Adipose cell maturation

Abstract

The present invention relates to a pluripotent stem cell comprising an expression construct for expressing a PPAR-gamma protein and an expression construct for expressing a CEBP alpha # protein. The invention further provides methods comprising the production of adipocytes using pluripotent stem cells and foods comprising adipocytes or pluripotent stem cells.

Description

Technical Field

[0001] The present invention relates to modified pluripotent cells and methods of differentiating said cells into adipocytes. Background Art

[0002] According to the latest estimates of the United Nations, in July 2022, the current world population was 7.9 billion [https: / / www.worldometers.info / es / poblacion-mundial / #ref-1], and is expected 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 in 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), it is estimated that global meat consumption will increase by 15% by 2031. On the other hand, the correlation between income growth and increased meat consumption is obvious 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 increasing interest in finding alternative protein sources that are ideally sustainable and contain the nutrients typically provided by 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 make up animal meat, but excluding the use of in vitro tissue and bioengineering techniques. Despite efforts to develop robust protocols for the scalable generation of animal cell types from easily accessible and renewable sources, the differentiation of animal (pluripotent) stem cells into specific cell types generally remains cumbersome, time-consuming, difficult to reproduce, and / or not yet established.

[0004] In addition, to date, plant-based and cultured meat alternatives have mainly focused on mimicking the muscle components of meat. However, fat is also an important component of meat, contributing to sensory / flavor, texture attributes, and palatability (Zhang, Shu et al. "DNA polymorphisms in bovine fatty acid synthase are associated with beef fatty acid composition 1." Animal genetics 39.1 (2008): 62-70.).

[0005] Tontonoz et al. (Cell, Volume 79, 1147-1156 – 30 - 12 - 1994) have previously described a modified cell line that can differentiate into adipocytes, in which PPARγ and CEBPα were co-expressed into a fibroblast cell line using a retroviral expression system, and subsequent spontaneous differentiation into adipocytes was observed. US2012219530 describes lentivirus-transduced human pluripotent cells with a differentiation efficiency of approximately 20%. However, these protocols have several limitations, including heterogeneous adipocyte maturity, lack of a scalable process, not being food-safe, and a culture time of up to 28 days.

[0006] Accordingly, there remains a need in the art to produce and culture mature adipocytes that are suitable for human consumption and can be produced in a scalable and cost-effective manner. Summary of the Invention

[0007] In a first aspect, the present invention relates to a pluripotent stem cell comprising:

[0008] i) an expression construct for expressing a transcriptional regulatory protein inserted into a first genetic safe harbor locus;

[0009] ii) an expression construct for expressing a PPAR-γ protein, wherein the coding sequence of the PPAR-γ protein is operably linked to an inducible promoter; and

[0010] iii) an expression construct for expressing a CEBPα protein, wherein the coding sequence of the CEBPα protein is operably linked to an inducible promoter;

[0011] wherein the expression constructs of ii) and iii) are inserted into at least one additional genetic safe harbor locus that is not the first genetic safe harbor locus,

[0012] and wherein the inducible promoter is regulated by the transcriptional regulatory protein.

[0013] In certain embodiments of the present invention, the expression constructs of ii) and iii) are inserted into a second genetic safe harbor locus different from the first genetic safe harbor locus. Preferably, the first and additional genomic safe harbor loci are selected from any two of the hROSA26 locus, the AAVS1 locus, the CLYBL gene, or the CCR5 gene, preferably where the genetic safe harbor loci are the hROSA26 locus and the AAVS1 locus.

[0014] In certain 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.

[0015] In certain embodiments of the present invention, the pluripotent stem cells belong to a livestock or poultry species. Preferably, the livestock species is pig or cattle, preferably pig. The pluripotent stem cells can belong to the family Suidae, such as the genus Sus, such as the species Sus domesticus.

[0016] In certain embodiments of the present invention, the expression construct inserted into the second genetic safe harbor locus encodes a PPAR-γ protein, a linker, and a CEBPα protein, preferably where the linker is P2A, more preferably where the linker contains the sequence of SEQ ID NO:3. Preferably, the construct contains the sequence of SEQ ID NO:4.

[0017] In certain embodiments of the present invention, the activity of the transcriptional regulatory protein is controlled by an exogenously supplied substance derivative. Preferably, the transcriptional regulatory protein is selected from the group consisting of: the tetracycline-responsive transcriptional activator protein (rtTa), the tetracycline repressor (TetR), the VgEcR synthetic receptor, or a chimeric transcriptional regulatory protein comprising the DNA-binding domain from the yeast GAL4 protein, the truncated ligand-binding domain from the human progesterone receptor, and the activation domain from the human NF-kB, preferably the transcriptional regulatory protein is rtTA.

[0018] In certain embodiments, the inducible promoter includes a Tet-responsive element (TRE).

[0019] In certain embodiments, the inducible promoter is the tetON promoter.

[0020] In a second aspect, the present invention provides a method for generating adipocytes, preferably white adipocytes, the method comprising

[0021] a) culturing the pluripotent stem cells according to any one of the preceding claims in a proliferation medium: subsequently

[0022] b) inducing adipocyte differentiation by adding the exogenous substances described herein.

[0023] In certain embodiments, the proliferation and / or differentiation medium does not contain or substantially does not contain at least one compound selected from the group consisting of insulin, dexamethasone, rosiglitazone, and isobutylmethylxanthine. In certain preferred embodiments, the proliferation and / or differentiation medium does not contain or substantially does not contain insulin. The proliferation and / or differentiation medium that does not contain or substantially does not contain insulin may optionally contain IGF-1 and / or LR3.

[0024] In certain embodiments, the differentiation period of the methods 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.

[0025] In certain embodiments, the adipocytes produced are for human and non-human dietary consumption.

[0026] In a further aspect, the present invention provides the use of the pluripotent stem cells described herein or the use of the method for producing adipocytes described herein for tissue engineering, optionally for the production of cultured meat.

[0027] In yet a further aspect, the present invention provides a food product comprising the pluripotent stem cells described herein or adipocytes obtained by the methods described herein. In certain embodiments, the food product is cultured meat. Detailed Description

[0028] Definition

[0029] Unless otherwise defined, the 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 many methods and materials similar or equivalent to those described herein, which can be used in the practice of the present invention. In fact, the present invention is in no way limited to the methods.

[0030] In this document and its claims, the verb "comprise" and its conjugations are used in their non-limiting sense, meaning including the items that follow the word, but not excluding items not specifically mentioned. Further, unless the context clearly requires the presence of one and only one element, the reference to an element by the indefinite article "a or an" does not exclude the possibility of the presence of more than one element. Thus, the indefinite article "a or an" generally means "at least one".

[0031] As used herein, the term "and / or" indicates that one or more of the stated circumstances may occur alone or in combination with at least one of the stated circumstances, up to and including all of the stated circumstances.

[0032] 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.).

[0033] 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 that value.

[0034] The term "heterologous" when used in reference to a nucleic acid (DNA or RNA) or a protein refers to such 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 is found in one or more positions in a cell or genome or DNA or RNA sequence different from that in which it is found in nature. A heterologous nucleic acid or protein is not endogenous to the cell into which it is introduced, but has been obtained from another cell or synthesized or recombinantly produced. Typically, although not necessarily, such nucleic acids encode proteins that are not normally produced by the cell that transcribes or expresses the DNA. Similarly, exogenous RNA encodes a protein that is not normally expressed in the cell in which the exogenous RNA is present. Heterologous 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 exogenous or foreign 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 in which at least two of the sequences are foreign to each other.

[0035] 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 sequences. These expression vectors typically include at least suitable transcriptional regulatory sequences and optionally 3' transcriptional termination signals. Additional factors necessary or helpful in effecting expression, such as expression enhancer elements, may also be present.

[0036] As used herein, the term "operably linked" refers to the joining of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when placed into 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, contiguous and in frame. Inducible promoter

[0037] As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, is located upstream with respect to the transcription direction of the transcription start site of the coding sequence, and is characterized structurally by the presence of a binding site for DNA-dependent RNA polymerase, a transcription start 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 those skilled in the art to directly or indirectly play a role in regulating the amount of transcription of 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 regulated by physiology or development, such as by the application of a chemical inducer. In the context of the present invention, the control is achieved through a transcriptional regulatory protein.

[0038] Any reference herein to a nucleotide or amino acid sequence accessible in a public sequence database refers to the version of the sequence entry available on the date of filing of this document.

[0039] All patents and references cited in this specification are hereby incorporated by reference in their entirety.

[0040] The inventors unexpectedly found that by using modified pluripotent stem cells, which comprise an expression construct for expressing a PPAR-γ protein and an expression construct for expressing a CEBPα protein, the time period required for the differentiation of pluripotent cells into mature adipocytes can be significantly shortened. As shown in the examples described herein, complete differentiation into mature adipocytes can be achieved in less than 10 days by using these modified pluripotent cell lines. In addition to significantly reducing the culture time and the associated costs involved, the use of the pluripotent cells described herein also provides a more reliable and scalable production of mature adipocytes compared to those previously described.

[0041] Accordingly, in a first aspect, the present invention relates to a pluripotent stem cell comprising:

[0042] i) an expression construct for expressing a transcriptional regulatory protein inserted into a first genetic safe harbor locus;

[0043] ii) an expression construct for expressing a PPAR-γ protein, wherein the coding sequence of the PPAR-γ protein is operably linked to an inducible promoter; and

[0044] iii) an expression construct for expressing a CEBPα protein, wherein the coding sequence of the CEBPα protein is operably linked to an inducible promoter;

[0045] wherein the expression constructs of ii) and iii) are inserted into at least one additional genetic safe harbor locus that is not the first genetic safe harbor locus,

[0046] and wherein the inducible promoter is regulated by the transcriptional regulatory protein.

[0047] Peroxisome proliferator-activated receptor gamma (PPAR-γ) is a type II nuclear receptor that functions as a transcription factor and is encoded by the PPARG gene in humans. PPARG is predominantly present in adipose tissue, colon, and macrophages. Two isoforms of PPARG have been detected in humans and mice: PPAR-γ1 (found in almost all tissues except muscle) and PPAR-γ2 (predominantly found in adipose tissue and the intestine). In certain embodiments, the coding sequence of PPAR-γ of the present invention encodes PPAR-γ2. PPARG regulates fatty acid storage and glucose metabolism. Genes activated by PPARG stimulate lipid uptake and adipogenesis in adipocytes. PPARG knockout mice lack adipose tissue, thus establishing PPARG as a major regulator of adipocyte differentiation. In certain embodiments, the coding sequence of PPAR-γ has the sequence of SEQ ID NO:1.

[0048] CCAAT / enhancer-binding protein alpha (CEBPα) is a protein encoded by the human CEBPA gene. The protein encoded by this intronless gene is a bZIP transcription factor that can bind to certain promoters and gene enhancers as a homodimer. It can also form heterodimers with related proteins CEBP-β and CEBP-γ, as well as different transcription factors such as c-Jun. The encoded protein is a key regulator of adipogenesis (the process of forming new adipocytes), lipid accumulation in these cells, and glucose and lipid metabolism in the liver. In certain embodiments, the coding sequence of CEBPα has the sequence of SEQ ID NO:2.

[0049] In certain embodiments, the nucleic acid molecule encoding the protein according to the present invention is 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 with a more preferred codon as compared to the wild-type sequence. Here, a non-preferred codon refers to 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 the organism than the non-preferred codon. The codon usage frequencies of a particular organism can be found in a codon frequency table, e.g., http: / / www.kazusa.or.jp / codon. Preferably, more than one non-preferred codon, preferably most or all non-preferred codons, are replaced with 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.

[0050] A transcriptional regulatory protein is a protein that binds to DNA, preferably binds sequence - specifically to a DNA site within or near a promoter, and facilitates the binding of the transcriptional machinery to the promoter, thereby transcribing a DNA sequence (transcriptional activator) or blocking this process (transcriptional repressor). Such entities are also referred to as transcription factors.

[0051] The DNA sequences to which transcriptional regulatory proteins bind are called transcription factor binding sites or response elements, which are found in or near the promoters of regulatory DNA sequences.

[0052] Transcriptional activator proteins bind to response elements and promote gene expression. Such proteins are preferred in the methods of the present invention for controlling the expression of inducible cassettes.

[0053] Genetic safe - harbor (GSH) loci are loci within the genome where genes or other genetic material can be inserted without any detrimental effects on the cell or the inserted genetic material. Most desirably, GSH loci are those where the expression of the inserted gene sequence is not perturbed by any read - through expression of neighboring genes and the expression of inducible cassettes minimally perturbs the endogenous transcriptional program. More formal criteria have been proposed that help to determine whether a particular locus will be a GSH locus in the future (Papapetrou et al., 2011, Nature Biotechnology, 29(1), 73 - 8. doi:10.1038 / nbt.1717). These criteria include loci that (i) are 50 kb or more from the 5′ end of any gene, (ii) are 300 kb or more from any cancer - related gene, (iii) are 300 kb or more from any microRNA (miRNA), (iv) are outside transcriptional units, and (v) are outside ultra - conserved regions (UCRs). It may not be necessary to meet all of these proposed criteria, as established GSHs do not meet all criteria. It is thought that a suitable GSH will meet at least 2, 3, 4, or all of these criteria.

[0054] In certain 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 certain embodiments, the first and additional genomic safe harbor sites are located at chr1:152,360,840-152,360,859, chr1:175,942,362 -175,942,381, chr1:231,999,396-231,999,415, chr2:45,708,354–45,708,373; chr8:68,720,172–68,720,191 of the human genome.

[0055] In certain embodiments of the present invention, the first and additional genomic safe harbor sites are selected from any two of the bovine safe harbor sites ROSA26, AAVS1, CLYBL gene, and CCR5 gene.

[0056] Preferably, the genetic safe harbor sites are the hROSA26 locus and the AAVS1 locus.

[0057] In certain embodiments of the present invention, the expression construct for expressing the PPAR-γ protein described herein and the expression construct for expressing the CEBPα protein described herein are inserted into a second genetic safe harbor site different from the first genetic safe harbor site. In certain embodiments, the expression construct inserted into the second genetic safe harbor site is capable of simultaneously expressing the PPAR-γ protein and the CEBPα protein.

[0058] 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 derivation of the pluripotent stem cell. Thus, in certain embodiments, the pluripotent stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines. In certain embodiments, the pluripotent stem cells are ectoderm-derived stem cells (EpiSC). In certain embodiments, the pluripotent stem cells express one or more markers selected from the group consisting of OCT-4, Sox2, Klf4, c-MYC, Nanog, Lin28, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Exemplary pluripotent stem cells can be generated using methods known in the art. "Induced pluripotent stem cells" (iPS cells or iPSCs) can be generated by protein transduction of reprogramming factors in somatic cells.

[0059] Pluripotent stem cells according to the present invention 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 other species. Thus, 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 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 giant pandas), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), etc.

[0060] Similarly, iPS cells can be from any species. These iPS cells have been successfully generated using mouse and human cells. In addition, iPS cells have been successfully generated using embryonic, fetal, neonatal, and adult tissues. Thus, iPS cells can be easily generated using donor cells from any species. Thus, iPS cells can be generated from any of the following species, including but not limited to humans, non-human primates, rodents (mice, rats), ungulates (cattle, sheep, etc.), dogs (domestic dogs and wild dogs), cats (domestic cats and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, marine mammals (dolphins, whales, etc.), etc.

[0061] In certain embodiments, the pluripotent stem cells according to the present invention or for use in the present invention are animal cells. In certain embodiments, the pluripotent stem cells according to the present invention or for use in the present invention are from an edible animal species.

[0062] Preferably, the pluripotent stem cells according to the present invention or for use in the present invention are from livestock or poultry animals. Livestock species include but are not limited to domestic cattle, pigs, sheep, goats, lambs, camels, buffalo, and rabbits.

[0063] Preferably, the pluripotent stem cells according to the present invention or for use in the present invention are porcine or bovine pluripotent stem cells. Most preferably, they are porcine pluripotent stem cells. In certain embodiments, the stem cells according to the present invention are porcine epiblast stem cells (pEpiSC).

[0064] Poultry species include but are not limited to domestic chickens, turkeys, ducks, geese, and pigeons. In certain embodiments, the cells are derived from common game species such as wild deer, galliformes, waterfowl, and hares. Preferably, the pluripotent stem cells according to the present invention or for use in the present invention are not human cells.

[0065] A transcriptional repressor binds to the response element and blocks gene expression.

[0066] Transcription regulatory proteins can be activated or inactivated by a variety of mechanisms, including ligand binding, interaction with other transcription factors (e.g., homodimerization or heterodimerization) or co-regulatory proteins, phosphorylation, and / or methylation. Transcription regulators can be controlled by activation or inactivation.

[0067] If the transcription regulatory protein is a transcriptional activator, preferably, the transcriptional activator requires activation. This activation can be carried out by any suitable means, but preferably, the transcription regulatory protein is activated by adding an exogenous substance to the cell. The supply of the exogenous substance to the cell can be controlled, so that the activation of the transcription regulatory protein can be controlled. Alternatively, an exogenous substance can be supplied to inactivate the transcription regulatory protein, and then the supply can be withdrawn to activate the transcription regulatory protein.

[0068] If the transcription regulatory protein is a transcriptional repressor, preferably, the transcriptional repressor requires inactivation. Thus, a substance is provided to prevent the transcriptional repressor from repressing transcription, thereby allowing transcription.

[0069] Any suitable transcription regulatory protein can be used, preferably a transcription regulatory protein that can be activated or inactivated. Preferably, an exogenous substance can be provided to control the transcription regulatory protein. Such a transcription regulatory protein is also referred to as an inducible transcription regulatory protein.

[0070] Thus, in certain embodiments, the pluripotent stem cells according to the present invention are controlled by an exogenously supplied substance.

[0071] In certain 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 (described by Goeke et al. Journal of molecular biology 416.1 (2012): 33-45; this document is incorporated herein by reference), aptamers (such as the RNA aptamers described by Hunsicker et al. "Chemistry & biology 16.2 (2009): 173-180; this document is incorporated herein by reference), tetracycline and anhydrotetracycline or derivatives thereof. Preferably, the exogenously supplied substance is doxycycline.

[0072] In certain embodiments, the transcriptional regulatory proteins described herein are selected from the group consisting of: tetracycline-responsive transcriptional activator protein (rtTa), tetracycline repressor (TetR), VgEcR synthetic receptor, or a hybrid transcriptional regulatory protein comprising the DNA-binding domain from the yeast GAL4 protein, the truncated ligand-binding domain from the human progesterone receptor, or the activation domain from the human NF-kB.

[0073] Tetracycline-controlled transcriptional activation is an inducible gene expression method well-known in the art, in which transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g., the more stable doxycycline). In this system, the transcriptional activator protein is the tetracycline-responsive transcriptional activator protein (rtTa) or its derivative. 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), and together they form the tetracycline-responsive element (TRE). This system has two forms, depending on whether the addition of tetracycline or its derivative activates (Tet-on) or inactivates (Tet-Off) the rTA protein.

[0074] In the Tet-Off system, tetracycline or its derivative binds to rTA and inactivates rTA, preventing it from binding to the TRE sequence and thus blocking transcription of the TRE-controlled gene. The Tet-On system consists of two components; (1) a constitutively expressed tetracycline-responsive transcriptional activator protein (rtTa) and an rtTa-sensitive inducible promoter (tetracycline-responsive element, TRE). This can bind to tetracycline or its more stable derivatives (including doxycycline (dox)), resulting in activation of rtTa, causing it to bind to the TRE sequence and induce expression of the TRE-controlled gene. In a preferred embodiment of the invention, the transcriptional regulatory protein is rtTA.

[0075] If the transcriptional regulatory protein is rtTA, the inducible promoter inserted into at least one additional GSH that is not the first GSH site comprises the tetracycline-responsive element (TRE). Thus, in certain embodiments, the inducible promoter comprises the Tet-responsive element (TRE).

[0076] In some embodiments, where the transcriptional regulatory protein is rtTA and comprises TRE, the exogenously supplied substance is the antibiotic tetracycline or one of its derivatives.

[0077] In certain embodiments of the present invention, the expression construct inserted into the second genetic safe harbor locus is a fusion protein encoding the PPAR-γ protein and the CEBPα protein, as described herein. In certain embodiments, the expression construct inserted into the second genetic safe harbor locus encodes the PPAR-γ protein, a linker, and the CEBPα protein, and in preferred embodiments, the construct comprises or consists of SEQ ID NO:4.

[0078] In certain embodiments, the linker sequence can be a cleavable linker. That is, the linker sequence can comprise an amino acid sequence that can be cleaved. For example, the linker sequence can comprise a sequence that can serve as a substrate for an enzyme that can cleave peptide bonds (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. A self-cleaving site is automatically cleaved without enzyme treatment. For example, the 2A self-cleaving peptides or 2A peptide family have been described, which includes 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; T2A is derived from Plutella xylostella virus 2A. In certain embodiments, the cleavable linker is thus selected from the group consisting of P2A, E2A, F2A, and T2A.

[0079] In some preferred embodiments, the expression construct comprises a picornavirus 2A (P2A) linker. Preferably, the expression construct comprises a linker that comprises or consists of the sequence of SEQ ID NO:3.

[0080] In certain embodiments, the expression construct inserted into the second genetic safe harbor locus encoding the PPAR-γ protein, a linker, and the CEBPα protein comprises or consists of the sequence of SEQ ID NO:4.

[0081] In certain embodiments, the inducible promoter operably linked to the PPAR-γ protein is different from the inducible promoter linked to the CEBPα protein. In certain embodiments, the inducible promoter operably linked to the PPAR-γ protein is the same as the inducible promoter linked to the CEBPα protein. Inducible promoters are well known in the art, and examples include but are not limited to CMV, CAG, CBh, PGK, SV40, ferritin heavy or light chain, etc.

[0082] In certain embodiments, the inducible promoter used in the present invention is the tetOn promoter. Preferably the 3rd generation TetOn promoter.

[0083] Cultivation method

[0084] The inventors of the present application unexpectedly found that by using the pluripotent cells described herein, the differentiation time required to obtain adipocytes can be significantly reduced. Accordingly, in a further aspect, the present invention relates to a method for generating adipocytes, the method comprising:

[0085] a) culturing the pluripotent stem cells described herein in a proliferation medium: subsequently

[0086] b) inducing adipocyte differentiation by adding the exogenous substances described herein.

[0087] In certain embodiments, the method of the present invention is an ex vivo method.

[0088] In certain embodiments, the method is used to generate mature adipocytes. Mature adipocytes are defined herein as adipocytes that display lipid accumulation and / or express detectable levels of PPARy, FABP4, PLIN1, and adiponectin.

[0089] In certain embodiments, the method of the present invention relates to a method for generating white adipocytes. In certain embodiments, the proliferation and / or differentiation medium does not contain or substantially does not contain at least one compound selected from the group consisting of insulin, dexamethasone, rosiglitazone, and isobutylmethylxanthine. In a preferred embodiment, the proliferation and / or differentiation medium does not contain or substantially does not contain insulin.

[0090] The proliferation and / or differentiation medium that does not contain or substantially does not contain insulin may optionally contain IGF-1 and / or LR3.

[0091] The proliferation and / or differentiation medium may contain up to about 20 μg / mL insulin, such as up to about 10 μg / mL insulin, such as about 5 μg / mL insulin, such as 1 μg / mL insulin.

[0092] The inventors unexpectedly discovered that the use of the pluripotent cells described herein obviates the need to culture cells with a commitment induction step. Typically, when culturing adipocytes, several culture periods can be distinguished. The commitment or determination period involves the formation of preadipocytes that have lost the potential to differentiate into other cell types. The differentiation of preadipocytes into adipocytes is facilitated by a highly regulated network of transcription factors expressed in chronological order to promote adipocyte morphology and biochemical characteristics such as insulin responsiveness, lipid transport and synthesis, and secretory capacity. The differentiation period 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 the methods described herein allows the pluripotent stem cells to differentiate into mature adipocytes without the need for a commitment induction step and without forced overexpression. The ability of the cells to skip this commitment induction step is particularly advantageous because it reduces the amount of compounds and small molecules that are typically required to be present in the differentiation medium. For example, it was found that the cells described herein are capable of differentiating in the absence of BMP-4, activin A, and FGF2, which are typically required for satisfactory differentiation. The ability to omit these compounds and small molecules from the differentiation medium reduces the cost of the medium and simplifies the manner of regulatory acceptance. The terms differentiation period and differentiation stage are used interchangeably herein.

[0093] Accordingly, in certain embodiments, the methods described herein do not include an additional commitment period induction step.

[0094] The methods described herein significantly reduce the time for the pluripotent cells described herein to differentiate into mature adipocytes. In certain embodiments, the time to produce mature adipocytes 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 in the methods described, the inventors observed a conversion rate of at least 95% by day 4 of culture, meaning that at least 95% of the cells were mature after 4 days of culture. Accordingly, in certain embodiments, the time to produce at least 95% mature adipocytes is at most 4 days.

[0095] In a further aspect, the invention provides adipocytes, preferably mature adipocytes, obtained by the methods described herein.

[0096] Culturing the cells as described herein can be carried out under so-called 2D culture conditions, which are considered a conventional method for culturing cells. However, the methods can also be readily adapted to allow culturing under 3D conditions, as shown in the examples below.

[0097] 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 person skilled in the art can easily perform 3D cell culture, for example, by using 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 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 use of scaffolds for growing cells. Instead, 3D spheroids can be established by using, for example, low-adhesion plates, hanging drop plates, micropatterned surfaces, rotating bioreactors, magnetic levitation, and magnetic 3D bioprinting.

[0098] Cells transduced with lentiviral vectors are not considered safe for food or unsafe for human and non-human dietary consumption. The pluripotent cells described herein of the methods described herein obviate the need for cells transduced with lentiviral vectors. Thus, in certain embodiments, the adipocytes produced according to the methods disclosed herein are for human and non-human dietary consumption. In certain embodiments, the adipocytes produced can be used to produce cultured meat for human consumption.

[0099] In a further aspect, the present invention provides the use of the pluripotent stem cells described herein or adipocytes obtained by the methods described herein for tissue engineering. In certain embodiments, the methods described herein are for ex vivo or in vivo tissue engineering.

[0100] In certain aspects, the use is for producing cultured meat. That is, the present invention provides the use of the pluripotent stem cells described herein or adipocytes obtained by the methods described herein for producing cultured meat.

[0101] In yet a further aspect, the present invention provides a food (also referred to as "foodstuff"), which comprises the pluripotent stem cells described herein or adipocytes produced and / or obtained by the methods described. In certain embodiments, the food is or 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 myocytes, mature muscle cells, minerals, synthetic substances, flavoring substances (such as, for example, herbs and spices), plant-based proteins, or proteins of microbial origin (such as yeast protein). Plant-based proteins and yeast proteins suitable for use in foods are known to those skilled in the art. In certain embodiments, the food is cultured meat or a cultured meat product.

[0102] In yet another aspect, the present invention provides a method for producing a food product, the method comprising combining the pluripotent stem cells described herein or the adipocytes produced and / or obtained with an edible composition for human or non-human consumption described herein. In certain embodiments, the food product is cultured meat.

[0103] Sequence Listing

[0104] Table 1: Sequences

[0105] SEQ ID NO: Name 1 DNA sequence encoding PPAR-γ protein 2 DNA sequence encoding CEBPα protein 3 GSG-P2A linker 4 PPARG-P2A-CEBPA construct 5 pMI014_PPARG-P2A-CEBPA_opti-ox-aavs1 Brief Description of the Drawings

[0106] Figure 1 : Comparison between EpiSC-PPARγ and EpiSC-PPARγ-CEBPα 2D differentiation. Undifferentiated (day 0) and differentiated (days 2, 4, 6, and 8) EpiSC-PPARγ and EpiSC CEBPα-PPARγ. Bright-field microscopy, 10x.

[0107] Figure 2 : Relative gene expression of mRNA of porcine PPARγ and CEBPα in Opti-Ox cells. A) PPARγ, B) CEBPα, C) endogenous PPARγ, D) endogenous CEBPα, E) adiponectin, F) LPL, G) perilipin-1, H) FABP4, i) CD36, and J) CEBPβ, K) ZBTB16, and L) Oct 4. EpiSC-PPARγ and EpiSC CEBPα-PPARγ were differentiated in 2D for 8 days.

[0108] Figure 3 : Quantification of total intracellular triglycerides in undifferentiated EpiSC-PPARγ and EpiSC-PPARγ-CEBPα and after 2, 4, 6, and 8 days of differentiation.

[0109] Figure 4 : Comparison between EpiSC-PPARγ and EpiSC-PPARγ-CEBPα 2D differentiation. Undifferentiated (day 0) and differentiated (days 7, 14, and 20). Fluorescence microscopy, 10x. Stained with Oil Red O (red, neutral lipids) and DAPI (blue, nuclei).

[0110] Figure 5 : Comparison between EpiSC-PPARγ and EpiSC-PPARγ-CEBPα 3D differentiation. EpiSC-PPARγ and EpiSC PPARγ-CEBPα in suspension differentiation (3D, day 6) stained with Oil Red O (red, neutral lipids) and DAPI (blue, nuclei). Fluorescence microscopy, 10x.

[0111] Figure 6 : EpiSC PPARγ-CEBPα differentiated according to Protocol D (mesoderm step + maturation step) or Protocol F (maturation step only) (days 4, 12, and 20). A) Bright-field microscopy, 40x. B) Quantification (absorbance) of intracellular Oil Red O staining of cells extracted from EpiSC CEBPα-PPARγ differentiated under Protocol D or Protocol F conditions for 20 days.

[0112] Figure 7 : EpiSC PPARγ-CEBPα differentiated (days 2, 7, and 13) under the following conditions: according to Protocol D (mesoderm step + maturation step), with maturation medium supplemented with (1) KSR, insulin, and dexamethasone, (2) KSR and dexamethasone, or (3) KSR and insulin; or according to Protocol F (maturation step only), with maturation medium supplemented according to conditions (1), (2), and (3). Bright-field microscopy, 20x.

[0113] Figure 8 : EpiSC PPARγ-CEBPα differentiated in suspension (3D, day 13) under the following conditions: according to Protocol D (mesoderm step + maturation step), with maturation medium supplemented with (1) KSR, insulin, and dexamethasone, (2) KSR and dexamethasone, or (3) KSR and insulin; or according to Protocol F (maturation step only), with maturation medium supplemented according to conditions (1), (2), and (3). Aggregates stained with Oil Red O (red, neutral lipids) and DAPI (blue, nuclei). Fluorescence microscopy, 10x.

[0114] Figure 9 ： EpiSC PPARγ-CEBPα differentiated in suspension (3D, day 7) in a bioreactor according to a fed-batch protocol similar to Protocol D (mesoderm step + maturation step), with medium supplemented with insulin and dexamethasone. Aggregates were collected on days 0, 3, 5, and 7 of differentiation, and total intracellular triglycerides were quantified.

[0115] Figure 10 : EpiSC PPARγ-CEBPα differentiated (day 7) according to Protocol F (maturation step only), with maturation medium supplemented with (A) insulin 20 μg / mL, (B) insulin 10 μg / mL, (C) insulin 5 μg / mL, or (D) insulin-free. In the absence of insulin, the maturation medium was also supplemented with (E) 0.05 μg / mL IGF-1 or (F) 0.05 μg / mL IGF-1 / LR3. Bright-field microscopy, 20x.

[0116] Examples

[0117] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the present invention.

[0118] Materials and Methods

[0119] Porcine ectoderm-derived stem cells (pEpISC) were differentiated into adipocytes.

[0120] Undifferentiated pEpISCs (Opti-Ox PPARγ and Opti-Ox CEBPα-PPARγ) were grown 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 2-mercaptoethanol (31350010, Thermo Fisher Scientific), 0.02 μg / mL activin A (QK001, Q-kine), 0.10 μg / mL FGF2 (QK002, Q-kine), 0.625 μg / mL XAV939 (X3004, Sigma-Aldrich)) on hESC-qualified geltrex (A1413301, Thermo Scientific)-coated plates. For 2D adipocyte differentiation, single cells were obtained after treatment with gentle cell dissociation reagent (07174, STEMCELL technologies), and cell number and viability were evaluated. Single pEpISC cells were seeded at a density of 50,000 - 150,000 cells / cm2 in the appropriate cell culture plates. After incubation overnight in proliferation medium containing 10 μM Rock inhibitor (Y-27632 (HBF2297, HelloBio)), the cells were cultured for 48 hours in StemPro-34 SFM medium (10639011, Thermo Scientific) supplemented with 25 ng / mL activin A (120-14E, PeproTech), 10 ng / mL BMP4 (120-05ET, PeproTech), 4 ng / mL FGF2 (Qk002, Qkine), and 50 mg / mL ascorbic acid (A8960, Sigma-Aldrich) to enhance stem cell commitment to the adipocyte lineage. On day 2, the medium was replaced with adipocyte maturation medium consisting of DMEM Ham F-12 (L0093-500, Biowest) containing 15% knockout serum replacement (10828-028, Thermo Fisher Scientific), 1 μg / mL insulin (12585014, Thermo Fisher Scientific), and 1 μM dexamethasone (D1756, Sigma-Aldrich). pEpISC differentiation was designated for a certain number of days. The medium was renewed 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.

[0121] For spheroid or aggregate differentiation experiments, undifferentiated single pEpISC (Opti-Ox PPARγ and Opti-Ox CEBPα-PPARγ) was seeded at 3 million cells / mL in 250 mL shake flasks containing 25 mL 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), 10 mM 2-mercaptoethanol (31350010, Thermo Fisher Scientific), 0.02 μg / mL activin A (QK001, Q-kine), 0.10 μg / mL FGF2 (QK002, Q-kine), 0.625 μg / mL XAV939 (X3004, Sigma-Aldrich, 2X% KnockOut Serum Replacement (10828-028, Thermo Fisher Scientific) KSR and 10 ng / mL FGF2). The next day, once spheroids had formed, the medium was replaced with supplemented StemPro-34 SFM. In the same manner, maturation medium was added on day 2 of differentiation and renewed every two days until the end of the experiment.

[0122] Oil Red O staining

[0123] As described previously, undifferentiated and differentiated pEpISC Opti-Ox PPARγ and pEpISC Opti-Ox CEBPα-PPARγ were stained with Oil Red O (O0625, Sigma). Briefly, cultured or aggregated cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) for 40 minutes at room temperature. After washing 3 times with PBS and permeabilizing with 60% isopropanol, the cells were stained with Oil Red O (0.5% in isopropanol) diluted to 40% with water for 30 minutes at room temperature. Thereafter, the pEpISC was washed several times with deionized water until only lipid droplets were correctly stained with the dye.

[0124] Undifferentiated and differentiated cells were imaged under an inverted bright-field microscope (Oxion Inverso, Euromex) or a fluorescence microscope (EVOS M7000, Thermo Fisher Scientific). At different checkpoints during adipogenesis, the triglyceride content of pEpISC Opti-Ox PPARγ and pEpISC Opti-Ox CEBPα-PPARγ was estimated by extracting Oil Red O dye from the cells with 100% isopropanol and spectrophotometrically measuring the resulting solution (Glomax Discover, Promega) at 540 mM. Oil Red O data were adjusted by estimating the cell number measured spectrophotometrically by HOESCH 33342 staining (H1399, Thermo Fisher Scientific) according to the manufacturer's instructions.

[0125] pEpISC Opti-Ox PPARγ and pEpISC Opti-Ox CEBPα-PPARγ differentiated in aggregates or spheroids were stained according to the same protocol described previously. Imaging acquisition was performed in two different ways. For a quick check of adipocyte quality, the stained aggregates were transferred onto a glass slide. After removing the excess buffer, the glass slide was mounted using a hard mounting medium (P36984, Thermo Fisher Scientific). During this process, the spheroid aggregates were flattened, thus obtaining an overview of the lipid presence and assessing its distribution. For a more accurate analysis, the unstained and fixed cell aggregates were cryopreserved in a sucrose solution. The cryopreserved samples were then sectioned at 20 μm using a cryostat (CM 1950, Leica). The cryosections were stained using the Oil Red O staining protocol described previously. Measurements included total cell number, total lipid number per cell, lipid size and shape, and total intensity (within the lipid object).

[0126] Triglyceride content

[0127] According to the manufacturer's instructions, the intracellular triglyceride content was quantified in undifferentiated and differentiated cultured / aggregated pEpISC Opti-Ox PPARγ and pEpISC Opti-Ox CEBPα-PPARγ using a Triglyceride Quantification Colorimetric / Fluorometric Kit (MAK266-1KT, Sigma-Aldrich). Briefly, total lipids were extracted with a solution of 5% Nonidet P40 substitute (11754599001, Sigma-Aldrich) in deionized water under heating. Glycerol was released from triglycerides by adding lipase, and subsequently, a color reaction was generated, which could be measured spectrophotometrically at 570 nm (Glomax Discover, Promega). The TG concentration was calculated based on a standard curve made from TG standards and normalized to the total cellular protein content.

[0128] Real-time quantitative PCR analysis (RT-qPCR)

[0129] According to the manufacturer's instructions, total RNA was extracted from undifferentiated and differentiated pEpISC Opti-Ox PPARγ and pEpISC Opti-Ox CEBPα-PPARγ using a Reliaprep Cell Miniprep System (Z6012, Promega). The RNA concentration and quality were determined using a micro-spectrophotometer DS-11 (DeNovix). First, 500 ng of purified total RNA from each sample was treated with DNase I to remove possible genomic DNA contamination, and subsequently, it was reverse-transcribed into cDNA using an iScript gDNA Clear cDNA Synthesis Kit (1725035BUN, BioRad). Specific primers for porcine pluripotency and mature adipocyte markers were designed for real-time quantitative PCR analysis (Table I). According to the manufacturer's guidelines, at least three samples from two independent experiments were amplified in triplicate using a PowerTrack SYBR Green Premix (A46112, Thermo Fisher Scientific) in a (thermal cycler) system. The RT-qPCR conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The YWHAZ gene was used as a housekeeping gene to normalize the target gene expression levels. Relative gene quantification was calculated by the 2-ΔΔCt method.

[0130] Results

[0131] Example 1: Development of an inducible transgenic overexpression method by dual GSH targeting in animal cells

[0132] To explore the potential of OPTi-OX for the forward programming of porcine (pPSC), we generated PPARG OPTi-OX pPSC. We sequentially targeted the rtTA cassette to the porcine ROSA26 GSH under the control of the CAG promoter and the PPARG transgene to the porcine AAVS1 GSH under the control of the doxycycline-inducible element and observed robust and homogeneous inducible transgene expression. Induction of PPARG expression after doxycycline treatment led to lipid accumulation in the cells. These results indicate that PPARG overexpression alone is sufficient to drive adipogenesis in pPSC. However, the pEpiSC PPARg lipid accumulation and cell morphology are far from comparable to those of porcine mature adipocytes, and some specific late adipogenesis markers (such as PLIN1 or adiponectin) are not expressed even after 21 days of differentiation.

[0133] After systematic screening of adipogenic factors by modulating key signaling cascades related to adipogenesis, PPARg and CEBPa were selected for a combined cell reprogramming strategy. We designed a knock-in that contains PPARg, a P2A "self-cleaving" peptide linker, and CEBPa in one open reading frame to simultaneously express PPARg and CEBPa 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 ribosome skipping during translation. Stable knock-in in AAVS1 GSH was selected by incorporation of a puromycin resistance cassette and selected by adding puromycin to the cell culture medium. After selection, single pEpiSC cells were plated, and clonal cell lines were isolated for growth and analysis. Subsequently, the incorporation of doxycycline-inducible PPARg-P2A-CEBPa was confirmed using PCR genomic analysis, Sanger sequencing, and RT-qPCR. Using a dual GSH targeting method, we selected clonal lines carrying two copies of each transgene and observed that homozygous targeting of both elements allowed inducible overexpression (data not shown). Importantly, the dual GSH targeting method did not affect SC self-renewal or differentiation, as determined by RT-qPCR (data not shown).

[0134] Example 2: Short-term differentiation of EpiSC-PPARγ-CEBPα into adipocytes.

[0135] pEpISC Opti-Ox PPARγ and pEpISC Opti-Ox PPARγ-CEBPα were differentiated with doxycycline for up to 8 days. Photographs of undifferentiated cells (day 0) and during differentiation (days 2, 4, 6, and 8) were taken using brightfield microscopy. As in Figure 1It can be seen that the lipid droplets produced by pEpISC containing Opti-Ox-PPARγ-CEBPα during differentiation are larger than those of pEpiSC PPARγ, indicating better cell maturation and more lipid accumulation. This difference was already visible on the 4th day after the start of differentiation, indicating that pEpISC containing Opti-Ox PPARγ-CEBPα differentiates much faster.

[0136] We used RT-qPCR to evaluate the relative gene expression of mRNAs of porcine endogenous PPARγ, CEBPα, adiponectin, LPL, perilipin-1, FABP4, CD36, and CEBPδ, and the results are shown in Figure 2. As expected, pEpiSC PPARγ-CEBPα cells already expressed Opti-Ox CEBPα and Opti-Ox PPARγ after 2 days of differentiation, which confirmed that the Opti-Ox system could stably express the transgene and confirmed the usability of the cell model. pEpiSC PPARγ-CEBPα cells had higher expression of multiple adipocyte markers (LPL and CD36, perilipin 1 (indicating lipid accumulation), CEBPδ and ZBTB16 (late adipogenesis markers), adiponectin, and FABP4 (markers of terminal cell differentiation)). Adiponectin and perilipin 1 were not expressed in pEpiSC PPARγ. This indicates that pEpiSC PPARγ-CEBPα has higher differentiation potential. In addition, we observed that Oct4 expression was induced in pEpiSC PPARγ-CEBPα on the 2nd day. This effect was not observed in pEpiSC PPARγ. Oct4 is known to be an important regulator for cell commitment to the adipocyte lineage, indicating that pEpiSC PPARγ-CEBPα does not require a commitment step during differentiation, thus allowing for faster differentiation.

[0137] Next, we used Oil Red O staining to quantify the total triglyceride content of undifferentiated and differentiated cells ( Figure 3 ). Oil Red O is a fat-soluble dye that stains neutral triglycerides and lipids. The results showed that pEpiSC PPARγ-CEBPα had significantly higher triglyceride content, indicating that they differentiated faster and more effectively compared to pEpiSC PPARγ.

[0138] Example 3: Long-term differentiation of EpiSC-PPARγ-CEBPα into adipocytes in 2D.

[0139] We repeated the experiment in Example 2, but now differentiated pEpISC Opti-Ox PPARγ and pEpISC Opti-Ox PPARγ-CEBPα cells for up to 20 days and collected the differentiated cells at days 7, 14, and 21 for Oil Red O staining ( Figure 4 ). Lipid droplets accumulated by pEpiSC CEBPα-PPARγ cells during adipogenesis were larger than those of pEpiSC PPARγ. This difference could already be detected at day 7, where pEpISC Opti-Ox PPARγ-CEBPα had a well-defined adipocyte-like shape and uniform lipid accumulation. Additionally, pEpiSC PPARγ-CEBPα cells did not need to reach confluence before starting differentiation (data not shown), indicating that pEpiSC PPARγ-CEBPα is less dependent on external adipogenic factors (ECM, secreted factors).

[0140] Example 4: EpiSC-PPARγ-CEBPα Skips the Commitment Step during Differentiation

[0141] To confirm that the commitment differentiation step is not required, EpiSC-PPARγ-CEBPα was differentiated in 2D using two differentiation protocols. Protocol D (including commitment + maturation steps) and Protocol F (only maturation steps). Cells were collected at days 4, 12, and 20 during differentiation and analyzed under bright-field microscopy ( Figure 6A ). Neutral lipid content was estimated by Oil Red O and adjusted by the number of cells measured by the HOESCH method. Both dyes were quantified by spectrophotometry ( Figure 6B ).

[0142] Although the commitment step is necessary to improve the differentiation capacity of pEpiSC PPARγ (data not shown), the adipogenic performance of EpiSC-PPARγ-CEBPα was comparable between Protocols D and F ( Figure 6A ). In fact, in the absence of the mesoderm step, the neutral lipid accumulation per cell was higher in differentiated EpiSCs-PPARγ-CEBPα ( Figure 6B ), making this model more promising in terms of cost reduction (process and medium) as several small molecules can be omitted for regulatory acceptance of cultured meat.

[0143] Example 5: EpiSC-PPARγ-CEBPα 2D Differentiation: Differentiation was performed using Protocol D and Protocol F in the presence of KSR + insulin + dexamethasone, KSR + dexamethasone (without additional insulin), and KSR + insulin (without dexamethasone).

[0144] To explore whether additional insulin and dexamethasone are required for the maturation steps of differentiation, EpiSC-PPARγ-CEBPα was differentiated in 2D using differentiation protocols D and F in the presence or absence of additional insulin and / or dexamethasone. Cells were collected on days 2, 7, and 13 during differentiation and analyzed under a bright-field microscope( Figure 7 A). The adipogenic performance was comparable between protocols D and F with insulin and dexamethasone and these protocols without insulin and / or dexamethasone. The ability to omit insulin and dexamethasone makes this model more promising in terms of cost reduction (process and medium), as several small molecules can be omitted for the regulatory acceptance of cultured meat.

[0145] Example 6: 3D culture of EpiSC-PPARγ-CEBPα

[0146] Suspension culture of cells in 3D is a necessary step to scale up to large volumes and produce the amount of adipocytes required for cultured meat products at a cost-competitive price (e.g., in an orbital shaker and / or bioreactor). Although suspension culture of cells in 3D allows cells to grow in an environment that more closely mimics the in vivo physiological environment of cells, a proper transition from 2D to 3D culture is required, and the differentiation data obtained from 2D experiments need to be validated in 3D.

[0147] Therefore, EpiSC-PPARγ-CEBPα was adapted to 3D suspension cell growth and grew as aggregates; according to the manufacturer's instructions, EpiSC-PPARγ-CEBPα amplified in a 6-well adherent cell plate was made into single cells with Accumax (00-4666-56, Thermo Fisher Scientific), and then transferred to 12.5 mL of medium and a RHO / ROCK pathway inhibitor in a 150 mL shake flask, and then amplified for at least 3 cycles. The medium was renewed daily during 3D amplification. Subsequently, 3D-adapted EpiSC-PPARγ-CEBPα was used for orbital shaker and bioreactor experiments.

[0148] To explore the adipogenic performance in 3D, 3 million cells / mL were seeded in a 250 mL shake flask containing 25 mL of medium, and EpiSC-PPARγ-CEBPα was differentiated using differentiation protocol D in the presence of additional insulin and dexamethasone. The medium was changed every other day. Cells were collected on day 6 during differentiation, stained with Oil Red O and DAPI, and visualized by fluorescence microscopy using the pancake method described previously( Figure 5) As previously described herein, analyze the relative gene expression of porcine PPARγ and CEBPα Opti-Ox-derived mRNA, as well as the mRNA of porcine endogenous PPARγ, CEBPα, adiponectin, LPL, perilipin-1, FABP4, CD36, and CEBPβ. The relative gene expression was not significantly different from that observed in cells grown in 2D culture (data not shown), confirming that adipocytes are suitable for growth and differentiation in 3D culture.

[0149] Example 7: EpiSC-PPARγ-CEBPα 3D suspension differentiation: Protocol optimization: Differentiation was performed using Protocol D and Protocol F in the presence of KSR + insulin + dexamethasone, KSR + dexamethasone (without additional insulin), and KSR + insulin (without dexamethasone).

[0150] To confirm that additional insulin and dexamethasone can also be omitted in 3D, EpiSC-PPARγ-CEBPα was differentiated in an orbital shaker and the setup of Example 5 was repeated in 3D. pEpiSC PPARγ-CEBPα was also subjected to 3D suspension differentiation according to Protocol D (mesoderm step + maturation step) and Protocol F (only maturation step) ( Figure 8 ). Intracellular triglycerides were also detected in the center of the aggregates, indicating sufficient nutrient and factor transport within the 3D structures (including large and small aggregates). Thus, pEpiSC PPARγ-CEBPα was also able to differentiate into adipocytes in the absence of additional insulin and dexamethasone in the maturation medium, again confirming the promise of this model in reducing costs (process and medium) as several small molecules can be omitted for regulatory acceptance of cultured meat.

[0151] Example 8: EpiSC-PPARγ-CEBPα 3D suspension differentiation in a bioreactor:

[0152] To explore whether pEpiSC PPARγ-CEBPα can grow and differentiate in a bioreactor, a fed-batch procedure was designed using an Ambr250 modular bioreactor system (Sartorius), in which 6x10 6Undifferentiated cells were seeded at 125,000 cells / mL in 120 mL of DMEM F / 12 medium (21041-025, Gibco), which was supplemented with KSR, BMP4, FGF2, activin A, Glutamax, and ascorbic acid at the same concentrations as described in previous experiments. The glucose concentration was maintained above 7 mM, the temperature was kept at physiological levels, and the stirring speed was sufficient to ensure that the aggregate size did not exceed the limits of nutrient transport. 7.5% sodium bicarbonate was added as needed to maintain the pH at 7.4 ± 0.5, and the dissolved oxygen set point was set between 30% - 70%. Additional insulin and doxycycline were added on days 0, 2, 4, and 6, while dexamethasone was added only once on day 2. Aggregates were collected on days 0, 3, 5, and 7 of differentiation for further analysis. The concentration of medium components was monitored every 24 hours to determine possible limitations (data not shown). Samples for neutral lipid quantification and gene expression analysis were obtained on days 0, 3, 5, and 7. The aggregate size was measured on days 0 and 6.

[0153] After 3 days of pEpiSC PPARγ-CEBPα differentiation, intracellular triglycerides were detected, and lipid accumulation increased continuously until the end of the experiment on day 7 of differentiation ( Figure 9 ). These results confirm that pEpiSC PPARγ-CEBPα can differentiate into adipocytes in a bioreactor, highlighting the potential of this model for scaling up the process.

[0154] Example 9: Differentiation of EpiSC-PPARγ-CEBPα in the absence of insulin

[0155] PPARγ-CEBPα pEpISC was seeded at 125,000 cells / cm2 in proliferation medium + 3.5 μM fasudil in 6-well plates pre-coated with Geltrex. The cells were evenly distributed in the wells and incubated overnight at 38.5 °C, 5% CO2 to ensure their attachment. The next day, adipocyte differentiation was induced by renewing the medium with maturation medium (Protocol F) supplemented with 0.5 μg / mL doxycycline and containing 20 μg / mL insulin (control, condition Figure 10 A), 10 μg / mL insulin (condition Figure 10 B), 5 μg / mL insulin (condition Figure 10 C), or in the absence of insulin (condition Figure 10 D). The maturation medium without insulin was also supplemented with 0.05 μg / mL IGF-1 (condition Figure 10 E) and 0.05 μg / mL IGF-1 / LR3 (condition Figure 10F). The maturation medium was changed every two days, and the cells were incubated at 38.5 °C and 5% CO2 during differentiation. Bright-field images were taken on day 8 of differentiation. These results confirmed that insulin was not required for efficient differentiation.

[0156] The following numbered statements (features) and examples of the methods and compositions disclosed herein are set forth. Each statement and example so defined of the invention disclosed herein may be combined with any other statement and / or example unless expressly stated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any one or more other features indicated as being preferred or advantageous.

[0157] Examples

[0158] The invention provides at least the following numbered statements / examples:

[0159] 1. A pluripotent stem cell comprising:

[0160] i) An expression construct for expressing a transcriptional regulatory protein inserted into a first genetic safe harbor locus;

[0161] ii) An expression construct for expressing a PPAR-γ protein, wherein the coding sequence of the PPAR-γ protein is operably linked to an inducible promoter; and

[0162] iii) An expression construct for expressing a CEBPα protein, wherein the coding sequence of the CEBPα protein is operably linked to an inducible promoter;

[0163] wherein these expression constructs of ii) and iii) are inserted into at least one additional genetic safe harbor locus that is not the first genetic safe harbor locus,

[0164] and wherein the inducible promoter is regulated by the transcriptional regulatory protein.

[0165] 2. The pluripotent stem cell according to Example 1, wherein these expression constructs of ii) and iii) are inserted into a second genetic safe harbor locus different from the first genetic safe harbor locus.

[0166] 3. The pluripotent stem cell according to Example 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.

[0167] 4. The pluripotent stem cell according to any one of Examples 1-3, wherein these pluripotent stem cells belong to a livestock or poultry species.

[0168] 5. The pluripotent stem cell according to Example 4, wherein these livestock species are pigs or cows, preferably pigs.

[0169] 6. The pluripotent stem cell according to any one of Embodiments 2-5, wherein the expression construct inserted into the second genetic safe harbor locus encodes a PPAR-γ protein, a linker, and a CEBPα protein, preferably wherein the linker is P2A, and more preferably wherein the linker comprises the sequence of SEQ ID NO:3.

[0170] 7. The pluripotent stem cell according to Embodiment 6, wherein the construct comprises the sequence of SEQ ID NO:4.

[0171] 8. The pluripotent stem cell according to any one of Embodiments 1-7, wherein the activity of the transcriptional regulatory protein is controlled by an exogenously supplied substance derivative.

[0172] 9. The pluripotent stem cell according to any one of Embodiments 1-8, wherein the transcriptional regulatory protein is selected from the group consisting of a tetracycline-responsive transcriptional activator protein (rtTa), a tetracycline repressor (TetR), a VgEcR synthetic receptor, or a chimeric transcriptional regulatory 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 the human NF-κB, preferably wherein the transcriptional regulatory protein is rtTA.

[0173] 10. The pluripotent stem cell according to any one of Embodiments 1-9, wherein the inducible promoter comprises a Tet-responsive element (TRE).

[0174] 11. The pluripotent stem cell according to any one of Embodiments 1-10, wherein the inducible promoter is a tetON promoter.

[0175] 12. The pluripotent stem cell according to any one of Embodiments 1-11, wherein the first and additional genomic safe harbor loci 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 loci are the hROSA26 locus and the AAVS1 locus.

[0176] 13. A method for producing adipocytes, preferably white adipocytes, the method comprising:

[0177] a) Culturing the pluripotent stem cell according to any one of the preceding claims in a proliferation medium: subsequently

[0178] b) Inducing adipocyte differentiation by adding the exogenous substance according to Claim 8, preferably wherein the proliferation and / or differentiation medium does not contain insulin and / or dexamethasone.

[0179] 14. The method according to embodiment 13, 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.

[0180] 15. The method according to embodiment 13 or 14, wherein the adipocytes produced are for human and non-human dietary consumption.

[0181] 16. Use of the pluripotent stem cells according to any one of embodiments 1-12 or use of the method for producing adipocytes according to any one of embodiments 13-15, the use being for tissue engineering, optionally for producing cultured meat.

[0182] 17. A food product comprising the pluripotent stem cells according to any one of embodiments 1-12 or adipocytes obtained by the method according to any one of claims 13-15.

[0183] 18. The food product according to embodiment 17, wherein the food product is cultured meat.

Claims

1. A pluripotent stem cell, comprising: i) An expression construct for expressing a transcriptional regulatory protein inserted into a first genetic safe harbor locus; ii) An expression construct for expressing a PPAR-γ protein, wherein the coding sequence of the PPAR-γ protein is operably linked to an inducible promoter; and iii) An expression construct for expressing a CEBPα protein, wherein the coding sequence of the CEBPα protein is operably linked to an inducible promoter; wherein these expression constructs of ii) and iii) are inserted into at least one additional genetic safe harbor locus that is not the first genetic safe harbor locus, and wherein the inducible promoter is regulated by the transcriptional regulatory protein.

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

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

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

5. The pluripotent stem cell according to claim 4, wherein these 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 locus encodes a PPAR-γ protein, a linker, and a CEBPα protein, preferably wherein the linker is P2A, more preferably wherein the linker comprises the sequence of SEQ ID NO:

3.

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

4.

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 substance derivative.

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 synthetic receptor, or a chimeric transcriptional regulatory protein comprising the DNA-binding domain from the yeast GAL4 protein, the truncated ligand-binding domain from the human progesterone receptor, and the activation domain from the human NF-kB, preferably the transcriptional regulatory protein is rtTA.

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

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

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

13. A method for generating adipocytes, preferably white adipocytes, the method comprising: a) culturing pluripotent stem cells according to any one of the preceding claims in a proliferation medium: subsequently b) inducing adipocyte differentiation by adding an exogenous substance according to claim 8, preferably the proliferation and / or differentiation medium does not contain insulin and / or dexamethasone.

14. The method according to claim 13, 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.

15. The method according to claim 13 or 14, wherein the generated adipocytes are used for human and non-human dietary consumption.

16. Use of pluripotent stem cells according to any one of claims 1-12 or use of a method for generating adipocytes according to any one of claims 13-15, the use being for tissue engineering, optionally for generating cultured meat.

17. A food product comprising pluripotent stem cells according to any one of claims 1-12 or adipocytes obtained by a method according to any one of claims 13-15.

18. The food product according to claim 17, wherein the food product is cultured meat.

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