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 a PAX7 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] This invention relates to modified pluripotent cells and methods for differentiating said cells into skeletal muscle cells. Background Technology

[0002] According to the latest UN estimates, as of July 2022, the world population was 7.9 billion [https: / / www.worldometers.info / es / poblacion-mundial / #ref-1], and is projected to reach 10 billion by around 2056. This growth will be unevenly distributed globally, with nine 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 projected to grow by 15% by 2031. On the other hand, the correlation between income growth and increased meat consumption is evident at lower income levels, 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 growing interest in finding alternative protein sources that are ideally sustainable and contain the nutrients typically found in meat in the human diet. Cultured meat has emerged as another alternative to traditional animal agriculture, aiming to produce the skeletal muscle and adipose tissue that typically constitute animal meat, but using in vitro tissue and bioengineering techniques. Despite efforts to develop robust protocols for scalably generating animal cell types from readily available and renewable sources, differentiating animal (pluripotent) stem cells into specific cell types often remains cumbersome, lengthy, difficult to reproduce, and / or not yet established.

[0004] It has been reported that porcine embryonic fibroblasts and porcine induced pluripotent stem cells (iPSCs) are used to reprogram skeletal muscle 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 porcine embryonic fibroblasts exhibited low differentiation efficiency, long culture times, and the need for serum-containing media. Induction of porcine iPSCs with MyoD1 for 8 days resulted in high transformation rates, but relied on the non-food-safe 5-aza-cytidine and CHIR99021. In summary, these protocols resulted in heterogeneous skeletal muscle maturation, lack of scalability, non-food-safety, and long culture times of up to 14 days.

[0005] Therefore, there remains a need in this field to produce and culture mature skeletal muscle cells that are suitable for human consumption and can be produced in a scalable and cost-effective manner. Summary of the Invention

[0006] This invention relates to the expression of transcriptional regulatory proteins MYOD and PAX7 in pluripotent stem cells. Therefore, in a first aspect, this invention relates to a pluripotent stem cell comprising:

[0007] i) An expression construct for expressing transcriptional regulatory proteins is inserted into the first genetic safe harbor site;

[0008] 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

[0009] iii) An expression construct for expressing the PAX7 protein, wherein the coding sequence of the PAX7 protein is operatively linked to an inducible promoter;

[0010] 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.

[0011] Furthermore, the inducible promoter is regulated by this transcriptional regulatory protein.

[0012] In some embodiments of the invention, the expression constructs of ii) and iii) are inserted into a second genetic safe harbor site, which is different from the first genetic safe harbor site. Preferably, the first and additional genetic safe harbor sites are selected from any two of the hROSA26 locus, AAVS1 locus, CLYBL gene, or CCR5 gene, and preferably, the genetic safe harbor sites are the hROSA26 locus and the AAVS1 locus.

[0013] In some embodiments of the present invention, the cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines and somatic cell lines.

[0014] In some embodiments of the invention, the pluripotent stem cells belong to livestock or poultry species. Preferably, the livestock species are pigs or cattle, and more preferably pigs.

[0015] In some embodiments of the invention, the expression construct inserted into the second genetic safe harbor site encodes the MYOD protein, the adapter, and the PAX7 protein, preferably wherein the adapter is a P2A adapter, more preferably wherein the adapter comprises the sequence of SEQ ID NO:6. Preferably, the construct comprises the sequence of SEQ ID NO:8.

[0016] In some embodiments of the 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 (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 rtTA.

[0017] In some embodiments, the inducible promoter includes a Tet reaction element (TRE).

[0018] In some embodiments, the induced promoter is the tetON promoter.

[0019] In a second aspect, the present invention provides a method for generating skeletal muscle cells (e.g., type I or type II muscle), the method comprising:

[0020] a) Culture pluripotent stem cells according to any one of the preceding claims in a proliferation medium: subsequently

[0021] b) Inducing skeletal muscle cell differentiation by adding the exogenous substances described herein. In some embodiments, the differentiation period of the methods described herein is up to 10 days, up to 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, or 2 days.

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

[0023] In some embodiments, the resulting skeletal muscle cells are intended for human and non-human dietary consumption.

[0024] In another aspect, the present invention provides the use of the pluripotent stem cells described herein or the methods described herein for generating skeletal muscle cells, which are used for tissue engineering and optionally for generating cultured meat.

[0025] In another aspect, the present invention provides a food comprising the pluripotent stem cells described herein or skeletal muscle cells obtained by the methods described herein. In some embodiments, the food is cultured meat. Detailed Implementation

[0026] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Those skilled in the art will recognize that many methods and materials similar to or equivalent to those described and used herein can be employed in the practice of this invention. In fact, this invention is by no means limited to these methods.

[0027] In this document and its claims, the verb "comprising" and its inflections are used in their non-limiting sense to mean including the items that follow the word, but not excluding items not specifically mentioned. Furthermore, unless the context explicitly requires the presence of one / a and only one / a type of element, mentioning an element by the indefinite article "a / a (a or an)" does not preclude the possibility of more than one / a type of element. Therefore, the indefinite article "a / a (a or an)" generally means "at least one / at least one".

[0028] As used herein, the term “and / or” indicates that one or more of the stated circumstances may occur individually or in combination with at least one of the stated circumstances, up to and including all of the stated circumstances.

[0029] As used in this article, “at least” a specific value means that specific value or more. For example, “at least 2” is understood as 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 conjunction with a numerical value (e.g., about 10) preferably means that the value can be a given value (10) plus or minus 0.1% of that value.

[0031] The term "heterogeneous" when used with respect to nucleic acids (DNA or RNA) or proteins refers to a nucleic acid or protein that is not naturally present as part of the organism, cell, genome, or DNA or RNA sequence in which it is found, or in one or more locations in a cell or genome or DNA or RNA sequence that is not found in nature. Heterogeneous nucleic acids or proteins are not endogenous to the cell in which they are introduced, but have been acquired from another cell or synthesized or recombined. Typically, although not always, such nucleic acids encode proteins that are not normally produced by cells that transcribe or express DNA. Similarly, exogenous RNA encodes proteins that are not normally expressed in cells in which the exogenous RNA is present. Heterogeneous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Those skilled in the art will recognize that any nucleic acid or protein that is foreign or foreign to the cell that expresses it is covered herein by the term heterogeneous nucleic acid or protein. The term heterogeneous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e., combinations in which at least two of the sequences are foreign relative to each other.

[0032] The term "expression vector" or "expression construct" refers to a nucleotide sequence capable of enabling gene expression in a host cell or host organism compatible with such a 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 achieving expression, such as expression enhancer elements, may 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 positioned to have a functional relationship with another nucleic acid sequence. For example, if a transcriptional regulatory sequence affects the transcription of a coding sequence, then the transcriptional regulatory sequence is operably linked to the coding sequence. Operable linkage means that the linked DNA sequences are typically contiguous, and in cases where binding of two protein-coding regions is required, contiguous and within the reading frame. Inducible promoters

[0034] As used herein, the term "promoter" refers to a nucleic acid fragment that controls the transcription of one or more coding sequences, is upstream of the transcription start site relative to the coding sequence, and is structurally characterized by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and any other DNA sequence, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequence known to those skilled in the art to directly or indirectly regulate the amount of transcription by 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 the application of a chemical inducer. In the case of this invention, control is achieved through transcriptional regulatory proteins.

[0035] Any references in this document to nucleotide or amino acid sequences accessible in public sequence databases refer to the version of the sequence entry available as of the date of this document's submission.

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

[0037] The inventors have unexpectedly discovered that by using modified pluripotent stem cells, the time required for pluripotent cells to differentiate into mature skeletal muscle cells can be significantly shortened. These modified pluripotent stem cells contain expression constructs for expressing the MYOD protein and an expression construct for expressing the PAX7 protein, or alternatively, expression constructs for expressing both MYOD and PAX7. As illustrated 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 (e.g., less than 5 days, such as 4 days or 3 days or faster). In addition to significantly reducing culture time and associated costs, the use of the pluripotent cells described herein provides a more scalable production of mature skeletal muscle cells compared to those previously described, resulting in a greater number of mature skeletal muscle cells.

[0038] Differentiated cells can form a spindle-like elongated morphology, undergo extensive cell fusion, and exhibit strong and homogeneous expression of myogenic markers based on mRNA and protein levels (MHC isotype / maturity, immunostaining MYOD / MYOG / MYH / TTN / TNNT / PAX3 / 7), as well as high fusion index, length, and thickness. Furthermore, spontaneous myofibril contraction can be observed, demonstrating the function of MYOD / PAX7.

[0039] The induction efficiency of the exogenous compound did not decrease during the extended culture period, demonstrating the robustness and reproducibility of the system (percentage of MHC-positive cells in different passages).

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

[0041] i) An expression construct for expressing transcriptional regulatory proteins is inserted into the first genetic safe harbor site;

[0042] 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

[0043] iii) An expression construct for expressing the PAX7 protein, wherein the coding sequence of the PAX7 protein is operatively linked to an inducible promoter;

[0044] 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.

[0045] Furthermore, the inducible promoter is regulated by this transcriptional regulatory protein.

[0046] The pluripotent stem cells of the present invention may optionally not contain expression constructs other than those listed in i), ii), and iii) above. Alternatively, the pluripotent stem cells of the present invention may optionally contain one or more expression constructs other than those listed in i), ii), and iii) above, such as 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 may provide proteins for expression, wherein the regulator is a transcription regulator, such as a transcription factor, rather than MYOD or PAX7.

[0047] If the pluripotent stem cells of the present invention containing one or more antibiotic resistance markers are generated, one or more such resistance markers can be removed. The pluripotent stem cells of the present invention may optionally not contain antibiotic resistance markers.

[0048] MYOD, also known as myoblast-determining 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 regulatory factors. MYOD is one of the earliest markers of myogenic commitment. MYOD is expressed at extremely low and essentially undetectable levels in resting satellite cells, but its expression is activated after exercise or muscle tissue injury. The effect of MYOD on satellite cells is dose-dependent: high MYOD expression inhibits cell renewal, promotes terminal differentiation, and can induce apoptosis. Although MYOD marks myoblast commitment, muscle development is not significantly inhibited in mouse mutants lacking the MYOD gene. This may be due to functional redundancy of Myf5 and / or Mrf4. However, the combination of MYOD and Myf5 is crucial for successful myogenesis. MYOD's function in development is to facilitate the entry of mesoderm cells into the skeletal myoblast lineage and then regulate this sustained state. MYOD also regulates muscle repair. MYOD mRNA levels have also been reported to be elevated in aging skeletal muscle. One of the main functions of MYOD is to remove cells from the cell cycle by enhancing the transcription of p21 and MYOD (stopping proliferation in differentiating muscle cells to achieve terminal cell cycle arrest). MYOD is inhibited by cyclin-dependent kinases (CDKs), which in turn are inhibited by p21. Therefore, MYOD enhances its own activity in cells in a feedforward manner. Sustained MYOD expression is necessary to preserve the expression of muscle-related genes. MYOD is also an important effector of the fast-twitch muscle fiber (types IIA, IIX, and IIB) phenotype.

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

[0050] PAX7 (pairing box protein) is a protein encoded by the PAX7 gene in humans. Pax-7 plays a role in neural crest development and gastrulation, and it is an important factor in the expression of neural crest markers such as Slug, Sox9, Sox10, and HNK-1. PAX7 is expressed in the palatine process of the maxilla, the McEllig cartilage, the midbrain, the nasal cavity, the nasal epithelium, the nasal sac, and the pons. Pax7 is a transcription factor that plays a role in myogenesis by regulating the proliferation of muscle precursor cells. It can bind to DNA as a heterodimer with PAX3. It also interacts with PAXBP1; this interaction links PAX7 to a WDR5-containing histone methyltransferase complex through similarity. It also interacts with DAXX. PAX7 serves as a marker for a rare subset of spermatogonial stem cells, particularly a subset of single spermatogonia. These PAX7+ spermatogonia are rare in adult testes but more prevalent in newborns, accounting for 28% of neonatal testicular germ cells. Unlike PAX7+ muscle satellite cells, PAX7+ spermatogonia proliferate rapidly and are not quiescent. PAX7+ spermatogonia are capable of inducing all stages of spermatogenesis and producing motile sperm. However, PAX7 is not essential for spermatogenesis, as mice lacking PAX7+ spermatogonia do not exhibit fertility defects. PAX7 may 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 did not survive. Furthermore, compared to control mice, mice lacking PAX7 showed delayed recovery of spermatogenesis after exposure to busulfan.

[0051] In some embodiments, the coding sequence of PAX7 has the sequence of SEQ ID NO:3 and the amino acid sequence of SEQ ID NO:4.

[0052] In some embodiments, the pluripotent stem cells described herein include (iv) an additional expression construct for expressing the MYOG protein, wherein the coding sequence of the MYOG protein is operatively linked to an inducible promoter, wherein the expression construct (iv) is inserted into at least one additional genetic safe harbor site that is not the first genetic safe harbor site.

[0053] MYOG, or myoblast, is a transcriptional activator encoded by the MYOG gene. Myoblasts are muscle-specific basic helical-loop-helical (bHLH) transcription factors involved in coordinating skeletal muscle development or myogenesis and repair. MYOG is a member of the MYOD family of transcription factors.

[0054] In mice, MYOG is essential for the development of functional skeletal muscle. Proper differentiation of most myogenic precursor cells during myogenesis requires MYOG. Severe skeletal muscle defects were observed when the DNA encoding myoblast was knocked out from the mouse genome. Mice lacking two copies of myoblast (homozygous-null) suffered perinatal death due to the absence of mature secondary skeletal muscle fibers throughout the body. In cell culture, myoblast can induce myogenesis in various non-myoblast types.

[0055] Therefore, in some embodiments, pluripotent stem cells may include expression constructs that provide expression of the proteins MYOD, PAX7, and MYOG.

[0056] In some embodiments, the nucleic acid molecule encoding the protein according to the invention can be codon-optimized for expression in mammalian cells. Methods for codon optimization are known and have been previously described (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 a wild-type sequence. Here, a non-preferred codon is a codon used less frequently in an organism than another codon encoding the same amino acid, and a more preferred codon is a codon used more frequently in an organism than the non-preferred codon. Codon usage frequencies in a particular organism can be found in codon frequency tables, for example, http: / / www.kazusa.or.jp / codon. Preferably, more than one, preferably most or all, of the non-preferred codons are replaced by more preferred codons. Preferably, the most commonly used codons in the organism are used in the codon-optimized sequence. Replacement with preferred codons generally results in higher expression.

[0057] Transcription regulatory proteins are DNA-binding proteins, preferably specifically binding to DNA sites located within or near the promoter, and promoting the binding of transcription mechanisms to the promoter, thereby transcribing the DNA sequence (transcription activators) or blocking this process (transcription repressors). Such entities are also known as transcription factors.

[0058] The DNA sequences to which transcription regulatory proteins bind are called transcription factor binding sites or response elements, and these are found in or near the promoters of the regulatory DNA sequences.

[0059] Transcriptional activating proteins bind to response elements and promote gene expression. Such proteins are preferred in the method of the present invention for controlling inducible cassette expression.

[0060] Genetic safe harbor (GSH) sites are loci within the genome where genes or other genetic material can be inserted without any harmful effects on the cell or the inserted genetic material. Most advantageous are GSH sites where the expression of the inserted gene sequence is not interfered with by any readthrough expression of neighboring genes, and the expression of the inducible cassette minimizes interference with endogenous transcriptional programs. More formal criteria have been proposed to 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 cancer-related gene, (iii) 300 kb or more from any microRNA (miRNA), (iv) located outside a transcription unit, and (v) located outside a highly conserved region (UCR). It may not be necessary to meet all of these proposed criteria, as the identified GSH does not meet all of them. It is believed that a suitable GSH will meet at least two, three, four, or all of these criteria.

[0061] In some embodiments of the present invention, the first and additional genomic safe harbor sites are selected from any two of the hROSA26 locus, AAVS1 locus, CLYBL gene, or CCR5 gene. In some embodiments, the first and additional genomic safe harbor sites are located in the human genome 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.

[0062] In some embodiments of the present invention, the first and additional genomic safe harbor sites are selected from any two of the safe harbor sites ROSA26, AAVS1, CLYBL gene or CCR5 gene.

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

[0064] In some embodiments of the invention, both the expression construct for expressing the MYOD protein and the expression construct for expressing the PAX7 protein described herein are inserted into a second genetic safe harbor site, different from the first genetic safe harbor site. In some embodiments, the expression construct inserted into the second genetic safe harbor site can simultaneously express both the MYOD protein and the PAX7 protein.

[0065] As used herein, the term "pluripotent stem cell" includes embryonic stem cells, embryo-derived stem cells, induced pluripotent stem cells, and somatic cells, regardless of the method of pluripotent stem cell derivation. Therefore, in some embodiments, 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 some embodiments, pluripotent stem cells are epiblast-derived stem cells (EpiSCs). In some embodiments, 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 transduction of reprogramming factors in somatic cells.

[0066] The pluripotent stem cells according to the present invention can be derived 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 other species. Therefore, those skilled in the art can generate embryonic stem cells and embryo-derived stem cells from any of the following species, including but not limited to humans, non-human primates, rodents (mice, rats), ungulates (cattle, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats, such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrels, guinea pigs, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), etc.

[0067] Similarly, iPS cells can originate from any species.

[0068] In some embodiments, the pluripotent stem cells according to the invention or used in the invention are animal cells. In some embodiments, the pluripotent stem cells according to the invention or used in the invention are derived from edible animal species.

[0069] Preferably, the pluripotent stem cells used in this invention are derived from livestock or poultry animals or marine animals. Livestock species include, but are not limited to, cattle, pigs, sheep, goats, lambs, camels, buffalo, and rabbits.

[0070] Preferably, the pluripotent stem cells according to the invention or used in the invention are porcine or bovine pluripotent stem cells. Most preferably, they are porcine pluripotent stem cells. In some embodiments, the stem cells according to the invention are porcine epiblast stem cells (pEpiSC).

[0071] Poultry species include, but are not limited to, chickens, turkeys, ducks, geese, and pigeons. In some embodiments, the cells are derived from common prey species such as wild deer, quails, waterfowl, and rabbits. Preferably, the pluripotent stem cells according to the invention or used in the invention are not human cells.

[0072] Seafood includes, but is 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 through a variety of mechanisms, including substance binding, interaction with other transcription factors (e.g., homodimerization or heterodimerization) or co-regulatory proteins, phosphorylation, and / or methylation. Transcriptional regulatory factors can be controlled through activation or inactivation.

[0075] If the transcriptional regulatory protein is a transcriptional activator, then preferably, the transcriptional activator needs to be activated. This activation can be achieved by any suitable means, but preferably by adding an exogenous substance to the cell. The supply of the exogenous substance to the cell can be controlled, thereby controlling the activation of the transcriptional regulatory protein. Alternatively, an exogenous substance can be supplied to inactivate the transcriptional regulatory protein, and then the supply can be withdrawn to activate the transcriptional regulatory protein.

[0076] If the transcriptional regulatory protein is a transcriptional repressor, it is preferable that the transcriptional repressor be inactivated. Therefore, a substance is provided to prevent the transcriptional repressor from blocking transcription, thereby allowing transcription.

[0077] Any suitable transcriptional regulatory protein can be used, preferably an activatable or inactivatable transcriptional regulatory protein. Preferably, exogenous substances can be provided to control the transcriptional regulatory protein. Such transcriptional regulatory proteins are also called inducible transcriptional regulatory proteins.

[0078] Therefore, in some embodiments, the pluripotent stem cells according to the invention are controlled by exogenously supplied substances.

[0079] In some embodiments, the exogenously supplied substance is selected from the group consisting of: peptides (such as those 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 inducers (such as those described by Goeke et al. in Journal of Molecular Biology 416.1(2012):33-45; which are incorporated herein by reference), aptamers (such as RNA aptamers described by Hunsicker et al. in Chemistry & Biology 16.2(2009):173-180; which are incorporated herein by reference), tetracyclines and dehydrated tetracyclines or derivatives thereof. Preferably, the exogenously supplied substance is doxycycline.

[0080] In some embodiments, the transcriptional regulatory proteins described herein are selected from the group consisting of: tetracycline-responsive transcriptional activator protein (rtTa), tetracycline repressor (TetR), VgEcR synthesis receptor, or hybrid transcriptional regulatory proteins comprising a DNA-binding domain from yeast GAL4 protein, a truncated ligand-binding domain from human progesterone receptor, or an activation domain from human NF-κB.

[0081] Tetracycline-controlled transcriptional activation is a well-known method of inducible gene expression in which transcription is reversibly turned on or off in the presence of an antibiotic, tetracycline, or one of its derivatives (e.g., the more stable doxycycline). In this system, the transcriptional activating protein is tetracycline-responsive transcriptional activator (rtTA) or a derivative thereof. The rtTA protein is capable of binding to DNA at a specific TetO operon sequence. Several repetitive sequences of such a TetO sequence are placed upstream of a minimal promoter (e.g., the CMV promoter), together forming a tetracycline-responsive element (TRE). This system exists in two forms, depending on whether the addition of tetracycline or its derivative activates (Tet-on) or inactivates (Tet-off) the rTA protein.

[0082] In the Tet-Off system, tetracycline or its derivatives bind to and inactivate rTA, preventing it from binding to TRE sequences and thus inhibiting the transcription of TRE-controlled genes. The Tet-On system consists of two components: (1) constitutively expressed tetracycline-responsive transcriptional activator protein (rtTa) and an rtTa-sensitive inducible promoter (Tet-responsive element, TRE). This can bind to tetracycline or its more stable derivatives (including doxycycline (dox)), leading to rtTa activation, which binds to TRE sequences and induces the expression of TRE-controlled genes. In a preferred embodiment of the invention, the transcriptional regulatory protein is rtTA.

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

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

[0085] In some embodiments of the invention, the expression construct inserted into the second genetic safe harbor site is a fusion protein encoding the MYOD and PAX7 proteins, as described herein. In some embodiments, the expression construct inserted into the second genetic safe harbor site encodes the MYOD protein, the adapter, and the PAX7 protein; in a preferred embodiment, the construct comprises or consists of SEQ ID NO:8. MYOD and PAX7 can be arranged in either order, i.e., MYOD is located at the 3' of PAX7, or vice versa.

[0086] In some embodiments, the adapter sequence may be a cleavable adapter. That is, the adapter sequence may contain an amino acid sequence that can be cleaved. For example, the adapter sequence may contain a sequence that can serve as a substrate for an enzyme capable of cleaving peptide bonds (i.e., cleavage sites). Many such cleavage sites are known and can be used by those skilled in the art of molecular biology. In some embodiments, the cleavable adapter may contain a self-cleaving site. The self-cleaving site is automatically cleaved without enzymatic treatment. For example, 2A self-cleaving peptides or the 2A peptide family have been described, including 2A peptides P2A, E2A, F2A, and T2A. F2A is derived from foot-and-mouth disease virus; E2A is derived from equine rhinitis virus type A; P2A is derived from porcine swine cyclovir-1 2A; and T2A is derived from tussock moth virus 2A. In some embodiments, the cleavable adapter is therefore selected from the group consisting of P2A, E2A, F2A, and T2A.

[0087] In some preferred embodiments, the expression construct comprises a small RNA virus 2A (P2A) adapter. Preferably, the expression construct comprises an adapter that contains or consists of the sequence of SEQ ID NO:6.

[0088] In some embodiments, the expression constructs described herein encoding the MYOD protein, adapter, and PAX7 protein at the insertion second genetic safe harbor site comprise or consist of the sequence of SEQ ID NO:8.

[0089] In some embodiments, the inducible promoter operatively linked to the MYOD protein is different from the inducible promoter linked to the PAX7 protein. In some embodiments, the inducible promoter operatively 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.

[0090] In some embodiments, the inducible promoter used in this invention is the tetOn promoter. Preferably, it is a third-generation TetOn promoter.

[0091] Cultivation methods

[0092] The inventors of this application unexpectedly discovered that by using the pluripotent cells described herein, the differentiation time required to obtain skeletal muscle cells can be significantly reduced. Therefore, in another aspect, the present invention relates to a method for generating skeletal muscle cells, the method comprising...

[0093] a) Culture the pluripotent stem cells described herein in a proliferation medium: subsequently

[0094] b) Inducing skeletal muscle cell differentiation by adding the exogenous substances described herein.

[0095] In some embodiments, the method of the present invention is an ex vivo method.

[0096] In some embodiments, the method is used to generate skeletal muscle cells. Mature skeletal muscle cells are defined herein as skeletal muscle cells exhibiting one or more of the following: multinucleation, formation of proteins involved in sarcomere formation (e.g., Titin and / or one or more myosin heavy chain isoforms), and final contractile function.

[0097] In some embodiments, the method of the present invention relates to a method for generating type 1 or type 2 muscles.

[0098] Proliferation and differentiation media can have the same composition. The same medium can be used for both proliferation and differentiation. That is, the same culture system can be used for both the proliferation and differentiation phases, for example, by altering the medium or by simply adding an inducer from the Opti-Ox system into the bioreactor. Adding an inducer for differentiation to the bioreactor is very attractive because it minimizes the capital investment in equipment, processing time, and cell manipulation.

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

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

[0101] The inventors unexpectedly discovered that using the pluripotent stem cells described herein eliminates the need for a typing induction step in cell culture. Typically, when culturing skeletal muscle cells, several culture phases can be distinguished. The typing or determination phase involves the formation of myoblasts that have lost their potential to differentiate into other cell types. The differentiation of myoblasts into skeletal muscle cells is facilitated by a highly regulated network of transcription factors expressed in a time-sequential manner 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. Sarcomere formation requires the expression of highly abundant muscle-specific proteins, which can account for up to 55% of the protein content of skeletal muscle cells. The differentiation phase is also divided into four stages: growth arrest, mitotic clonal expansion, early differentiation, and terminal differentiation. Using the pluripotent stem cells described herein in this method allows pluripotent stem cells to differentiate into mature skeletal muscle cells with a very short (2–4 days) myoblast-like cell state and can efficiently differentiate into mature skeletal muscle cells. This short state is particularly advantageous because it reduces the amount of compounds and small molecules typically required in myoblast proliferation media. The cells described in this article can differentiate in the absence of fetal bovine serum (FBS), dexamethasone, and EGF or FGF2 (which are typically necessary for satisfactory differentiation). The ability to eliminate these compounds from the differentiation medium reduces medium costs and simplifies regulatory acceptance.

[0102] The method described herein significantly reduces the time required for the pluripotent cells described herein to differentiate into mature skeletal muscle cells. In some embodiments, the time required to generate skeletal muscle cells using the claimed method is up to 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, or 2 days. Using the described pluripotent cells in the method, the inventors observed a conversion rate of at least 95% on day 4 of culture, meaning that at least 95% of the cells mature after 4 days of culture. Therefore, in some embodiments, the time required to generate at least 95% mature skeletal muscle cells is up to 4 days.

[0103] In another aspect, the present invention provides skeletal muscle cells, preferably mature skeletal muscle cells, obtained by the methods described herein.

[0104] Cell culture as described herein can be performed under so-called 2D culture conditions, which is considered the standard method for culturing cells. However, the method can also be readily adapted to allow culture under 3D conditions, as illustrated in the examples below.

[0105] 3D cell culture is an artificially created environment that allows cells to grow or interact with their surroundings in three dimensions. In such cultures, cells typically form 3D colonies, often referred to as "spheroids." 3D culture methods allow for more accurate modeling of cell growth and behavior within the body. Technicians can easily perform 3D cell culture, for example, by utilizing any of a variety of commercially available culture tools. For instance, 3D culture can be performed using scaffold-based or scaffold-free techniques. Scaffold-based techniques utilize supports (such as solid scaffolds and hydrogels) to enable cells to form 3D cultures. Such scaffolds may be designed to mimic the natural extracellular matrix (ECM) present in vivo. Scaffold-free techniques eliminate the need for scaffolds to grow cells. Instead, 3D spheroids can be created using, for example, low-adhesion plates, pendant plates, micropatterned surfaces, rotating bioreactors, magnetic levitation, and magnetic 3D bioprinting.

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

[0107] In another 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. In some aspects, the use is for producing cultured meat.

[0108] In another 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 (e.g., in therapeutic applications).

[0109] In another aspect, the present invention provides a food (also referred to as "food") comprising the pluripotent stem cells described herein or skeletal muscle cells generated and / or obtained by the methods described herein. In some embodiments, the food 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 skeletal muscle cells, mature muscle cells, minerals, synthetic substances, flavoring substances (e.g., herbs and spices), plant-based proteins, or microbial-derived proteins (e.g., yeast proteins). Plant-based proteins and yeast proteins suitable for use in food are known to those skilled in the art. In some embodiments, the food is cultured meat.

[0110] In another aspect, the present invention provides a method for producing food, the method comprising combining the pluripotent stem cells described herein, or the resulting and / or obtained skeletal muscle cells, with an edible composition described herein for human or non-human consumption. In some embodiments, the food is cultured meat.

[0111] Sequence Description

[0112] Table 1: Sequences

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] Attached Figure Description

[0119] Figure 1 EpiSC-MYOD1 (left) and EPISC-PAX7-MYOD1 (right) differentiated for 8 days in a 2D monolayer culture. The cultures were stained with myomalacin, and the nuclei were visualized using DAPI.

[0120] Figure 2 Monolayer differentiation of EpiSC-PAX7-MyoD1 cells and magnified skeletal muscle cells showed extensive striped patterns, indicating complete differentiation into functional skeletal muscle cells. Eight days after differentiation, cells were labeled with an anti-myomacroprotein antibody, and the nuclei were visualized using DAPI.

[0121] Figure 3 Comparison of the effects of doxycycline duration on the differentiation capacity of EpiSC-PAX7-MYOD1: 4 days (left) and 2 days (right), with a total culture time of 6 days. Cultures were stained with myomalacin, and nuclei were visualized using DAPI.

[0122] Figure 4 3D suspension differentiation 4 days after doxycycline addition. Aggregates were stained with Pax7 and nuclei were visualized using DAPI.

[0123] Figure 5 Comparison between EpiSC-MyoD1 (left) and EpiSC-Pax7-MyoD1 (right) 8 days after 3D suspension differentiation. Aggregates were fixed and labeled with anti-myoma macroprotein antibody and DAPI nuclear staining.

[0124] Figure 6 Comparison between EpiSC-MyoD1 (left) and EpiSC-Pax7-MyoD1 (right) 8 days after 3D suspension differentiation. (Magnified) Figure 5 The arrow indicates sarcomere formation.

[0125] Figure 7 EpiSC-PAX7 (A) and EPISC-PAX7-MYOD1 (B) were differentiated in 2D monolayer cultures for 18 days and 8 days, respectively. The cultures were stained with myomalacin, and the nuclei were visualized using DAPI.

[0126] Figure 8 ： Comparison between EpiSC-PAX7 (left) and EpiSC-Pax7-MyoD1 (right) 18 or 8 days after 3D suspension differentiation. Aggregates were fixed and labeled with anti-myomacroprotein antibody and DAPI nuclear staining.

[0127] Example

[0128] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

[0129] Materials and methods

[0130] Porcine epiblast-derived stem cells (pEpISC) differentiate into skeletal muscle cells.

[0131] Undifferentiated pEpISCs (Opti-Ox MyoD1 and Opti-Ox Pax7-MyoD1) were propagated on hESC-qualified geltrex (A1413301, Thermo Scientific) plates in N2B27 proliferation medium (50% DMEM Ham F-12 (L0093-500, Biowest), 50% Neurobasal medium (21103049, Thermo Fisher Scientific), B27 supplement (17504044, Thermo Fisher Scientific), N2 supplement (17502001, Thermo Fisher Scientific), glutamax (35050061, Thermo Fisher Scientific), 10 mM Cells were grown in 2-mercaptoethanol (31350010, Thermo Fisher Scientific), 0.02 μg / mL activin A (QK001, Q-kine), 0.10 μg / mL LFGF2 (QK002, Q-kine), and 0.625 μg / mL XAV939 (X3004, Sigma-Aldrich). For 2D skeletal muscle differentiation, single cells were obtained after treatment with a mild cell dissociation reagent (07174, STEMCELL Technologies), and cell number and viability were assessed. Single pEpISCs were seeded in appropriate cell culture plates at a density of 100,000–200,000 cells / cm². After incubating overnight in proliferation medium with 10 μm 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 substitute (10828-028, Thermo Fisher Scientific) and 1x insulin-transferrin-selenium (ITS-G) (41400045, Thermo Fisher Scientific) for the entire differentiation duration. The medium was refreshed every two 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 shown in the figure or example. On day 2 or 4, the medium was completely replaced with doxycycline-free medium.

[0132] For spheroid or aggregate differentiation experiments, undifferentiated single pEpISCs (Opti-Ox MyoD1 and Opti-OxPax7-MyoD1) were seeded at 3 million cells / mL in 150mL shake flasks containing 12.5mL of proliferation medium (containing 50% DMEM Ham F-12 (L0093-500, BioWest), 50% Neurobasal medium (21103049, Thermo Fisher Scientific), B27 supplement (17504044, Thermo Fisher Scientific), N2 supplement (17502001, Thermo Fisher Scientific), glutamax (35050061, Thermo Fisher Scientific), 10mM 2-mercaptoethanol (31350010, Thermo Fisher Scientific), 0.02μg / mL activin A (QK001, Q-kine), and 0.10μg / mL... FGF2 (QK002, Q-kine), 0.625 μg / mL XAV939 (X3004, Sigma-Aldrich), 2X% knockout serum substitute (10828-028, Thermo Fisher Scientific), KSR, and 10 ng / mL FGF2 were used. Two days later, the aggregate formation medium was replaced with the skeletal muscle differentiation medium as described above, and the medium was refreshed every two days until the end of the experiment.

[0133] result

[0134] Example 1: Development of an inducible transgene overexpression method in animal cells using dual genetic safe harbor (GSH) targeting.

[0135] These examples employ the Opti-ox method for generating cells with controllable transcription, as described in WO 2018096343 A1. This method involves inserting an inducible cassette, allowing control over the transcription of genetic material within the inducible cassette using a dual-genome safe harbor targeting system.

[0136] To explore the potential of OPTI-OX for myogenic reprogramming of porcine pluripotent stem cells (pPSCs), we generated MYODOPTi-OX pPSCs. We sequentially targeted the rtTA box to porcine ROSA26 GSH under the control of the CAG promoter, and then targeted the MYOD transgene to porcine AAVS1 GSH under the control of the doxycycline inducible element. We observed robust and homogeneous inducible transgene expression. Furthermore, we observed that MYOD reprogramming of pPSCs directly generated myotubes 8 days after expression. Figure 1 (Left). This discovery demonstrates that MYOD can directly reprogram pPSCs into myotubes in 2D culture.

[0137] MYOD and PAX7 were selected for a combined cell reprogramming strategy. We designed a targeting vector containing MYOD, a P2A “self-cleaving” peptide linker, and PAX7 within an open reading frame to simultaneously express MYOD and PAX7 via doxycycline induction. The P2A peptide linker is a well-characterized short peptide linker of 18–22 amino acids that, due to ribosome jumping during translation, produces the expression of two separate gene products from a single open reading frame. Stable knock-in in AAVS1GSH was selected by incorporation of a puromycin resistance cassette, and selection was further achieved by incorporating puromycin into the cell culture medium. Following selection, single pEpiSC cells were plated, and clonal cell lines were isolated for growth and analysis. The incorporation of doxycycline-inducible MYOD-P2A-PAX7 was subsequently confirmed using PCR genomic analysis, Sanger sequencing, and RT-qPCR. Using a 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 does not affect the SC self-renewal or differentiation capacity of pEpiSCs, as determined by RT-qPCR (data not shown).

[0138] Example 2: EpiSC-MYOD-PAX7 2D differentiates into multinucleated skeletal muscle cells

[0139] pEpISC Opti-Ox MYOD and pEpISC Opti-Ox PAX7-MYOD were expanded in 3D suspension culture, unicellularized, and produced at 200,000 cells / cm². 2 Inoculated into coated plastic wells. One day after inoculation, doxycycline was added to the culture medium to initiate differentiation. In pEpISC Opti-Ox PAX7-MYOD culture, doxycycline was removed on day 4. Both lines showed formation of myomalacin-positive skeletal muscle cells; however, Pax7-MyoD1 produced thicker skeletal muscle cells. Figure 1 And it formed a powerful sarcomere structure, such as Figure 2 As shown. Next, the timing of discontinuing doxycycline to reduce differentiation time was tested. Discontinuation was tested on days 2 and 4 of culture, as shown. Figure 3 As shown, multinucleated skeletal muscle cells were formed under both conditions, indicating that 2 days of doxycycline induction was sufficient to trigger complete differentiation, as observed by the high similarity in myomatin expression and morphology. Figure 3 ).

[0140] Example 3: 3D culture of EpiSC-MYOD and EpiSC-MYOD-PAX7

[0141] For the expansion (proliferation) phase of cell culture in cultured meat production, shakers and / or bioreactors are typically used to scale up to large volumes and generate the amount of cell clusters required for cultured meat products at a cost-competitive price. Primary cells derived from muscle biopsies are usually used. However, primary cultures have limited self-renewal, lose differentiation capacity during expansion, and their quality varies between biopsies. Other sources (e.g., ESCs and iPSCs) have high expansion capacity but are undifferentiated and differentiation is induced using small molecules to mimic embryonic development (review Chal et al., June 15, 2017; 144(12):2104-2122. doi:10.1242 / dev.151035.) or via ectopic expression of the transcription factors MYOD or PAX7. For meat production, cell differentiation into myotubes and subsequently myofibrils is an integral part of the process, which typically occurs in subsequent separate steps. Skeletal muscle cell differentiation generally requires different conditions in terms of nutrition and physical environment. The necessary nutrients can be provided by switching from a nutrient-rich proliferation medium to a differentiation medium composed of low serum, but providing the physical environment required for cell differentiation is more challenging. Furthermore, the substrate requirements for the proliferation and differentiation phases typically differ in surface chemistry and morphology. For skeletal muscle cells in suspension, a particular challenge is that they need to attach laterally to rods, scaffolds, or surfaces to form elongated, stretchable, multinucleated myotubes.

[0142] In this example, EpiSC-MYOD and EpiSC-PAX7-MYOD were adapted for 3D suspension cell growth and grew as aggregates. EpiSCs expanded in adherent 6-well cell plates were single-celled using Accumax (00-4666-56, Thermo Fisher Scientific) according to the manufacturer's instructions, then transferred to 12.5 mL of culture medium and RHO / ROCK pathway inhibitor in 150 mL shake flasks, and expanded for at least 3 cycles. The culture medium was changed daily during 3D expansion. Subsequently, the 3D-adapted EpiSCs were used in shaker and bioreactor experiments.

[0143] To explore skeletal muscle differentiation in 3D suspension culture, 500,000 cells / mL were seeded in 150 mL shake flasks containing 12.5 mL of culture medium and differentiated into EpiSC-MYOD or EpiSC-PAX7-MYOD using the specified differentiation protocol. The culture medium was changed every other day. After inducing PAX7 / MYOD with doxycycline, we detected PAX7 expression in the aggregates within 2 days. Figure 4 Eight days after differentiation, the two EpiSC lines ( Figure 5Both lines showed skeletal muscle cell formation, but aggregates containing EpiSC-PAX7-MYOD showed larger and longer skeletal muscle cells including many nuclei. Monocytes were predominantly detected in the EpiSC-MYOD aggregates. As observed by immunofluorescence staining, both lines produced myomagrain at this time point, but a myomagrain stripe pattern was detected only in EpiSC-PAX7-MYOD, indicating the formation of functional sarcomeres and improved maturity. Figure 6 arrow).

[0144] The simplified biological process shown is very attractive, in which the same culture system can be used for both the proliferation and differentiation phases by altering the culture medium or by adding only the Opti-ox system inducer to the bioreactor / only adding the inducer for differentiation to the bioreactor, as it minimizes the capital investment in equipment, processing time, and cell manipulation.

[0145] Example 4: Comparison of differentiation rates between EpiSC-PAX7 and EpiSC-PAX7-MYOD

[0146] pEpISC Opti-Ox PAX7 cells were cultured in suspension for 2 days in Pax7 induction medium (DMEM / F12, 1xITS, and 15% KSR) containing 3 μM CHIR99021, and then transferred to Pax7 induction medium containing 1 μg / ml DOX and 20 ng / ml FGF2 for 10 days. After 10 days, cells differentiated directly into skeletal muscle cells as aggregates, or as single cells on geltrex-coated culture dishes. Differentiation was initiated by discontinuing DOX using Pax7 differentiation medium (DMEM HG, 1xITS, 1% KSR). For pEpISC, Opti-Ox PAX7-MYOD cells were first induced for 4 days in PAX7 / MYOD medium containing DMEM / HG, 1xITS, 15% KSR, and 1 μg / ml DOX. On day 4 of culture, DOX was discontinued and cells were transferred to PAX7 / MYOD differentiation medium (DMEM HG, 1x ITS, and 1% KSR). Cell differentiation of Pax7 cells was measured on day 18, as shorter differentiation times do not result in significantly differentiated cells, and differentiation of Pax7-MyoD1 cells was measured on day 8. Figure 7 ).

[0147] Eight days after differentiation, Pax7-MyoD1 cells showed the formation of myomagrain-positive skeletal muscle cells and even thicker skeletal muscle cells than those observed 18 days after differentiation with Pax7 cells. Figure 7A). Pax7-MyoD1 cells further showed strong formation of sarcomere structures, such as Figure 7 As shown in Figure B, even 18 days after differentiation, the differences in differentiation remained significant, as EpiSC-PAX7 cells still did not achieve the same thickness and sarcomere structure. Furthermore, aggregates containing EpiSC-PAX7-MYOD showed larger and longer skeletal muscle cells including numerous nuclei. Figure 8 ).

Claims

1. A pluripotent stem cell comprising: i) an expression construct for expressing a transcriptional regulator inserted in a first genetic safe harbor site; 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 iii) an 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 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, and wherein the inducible promoter is regulated by the transcriptional regulator.

2. The pluripotent stem cell of claim 1, wherein 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.

3. The pluripotent stem cell of claim 1 or 2, wherein 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.

4. The pluripotent stem cell of any one of claims 1-3, wherein the pluripotent stem cells belong to a livestock or poultry species.

5. The pluripotent stem cell of claim 4, wherein the livestock species is a pig or a cow, preferably a pig.

6. The pluripotent stem cell of any one of claims 2-5, wherein the expression construct inserted in 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 of any one of the preceding claims, wherein the activity of the transcriptional regulator is controlled by an exogenously supplied derivative of a substance.

9. The pluripotent stem cell of any one of the preceding claims, wherein the transcriptional regulator 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 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 is rtTA.

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

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

12. The pluripotent stem cell of any one of the preceding claims, wherein 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.

13. A method for producing skeletal muscle cells, the method comprising a) culturing the pluripotent stem cell according to any one of the preceding claims in a proliferation medium: followed by b) inducing skeletal muscle cell differentiation by addition of the exogenous substance according to claim 8.

14. The method according to claim 13, wherein the proliferation medium and 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 produced skeletal muscle cells are for human and non-human dietary consumption.

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

18. A food product comprising the pluripotent stem cell 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. The food product according to claim 18, wherein the food product is cultured meat.

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