Maturation of skeletal muscle cells

Abstract

The present invention relates to a pluripotent stem cell comprising an expression construct for expressing an MYOD protein and an expression construct for expressing an MYOG # protein. The invention further provides methods comprising the production of skeletal muscle cells using pluripotent stem cells and foods comprising skeletal muscle cells or pluripotent stem cells.

Description

TECHNICAL FIELD

[0001] The present invention relates to modified pluripotent cells and methods of differentiating said cells into skeletal muscle cells. BACKGROUND

[0002] According to the latest estimates by the United Nations, the world population is currently 7.9 billion in 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 globally, with 9 countries including India, Nigeria, Pakistan, Egypt and the United States accounting for half of the projected global population growth over the next 30 years. Population and economic growth are the main 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 expected to increase by an estimated 15% by 2031. On the other hand, the correlation between income growth and meat consumption increases is evident at lower income rates, but once consumers reach a sufficient standard of living, they become more sensitive to environmental, ethical, animal welfare and health issues.

[0003] Therefore, there is an increasing interest in finding alternative protein sources that are, ideally, sustainable and contain the nutrients that meat typically provides in the human diet. Cultured meat is an alternative to traditional animal agriculture that aims to produce skeletal muscle and adipose tissue that typically make up animal meat, but with the exception of using in vitro tissue and bioengineering techniques. Despite efforts to develop robust protocols to generate animal cell types scalable from readily accessible and renewable sources, the differentiation of animal (pluripotent) stem cells into specific cell types is often still tedious, lengthy and difficult to reproduce and / or has not been established.

[0004] Pig embryonic fibroblasts and pig induced pluripotent stem cells have been reported to skeletal muscle reprogram cells derived from livestock (Jeong et al., 2021 and Genovese et al., 2017). In both studies, MyoD1 overexpression was used to reprogram skeletal muscle cells. Lentiviral overexpression of MyoD1 in pig embryonic fibroblasts exhibited low differentiation efficiency, long culture times and the need for serum-containing media. Induction of pig induced pluripotent stem cells with MyoD1 for 8 days resulted in high conversion rates but relied on 5-aza-cytidine and CHIR99021, which are not food-safe. In summary, these protocols resulted in heterogeneous skeletal muscle maturation, lack of scalability, were not food-safe and had long culture times of up to 14 days.

[0005] Therefore, there is still a need in the art for producing and cultivating mature skeletal muscle cells that are suitable for human consumption and that can be produced in a scalable and cost-effective manner. SUMMARY

[0006] The present invention relates to the expression of the transcriptional regulatory proteins MYOD and MYOG in pluripotent stem cells. Therefore, in a first aspect, the present invention relates to a pluripotent stem cell comprising:

[0007] i) an expression construct for expressing a transcriptional regulatory protein inserted in a first genetic safe harbor site;

[0008] ii) an expression construct for expressing a MYOD protein, wherein the coding sequence of the MYOD protein is operably linked to an inducible promoter; and

[0009] iii) an expression construct for expressing a MYOG protein, wherein the coding sequence of the MYOG protein is operably linked to an inducible promoter;

[0010] wherein the expression constructs of ii) and iii) are inserted in at least one additional genetic safe harbor site that is not the first genetic safe harbor site,

[0011] and wherein the inducible promoter is regulated by the transcriptional regulatory protein.

[0012] In certain embodiments of the present invention, the expression constructs of ii) and iii) are inserted in a second genetic safe harbor site that is different from the first genetic safe harbor site. Preferably, 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, preferably wherein the genetic safe harbor sites are the hROSA26 locus and the AAVS1 locus.

[0013] In certain embodiments of the present invention, the cell is selected from the group consisting of an embryonic stem cell, an induced pluripotent stem cell, an embryonic cell line and a somatic cell line.

[0014] In certain embodiments of the present invention, the pluripotent stem cell belongs to a livestock or poultry species. Preferably, the livestock species is a pig or a cow, preferably a pig.

[0015] In certain embodiments of the present invention, the expression construct inserted in the second genetic safe harbor site encodes a MYOD protein, a linker and a MYOG protein, preferably wherein the linker is a P2A linker, more preferably wherein the linker comprises the sequence of SEQ ID NO: 6. Preferably, the construct comprises the sequence of SEQ ID NO: 8.

[0016] In certain embodiments of the application, the activity of the transcriptional regulator protein is controlled by an exogenously supplied substance derivative. Preferably, the transcriptional regulator protein is selected from the group consisting of a tetracycline-responsive transcriptional activator (rtTa), a tetracycline repressor (TetR), a VgEcR synthetic receptor, or a hybrid transcriptional regulator protein comprising a DNA-binding domain from the yeast GAL4 protein, a truncated ligand-binding domain from the human progesterone receptor, and an activation domain from human NF-kB, preferably the transcriptional regulator protein is rtTA.

[0017] In certain embodiments, the inducible promoter comprises a Tet response element (TRE).

[0018] In certain embodiments, the inducible promoter is a tetON promoter.

[0019] In a second aspect, the application provides a method for producing skeletal muscle cells (e.g. muscle type 1 or 2), the method comprising

[0020] a) culturing the pluripotent stem cell according to any one of the preceding claims in a proliferation medium: followed by

[0021] b) inducing skeletal muscle cell differentiation by addition of an exogenous substance as described herein. In certain embodiments, the differentiation phase of the method described herein is at most 10 days, at most 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days or 2 days.

[0022] The medium used for proliferation and the medium used for differentiation can be the same medium or different media.

[0023] In certain embodiments, the produced skeletal muscle cells are for human and non-human dietary consumption.

[0024] In a further aspect, the application provides the use of a pluripotent stem cell as described herein or the use of a method for producing skeletal muscle cells as described herein, for tissue engineering, optionally for producing cultured meat.

[0025] In yet a further aspect, the application provides a food product comprising a pluripotent stem cell as described herein or a skeletal muscle cell obtained by a method as described herein. In certain embodiments, the food product is cultured meat. DETAILED DESCRIPTION

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation depending on the disclosure presented herein, many methods and materials similar or equivalent to those described herein for the practice of the present application. Indeed, the present application is in no way limited to the methods described herein.

[0027] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but not to the exclusion of other items that are not specifically recited. Additionally, unless the context clearly requires otherwise, the use of the singular "a", "an" and "the" in the claims and the specification including the words "comprising", "comprises" and "comprised of" includes the plural and vice versa unless the context clearly requires otherwise. Thus, the use of the words "a" and "an" and "the" is intended to be inclusive of the singular and plural. The term "at least" preceding a recited item or a group of recited items is intended to mean that the item or group of items is present, but not necessarily more than one. Thus, the term "at least" is understood to be synonymous with the term "one or more."

[0028] As used herein, the term "and / or" means that one or more of the stated circumstances can occur or occur in combination with at least one of the stated circumstances.

[0029] As used herein, "at least" a particular value means that particular value or more. 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.).

[0030] The word "about" or "approximately" when used in connection with a numerical value (e.g., about 10) preferably means that the value can be the given value (10) plus or minus 0.1% of the value.

[0031] The term "heterologous" when used in reference to a nucleic acid (DNA or RNA) or protein means that the nucleic acid or protein is not naturally present as part of the organism, cell, genome or DNA or RNA sequence in which it is found, or is found in a location in the cell or genome or DNA or RNA sequence that is 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 has been obtained or synthesized or recombinantly produced from another cell. Typically, although not necessarily, such a nucleic acid encodes a protein that is not normally produced by the cell in which the DNA is transcribed or expressed. Similarly, an exogenous RNA encodes a protein that is not normally expressed in the cell in which the exogenous RNA is present. A heterologous nucleic acid and protein can also be referred to as a foreign nucleic acid or protein. One skilled in the art will recognize that any nucleic acid or protein that is foreign or exogenous to the cell in which it is expressed is encompassed by the term heterologous nucleic acid or protein herein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e., combinations of at least two of the sequences are foreign to each other.

[0032] The term "expression vector" or "expression construct" refers to a nucleotide sequence capable of effecting expression of a gene in a host cell or host organism compatible with such sequence. These expression vectors typically include at least a suitable transcriptional regulatory sequence and optionally a 3' transcription termination signal. Additional factors necessary or helpful in effecting expression, such as expression enhancer elements, can also be present.

[0033] 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 in a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if the transcriptional regulatory sequence affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, are contiguous and in reading frame. Inducible promoters

[0034] As used herein, the term "promoter" refers to a nucleic acid fragment, which functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcriptional start site of the coding sequence, and has structural features that provide for binding of DNA-dependent RNA polymerase, a transcriptional initiation site, and any other DNA sequences, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequences known to one of skill in the art that function either directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is one that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is one that is physiologically or developmentally regulated, for example by the application of a chemical inducer. In the context of the present application, control is effected by transcriptional regulatory proteins.

[0035] Any reference herein to nucleotide or amino acid sequences accessible in public sequence databases refers to the version of the sequence entry available on the date of filing of this document.

[0036] All patents and references cited in this specification are hereby incorporated by reference in their entirety.

[0037] The inventors have surprisingly found that by using modified pluripotent stem cells comprising an expression construct for expressing a MYOD protein and an expression construct for expressing a MYOG protein, or alternatively an expression construct expressing both MYOD and MYOG, the time period required for the differentiation of pluripotent cells into mature skeletal muscle cells can be significantly reduced. As shown in the examples described herein, by using these modified pluripotent cell lines, complete differentiation into mature skeletal muscle cells can be achieved in 6 days or less, for example less than 5 days, for example 4 days or 3 days or faster. In addition to significantly reducing the culturing time and associated costs involved, the use of the pluripotent cells described herein also provides for a more scalable production of mature skeletal muscle cells compared to previously described, allowing for the generation of more mature skeletal muscle cells.

[0038] The differentiated cells can form spindle-like elongated morphology, undergo extensive cell fusion and exhibit strong and uniform myogenic marker expression based on mRNA and protein levels (MHC isoforms / maturity, immunostaining MYOD / MYOG / MYH / TTN / TNNT / PAX3 / 7) and high fusion index, length and thickness. In addition, spontaneous muscle fiber contractions can also be observed, demonstrating functionality of MYOD / PAX7.

[0039] The induction efficiency of exogenous compounds is not reduced over extended culturing periods, demonstrating the robustness and reproducibility of the system (percentage of MHC positive cells across different passages).

[0040] Thus, in a first aspect, the present application relates to a pluripotent stem cell comprising:

[0041] i) an expression construct for expressing a transcriptional regulator protein inserted into a first genetic safe harbor site;

[0042] ii) an expression construct for expressing a MYOD protein, wherein the coding sequence of the MYOD protein is operably linked to an inducible promoter; and

[0043] iii) an expression construct for expressing a MYOG protein, wherein the coding sequence of the MYOG protein is operably linked to an inducible promoter;

[0044] wherein the expression constructs of ii) and iii) are inserted into at least one further genetic safe harbor site which is not the first genetic safe harbor site,

[0045] and wherein the inducible promoter is regulated by the transcriptional regulator protein.

[0046] The pluripotent stem cells of the application can optionally not comprise expression constructs other than those listed in i), ii) and iii) above. Alternatively, the pluripotent stem cells of the application can optionally comprise one or more expression constructs other than those listed in i), ii) and iii) above, for example one or more expression constructs inserted into at least one additional genetic safe harbor site that is not the first genetic safe harbor site. Such additional expression constructs can provide a protein for expression, wherein the modulator is a transcriptional modulator, for example a transcription factor, other than MYOD or MYOG.

[0047] If the pluripotent stem cells of the application comprising one or more antibiotic resistance markers are generated, the one or more such resistance markers can be removed. The pluripotent stem cells of the application can optionally not comprise antibiotic resistance markers.

[0048] MYOD, also known as myoblast determination protein 1, is a protein that plays a major role in regulating muscle differentiation in animals. MYOD belongs to a family of proteins known as myogenic regulators. MYOD is one of the earliest markers of myogenic commitment. MYOD is expressed at very low and essentially undetectable levels in quiescent satellite cells, but MYOD expression is activated after exercise or muscle tissue injury. The effect of MYOD on satellite cells is dose dependent: high MYOD expression suppresses cell renewal, promotes terminal differentiation and can induce apoptosis. Although MYOD marks myoblast commitment, muscle development is not significantly inhibited in mice mutants lacking the MYOD gene. This can be due to functional redundancy of Myf5 and / or Mrf4. However, the combination of MYOD and Myf5 is essential for successful myogenesis. The function of MYOD in development is to commit mesodermal cells to the skeletal myoblast lineage and then to regulate this persistent state. MYOD can also regulate muscle repair. MYOD mRNA levels have also been reported to be elevated in aged skeletal muscle. One of the main roles of MYOD is to remove cells from the cell cycle (stop proliferation in differentiating myocytes for terminal cell cycle arrest) by enhancing the transcription of p21 and MYOD. MYOD is inhibited by cyclin-dependent kinases (CDKs). CDKs, in turn, are inhibited by p21. Thus, MYOD enhances its own activity in the cell in a feed-forward manner. Persistent MYOD expression is necessary to preserve the expression of muscle-related genes. MYOD is also an important effector of fast muscle fiber (types IIA, IIX, and IIB) phenotypes.

[0049] In certain embodiments, the coding sequence for MYOD has the sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2.

[0050] 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 coordinating skeletal muscle development or myogenesis and repair. MYOG is a member of the MYOD family of transcription factors.

[0051] In mice, MYOG is essential for the development of functional skeletal muscle. MYOG is required for the proper differentiation of most muscle-derived precursor cells during the process of myogenesis. When the DNA encoding myogenin is knocked out from the mouse genome, severe skeletal muscle defects are observed. Mice lacking two copies of myogenin (homozygous-null) suffer perinatal death due to a 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.

[0052] In certain embodiments, the coding sequence of MYOG has the sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4.

[0053] In certain embodiments, the pluripotent stem cell described herein comprises iv) a further expression construct for expressing a PAX7 protein, wherein the coding sequence of the PAX7 protein is operably linked to an inducible promoter, wherein the expression construct of iv) is inserted into at least one further genetic safe harbor site which is not the first genetic safe harbor site.

[0054] PAX7 (Paired box protein) is a protein in humans that is encoded by the PAX7 gene. Pax-7 plays a role in neural crest development and primitive gut formation, and it is an important factor for expression of neural crest markers such as Slug, Sox9, Sox10, and HNK-1. PAX7 is expressed in the maxillary process palatine, Meckel's cartilage, midbrain, nasal cavity, nasal epithelium, nasal placode, and pons. Pax7 is a transcription factor that plays a role in myogenesis by regulating muscle precursor cell proliferation. It can bind to DNA as a heterodimer with PAX3. Also interacts with PAXBP1; this interaction links PAX7 to the WDR5-containing histone methyltransferase complex through similarity. Also interacts with DAXX. PAX7 is a marker for a rare subpopulation of spermatogonial stem cells, specifically a subpopulation of single spermatogonia. These PAX7+ spermatogonia are rare in the adult testis but are more prevalent in the neonatal testis, accounting for 28% of the neonatal testis germ cells. Unlike PAX7+ muscle satellite cells, PAX7+ spermatogonia proliferate rapidly and are not quiescent. PAX7+ spermatogonia are capable of all stages of spermatogenesis and produce motile sperm. However, PAX7 is not essential for spermatogenesis, as mice without PAX7+ spermatogonia show no defects in fertility. PAX7 can also play a role in the recovery of spermatogenesis. Unlike other spermatogonia, PAX7+ spermatogonia are resistant to radiation and chemotherapy. Surviving PAX7+ spermatogonia are able to increase in number after these therapies and differentiate into other forms of spermatogonia that do not survive. Additionally, mice lacking PAX7 have a delayed recovery of spermatogenesis after exposure to busulfan compared to control mice.

[0055] Thus, in certain embodiments, the pluripotent stem cells can comprise expression constructs that provide expression of the proteins MYOD, MYOG, and PAX7.

[0056] In certain embodiments, the nucleic acid molecule encoding a protein according to the application can be codon-optimized for expression in mammalian cells. Methods for codon-optimization are known and have been described previously (e.g. WO 96 / 09378 for mammalian cells). A sequence is considered codon-optimized if at least one non-preferred codon is replaced by a more preferred codon compared to the wild-type sequence. 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 the non-preferred codon. The codon usage frequency of a particular organism can be found in codon frequency tables, e.g. http: / / www.kazusa.or.jp / codon. Preferably, more than one non-preferred codon, preferably most or all non-preferred codons are replaced by more preferred codons. Preferably, the most frequently used codons in an organism are used in the codon-optimized sequence. Replacement by preferred codons typically results in higher expression.

[0057] A transcriptional regulatory protein is a protein that binds to DNA, preferably to a DNA site sequence located within or near a promoter, and facilitates the binding of the transcription machinery to the promoter, thereby transcribing the DNA sequence (transcriptional activator) or blocking this process (transcriptional repressor). Such entities are also referred to as transcription factors.

[0058] The DNA sequence to which a transcriptional regulatory protein binds is referred to as a transcription factor binding site or response element, which is found in or near the promoter of the regulated DNA sequence.

[0059] A transcriptional activator protein binds to a response element and facilitates gene expression. Such proteins are preferred in the methods of the application for controlling the expression of an inducible cassette.

[0060] Genetic safe harbor (GSH) sites are loci within the genome where genes or other genetic material can be inserted without any deleterious effects on the cell or the inserted genetic material. Most beneficial are GSH sites where expression of the inserted gene sequence is not interfered with by any read-through expression of neighboring genes, and expression of the inducible cassette minimizes interference with endogenous transcriptional programs. More formal criteria have been proposed that help determine whether a particular locus will be a GSH site in the future (Papapetrou et al., 2011, Nature Biotechnology, 29(1), 73-8. doi: 10.1038 / nbt.1717.). These criteria include sites that are (i) 50 kb or more from the 5’ end of any gene, (ii) 300 kb or more from any gene associated with cancer, (iii) 300 kb or more from any microRNA (miRNA), (iv) outside of a transcriptional unit, and (v) outside of an ultraconserved region (UCR). It can not be necessary to meet all of these proposed criteria, as GSHs that have been identified do not meet all of the criteria. It is believed that a suitable GSH will meet at least 2, 3, 4, or all of these criteria.

[0061] In certain embodiments of the application, the first and additional genomic safe harbor site is 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 site is 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.

[0062] In certain embodiments of the application, the first and additional genomic safe harbor site is selected from any two of the safe harbor sites ROSA26, AAVS1, the CLYBL gene, or the CCR5 gene.

[0063] Preferably, the genetic safe harbor sites are the hROSA26 locus and the AAVS1 locus.

[0064] In certain embodiments of the application, the expression construct for expressing a MYOD protein described herein and the expression construct for expressing a MYOG protein described herein are 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 expressing both a MYOD protein and a MYOG protein.

[0065] As used herein, the term "pluripotent stem cell" includes embryonic stem cells, embryonic-derived stem cells, induced pluripotent stem cells, and somatic cells, regardless of the method of derivation of the pluripotent stem cell. Thus, in certain embodiments, the pluripotent stem cell is selected from the group consisting of an embryonic stem cell, an induced pluripotent stem cell, an embryonic cell line, and a somatic cell line. In certain embodiments, the pluripotent stem cell is an epiblast-derived stem cell (EpiSC). In certain embodiments, the pluripotent stem cell expresses 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 transduction of reprogramming factors in somatic cells.

[0066] The pluripotent stem cells according to the present application can be from any species. For example, embryonic stem cells have been successfully derived in mice, various non-human primates, and humans, and embryonic stem-like cells have been generated from many additional species. Thus, one of skill in the art can generate embryonic stem cells and embryonic-derived stem cells from any of the following species, including but not limited to, humans, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic dogs and wild dogs), cats (domestic cats and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrels, guinea pigs, goats, elephants, pandas (including the giant panda), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), and the like.

[0067] Likewise, iPS cells can be from any species.

[0068] In certain embodiments, the pluripotent stem cell according to the present application or for use in the present application is an animal cell. In certain embodiments, the pluripotent stem cell according to the present application or for use in the present application is from an edible animal species.

[0069] Preferably, the pluripotent stem cell according to the present application or for use in the present application is from a livestock or poultry animal or a seafood animal. Livestock species include, but are not limited to, cattle, pigs, sheep, goats, lambs, camels, water buffalo, and rabbits.

[0070] Preferably, the pluripotent stem cell according to the present application or for use in the present application is a pig or bovine pluripotent stem cell. Most preferably, the pluripotent stem cell is a pig pluripotent stem cell. In certain embodiments, the stem cell according to the present application is a pig epiblast stem cell (pEpiSC).

[0071] Poultry species include, but are not limited to, chickens, turkeys, ducks, geese and pigeons. In certain embodiments, the cells are derived from common game species, such as deer, partridges, waterfowl and hares. Preferably, the pluripotent stem cells according to the present invention or for use in the present invention are not human cells.

[0072] Seafood species include, but are not limited to, fish and shellfish.

[0073] Transcriptional repressor proteins bind to response elements and prevent gene expression.

[0074] Transcriptional regulatory proteins can be activated or inactivated by a variety of mechanisms, including binding of a substance, interaction with other transcription factors (e.g., homo- or hetero-dimerization) or co-regulatory proteins, phosphorylation and / or methylation. Transcriptional regulatory factors can be controlled by activation or inactivation.

[0075] If the transcriptional regulatory protein is a transcriptional activator protein, it is preferred that the transcriptional activator protein requires activation. This activation can be performed by any suitable means, but it is preferred that the transcriptional regulatory protein is activated by the addition of 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 is withdrawn to activate the transcriptional regulatory protein.

[0076] If the transcriptional regulatory protein is a transcriptional repressor protein, it is preferred that the transcriptional repressor protein requires inactivation. Thus, a substance is provided to prevent the transcriptional repressor protein from repressing transcription, and thus allowing transcription.

[0077] Any suitable transcriptional regulatory protein can be used, preferably a transcriptional regulatory protein that can be activated or inactivated. It is preferred that an exogenous substance can be provided to control the transcriptional regulatory protein. Such transcriptional regulatory proteins are also referred to as inducible transcriptional regulatory proteins.

[0078] Thus, in certain embodiments, the pluripotent stem cells according to the present invention are controlled by an exogenously supplied substance.

[0079] In certain embodiments, the exogenously supplied substance is selected from the group consisting of a peptide (such as described by 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 an inducer (as described in Goeke et al. Journal of molecular biology 416.1 (2012): 33-45; this document is incorporated by reference herein), an aptamer (such as the RNA aptamer described in Hunsicker et al. Chemistry & biology 16.2 (2009): 173-180; this document is incorporated by reference herein), tetracycline and anhydrotetracycline or a derivative thereof. Preferably, the exogenously supplied substance is doxycycline.

[0080] In certain embodiments, the transcriptional regulator protein described herein is selected from the group consisting of a tetracycline-responsive transcriptional activator (rtTA), a tetracycline repressor (TetR), a VgEcR synthetic receptor, or a hybrid transcriptional regulator protein comprising a DNA-binding domain from the yeast GAL4 protein, a truncated ligand-binding domain from the human progesterone receptor, or an activation domain from human NF-kB.

[0081] Tetracycline-controlled transcriptional activation is a well-known method for inducible gene expression in the art, in which transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives, such as the more stable doxycycline. In this system, the transcriptional activator protein is a tetracycline-responsive transcriptional activator (rtTA) or a derivative thereof. The rtTA protein is capable of binding 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), which together form a tetracycline-responsive element (TRE). The system has two forms, depending on whether the addition of tetracycline or derivative activates (Tet-on) or inactivates (Tet-off) the rTA protein.

[0082] In the Tet-Off system, tetracycline or a derivative thereof binds to rTA and inactivates rTA, preventing it from binding to the TRE sequence and thereby preventing transcription of the TRE-controlled gene. The Tet-On system consists of two components; (1) constitutively expressed tetracycline-responsive transcriptional activator (rtTa) and rtTa-sensitive inducible promoter (Tet response element, TRE). This can be bound by tetracycline or its more stable derivatives, including doxycycline (dox), resulting in activation of rtTa, which binds to the TRE sequence and induces expression of the TRE-controlled gene. In preferred embodiments of the present invention, the transcriptional regulatory protein is rtTA.

[0083] If the transcriptional regulatory protein is rtTA, the inducible promoter inserted in at least one additional GSH that is not the first GSH site comprises a tetracycline response element (TRE). Thus, in certain embodiments, the inducible promoter comprises a Tet response element (TRE).

[0084] In some embodiments, where the transcriptional regulatory protein is rtTA and comprises a TRE, the exogenously supplied substance is the antibiotic tetracycline or one of its derivatives.

[0085] In certain embodiments of the present invention, the expression construct inserted into the second genetic safe harbor site is a fusion protein encoding a MYOD protein and a MYOG protein, as described herein. In certain embodiments, the expression construct inserted into the second genetic safe harbor site encodes a MYOD protein, a linker, and a MYOG protein, and in preferred embodiments, the construct comprises or consists of SEQ ID NO: 8. MYOD and MYOG can be arranged in either order, i.e., MYOD is located 3’ of MYOG, or vice versa.

[0086] In certain embodiments, the linker sequence can be a cleavable linker. That is, the linker sequence can comprise an amino acid sequence that is capable of being cleaved. For example, the linker sequence can comprise a sequence that is capable of being a substrate for an enzyme that is capable of cleaving a peptide bond, i.e., a cleavage site. Many such cleavage sites are known to and can be used by those skilled in the art of molecular biology. In some embodiments, the cleavable linker can comprise a self-cleaving site. Self-cleaving sites are cleaved automatically, without the need for enzyme treatment. For example, the 2A self-cleaving peptides or 2A peptide family have been described, which include 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 the 2A of the Andes virus. In certain embodiments, the cleavable linker is thus selected from the group consisting of P2A, E2A, F2A, and T2A.

[0087] In some preferred embodiments, the expression construct comprises a picornavirus 2A (P2A) linker. Preferably, the expression construct comprises a linker comprising or consisting of the sequence of SEQ ID NO: 6.

[0088] In certain embodiments, the expression construct encoding the MYOD protein, the linker and the MYOG protein inserted into the second genetic safe harbor site described herein comprises or consists of the sequence of SEQ ID NO: 8.

[0089] In certain embodiments, the inducible promoter operably linked to the MYOD protein is different from the inducible promoter linked to the MYOG protein. In certain embodiments, the inducible promoter operably linked to the MYOD protein and the inducible promoter linked to the MYOG protein are the same. Inducible promoters are well known in the art, examples include but are not limited to CMV, CAG, CBh, PGK, SV40, ferritin heavy chain or light chain, etc.

[0090] In certain embodiments, the inducible promoter used in the present application is a tetOn promoter. Preferably a 3rdgeneration TetOn promoter.

[0091] Method of cultivation

[0092] The inventors of the present application have surprisingly found that by using the pluripotent cells described herein, the differentiation time required to obtain skeletal muscle cells can be significantly reduced. Thus, in a further aspect, the present application relates to a method for producing skeletal muscle cells, the method comprising

[0093] a) culturing the pluripotent stem cell described herein in a proliferation medium: followed by

[0094] b) inducing skeletal muscle cell differentiation by adding an exogenous substance described herein.

[0095] In certain embodiments, the method of the present application is an ex vivo method.

[0096] In certain embodiments, the method is for producing skeletal muscle cells. A mature skeletal muscle cell is defined herein as a skeletal muscle cell that displays one or more of multinucleation, formation of proteins involved in sarcomere formation (e.g. Titin and / or one or more myosin heavy chain isoforms) and eventual contractile function.

[0097] In certain embodiments, the method of the present application relates to a method for producing muscle of type 1 or type 2.

[0098] The proliferation and differentiation media can have the same composition. The same media can be used for proliferation and differentiation. That is, the same culture system can be used for the proliferation and differentiation phases, for example via media changes or by adding the induction agent of the opti-ox system within the bioreactor only. The addition of the induction agent for differentiation into the bioreactor is very attractive as it minimizes the equipment, processing time and capital investment for cell handling.

[0099] Optionally, the proliferation and / or differentiation can be performed in the absence of insulin and / or retinoic acid.

[0100] Optionally, the proliferation and / or differentiation can be performed as a single cell suspension. Alternatively, the proliferation and / or differentiation can comprise aggregates.

[0101] The inventors have surprisingly found that the use of the pluripotent cells described herein avoids the need to culture the cells with a commitment induction step. Typically, when culturing skeletal muscle cells, several culture phases can be distinguished. The commitment or determination phase involves the formation of myoblasts, which have lost the potential to differentiate into other cell types. The differentiation of myoblasts to skeletal muscle cells is facilitated by a highly regulated network of transcription factors expressed in temporal order to promote skeletal muscle morphological and biochemical characteristics, such as the fusion of single myoblasts into multinucleated skeletal muscle cells and the formation of sarcomeres, which requires the expression of highly abundant muscle-specific proteins, which can account for up to 55% of the skeletal muscle cell protein content. The differentiation phase is also 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 differentiates the pluripotent stem cells into mature skeletal muscle cells with a very short (2-4 days) myoblast-like cell state, and can effectively differentiate into mature skeletal muscle. This short state is particularly advantageous as it reduces the amount of compounds and small molecules that would normally need to be present in the myoblast proliferation media. The cells described herein can be able to differentiate in the absence of fetal bovine serum (FBS), dexamethasone and EGF or FGF2, which are normally required for satisfactory differentiation. The ability to omit these compounds from the differentiation media reduces media costs and simplifies the way regulatory approvals are obtained.

[0102] The methods described herein significantly reduce the time for the pluripotent cells described herein to differentiate into mature skeletal muscle cells. In certain embodiments, the time to produce 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 have observed a conversion rate of at least 95% by day 4 of culture, meaning that at least 95% of the cells are mature after 4 days of culture. Thus, in certain embodiments, the time to produce at least 95% mature skeletal muscle cells is at most 4 days.

[0103] In a further aspect, the present application provides a skeletal muscle cell, preferably a mature skeletal muscle cell, obtained by the methods described herein.

[0104] Culturing cells as described herein can be performed under so-called 2D culture conditions, which are considered the conventional method of culturing cells. However, the methods can also readily be adapted to allow culturing under 3D conditions, as shown in the examples below.

[0105] 3D cell culture is an artificially created environment that enables cells to grow in three dimensions or interact with their surroundings. In such cultures, cells typically form 3D colonies, which can be referred to as“spheroids”. 3D culture methods can more accurately model cell growth and behavior in vivo. A skilled person is readily able to perform 3D cell culture, for example by making use of any of a variety of commercially available culture tools. For example, 3D culture can be performed using scaffold-based or scaffold-free techniques. Scaffold-based techniques make use of supports (e.g. solid scaffolds and hydrogels) to enable cells to form 3D cultures. Such scaffolds can aim to mimic the natural extracellular matrix (ECM) present in vivo. Scaffold-free techniques omit the use of scaffolds to grow cells. Instead, 3D spheroids can be established by using, for example, low-attachment plates, hanging drop plates, micro-patterned surfaces, rotating bioreactors, magnetic levitation and magnetic 3D bioprinting.

[0106] Cells transduced with lentiviral vectors are not considered food-safe or safe for human and non-human dietary consumption. The pluripotent cells described herein for the methods described herein avoid the need for lentiviral transduced cells. Accordingly, in certain embodiments, the skeletal muscle cells produced according to the methods disclosed herein are suitable for human and non-human dietary consumption. In certain embodiments, the produced skeletal muscle cells can be used to produce cultured meat for human consumption. The meat according to the present application can be derived from any non-human animal, including any of the species mentioned herein.

[0107] In a further aspect, the present application provides the use of a pluripotent stem cell described herein or a skeletal muscle cell obtained by a method described herein for tissue engineering. In certain aspects, the use is for the production of cultured meat.

[0108] In a further aspect, the present application provides the use of a pluripotent stem cell described herein or a skeletal muscle cell obtained by a method described herein for tissue engineering, for example in a therapeutic method.

[0109] In yet a further aspect, the present application provides a food product (also referred to as "foodstuff") comprising the pluripotent stem cell described herein or the skeletal muscle cell produced and / or obtained by the method. In certain embodiments, the food product further comprises an edible composition for human or non-human consumption. For example, the edible composition for human or non-human consumption comprises at least one of a skeletal muscle cell, a mature muscle cell, a mineral, a synthetic substance, a flavoring substance (such as, for example, herbs and spices), a plant-based protein, or a microbially-derived protein (such as, for example, a yeast protein). Plant-based proteins and yeast proteins suitable for use in food products are known to the person skilled in the art. In certain embodiments, the food product is a cultured meat.

[0110] In yet a further aspect, the present application provides a method of producing a food product, the method comprising combining the pluripotent stem cell described herein or the skeletal muscle cell produced and / or obtained with an edible composition for human or non-human consumption described herein. In certain embodiments, the food product is a cultured meat.

[0111] SEQUENCE DESCRIPTION

[0112] Table 1 : Sequences

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] BRIEF DESCRIPTION OF DRAWINGS

[0119] Figure 1 EpiSC-MYOD1 (left) and EpISC-MYOD1-MYOG (right) differentiated for 8 days in 2D monolayer culture. Cultures were stained for myosin and nuclei were visualized with DAPI.

[0120] Figure 2 Comparison between EpiSC-MyoD1 (left) and EpiSC-MyoD1-MyoG (right) after 5 days of 3D suspension differentiation. Aggregates were stained for myosin and nuclei were visualized with DAPI.

[0121] Figure 3 Comparison between EpiSC-MyoD1 (left) and EpiSC-MyoD1-MyoG (right) after 5 days of 3D suspension differentiation. Zoomed inFigure 1 , highlighting (white arrows) myotome formation, indicating full differentiation into functional skeletal muscle cells. The striated pattern is only visible in EpiSC-MyoD1-MyoG cell line.

[0122] Figure 4 Comparison between EpiSC-MyoD1 (left) and EpiSC-MyoD1-MyoG (right) after 8 days of 3D suspension differentiation. Aggregates were stained with myosin and nuclei were visualized with DAPI.

[0123] Figure 5 ： 3D suspension differentiation of EpiSC-MYOD1-MYOG for 3 days (left), 4 days (middle) and 5 days (right). Aggregates were fixed and labeled with anti-myosin antibodies while nuclei were stained with DAPI.

[0124] Figure 6: (A) Comparison between EpiSC-MyoD1 (left) and EpiSC-MyoD1-MyoG1 (right) after 7 days and 2 days of 3D suspension differentiation showing representative aggregates of frozen sections of complete fixation staining (external staining) and staining with myotilin and Dapi in the middle. (B) Real time qRT-PCR analysis of MYH3 expression during 2D differentiation of EpiSC-MYOD1. (C) MYH3 expression of EpiSC-MYOD1 aggregates differentiated for 7 days and EpiSC-MYOD1-MYOG aggregates differentiated for 2 days. Expression data were normalized to RPL27 expression.

[0125] Figure 7 Proliferation of EpiSC-MyoD1-MyoG aggregates followed by 2 days of differentiation in bioreactor or shake flask, then fixation and labeling with anti-myosin and anti-myogenin antibodies while nuclei were stained with DAPI.

[0126] Examples

[0127] The present application is further illustrated by the following examples, which should not be construed as limiting the scope of the present application.

[0128] Materials and methods

[0129] Porcine epidermal derived stem cells (pEpISC) differentiation into skeletal muscle cells.

[0130] Undifferentiated pEpISCs (Opti-Ox MyoD1 and Opti-Ox MyoD1-MyoG) were grown in N2B27 proliferation medium (50% DMEM Ham 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 pg / mL Activin A (QK001, Q-kine), 0.10 pg / mL FGF2 (QK002, Q-kine), 0.625 pg / mL XAV939 (X3004, Sigma Aldrich)) on hESC-qualified geltrex (A1413301, Thermo Scientific) coated plates. For 2D skeletal muscle differentiation, single cells were obtained after treatment with gentle cell dissociation reagent (07174, STEMCELL technologies) and cell number and viability were assessed. Single pEpISCs were seeded in corresponding cell culture plates at a density of 100000-200000 cells / cm2. After overnight incubation in proliferation medium with 10 pm Rock inhibitor (Y-27632 (HBF2297, HelloBio)) cells were cultured in DMEM high glucose medium or DMEM Ham F-12 (L0093-500, Biowest) supplemented with 1% Knockout Serum Replacement (10828-028, Thermofisher) and 1x Insulin-Transferrin-Selenium (ITS-G) (41400045, Thermofisher) for the first 4 days of differentiation. On day 4, the Knockout Serum Replacement concentration was increased to 15% to initiate maturation of skeletal muscle cells. The medium was refreshed every two days. Doxycycline (1 pg / mL, D9891, Sigma Aldrich) was added to the differentiation medium to activate the Opti-OX system in these cells.

[0131] For spheroid or aggregate differentiation experiments, undifferentiated single pEpISC (Opti-Ox MyoD1 and Opti-Ox MyoD1-MyoG) were seeded at 3 million cells / mL in 150 mL shake flasks containing 12.5 mL proliferation medium (containing 50% DMEM Ham 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 pg / mL Activin A (QK001, Q-kine), 0.10 pg / mL FGF2 (QK002, Q-kine), 0.625 pg / mL XAV939 (X3004, Sigma Aldrich), 2X% Knockout Serum Replacement (10828-028, ThermoFisher) KSR and 10 ng / mL FGF2). Two days later, the aggregate formation medium was replaced with skeletal muscle differentiation medium as described above and refreshed every two days until the end of the experiment.

[0132] Results

[0133] Example 1: Development of an inducible transgene overexpression method by dual genetic safe harbor (GSH) targeting in animal cells

[0134] The Opti-ox method for generating controllable transcriptional cells as described in WO 2018096343 Al was employed in these examples. This method involves the insertion of an inducible cassette such that transcription of genetic material within this inducible cassette can be controlled by using a dual genome safe harbor targeting system.

[0135] To explore the myogenic reprogramming potential of OPTi-OX on porcine pluripotent stem cells (pPSCs), we generated MYODOPTi-OX pPSCs. We targeted the rtTA cassette under the control of the CAG promoter to the pig ROSA26 GSH and the MYOD transgene under the control of the doxycycline inducible element to the pig AAVS1 GSH sequentially. We observed robust and homogeneous inducible transgene expression. In addition, we observed direct generation of myotubes from MYOD reprogrammed pPSCs after 8 days of expression Figure 1 (left). This finding demonstrates that MYOD can directly reprogram pPSCs to myotubes in 2D culture.

[0136] MYOD and MYOG were chosen for the combined cell reprogramming strategy. We designed a targeting vector with MYOD, a P2A "self-cleaving" peptide linker, and MYOG in one open reading frame to express MYOD and MYOG simultaneously by doxycycline induction. The 2A peptide linker is a well-characterized short peptide linker of 18-22 amino acids that produces two separate gene products from a single open reading frame due to ribosomal skipping during translation. Stable knock-ins in AAVS1 GSH were selected by incorporation of a puromycin resistance cassette and selected by incorporation of puromycin in the cell culture media. After selection, single pEpiSC cells were plated and clonal cell lines were isolated for growth and analysis. Incorporation of doxycycline-inducible MYOD-P2A-MYOG was subsequently confirmed using PCR genomic analysis, Sanger sequencing, and RT-qPCR. Using the dual GSH targeting approach, we selected clones that targeted both alleles and thus carried two copies of each transgene, and observed that homozygous targeting of both elements allowed for inducible overexpression (data not shown). Importantly, the dual GSH targeting approach did not affect the SC self-renewal or differentiation capacity of pEpiSCs, as determined by RT-qPCR (data not shown).

[0137] The advantage of using the MyoD / MyoG line compared to MyoD alone is that the differentiation time is reduced from 8 days to 3 days and the amount of multinucleated skeletal muscle cells is increased, indicating improved maturity. All of this indicates that the skeletal muscle cells generated by MyoD / MyoG have a more similar protein profile to natural meat.

[0138] Example 2: EpiSC-MYOD-MYOG 2D differentiation into multinucleated skeletal muscle cells

[0139] pEpISC Opti-Ox MYOD and pEpISC Opti-Ox MYOD-MYOG were expanded in 3D suspension culture, single-cellized and plated at 200,000 cells / cm2 2 in coated plastic wells. One day after attachment, doxycycline was added to the culture media to start differentiation. Cells were differentiated for 8 days. Both lines showed the formation of myosin positive skeletal muscle cells, however MyoD1-MyoG produced thicker skeletal muscle cells Figure 1 ).

[0140] Example 3: 3D culture of EpiSC-MYOD and EpiSC-MYOD-MYOG

[0141] For the expansion (proliferation) phase of cell culture in cultured meat production, typically a shaker and / or bioreactor is used to scale up to large volumes and generate the amount of cell mass needed to produce cultured meat products at a cost-competitive price. Typically, primary cells derived from muscle biopsies are used. However, primary cultures have limited self-renewal, lose differentiation capacity during expansion, and are variable in quality between biopsies. Other sources, such as ESCs and iPSCs, have high expansion capacity but are in an undifferentiated state and differentiation is induced using small molecules that mimic embryonic development (reviewed in Chal et al., 2017 Jun 15; 144(12):2104-2122. doi: 10.1242 / dev.151035) or via ectopic expression of the transcription factors MYOD1 or PAX7. For meat production, cell differentiation into myotubes and subsequent differentiation into myofibers is an integral part of the process, which typically occurs in a separate step afterwards. Differentiation of skeletal muscle cells typically requires different conditions in terms of nutritional and physical environment. The necessary nutrition can be provided by switching from a proliferation medium rich in nutrients to a differentiation medium consisting of low serum, but providing the physical environment required for cell differentiation is more challenging. In addition, the substrate requirements for the proliferation and differentiation phases are typically different in terms of surface chemistry and topography. One particular challenge for skeletal muscle cells in suspension is that skeletal muscle cells need to attach to a side of a rod, scaffold or surface to form elongated stretched multinucleated myotubes.

[0142] In this example, EpiSC-MYOD and EpiSC-MYOD-MYOG adapted to 3D suspension cell growth and grew as aggregates; EpiSC expanded in adherent 6-well cell plates were single-cellized with Accumax (00-4666-56, Thermo Fisher Scientific) according to the manufacturer’s instructions and then transferred to 12.5 mL of medium and RHO / ROCK pathway inhibitors in a 150 mL shake flask for at least 3 cycles of expansion. Medium was refreshed daily during 3D expansion. Subsequently, 3D adapted EpiSC were used for shaker and bioreactor experiments.

[0143] To explore skeletal muscle differentiation in 3D suspension culture, 500,000 cells / mL were seeded in a 150 mL shake flask containing 12.5 mL of medium and EpiSC-MYOD or EpiSC-MYOD-MYOG were differentiated using the differentiation protocol. Medium was changed every other day. After 5 days of EpiSC-MYOD-MYOG differentiation, multinucleated skeletal muscle cells with elongated morphology had formed, while only round morphology was observed in EpiSC-MYOD Figure 2). Both lines produced myosin at this time point as observed by immunofluorescent staining, but only the EpiSC-MYOD-MYOG showed a striated pattern of myosin, indicating that functional myotubes have been formed and maturation is improved Figure 3 Prolonging the differentiation period to 8 days further matured both EpiSC lines in skeletal muscle cells ( Figure 4 ), but the aggregates containing EpiSC-MYOD-MYOG showed larger and longer skeletal muscle cells including many nuclei. In the EpiSC-MYOD aggregates, mainly single-nucleated cells were detected. Since we observed maturation of skeletal muscle cells after 5 days of differentiation with EpiSC-MYOD-MYOG aggregates, we further shortened the cultivation time to 3 and 4 days and compared it to 5 days of differentiation ( Figure 5 ). At all time points, we observed the formation of myosin-positive multinucleated skeletal muscle cells, indicating that 3 days of differentiation are sufficient to induce correct skeletal muscle cell formation from EpiSCs exogenously expressing MYOD-MYOG. MYH3 is the major contractile protein and is a downstream target of MYOD1. Next, we determined MYH3 expression at different time points during 2D differentiation of EpiSC-MYOD1 cells ( Figure 6B ). MYH3 expression increased during differentiation and we detected a 7-fold increase in MYH3 expression between day 2 and day 8. Next, we compared MYH3 expression between MyoD and MyoD / MyoG lines at day 7 and day 2 of differentiation, respectively ( Figure 6C ). We detected similar expression in both lines, showing that MYH3 upregulation is faster in MyoD / MyoG cells and confirmed the results shown in Figure 6A Comparison of cells grown in bioreactor or shake flask ( Figure 7 ) showed that formation of myosin-positive multinucleated skeletal muscle cells and expression of MHC and MyoG could be detected in both culture conditions.

[0144] The simplified bioprocess shown is very attractive, where the same cultivation system can be used for the proliferation and differentiation phase by either medium change or by adding the inducer of the Opti-ox system only within the bioreactor / adding the inducer for differentiation to the bioreactor only, as it minimizes equipment, processing time and capital investment for cell handling.

Claims

1. A pluripotent stem cell, comprising: i) An expression construct for expressing transcriptional regulatory proteins is inserted into the first genetic safe harbor site; ii) an expression construct for expressing the MYOD protein, wherein the coding sequence of the MYOD protein is operatively linked to an inducible promoter; and iii) An expression construct for expressing the MYOG protein, wherein the coding sequence of the MYOG protein is operatively 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. Furthermore, the inducible promoter is regulated by this transcriptional regulatory protein.

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

3. The pluripotent stem cell according to 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 1-3, wherein such pluripotent stem cells belong to a livestock or poultry species.

5. The pluripotent stem cells according to claim 4, wherein the livestock species are pigs or cattle, preferably pigs.

6. The pluripotent stem cell according to any one of claims 2-5, wherein the expression construct inserted into the second genetic safe harbor site encodes the MYOD protein, the adapter, and the MYOG protein, preferably wherein the adapter is a P2A adapter, more preferably wherein the adapter 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 the preceding claims, wherein the activity of the transcriptional regulatory protein is controlled by an exogenously supplied derivative of the substance.

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

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

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

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

13. A method for generating skeletal muscle cells, the method comprising: a) Culture pluripotent stem cells according to any one of the preceding claims in a proliferation medium: subsequently b) Inducing skeletal muscle cell differentiation by adding the exogenous substance as described in claim 8.

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

15. The method according to claim 13 or 14, wherein the differentiation period 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 resulting skeletal muscle cells are intended for human and non-human dietary consumption.

17. Use of pluripotent stem cells according to any one of claims 1 to 12 or use of a method for generating skeletal muscle cells according to any one of claims 13 to 16, the use being for tissue engineering, optionally for generating cultured meat.

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

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

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