably 0.05 to 0.18 μM T3, even more preferably 0.06 to 0.15 μM T3, even more preferably 0.08 to 0.12 μM T3, even more preferably about 0.1 μM T3.

88. 88. The method of any one of claims 1-60 or 80-87, wherein the skeletal muscle tissue has the property of self-renewal.

89. 89. The method of claim 88, wherein the regenerative properties are characterized by restored contractility and / or muscle recovery, preferably wherein said restored contractility and / or muscle recovery is measured after irreversible muscle damage using a cardiotoxin, more preferably wherein said restored contractility and / or muscle recovery is measured 10 to 30 days after incubation with the cardiotoxin.

90. 90. The method of any one of claims 1 to 60 or 80 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. 91. Engineered skeletal muscle tissue produced by the method of any one of claims 1-60 or 80-90.

92. 92. The engineered skeletal muscle tissue of any one of claims 61-64 or 91, wherein the engineered skeletal muscle tissue produces a contractile force at 100 Hz 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.

93. 93. The engineered skeletal muscle tissue of any one of claims 61-64 or 91-92, having a contraction velocity 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 at 100 Hz stimulation.

94. 94. The engineered skeletal muscle tissue of any one of claims 61-64 or 91-93, having a relaxation rate at the end of 100 Hz stimulation 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.

95. Use of skeletal muscle tissue according to any one of claims 61 to 64 or 91 to 94, and / or cells according to any one of claims 65 to 69, and / or skeletal myotube cells according to claim 70 in an in vitro drug assay; particularly, the drug assay is a toxicity assay or an assay of skeletal muscle tissue function under the influence of pharmacological drug candidates and gene therapy drug candidates.

96. A skeletal muscle tissue according to any one of claims 61 to 64 and 91 to 94, and / or a cell according to any one of claims 65 to 69, and / or a skeletal myotube cell according to claim 70, for use in medicine.

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