Skeletal muscle cell maturation

Abstract

The present invention relates to pluripotent stem cells comprising an expression construct for expressing a MYOD protein and an expression construct for expressing a PAX7 protein. The present invention further provides a method for producing skeletal muscle cells comprising the pluripotent stem cells, and a food product comprising the skeletal muscle cells or the pluripotent stem cells.

Description

[Technical Field]

[0001] The present invention relates to modified pluripotent cells and methods for differentiating said cells into skeletal muscle cells. [Background technology]

[0002] According to the latest United Nations estimates, the current world population is 7.9 billion as of July 2022 [https: / / www.worldometers.info / es / poblacion-mundial / #ref-1] and is expected to reach 10 billion around 2056. This growth will be unevenly distributed around the world, with nine countries—India, Nigeria, Pakistan, Egypt, and the United States—accounting for half of the projected global population increase over the next 30 years. Population and economic growth are the primary drivers of increased meat consumption. According to the Food and Agriculture Organization of the United Nations (FAO, https: / / www.oecd-ilibrary.org / agriculture-and-food / oecd-fao-agricultural-outlook-2022-2031_f1b0b29c-en), global meat consumption is projected to increase by 15% by 2031. On the other hand, the correlation between rising income and higher meat consumption is clearly demonstrated at lower income levels, but once consumers reach a decent standard of living, they become more sensitive to environmental, ethical, and animal welfare and health concerns.

[0003] For this reason, there is growing interest in finding alternative protein sources that are ideally sustainable and contain the nutrients typically provided by meat in the human diet. Cultured meat emerged as another alternative to traditional animal agriculture, aiming to generate the skeletal muscle and adipose tissue that typically constitutes animal meat, but using in vitro tissue and biological engineering techniques. Despite efforts to develop robust protocols for the scalable generation of animal cell types from easily accessible and renewable sources, the differentiation of animal (pluripotent) stem cells into specific cell types is often laborious, time-consuming, difficult to reproduce, and / or has yet to be established.

[0004] Skeletal muscle reprogramming of livestock-derived cells has been reported for porcine embryonic fibroblasts and porcine induced pluripotent stem cells (Jeong et al., 2021 & Genovese et al., 2017). In both studies, skeletal muscle cells were reprogrammed using MyoD1 overexpression. Lentiviral overexpression of MyoD1 in porcine embryonic fibroblasts resulted in low differentiation efficiency, long culture times, and required serum-containing medium. Induction of porcine induced pluripotent stem cells with MyoD1 for 8 days resulted in a high conversion rate but relied on 5-azacytidine and CHIR99021, which are unsafe for food use. In conclusion, these protocols resulted in uneven skeletal muscle maturation, were not feasible for scalability, were unsafe for food use, and required long culture periods of up to 14 days.

[0005] Thus, there remains a need in the art for the production and culture of mature skeletal muscle cells that are suitable for human consumption and can be produced in a scalable, cost-effective manner. Summary of the Invention [Means for solving the problem]

[0006] The present invention relates to the expression of transcriptional regulatory proteins MYOD and PAX7 in pluripotent stem cells. Thus, in a first aspect, the present invention relates to pluripotent stem cells, which pluripotent stem cells comprise: i) an expression construct for expression of a transcriptional regulatory protein inserted into a first genetic safe harbor site; ii) an expression construct for expression of a MYOD protein, wherein the coding sequence for the MYOD protein is operably linked to an inducible promoter; and iii) an expression construct for expression of a PAX7 protein, wherein the coding sequence for the PAX7 protein is operably linked to an inducible promoter; The expression constructs of ii) and iii) are inserted into at least one additional genetic safe harbor site that is not the first genetic safe harbor site, and Inducible promoters are regulated by transcriptional regulatory proteins.

[0007] In certain embodiments of the invention, the expression constructs of ii) and iii) are both inserted into a second genetic safe harbor site different from the first genetic safe harbor site. Preferably, said first and further genetic safe harbor sites are selected from any two of the hROSA26 locus, the AAVS1 locus, the CLYBL gene, or the CCR5 gene, and preferably the genetic safe harbor sites are the hROSA26 locus and the AAVS1 locus.

[0008] In certain embodiments of the invention, the cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines.

[0009] In a particular embodiment of the invention, the pluripotent stem cells are pluripotent stem cells of a livestock or poultry species. Preferably, the livestock species is porcine or bovine, preferably porcine.

[0010] In certain embodiments of the invention, the expression construct inserted into the second genetic safe harbor site encodes a MYOD protein, a linker, and a PAX7 protein, preferably the linker is a P2A linker, more preferably the linker comprises the sequence of SEQ ID NO: 6. Preferably, the expression construct comprises the sequence of SEQ ID NO: 8.

[0011] In a specific embodiment of the present invention, the activity of the transcriptional regulatory protein is controlled by an exogenously supplied derivative. Preferably, the transcriptional regulatory protein is selected from the group consisting of tetracycline-responsive transcriptional activator protein (rtTA), tetracycline repressor (TetR), VgEcR synthetic receptor, or a hybrid transcriptional regulatory protein comprising a DNA-binding domain derived from yeast GAL4 protein, a truncated ligand-binding domain derived from human progesterone receptor, and an activation domain derived from human NF-kB; preferably, the transcriptional regulatory protein is rtTA.

[0012] In certain embodiments, the inducible promoter comprises a Tet-responsive element (TRE).

[0013] In certain embodiments, the inducible promoter is a tetON promoter.

[0014] In a second aspect, the present invention provides a method for producing skeletal muscle cells, such as type 1 or type 2 skeletal muscle cells, comprising: a) culturing the pluripotent stem cells according to any one of the preceding claims in a growth medium; followed by b) inducing differentiation of skeletal muscle cells by adding an exogenous substance as described herein. In certain embodiments, the differentiation stage of the methods described herein is for up to 10 days, up to 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, or 2 days.

[0015] The medium used for proliferation and the medium used for differentiation may be the same or different.

[0016] In certain embodiments, the skeletal muscle cells produced are for human and non-human nutritional use.

[0017] In a further aspect, the invention provides for the use of the pluripotent stem cells described herein, or the methods for producing skeletal muscle cells described herein, for tissue engineering, and optionally for the production of cultivated meat.

[0018] In yet a further aspect, the present invention provides a food product comprising the pluripotent stem cells described herein or skeletal muscle cells obtained by the methods described herein. In certain embodiments, the food product is cultured meat. DETAILED DESCRIPTION OF THE INVENTION

[0019] Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is not limited to such methods in any way.

[0020] In this specification and its claims, the verb "comprise" and its conjugations are used in their open-ended sense so that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that a plurality of elements may be present, unless the context clearly requires the presence of only one element. Thus, the indefinite article "a" or "an" typically means "at least one."

[0021] As used herein, the term "and / or" indicates that one or more of the specified instances may occur alone or in combination with at least one and up to all of the specified instances.

[0022] As used herein, "at least" a particular value means greater than or equal to the particular value. For example, "at least 2" is understood to be the same as "2 or more," i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ... etc.

[0023] The terms "about" or "approximately," when used in connection with a numerical value (e.g., about 10), preferably mean that the value can be a given value from minus 0.1% to plus 0.1% (of 10) of the value.

[0024] The term "heterologous," when used with reference to a nucleic acid (DNA or RNA) or protein, refers to a nucleic acid or protein that is not naturally occurring as part of the organism, cell, genome, or DNA or RNA sequence in which it is found, or to a nucleic acid or protein that is found in a cell, or location within the genome, DNA, or RNA sequence, different from that in which it is found in nature. A heterologous nucleic acid or protein is not endogenous to the cell into which it is introduced, but is obtained from another cell, or is synthetically or recombinantly produced. Generally, although not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly, exogenous RNA encodes proteins that are not normally expressed in the cell in which the exogenous RNA is present. Heterologous nucleic acids and proteins are sometimes referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is encompassed herein by the term heterologous nucleic acid or protein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e., combinations in which at least two linked sequences are foreign to each other.

[0025] The term "expression vector" or "expression construct" refers to a nucleotide sequence capable of effecting the expression of a gene in a host cell or host organism compatible with such sequence. These expression vectors typically contain at least appropriate transcription regulatory sequences and, optionally, a 3' transcription termination signal. Additional factors necessary or helpful for expression may also be present, for example, expression enhancer elements.

[0026] As used herein, the term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it effects transcription of the coding sequence. Operably linked means that the linked DNA sequences are typically contiguous, and, where necessary, join two adjacent protein-coding regions in reading frame. Inducible promoters

[0027] As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control transcription of one or more coding sequences, is located upstream in the direction of transcription from the transcription start site of that coding sequence, and is structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription initiation site, and other DNA sequences (including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and other sequences of nucleotides known to those skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter). A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, for example, by application of a chemical inducer. In the present invention, regulation is exerted by a transcriptional regulatory protein.

[0028] References herein to nucleotide or amino acid sequences accessible in public sequence databases refer to the versions of the sequence entries available on the filing date of this application.

[0029] All patent and literature references cited herein are hereby incorporated by reference in their entirety.

[0030] The inventors have surprisingly found that the time required for differentiation of pluripotent cells into mature skeletal muscle cells can be dramatically reduced by using modified pluripotent stem cells containing an expression construct for expression of the MYOD protein and an expression construct for expression of the PAX7 protein, or an expression construct that expresses both MYOD and PAX7. As shown in the Examples described herein, by using these modified pluripotent cell lines, complete differentiation into mature skeletal muscle cells can be rapidly achieved within 6 days, e.g., less than 5 days, e.g., 4 days or 3 days or more. Aside from significantly reducing culture time and associated costs, the use of the pluripotent cells described herein also provides for larger-scale production of mature skeletal muscle cells, resulting in the generation of more mature skeletal muscle cells, compared to those previously described.

[0031] The differentiated cells grew into elongated spindle-like morphologies, were capable of extensive cell fusion, and showed strong and homogeneous expression of myogenic markers based on mRNA and protein levels (MHC isoforms / maturity, immunostaining for MYOD / MYOG / MYH / TTN / TNNT / PAX3 / 7), as well as high fusion index, length, and thickness. Furthermore, spontaneous muscle fiber contraction could be observed, demonstrating the function of MYOD / PAX7.

[0032] The induction efficiency of exogenous compounds did not decrease with increasing culture time, demonstrating the robustness and reproducibility (proportion of MHC-positive cells at different passages) of this system.

[0033] Thus, in a first aspect, the present invention relates to a pluripotent stem cell, which pluripotent stem cell comprises: i) an expression construct for expression of a transcriptional regulatory protein inserted into a first genetic safe harbor site; ii) an expression construct for expression of a MYOD protein, wherein the coding sequence for the MYOD protein is operably linked to an inducible promoter; and iii) an expression construct for expression of a PAX7 protein, wherein the coding sequence for the PAX7 protein is operably linked to an inducible promoter; the expression constructs of ii) and iii) are inserted into at least one additional genetic safe harbor site that is not the first genetic safe harbor site; And, the inducible promoter is regulated by a transcriptional regulatory protein.

[0034] The pluripotent stem cells of the present invention may optionally not contain any expression constructs other than those determined in i), ii), and iii) above. Alternatively, the pluripotent stem cells of the present invention may optionally contain one or more expression constructs in addition to those determined in i), ii), and iii) above, e.g., in at least one additional genetic safe harbor site that is not the first genetic safe harbor site. Such additional expression constructs may be provided for the expression of regulatory factors or transcription factors, e.g., proteins that regulate transcription factors other than MYOD or PAX7.

[0035] When pluripotent stem cells of the present invention are produced that contain one or more antibiotic resistance markers, one or more of such resistance markers may be removed.The pluripotent stem cells of the present invention may optionally not contain antibiotic resistance markers.

[0036] MYOD, also known as myoblast determination protein 1, is an animal protein that plays a major role in regulating muscle differentiation. MYOD belongs to a family of proteins known as myogenic regulatory factors. MYOD is one of the early markers of myogenic commitment. MYOD is expressed at extremely low, essentially undetectable, levels in quiescent satellite cells, but its expression is activated in response to exercise and muscle tissue injury. The effect of MYOD on satellite cells is dose-dependent: high MYOD expression can suppress cell renewal, promote terminal differentiation, and induce apoptosis. Although MYOD signals myoblast commitment, muscle development is not dramatically impaired in mouse mutants lacking the MYOD gene. This may be due to functional redundancy from Myf5 and / or Mrf4. Nevertheless, the combination of MYOD and Myf5 is essential for successful myogenesis. The function of MYOD during development is to commit mesodermal cells to the skeletal myoblast lineage and regulate its ongoing state. MYOD may also regulate muscle repair. MYOD mRNA levels have also been reported to increase in aging skeletal muscle. One of the major functions of MYOD is to withdraw cells from the cell cycle (arrest of proliferation due to terminal cell cycle arrest in differentiated muscle cells) by promoting the transcription of p21 and MYOD. MYOD is inhibited by cyclin-dependent kinases (CDKs). CDKs are inhibited by p21. Thus, MYOD enhances its own activity within the cell in a feedforward manner. Sustained MYOD expression is required to maintain the expression of muscle-related genes. MYOD is also an important effector of the fast-twitch muscle fiber (types IIA, IIX, and IIB) phenotype.

[0037] In a particular embodiment, the coding sequence of MYOD has the coding sequence of SEQ ID NO:1 and the amino acid sequence of SEQ ID NO:2.

[0038] PAX7 (paired box protein) is a protein encoded by the PAX7 gene in humans. Pax7 plays a role in neural crest development and gastrulation and is an important factor in the expression of neural crest markers such as Slug, Sox9, Sox10, and HNK-1. PAX7 is expressed in the palatal shelves of the maxilla, Meckel's cartilage, midbrain, nasal cavity, nasal epithelium, nasal alveoli, and nasal bridge. Pax7 is a transcription factor that plays a role in myogenesis by regulating the proliferation of muscle progenitor cells. Pax7 can bind to DNA as a heterodimer with PAX3. It also interacts with PAXBP1; this interaction links PAX7 to the WDR5-containing histone methyltransferase complex by similarity. It also interacts with DAXX. PAX7 functions as a marker for a rare subset of spermatogonial stem cells, specifically a subset of single spermatogonia. These PAX7+ spermatogonia are rare in adult testes but are much more common in newborns, accounting for 28% of germ cells in neonatal testes. Unlike PAX7+ myosatellite cells, PAX7+ spermatogonia proliferate rapidly and are not quiescent. PAX7+ spermatogonia can initiate all stages of spermatogenesis and produce motile sperm. However, PAX7 is not required for spermatogenesis, as mice lacking PAX7+ spermatogonia do not exhibit fertility defects. PAX7 may also function to restore spermatogenesis. Unlike other spermatogonia, PAX7+ spermatogonia are resistant to radiation and chemotherapy. Surviving PAX7+ spermatogonia increase in number after these treatments and can differentiate into other forms of spermatogonia that do not survive. In addition, mice lacking PAX7 showed delayed spermatogenesis recovery after exposure to busulfan compared with control mice.

[0039] In certain embodiments, the coding sequence for PAX7 has the sequence of SEQ ID NO:3 and the amino acid sequence of SEQ ID NO:4.

[0040] In certain embodiments, the pluripotent stem cells described herein comprise iv) an additional expression construct for expression of a MYOG protein, wherein the coding sequence for the MYOG protein is operably linked to an inducible promoter, and wherein the expression construct of iv) is inserted into at least one additional genetic safe harbor site that is not the first genetic safe harbor site.

[0041] MYOG, or myogenin, is a transcriptional activator encoded by the MYOG gene. Myogenin is a muscle-specific basic helix-loop-helix (bHLH) transcription factor involved in regulating skeletal muscle development, or myogenesis and repair. MYOG is a member of the MYOD family of transcription factors.

[0042] In mice, MYOG is essential for the development of functional skeletal muscle. MYOG is required for the proper differentiation of most myogenic progenitor cells during myogenesis. When the DNA encoding myogenin was knocked out of the mouse genome, severe skeletal muscle defects were observed. Mice lacking both copies of myogenin (homozygous null) are perinatal lethal due to the lack of mature secondary skeletal muscle fibers throughout the body. In cell culture, myogenin can induce myogenesis in a variety of non-muscle cell types.

[0043] Thus, in certain embodiments, the pluripotent stem cells may comprise expression constructs that provide for the expression of the proteins MYOD, PAX7, and MYOG.

[0044] In certain embodiments, nucleic acid molecules encoding proteins according to the present invention may be codon-optimized for expression in mammalian cells. Methods for codon optimization are known and have been described (e.g., for mammalian cells, see WO 96 / 09378). A sequence is considered codon-optimized if at least one non-preferred codon has been replaced with a more preferred codon compared to the wild-type sequence. As used herein, a non-preferred codon is a codon that is used less frequently in an organism than another codon encoding the same amino acid, and a more preferred codon is a codon that is used more frequently in an organism than a non-preferred codon. The codon usage frequency for a particular organism can be found in a codon frequency table, such as http: / / www.kazusa.or.jp / codon. Preferably, two or more non-preferred codons, preferably most or all non-preferred codons, are replaced with more preferred codons. Preferably, the codon most frequently used in the organism is used in the codon-optimized sequence. Replacement with a preferred codon generally results in higher expression.

[0045] Transcriptional regulatory proteins are proteins that bind in a sequence-specific manner to DNA, preferably to DNA sites located within or near a promoter, and promote the binding of the transcription machinery to the promoter, thus either promoting transcription of the DNA sequence (transcriptional activators) or blocking this process (transcriptional repressors). Such entities are also known as transcription factors.

[0046] DNA sequences to which transcriptional regulatory proteins bind are called transcription factor binding sites or response elements, and they are found within or near the promoter of the regulated DNA sequence.

[0047] Transcriptional activator proteins bind to response elements and promote gene expression. Such proteins are preferred in the methods of the present invention for controlling inducible cassette expression.

[0048] A genetic safe harbor (GSH) site is a locus within the genome where genes or other genetic material can be inserted without any deleterious effects on the cell or the inserted genetic material. The most beneficial GSH sites are those where expression of the inserted genetic sequence is not disrupted by any read-through expression from adjacent genes and where expression of the inducible cassette minimizes interference with the endogenous transcription program. More formal criteria have been proposed to help determine whether a particular locus is a GSH site in the future (Papapetrou et al., 2011, Nature Biotechnology, 29(1), 73-8. doi:10.1038 / nbt.1717). These criteria include: (i) more than 50 kb from the 5' end of any gene; (ii) more than 300 kb from any gene associated with cancer; (iii) more than 300 kb from any microRNA (miRNA); (iv) located outside of a transcription unit; and (v) located outside of an ultraconserved region (UCR). It is not necessary to meet all of these proposed criteria, as previously identified GSHs do not meet all criteria. A suitable GSH would likely meet at least two, three, four, or all of these criteria.

[0049] In certain embodiments of the invention, the first and additional genomic safe harbor sites are selected from any two of the hROSA26 locus, the AAVS1 locus, the CLYBL gene, or the CCR5 gene. In certain embodiments, the first and additional genomic safe harbor sites are located at chr1:152,360,840-152,360,859, chr1:175,942,362-175,942,381, chr1:231,999,396-231,999,415, chr2:45,708,354-45,708,373; chr8:68,720,172-68,720,191 of the human genome.

[0050] In certain embodiments of the invention, the first and further genomic safe harbor sites are selected from any two of the safe harbor sites ROSA26, AAVS1, the CLYBL gene, or the CCR5 gene.

[0051] Preferably, the genetic safe harbor sites are the hROSA26 locus and the AAVS1 locus.

[0052] In certain embodiments of the invention, an expression construct for expression of a MYOD protein described herein and an expression construct for expression of a PAX7 protein described herein are both inserted into a second genetic safe harbor site that is different from the first genetic safe harbor site. In certain embodiments, the expression construct inserted into the second genetic safe harbor site is capable of simultaneously expressing both the MYOD protein and the PAX7 protein.

[0053] As used herein, the term "pluripotent stem cells" includes embryonic stem cells, embryo-derived stem cells, induced pluripotent stem cells, and somatic cells, regardless of the method by which the pluripotent stem cells are derived. Thus, in certain embodiments, the pluripotent stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines. In certain embodiments, the pluripotent stem cells are epiblast-derived stem cells (EpiSCs). In certain embodiments, the pluripotent stem cells express one or more markers selected from the group consisting of OCT-4, Sox2, Klf4, c-MYC, Nanog, Lin28, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Exemplary pluripotent stem cells can be generated using methods known in the art. "Induced pluripotent stem cells" (iPS cells or iPSCs) can be generated by protein introduction of reprogramming factors into somatic cells.

[0054] Pluripotent stem cells according to the present invention can be derived from any species. Embryonic stem cells have been successfully derived in, for example, mice, several species of non-human primates, and humans, and embryonic stem-like cells have been generated from numerous additional species. Thus, those skilled in the art can generate embryonic stem cells and embryo-derived stem cells from any species, including, but not limited to, humans, non-human primates, rodents (mice, rats), ungulates (such as cows and sheep), dogs (domestic and wild), cats (domestic and wild cats such as lions, tigers, and cheetahs), rabbits, hamsters, gerbils, squirrels, guinea pigs, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, and marine mammals (such as dolphins and whales).

[0055] Similarly, iPS cells can be derived from any species.

[0056] In certain embodiments, the pluripotent stem cells according to the invention or for use in the invention are animal cells, hi certain embodiments, the pluripotent stem cells according to the invention or for use in the invention are derived from food animal species.

[0057] Preferably, the pluripotent stem cells according to or for use in the present invention are derived from livestock or poultry animals or marine animals, including but not limited to domestic cattle, pigs, sheep, goats, lambs, camels, buffalo, and rabbits.

[0058] Preferably, the pluripotent stem cells according to the present invention or for use in the present invention are porcine or bovine pluripotent stem cells. Most preferably, they are porcine pluripotent stem cells. In a specific embodiment, the stem cells according to the present invention are porcine epiblast stem cells (pEpiSCs).

[0059] Poultry species include, but are not limited to, domestic chickens, turkeys, ducks, geese, and pigeons. In certain embodiments, the cells are derived from common game birds such as wild deer, domestic chickens, waterfowl, and hares. Preferably, the pluripotent stem cells according to or for use in the present invention are not human cells.

[0060] Seafood includes, but is not limited to, fish and shellfish.

[0061] Transcriptional repressor proteins bind to response elements and prevent gene expression.

[0062] Transcriptional regulatory proteins can be activated or inactivated by several mechanisms, including binding of substances, interaction with other transcription factors (e.g., homodimerization or heterodimerization) or coregulatory proteins, phosphorylation, and / or methylation. Transcriptional regulatory factors can be controlled by activation or inactivation.

[0063] When the transcriptional regulatory protein is a transcriptional activator protein, it is preferred that the transcriptional activator protein requires activation. This activation can be achieved by any suitable means, but it is preferred that the transcriptional regulatory protein is activated by adding an exogenous substance to the cell. The supply of the exogenous substance to the cell can be controlled, and thus the activation of the transcriptional regulatory protein can be controlled. Alternatively, the exogenous substance can be supplied to inactivate the transcriptional regulatory protein, and then the supply can be stopped to activate the transcriptional regulatory protein.

[0064] When the transcriptional regulatory protein is a transcriptional repressor protein, it is preferred that the transcriptional repressor protein requires inactivation, and therefore a substance is provided to prevent the transcriptional repressor protein from repressing transcription, thus allowing transcription to occur.

[0065] Any suitable transcriptional regulatory protein can be used, preferably an activatable or inactivatable transcriptional regulatory protein. Preferably, the transcriptional regulatory protein is obtained by supplying an exogenous substance to control the transcriptional regulatory protein. Such transcriptional regulatory proteins are also called inducible transcriptional regulatory proteins.

[0066] Thus, in certain embodiments, pluripotent stem cells according to the present invention are regulated by exogenously supplied substances.

[0067] In certain embodiments, the exogenously supplied substance is selected from the group consisting of a peptide (e.g., those described in Klotzsche, et al., Journal of Biological Chemistry 280.26 (2005): 24591-24599 or Schlicht et al., Applied and environmental microbiology 72.8 (2006): 5637-5642, or the inducer described in Goeke, et al., Journal of molecular biology 416.1 (2012): 33-45, which are incorporated herein by reference), an aptamer (e.g., the RNA aptamer described in Hunsicker et al., "Chemistry & biology" 16.2 (2009): 173-180, which are incorporated herein by reference), tetracycline, and anhydroteracycline or a derivative thereof. Preferably, the exogenously supplied substance is doxycycline.

[0068] In certain embodiments, the transcriptional regulatory proteins described herein are selected from the group consisting of tetracycline-responsive transcriptional activator protein (rtTa), tetracycline repressor (TetR), VgEcR synthetic receptor, or hybrid transcriptional regulatory proteins comprising a DNA-binding domain from yeast GAL4 protein, a truncated ligand-binding domain from the human progesterone receptor, or an activation domain from human NF-kB.

[0069] Tetracycline-controlled transcriptional activation is a method of inducible gene expression well known in the art, in which transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g., the more stable doxycycline). In this system, the transcriptional activator protein is the tetracycline-responsive transcriptional activator protein (rtTA) or its derivatives. The rtTA protein can bind to DNA at specific TetO operator sequences. Several repeats of such TetO sequences are placed upstream of a minimal promoter (such as the CMV promoter), and together they form a tetracycline response element (TRE). This system has two forms, depending on whether the addition of tetracycline or a derivative activates (Tet-On) or inactivates (Tet-Off) the rTA protein.

[0070] In the Tet-Off system, tetracycline or its derivatives bind to rTA, inactivating it and rendering it unable to bind to the TRE sequence, thereby preventing transcription of the TRE-controlled gene. The Tet-On system consists of two components: (1) a constitutively expressed tetracycline-responsive transcription activator protein (rtTa) and an rtTa-sensitive inducible promoter (Tet Responsive Element, TRE). This can be bound by tetracycline or its more stable derivatives (including doxycycline (dox)), causing activation of rtTa, allowing it to bind to the TRE sequence and inducing expression of the TRE-controlled gene. In a preferred embodiment of the present invention, the transcriptional regulatory protein is rtTA.

[0071] When the transcriptional regulatory protein is rtTA, the inducible promoter inserted into at least one additional GSH site that is not the first GSH site contains a tetracycline responsive element (TRE). Thus, in certain embodiments, the inducible promoter contains a Tet Responsive Element (TRE).

[0072] In some embodiments, the transcriptional regulatory protein is rtTA and, when it contains a TRE, the exogenously supplied substance is the antibiotic tetracycline or one of its derivatives.

[0073] In certain embodiments of the invention, the expression construct inserted into the second genetic safe harbor site is a fusion protein encoding both the MYOD protein and the PAX7 protein described herein. In certain embodiments, the expression construct inserted into the second genetic safe harbor site encodes the MYOD protein, a linker, and the PAX7 protein, and in preferred embodiments, the construct comprises or consists of SEQ ID NO: 8. MYOD and PAX7 may be positioned in either order, i.e., MYOD may be positioned 3' to PAX7, or vice versa.

[0074] In certain embodiments, the linker sequence may be a cleavable linker. That is, the linker sequence may include a sequence of amino acids that can be cleaved. For example, the linker sequence may include a sequence that can act as a substrate for an enzyme capable of cleaving a peptide bond, i.e., a cleavage site. Many such cleavage sites are known to and available to those skilled in the art of molecular biology. In some embodiments, the cleavable linker may include a self-cleavage site. A self-cleavage site is automatically cleaved without the need for enzymatic treatment. For example, a family of 2A self-cleaving peptides, or 2A peptides, has been described, including the 2A peptides P2A, E2A, F2A, and T2A. F2A is derived from foot-and-mouth disease virus; E2A is derived from equine rhinitis A virus; P2A is derived from porcine teschovirus-1 2A; and T2A is derived from thosea asigna virus 2A. In certain embodiments, the cleavable linker is therefore selected from the group consisting of P2A, E2A, F2A, and T2A.

[0075] In some preferred embodiments, the expression construct comprises a picornavirus 2A (P2A) linker. Preferably, the expression construct comprises a linker that comprises or consists of the sequence of SEQ ID NO:6.

[0076] In certain embodiments, the expression construct inserted into the second genetic safe harbor site encoding the MYOD protein, linker, and PAX7 protein described herein comprises or consists of the sequence of SEQ ID NO:8.

[0077] In certain embodiments, the inducible promoter operably linked to the MYOD protein is different from the inducible promoter linked to the PAX7 protein. In certain embodiments, the inducible promoter operably linked to the MYOD protein is the same as the inducible promoter linked to the PAX7 protein. Inducible promoters are well known in the art, and examples include, but are not limited to, CMV, CAG, CBh, PGK, SV40, ferritin heavy chain or light chain, etc.

[0078] In a particular embodiment, the inducible promoter used in the present invention is the tetOn promoter. The third generation TetOn promoter is preferred.

[0079] Culture method The inventors of the present application have surprisingly found that the differentiation time required to obtain skeletal muscle cells can be dramatically reduced by using the pluripotent cells described herein. Thus, in a further aspect, the present invention relates to a method for producing skeletal muscle cells, the method comprising: a) culturing the pluripotent stem cells described herein in a growth medium; followed by b) inducing differentiation of skeletal muscle cells by adding an exogenous substance as described herein.

[0080] In certain embodiments, the methods of the present invention are ex vivo methods.

[0081] In certain embodiments, the method is a method for producing a skeletal muscle cell. A mature skeletal muscle cell is defined herein as a skeletal muscle cell that exhibits one or more of the following: multinucleation, sarcomere formation, and formation of proteins involved in ultimate contractile function (e.g., titin and / or one or more myosin heavy chain isoforms).

[0082] In certain embodiments, the methods of the present invention relate to methods for producing type 1 or type 2 muscles.

[0083] The proliferation medium and differentiation medium may have the same composition. The same medium may be used for proliferation and differentiation. That is, the same culture system can be used for both the proliferation and differentiation stages, for example, by changing the medium or by adding only inducers of the optio-ox system to the bioreactor. Adding inducers for differentiation to the bioreactor is very attractive because it minimizes capital investment in equipment, processing time, and cell manipulation.

[0084] Optionally, proliferation and / or differentiation may be carried out in the absence of insulin and / or retinoic acid.

[0085] Optionally, expansion and / or differentiation may be performed as a single cell suspension, or alternatively, expansion and / or differentiation may involve aggregates.

[0086] The inventors surprisingly found that the use of the pluripotent cells described herein eliminates the need for culturing cells using a commitment induction step. Typically, when culturing skeletal muscle cells, several culture stages can be distinguished. The commitment or decision stage involves the formation of myoblasts, which have lost the potential to differentiate into other cell types. Differentiation of myoblasts into skeletal muscle cells is driven by a highly regulated network of transcription factors expressed in a sequential manner, which promotes the morphological and biochemical characteristics of skeletal muscle, such as the fusion of single myoblasts into multinucleated skeletal muscle cells and the formation of sarcomeres, which require the expression of highly abundant muscle-specific proteins that can account for up to 55% of the protein content of skeletal muscle cells. The differentiation stage can also be divided into four stages: growth arrest, mitotic clonal expansion, early differentiation, and terminal differentiation. The use of the pluripotent stem cells described herein in this method allows for the efficient differentiation of pluripotent stem cells into mature skeletal muscle cells, with a very short myoblast-like cell state (2-4 days). This short cell state is particularly advantageous for reducing the amount of compounds and small molecules typically required to be present in myoblast growth media. The cells described herein may be capable of differentiation in the absence of fetal bovine serum (FBS), dexamethasone, and EGF or FGF2, which are typically required for full differentiation. The ability to omit these compounds from the differentiation media reduces media costs and facilitates regulatory approval.

[0087] The methods described herein dramatically reduce the time to differentiate the pluripotent cells described herein into mature skeletal muscle cells. In certain embodiments, the time to generate skeletal muscle cells using the claimed methods is at most 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, or 2 days. Using the pluripotent cells described in the described methods, the inventors observed a conversion rate of at least 95% by the fourth day of culture, meaning that at least 95% of the cells are mature after four days of culture. Thus, in certain embodiments, the time to generate at least 95% mature skeletal muscle cells is at most 4 days.

[0088] In a further aspect, the present invention provides a skeletal muscle cell, preferably a mature skeletal muscle cell, obtainable by the methods described herein.

[0089] The cell culture described herein can be performed under so-called 2D culture conditions, which are considered the conventional approach to culturing cells. However, the described methods can also be easily adapted to allow culturing under 3D conditions, as shown in the examples below.

[0090] 3D cell culture is an artificially created environment that allows cells to grow and interact with their surroundings in three dimensions. In such cultures, cells typically form 3D colonies, sometimes called "spheroids." 3D culture approaches can more accurately model in vivo cell growth and behavior. Those skilled in the art can easily perform 3D cell culture, for example, by utilizing several commercially available culture tools. For example, 3D culture can be performed using scaffolding or scaffold-free techniques. Scaffold-based techniques utilize supports such as solid scaffolds and hydrogels to culture cells in 3D. Such scaffolds can be designed to mimic the natural extracellular matrix (ECM) present in vivo. Scaffold-free techniques do not require the use of a scaffold for cell growth. Instead, 3D spheroids can be established using, for example, low-attachment plates, hanging drop plates, micropatterned surfaces, rotating bioreactors, magnetic levitation, and magnetic 3D bioprinting.

[0091] Cells transduced with lentiviral vectors have not been recognized as safe for use in food or for human or non-human consumption. The pluripotent cells described herein in the methods described herein do not require the use of lentiviral-transduced cells. Thus, in certain embodiments, skeletal muscle cells produced according to the methods disclosed herein are suitable for human and non-human consumption. In certain embodiments, the skeletal muscle cells produced can be used to produce cultured meat for human consumption. Meat according to the present invention may be derived from any non-human animal, including any of the species described herein.

[0092] In a further aspect, the present invention provides the use of the pluripotent stem cells described herein or skeletal muscle cells obtained by the methods described herein for tissue engineering, hi a particular aspect, the use is for the production of cultivated meat.

[0093] In a further aspect, the present invention provides the use of pluripotent stem cells as described herein or skeletal muscle cells obtained by the methods described herein in tissue engineering, for example in therapy.

[0094] In yet a further aspect, the present invention provides food products (also referred to as "foodstuffs") comprising the pluripotent stem cells described herein or skeletal muscle cells produced and / or obtainable by the methods described. In certain embodiments, the food products further comprise an edible composition for human or non-human consumption. For example, an edible composition for human or non-human consumption comprises at least one of skeletal muscle cells, mature muscle cells, minerals, synthetic substances, flavoring substances (e.g., herbs and spices), plant-based proteins, or proteins of microbial origin, such as yeast proteins. Plant-based proteins and yeast proteins suitable for use in foods are known to those of skill in the art. In certain embodiments, the food product is cultured meat.

[0095] In yet a further aspect, the invention provides a method of producing a food product, the method comprising combining the pluripotent stem cells and / or resulting skeletal muscle cells described or produced herein with an edible composition for human or non-human consumption described herein. In certain embodiments, the food product is cultured meat.

[0096] Array Description

[0097] [Table 1]

[0098] [Table 2]

[0099] [Table 3]

[0100] [Table 4]

[0101] [Table 5]

[0102] [Table 6]

[0103] [Table 7] [Brief explanation of the drawings]

[0104] [Figure 1]Figure 1: EpISCs-MYOD1 (left) and EpISCs-PAX7-MYOD1 (right) differentiated in 2D monolayer culture for 8 days. Cultures were stained with titin and nuclei were visualized with DAPI. [Figure 2] Figure 2: Monolayer differentiation of EpiSCs-PAX7-MyoD1 and expansion of skeletal muscle cells, showing extensive striated patterns indicating complete differentiation into functional skeletal muscle cells. Cells were labeled with anti-titin antibody after 8 days of differentiation, and nuclei were visualized with DAPI. [Figure 3] Figure 3: Comparison of doxycycline duration on the differentiation potential of EpiSCs-PAX7-MYOD1 for 4 days (left) and 2 days (right) over a total culture period of 6 days. Cultures were stained with titin and nuclei were visualized with DAPI. [Figure 4] Figure 4: 3D suspension differentiation after 4 days of doxycycline addition. Aggregates were stained with Pax7 and nuclei were visualized with DAPI. [Figure 5] Figure 5: Comparison of EpiSCs-MyoD1 (left) and EpiSCs-Pax7-MyoD1 (right) after 8 days of 3D suspension differentiation. Aggregates were fixed and labeled with anti-titin antibody, and nuclei were stained with DAPI. [Figure 6] Figure 6: Comparison of EpiSCs-MyoD1 (left) and EpiSCs-Pax7-MyoD1 (right) after 8 days of 3D suspension differentiation. Enlarged view of Figure 5. Arrows indicate sarcomere formation. [Figure 7] Figure 7: EpiSCs-PAX7 (A) and EpISCs-PAX7-MYOD1 (B) differentiated for 18 days and 8 days in 2D monolayer culture. Cultures were stained with titin and nuclei were visualized with DAPI. [Figure 8] Figure 8: Comparison of EpiSCs-PAX7 (left) and EpiSCs-Pax7-MyoD1 (right) after 18 days or 8 days of 3D suspension differentiation. Aggregates were fixed and labeled with anti-titin antibody, and nuclei were stained with DAPI. [Example]

[0105] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

[0106] Materials and Methods Differentiation of porcine epiblast-derived stem cells (pEpISCs) into skeletal muscle cells Undifferentiated pEpISCs (Opti-Ox MyoD1 and Opti-Ox Pax7-MyoD1) were cultured in N2B27 growth medium (50% DMEM Ham's) on hESC-qualified geltrex (A1413301, Thermo Scientific)-coated plates. Cells were grown in F-12 (L0093-500, Biowest), 50% Neurobasal medium (21103049, Thermofisher), B27 supplement (17504044, Thermofisher), N2 supplement (17502001, Thermofisher), glutamax (35050061, Thermofisher), 10 mM 2-mercaptoethanol (31350010, Thermofisher), 0.02 μg / mL activin A (QK001, Q-kine), 0.10 μg / mL FGF2 (QK002, Q-kine), and 0.625 μg / mL XAV939 (X3004, Sigma-Aldrich). For 2D skeletal muscle differentiation, gentle cell dissociation reagent (07174, STEMCELL) was used. After treatment with the EpISC-derived single cells, cell number and viability were assessed. Single pEpISCs were cultured at 100,000–200,000 cells / cm. 2Cells were seeded at a density of 10 μM onto corresponding cell culture plates. After overnight incubation in growth medium containing 10 μM Rock inhibitor (Y-27632 (HBF2297, HelloBio)), the cells were cultured in DMEM high glucose medium or DMEM Ham's F-12 (L0093-500, Biowest) supplemented with 1% Knockout Serum Replacement (10828-028, Thermofisher) and 1× Insulin-Transferrin-Selenium (ITS-G) (41400045, Thermofisher) for the complete differentiation period. The medium was refreshed every 2 days. Doxycycline (1 μg / mL, D9891, Sigma-Aldrich) was added to the differentiation medium to activate the Opti-OX system in these cells only on days 0 and 2, as indicated in the figures or examples. On days 2 and 4, the medium was completely refreshed with doxycycline-free medium.

[0107] For spheroid or aggregate differentiation experiments, undifferentiated single-pEpISCs (Opti-OxMyoD1 and Opti-Ox Pax7-MyoD1) were cultured in 12.5 mL of growth medium (50% DMEM Ham's F-12 (L0093-500, Biowest), 50% Neurobasal medium (21103049, Thermofisher), B27 supplement (17504044, Thermofisher), N2 supplement (17502001, Thermofisher), Glutamax (35050061, Thermofisher), 10 mM 2-mercaptoethanol (31350010, Thermofisher), 0.02 μg / mL activin A (QK001, Q-kine), 0.10 μg / mL Three million cells / mL were seeded into 150 mL shake flasks containing 0.625 μg / mL FGF2 (QK002, Q-kine), 0.625 μg / mL XAV939 (X3004, Sigma-Aldrich), 2% Knockout Serum Replacement (10828-028, Thermofisher) KSR, and 10 ng / mL X FGF2. After 2 days of aggregate formation, the medium was changed to skeletal muscle differentiation medium as described above and refreshed every 2 days until the end of the experiment.

[0108] result Example 1 Development of an inducible transgene overexpression method by double genomic safe harbor (GSH) targeting in animal cells The Opti-ox approach to generating cells capable of controlled transcription as described in WO2018096343A1 was employed in these examples, where an inducible cassette was inserted such that transcription of the genetic material within the inducible cassette could be controlled by using a dual genomic safe harbor targeting system.

[0109] To explore the potential of OPTi-OX for myogenic reprogramming of porcine pluripotent stem cells (pPSCs), we generated MYOD OPTi-OX pPSCs. We sequentially targeted an rtTA cassette to porcine ROSA26 GSH under the control of a CAG promoter and a MYOD transgene to porcine AAVS1 GSH under the control of a doxycycline-inducible element. We observed robust and homogeneous inducible transgene expression. Furthermore, we observed that MYOD reprogramming of pPSCs directly formed myotubes 8 days after expression (Figure 1, left). This finding demonstrated that MYOD can directly reprogram pPSCs into myotubes in 2D culture.

[0110] MYOD and PAX7 were selected for use in a combinatorial cell reprogramming strategy. We designed a targeting vector containing MYOD, a P2A "self-cleaving" peptide linker, and PAX7 all in a single open reading frame to simultaneously express MYOD and PAX7 upon doxycycline induction. The P2A peptide linker is a well-characterized short peptide linker of 18-22 amino acids that allows ribosomal skipping during translation, resulting in the expression of two separate gene products from a single open reading frame. Stable knock-ins in AAVS1 GSH were selected by the incorporation of a puromycin resistance cassette and the addition of puromycin to the cell culture medium. After selection, single pEpiSC cells were plated, and clonal cell lines were isolated for expansion and analysis. The incorporation of doxycycline-inducible MYOD-P2A-PAX7 was then confirmed using PCR genomic analysis, Sanger's method, and RT-qPCR. Using the dual GSH targeting approach, we targeted both alleles and selected clones carrying two copies of each transgene, and observed that homozygous targeting of both elements allowed inducible overexpression (data not shown). Importantly, the dual GSH targeting approach did not affect SC self-renewal or the differentiation capacity of pEpiSCs, as determined by RT-qPCR (data not shown).

[0111] Example 2 2D Differentiation of EpiSCs-MYOD-PAX7 into Multinucleated Skeletal Muscle Cells pEpISCs Opti-OxMYOD and pEpISCs Opti-OxPAX7-MYOD were grown in 3D suspension culture, singled, and plated on coated plastic wells at 200k cells / cm. 2One day after attachment, doxycycline was added to the medium to initiate differentiation. On day 4, doxycycline was removed from the pEpISCs Opti-OxPAX7-MYOD cultures. Both strains demonstrated the formation of titin-positive skeletal muscle cells, but Pax7-MyoD1 resulted in thicker skeletal muscle cells (Figure 1) and stronger formation of sarcomere structures, as visualized in Figure 2. Next, we examined the timing of doxycycline withdrawal to shorten differentiation time. As shown in Figure 3, withdrawal was examined on days 2 and 4 of culture. Both conditions resulted in the formation of multinucleated skeletal muscle cells, as observed by the high similarity in titin expression and morphology, indicating that 2 days of doxycycline induction was sufficient to induce complete differentiation (Figure 3).

[0112] Example 3 3D culture of EpiSCs-MYOD and EpiSCs-MYOD-PAX7 The cell culture expansion (proliferation) stage of cultivated meat production is often scaled up using shakers and / or bioreactors to produce the required quantities of cell mass for cultivated meat products at a cost-competitive price. Primary cells derived from muscle biopsies are typically used. However, primary cultures have limited self-renewal, lose the ability to differentiate during expansion, and vary in quality between biopsies. Other sources, such as ESCs and iPSCs, have high proliferation capacity but remain undifferentiated. Differentiation is induced using small molecules to mimic embryonic development (reviewed in Chal et al., 2017 Jun 15;144(12):2104-2122. doi: 10.1242 / dev.151035.) or through ectopic expression of the transcription factors MYOD or PAX7. In meat production, differentiation of cells into myotubes and subsequent myofibers is an integral part of the process and typically occurs in separate subsequent steps. Differentiation of skeletal muscle cells often requires different conditions in terms of nutrients and physical environment. While the necessary nutrients can be provided by switching from a nutrient-rich growth medium to a differentiation medium consisting of low serum, providing the physical environment necessary for cells to differentiate is more difficult. In addition, the substrate requirements for the proliferation and differentiation phases typically differ in terms of surface chemistry and topography. A particular challenge for skeletal muscle cells in suspension is that they require a pole, scaffold, or surface attachment surface to form elongated, multinucleated myotubes.

[0113] In this example, EpiSCs-MYOD and EpiSCs-PAX7-MYOD were adapted to 3D suspension cell growth and grown as aggregates; EpiSCs grown in adherent 6-well cell plates were dissociated into single cells using Accumax (00-4666-56, ThermoFisher Scientific) according to the manufacturer's instructions, transferred to 150 mL shake flasks containing 12.5 mL medium and a RHO / ROCK pathway inhibitor, and then expanded for at least three cycles. The medium during 3D growth was refreshed daily. The 3D-adapted EpiSCs were then used in shaker and bioreactor experiments.

[0114] To investigate skeletal muscle differentiation in 3D suspension culture, 500,000 cells / mL were seeded into 150 mL shake flasks containing 12.5 mL of medium, and EpiSCs-MYOD or EpiSCs-PAX7-MYOD were differentiated using the differentiation protocol. The medium was changed every other day. We detected PAX7 expression in the aggregates within two days of inducing PAX7 / MYOD expression with doxycycline (Figure 4). After 8 days of differentiation, both EpiSC lines (Figure 5) showed skeletal muscle cell formation, but the aggregates containing EpiSCs-PAX7-MYOD showed much larger and elongated skeletal muscle cells with many nuclei. In EpiSCs-MYOD aggregates, primarily mononuclear cells were detected. As observed by immunofluorescence staining, both lines produced titin protein at this time point, but only in EpiSCs-PAX7-MYOD was a striated pattern of titin detected, indicating that functional sarcomeres had already formed and improved maturation ( Figure 6 , arrowheads).

[0115] The simplified bioprocess presented is very attractive because it allows the use of the same culture system for both the proliferation and differentiation stages via medium exchange or by simply adding the Opti-ox inducer into the bioreactor / for differentiation, minimizing capital investments in equipment, processing time, and cell manipulation.

[0116] Example 4 Comparison of differentiation rates between EpiSCs-PAX7 and EpiSCs-PAX7-MYOD pEpISCs Opti-OxPAX7 were cultured in suspension in Pax7 induction medium (DMEM / F12, 1x ITS, and 15% KSR) containing 3 μM CHIR99021 for 2 days, then switched to Pax7 induction medium containing 1 μg / ml DOX and 20 ng / ml FGF2 for 10 days. After 10 days, cells were differentiated directly into skeletal muscle cells within aggregates or as single cells plated on Geltrex-coated dishes. Differentiation was initiated using Pax7 differentiation medium (DMEM HG, 1x ITS, 1% KSR) with DOX withdrawal. For pEpISCs, Opti-OxPAX7-MYOD cells were first induced for 4 days in PAX7 / MYOD medium containing DMEM / HG, 1x ITS, 15% KSR, and 1 μg / ml DOX. On day 4 of culture, DOX was discontinued and the cells were switched to PAX7 / MYOD differentiation medium (DMEM HG, 1x ITS, and 1% KSR). Because shorter differentiation times did not yield visibly differentiated cells, differentiation of Pax7 cells was measured on day 18, and differentiation of Pax7-MyoD1 cells was measured on day 8 (Figure 7).

[0117] After 8 days of differentiation, Pax7-MyoD1 cells showed the formation of titin-positive skeletal muscle cells, which were thicker than those observed after 18 days of differentiation in Pax7 cells (Figure 7A). Pax7-MyoD1 cells also showed strong formation of sarcomere structures, as visualized in Figure 7B. Even after 18 days of differentiation, the difference in differentiation is still significant, as EpiSCs-PAX7 cells have not yet reached the same thickness and sarcomere structure. Furthermore, aggregates containing EpiSCs-PAX7-MYOD showed much larger and longer skeletal muscle cells containing many nuclei (Figure 8).

Claims

1. (i) an expression construct for expression of a transcriptional regulatory protein inserted into a first genetic safe harbor site; (ii) an expression construct for expression of a MYOD protein, wherein the coding sequence of the MYOD protein is operably linked to an inducible promoter; and (iii) an expression construct for expression of a PAX7 protein, wherein the coding sequence for the PAX7 protein is operably linked to an inducible promoter. A pluripotent stem cell comprising: (ii) and (iii) the expression constructs are inserted into at least one additional genetic safe harbor site that is not the first genetic safe harbor site; the inducible promoter is regulated by the transcriptional regulatory protein; Pluripotent stem cells.

2. The pluripotent stem cell of claim 1, wherein both of the expression constructs (ii) and (iii) are inserted into a second genetic safe harbor site that is different from the first genetic safe harbor site.

3. The pluripotent stem cell of claim 1 or 2, wherein the cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines.

4. The pluripotent stem cells according to any one of claims 1 to 3, which are of a livestock species or a poultry species.

5. The pluripotent stem cells according to claim 4, wherein the livestock species is porcine or bovine, preferably porcine.

6. The pluripotent stem cell of any one of claims 2 to 5, wherein the expression construct inserted into the second genetic safe harbor site encodes a MYOD protein, a linker, and a PAX7 protein, preferably wherein the linker is a P2A linker, more preferably wherein the linker comprises the sequence of SEQ ID NO:

6.

7. The pluripotent stem cell of claim 6, wherein the construct comprises the sequence of SEQ ID NO:

8.

8. The pluripotent stem cell according to any one of claims 1 to 7, wherein the activity of the transcriptional regulatory protein is controlled by an exogenously supplied substance inducer.

9. The pluripotent stem cell according to any one of claims 1 to 8, wherein the transcriptional regulatory protein is selected from the group consisting of tetracycline-responsive transcriptional activator protein (rtTa), tetracycline repressor (TetR), VgEcR synthetic receptor, or a hybrid transcriptional regulatory protein, the hybrid transcriptional regulatory protein comprising a DNA-binding domain derived from yeast GAL4 protein, a truncated ligand-binding domain derived from human progesterone receptor, and an activation domain derived from human NF-kB, and preferably the transcriptional regulatory protein is rtTA.

10. The pluripotent stem cell according to any one of claims 1 to 9, wherein the inducible promoter comprises a Tet-responsive element (TRE).

11. The pluripotent stem cell according to any one of claims 1 to 10, wherein the inducible promoter is a tetON promoter.

12. The pluripotent stem cell according to any one of claims 1 to 11, wherein the first and further genetic safe harbor sites are selected from any two of the hROSA26 locus, the AAVS1 locus, the CLYBL gene, or the CCR5 gene, and preferably the genetic safe harbor sites are the hROSA26 locus and the AAVS1 locus.

13. A method for producing skeletal muscle cells, comprising: a) culturing the pluripotent stem cells according to any one of claims 1 to 12 in a growth medium; and b) inducing differentiation of skeletal muscle cells by adding the exogenous substance of claim 8.

14. The method of claim 13, wherein the proliferation medium and the differentiation medium have the same composition.

15. 15. The method of claim 13 or 14, wherein the differentiation stage is at most 10 days, at most 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, or 3 days.

16. The method according to any one of claims 13 to 15, wherein the skeletal muscle cells produced are for human and non-human nutrition.

17. Use of the pluripotent stem cells of any one of claims 1 to 12, or the method for producing skeletal muscle cells of any one of claims 13 to 16, for tissue engineering, optionally for the production of cultivated meat.

18. A food product comprising the pluripotent stem cells according to any one of claims 1 to 12 or skeletal muscle cells obtained by the method according to any one of claims 13 to 16.

19. 20. The food product of claim 18, wherein the food product is cultured meat.

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