Adipocyte maturation

Abstract

There remains a need in the art for the production and culture of mature adipocytes that are suitable for human consumption and can be produced in a scalable, cost-effective manner. The present invention relates to pluripotent stem cells comprising an expression construct for the expression of PPAR-γ protein and an expression construct for the expression of CEBPα protein. The present invention further provides a method for producing adipocytes comprising the pluripotent stem cells, and a food product comprising the adipocytes or the pluripotent stem cells.

Description

[Technical Field]

[0001] [Field of the Invention] The present invention relates to modified pluripotent cells and methods for differentiating said cells into adipocytes. [Background technology]

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

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

[0004] Additionally, to date, plant-based cultured meat substitutes have primarily focused on mimicking the muscle component of meat. However, fat is also an important component of meat, contributing to sensory / flavor, texture characteristics, 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] Engineered cell lines capable of differentiating into adipocytes were previously described by Tontonoz et al. (Cell, Vol. 79, pp. 1147-1156 - 30-12-1994), who used a retroviral expression system to coexpress PPARγ and CEBPα in a fibroblast cell line, where spontaneous differentiation into adipocytes was observed. U.S. Patent Application Publication No. 2012219530 describes lentivirally transduced human pluripotent cells with a differentiation efficiency of approximately 20%. However, these protocols have several limitations, including heterogeneous adipocyte maturity, lack of scalable processes, food safety issues, and long culture times of up to 28 days.

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

[0007] [Summary of the Invention] In a first aspect, the present invention relates to a pluripotent stem cell, the pluripotent stem cell comprising: i) an expression construct for expression of a transcriptional regulatory protein inserted into a first genetic safe harbor site; ii) an expression construct for the expression of 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 the expression of a CEBPα protein, wherein the coding sequence for the CEBPα protein is operably linked to an inducible promoter. Including, 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; Inducible promoters are regulated by transcriptional regulatory proteins.

[0008] In certain embodiments of the invention, the expression constructs of ii) and iii) are both inserted into a second genetic safe harbor site different from the first genetic safe harbor site. Preferably, said first and further genetic safe harbor sites are selected from any two of the hROSA26 locus, the AAVS1 locus, the CLYBL gene, or the CCR5 gene, and preferably the genetic safe harbor sites are the hROSA26 locus and the AAVS1 locus.

[0009] In certain embodiments of the invention, the cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines.

[0010] In a specific embodiment of the present invention, the pluripotent stem cells are pluripotent stem cells of a livestock or poultry species. Preferably, the livestock species is porcine or bovine, preferably porcine. The pluripotent stem cells may be, for example, from the genus Sus, such as the species S. domesticus.

[0011] In certain embodiments of the invention, the expression construct inserted into the second genetic safe harbor site encodes a PPAR-γ protein, a linker, and a CEBPα protein, preferably, the linker is P2A, and more preferably, the linker comprises the sequence of SEQ ID NO: 3. Preferably, the expression construct comprises the sequence of SEQ ID NO: 4.

[0012] In a specific embodiment of the present invention, the activity of the transcriptional regulatory protein is controlled by an exogenously supplied derivative. Preferably, the transcriptional regulatory protein is selected from the group consisting of tetracycline-responsive transcriptional activator protein (rtTA), tetracycline repressor (TetR), VgEcR synthetic receptor, or a hybrid transcriptional regulatory protein comprising a DNA-binding domain derived from yeast GAL4 protein, a truncated ligand-binding domain derived from human progesterone receptor, and an activation domain derived from human NF-kB; preferably, the transcriptional regulatory protein is rtTA.

[0013] In certain embodiments, the inducible promoter comprises a Tet-responsive element (TRE).

[0014] In certain embodiments, the inducible promoter is a tetON promoter.

[0015] In a second aspect, the present invention provides a method for producing adipocytes, preferably white adipocytes, comprising: a) culturing pluripotent stem cells according to any one of the preceding claims in a growth medium; followed by b) Inducing adipocyte differentiation by adding an exogenous substance as described herein.

[0016] In certain embodiments, the proliferation and / or differentiation medium is free or substantially free of 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 is free or substantially free of insulin. A proliferation and / or differentiation medium that is free or substantially free of insulin may optionally contain IGF-1 and / or LR3.

[0017] In certain embodiments, the differentiation stage of the methods described herein is for a maximum of 10 days, a maximum of 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, or 2 days.

[0018] In certain embodiments, the adipocytes produced are for human and non-human nutritional use.

[0019] In a further aspect, the invention provides for the use of the pluripotent stem cells described herein, or the methods for producing adipocytes described herein, for tissue engineering, and optionally for the production of cultivated meat.

[0020] 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 a particular embodiment, the food product is cultured meat. [Brief explanation of the drawings]

[0021] [Figure 1] Figure 1: Comparison of 2D differentiation of EpiSCs-PPARγ and EpiSCs-PPARγ-CEBPα. Undifferentiated (day 0) and differentiated (days 2, 4, 6, and 8) EpiSCs-PPARγ and EpiSCs-CEBPα-PPARγ. Brightfield microscope, 10x magnification. [Figure 2A]Figure 2: mRNA relative gene expression of porcine PPARγ and CEBPα 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) Oct4. EpiSCs-PPARγ and EpiSCs-CEBPα-PPARγ were differentiated in 2D for 8 days. [Figure 2B] Figure 2: mRNA relative gene expression of porcine PPARγ and CEBPα 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) Oct4. EpiSCs-PPARγ and EpiSCs-CEBPα-PPARγ were differentiated in 2D for 8 days. [Figure 2C] Figure 2: mRNA relative gene expression of porcine PPARγ and CEBPα 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) Oct4. EpiSCs-PPARγ and EpiSCs-CEBPα-PPARγ were differentiated in 2D for 8 days. [Figure 2D] Figure 2: mRNA relative gene expression of porcine PPARγ and CEBPα 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) Oct4. EpiSCs-PPARγ and EpiSCs-CEBPα-PPARγ were differentiated in 2D for 8 days. [Figure 2E]Figure 2: mRNA relative gene expression of porcine PPARγ and CEBPα 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) Oct4. EpiSCs-PPARγ and EpiSCs-CEBPα-PPARγ were differentiated in 2D for 8 days. [Figure 2F] Figure 2: mRNA relative gene expression of porcine PPARγ and CEBPα 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) Oct4. EpiSCs-PPARγ and EpiSCs-CEBPα-PPARγ were differentiated in 2D for 8 days. [Figure 2G] Figure 2: mRNA relative gene expression of porcine PPARγ and CEBPα 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) Oct4. EpiSCs-PPARγ and EpiSCs-CEBPα-PPARγ were differentiated in 2D for 8 days. [Figure 2H] Figure 2: mRNA relative gene expression of porcine PPARγ and CEBPα 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) Oct4. EpiSCs-PPARγ and EpiSCs-CEBPα-PPARγ were differentiated in 2D for 8 days. [Figure 2I]Figure 2: mRNA relative gene expression of porcine PPARγ and CEBPα 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) Oct4. EpiSCs-PPARγ and EpiSCs-CEBPα-PPARγ were differentiated in 2D for 8 days. [Figure 2J] Figure 2: mRNA relative gene expression of porcine PPARγ and CEBPα 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) Oct4. EpiSCs-PPARγ and EpiSCs-CEBPα-PPARγ were differentiated in 2D for 8 days. [Figure 2K] Figure 2: mRNA relative gene expression of porcine PPARγ and CEBPα 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) Oct4. EpiSCs-PPARγ and EpiSCs-CEBPα-PPARγ were differentiated in 2D for 8 days. [Figure 2L] Figure 2: mRNA relative gene expression of porcine PPARγ and CEBPα 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) Oct4. EpiSCs-PPARγ and EpiSCs-CEBPα-PPARγ were differentiated in 2D for 8 days. [Figure 3] Figure 3: Quantification of intracellular total triglycerides in undifferentiated EpiSCs-PPARγ and EpiSCs-PPARγ-CEBPα after 2, 4, 6, and 8 days of differentiation. [Figure 4] Figure 4: Comparison of 2D differentiation of EpiSCs-PPARγ and EpiSCs-PPARγ-CEBPα. Undifferentiated (day 0) and differentiated (days 7, 14, and 20). Fluorescence microscope, 10x magnification. Stained with Oil Red O (red, neutral lipids) and DAPI (blue, nuclei). [Figure 5] Figure 5: Comparison of 3D differentiation of EpiSCs-PPARγ and EpiSCs-PPARγ-CEBPα. EpiSCs-PPARγ and EpiSCs-PPARγ-CEBPα were differentiated in suspension (3D, day 6) stained with Oil Red O (red, neutral lipids) and DAPI (blue, nuclei). Fluorescence microscope, 10x magnification. [Figure 6A] Figure 6: EpiSCs PPARγ-CEBPα differentiated under Protocol D (mesoderm step + maturation step) or Protocol F (maturation step only) (days 4, 12, and 20). (A) Brightfield microscope, 40x magnification. (B) Quantification (absorbance) of intracellular Oil Red O staining extracted from EpiSCs CEBPα-PPARγ differentiated under Protocol D or Protocol F for 20 days. [Figure 6B] Figure 6: EpiSCs PPARγ-CEBPα differentiated under Protocol D (mesoderm step + maturation step) or Protocol F (maturation step only) (days 4, 12, and 20). (A) Brightfield microscope, 40x magnification. (B) Quantification (absorbance) of intracellular Oil Red O staining extracted from EpiSCs CEBPα-PPARγ differentiated under Protocol D or Protocol F for 20 days. [Figure 7] Figure 7: EpiSCs PPARγ-CEBPα differentiated under Protocol D (mesoderm step + maturation step) using maturation medium supplemented with (1) KSR, insulin, and dexamethasone, (2) KSR and dexamethasone, or (3) KSR and insulin; or under Protocol F (maturation step only) using maturation medium supplemented according to conditions (1), (2), and (3) (days 2, 7, and 13). Brightfield microscope, 20x magnification. [Figure 8]Figure 8: EpiSCs PPARγ-CEBPα differentiated in suspension (3D, day 13) under Protocol D (mesoderm step + maturation step) using maturation medium supplemented with (1) KSR, insulin, and dexamethasone, (2) KSR and dexamethasone, or (3) KSR and insulin; or under Protocol F (maturation step only) using maturation medium supplemented according to conditions (1), (2), and (3). Aggregates stained with Oil Red O (red, neutral lipids) and DAPI (blue, nuclei). Fluorescence microscope, 10x magnification. [Figure 9] Figure 9: EpiSCs PPARγ-CEBPα differentiated in suspension in a bioreactor under a fed-batch protocol similar to Protocol D (mesoderm step + maturation step) using medium supplemented with insulin and dexamethasone (3D, day 7). Aggregates were harvested on days 0, 3, 5, and 7 of differentiation, and total intracellular triglycerides were quantified. [Figure 10] Figure 10: EpiSCs PPARγ-CEBPα (day 7) differentiated under protocol F (maturation step only) using maturation medium supplemented with (A) 20 μg / mL, (B) 10 μg / mL, (C) 5 μg / mL insulin, or (D) maturation medium without insulin. Maturation medium was also supplemented with (E) 0.05 μg / mL IGF-1 or (F) 0.05 μg / mL IGF-1 / LR3 in the absence of insulin. Brightfield microscope, 20x magnification. DETAILED DESCRIPTION OF THE INVENTION

[0022] [Description of the Invention] [Definition] Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is not limited to such methods in any way.

[0023] In this specification and its claims, the verb "comprise" and its conjugations are used in their open-ended sense so that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that a plurality of elements may be present, unless the context clearly requires the presence of only one element. Thus, the indefinite article "a" or "an" typically means "at least one."

[0024] As used herein, the term "and / or" indicates that one or more of the specified instances may occur alone or in combination with at least one and up to all of the specified instances.

[0025] As used herein, "at least" a particular value means greater than or equal to the particular value. For example, "at least 2" is understood to be the same as "2 or more," i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ... etc.

[0026] The terms "about" or "approximately," when used in connection with a numerical value (e.g., about 10), preferably mean that the value can be a given value from minus 0.1% to plus 0.1% (of 10) of the value.

[0027] The term "heterologous," when used with reference to a nucleic acid (DNA or RNA) or protein, refers to a nucleic acid or protein that is not naturally occurring as part of the organism, cell, genome, or DNA or RNA sequence in which it is found, or to a nucleic acid or protein that is found in a cell, or location within the genome, DNA, or RNA sequence, different from that in which it is found in nature. A heterologous nucleic acid or protein is not endogenous to the cell into which it is introduced, but is obtained from another cell, or is synthetically or recombinantly produced. Generally, although not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly, exogenous RNA encodes proteins that are not normally expressed in the cell in which the exogenous RNA is present. Heterologous nucleic acids and proteins are sometimes referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is encompassed herein by the term heterologous nucleic acid or protein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e., combinations in which at least two linked sequences are foreign to each other.

[0028] The term "expression vector" or "expression construct" refers to a nucleotide sequence capable of effecting the expression of a gene in a host cell or host organism compatible with such sequence. These expression vectors typically contain at least appropriate transcription regulatory sequences and, optionally, a 3' transcription termination signal. Additional factors necessary or helpful for expression may also be present, for example, expression enhancer elements.

[0029] As used herein, the term "operably linked" refers to the linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it effects transcription of the coding sequence. Operably linked means that the linked DNA sequences are typically contiguous, and, where necessary, join two adjacent protein-coding regions in reading frame. Inducible promoters

[0030] As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control transcription of one or more coding sequences, is located upstream in the direction of transcription from the transcription start site of that coding sequence, and is structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription initiation site, and other DNA sequences (including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and other sequences of nucleotides known to those skilled in the art to act directly or indirectly to regulate the amount of transcription from the promoter). A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, for example, by application of a chemical inducer. In the present invention, regulation is exerted by a transcriptional regulatory protein.

[0031] References herein to nucleotide or amino acid sequences accessible in public sequence databases refer to the versions of the sequence entries available on the filing date of this application.

[0032] All patent and literature references cited herein are hereby incorporated by reference in their entirety.

[0033] The present inventors have surprisingly found that by using modified pluripotent stem cells containing an expression construct for the expression of PPAR-γ protein and an expression construct for the expression of CEBPα protein, the time period required for the differentiation of pluripotent cells into mature adipocytes can be dramatically reduced. As shown in the Examples described herein, by using these modified pluripotent cell lines, complete differentiation into mature adipocytes can be achieved in less than 10 days. Aside from significantly reducing culture time and associated costs, the use of the pluripotent cells described herein also provides for more reliable and scalable production of mature adipocytes compared to those previously described.

[0034] Thus, in a first aspect, the present invention relates to a pluripotent stem cell, which pluripotent stem cell comprises: i) an expression construct for expression of a transcriptional regulatory protein inserted into a first genetic safe harbor site; ii) an expression construct for the expression of a PPAR-γ protein, wherein the coding sequence for the PPAR-γ protein is operably linked to an inducible promoter; and iii) an expression construct for expression of a CEBPα protein, wherein the coding sequence for the CEBPα protein is operably linked to an inducible promoter; the expression constructs of ii) and iii) are inserted into at least one additional genetic safe harbor site that is not the first genetic safe harbor site; And, the inducible promoter is regulated by a transcriptional regulatory protein.

[0035] 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 primarily found 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 (found primarily in adipose tissue and intestine). In certain embodiments, the PPAR-γ coding sequence of the present invention encodes PPAR-γ2. PPARG regulates fatty acid storage and glucose metabolism. Genes activated by PPARG stimulate lipid uptake and adipogenesis by adipocytes. PPARG knockout mice lack adipose tissue, establishing PPARG as a master regulator of adipocyte differentiation. In certain embodiments, the PPAR-γ coding sequence has the sequence of SEQ ID NO: 1.

[0036] CCAAT / enhancer-binding protein alpha (CEBPα) is a protein encoded by the human CEBPα gene. The protein encoded by this intronless gene is a bZIP transcription factor that can bind to specific promoters and gene enhancers as a homodimer. It can also form heterodimers with different transcription factors, such as the related proteins CEBP-β and CEBP-γ, and c-Jun. The encoded protein is a key regulator of adipogenesis (the process of forming new fat cells) and the accumulation of lipids in those cells, as well as glucose and lipid metabolism in the liver. In a specific embodiment, the coding sequence for CEBPα has the sequence of SEQ ID NO:2.

[0037] In certain embodiments, nucleic acid molecules encoding proteins according to the present invention are codon-optimized for expression in mammalian cells. Methods for codon optimization are known and have been described (e.g., for mammalian cells, see WO 96 / 09378). A sequence is considered codon-optimized if at least one non-preferred codon has been replaced with a more preferred codon compared to the wild-type sequence. As used herein, a non-preferred codon is a codon that is used less frequently in an organism than another codon encoding the same amino acid, and a more preferred codon is a codon that is used more frequently in an organism than a non-preferred codon. The codon usage frequency for a particular organism can be found in a codon frequency table, such as http: / / www.kazusa.or.jp / codon. Preferably, two or more non-preferred codons, preferably most or all non-preferred codons, are replaced with more preferred codons. Preferably, the codon most frequently used in the organism is used in the codon-optimized sequence. Replacement with a preferred codon generally results in higher expression.

[0038] Transcriptional regulatory proteins are proteins that bind in a sequence-specific manner to DNA, preferably to DNA sites located within or near a promoter, and promote the binding of the transcription machinery to the promoter, thus either promoting transcription of the DNA sequence (transcriptional activators) or blocking this process (transcriptional repressors). Such entities are also known as transcription factors.

[0039] DNA sequences to which transcriptional regulatory proteins bind are called transcription factor binding sites or response elements, and they are found within or near the promoter of the regulated DNA sequence.

[0040] Transcriptional activator proteins bind to response elements and promote gene expression. Such proteins are preferred in the methods of the present invention for controlling inducible cassette expression.

[0041] A genetic safe harbor (GSH) site is a locus within the genome where genes or other genetic material can be inserted without any deleterious effects on the cell or the inserted genetic material. The most beneficial GSH sites are those where expression of the inserted genetic sequence is not disrupted by any read-through expression from adjacent genes and where expression of the inducible cassette minimizes interference with the endogenous transcription program. More formal criteria have been proposed to help determine whether a particular locus is a GSH site in the future (Papapetrou et al., 2011, Nature Biotechnology, 29(1), 73-8. doi:10.1038 / nbt.1717). These criteria include: (i) more than 50 kb from the 5' end of any gene; (ii) more than 300 kb from any gene associated with cancer; (iii) more than 300 kb from any microRNA (miRNA); (iv) located outside of a transcription unit; and (v) located outside of an ultraconserved region (UCR). It is not necessary to meet all of these proposed criteria, as previously identified GSHs do not meet all criteria. A suitable GSH would likely meet at least two, three, four, or all of these criteria.

[0042] In certain embodiments of the invention, the first and additional genomic safe harbor sites are selected from any two of the hROSA26 locus, the AAVS1 locus, the CLYBL gene, or the CCR5 gene. In certain embodiments, the first and additional genomic safe harbor sites are located at chr1:152,360,840-152,360,859, chr1:175,942,362-175,942,381, chr1:231,999,396-231,999,415, chr2:45,708,354-45,708,373; chr8:68,720,172-68,720,191 of the human genome.

[0043] In certain embodiments of the invention, the first and further genomic safe harbor sites are selected from any two of the bovine safe harbor sites ROSA26, AAVS1, the CLYBL gene, and the CCR5 gene.

[0044] Preferably, the genetic safe harbor sites are the hROSA26 locus and the AAVS1 locus.

[0045] In certain embodiments of the invention, an expression construct for expression of a PPAR-γ protein described herein and an expression construct for expression of a CEBPα protein described herein are both inserted into a second genetic safe harbor site that is different from the first genetic safe harbor site. In certain embodiments, the expression construct inserted into the second genetic safe harbor site is capable of simultaneously expressing both a PPAR-γ protein and a CEBPα protein.

[0046] As used herein, the term "pluripotent stem cells" includes embryonic stem cells, embryo-derived stem cells, induced pluripotent stem cells, and somatic cells, regardless of the method by which the pluripotent stem cells are derived. Thus, in certain embodiments, the pluripotent stem cells are selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines. In certain embodiments, the pluripotent stem cells are epiblast-derived stem cells (EpiSCs). In certain embodiments, the pluripotent stem cells express one or more markers selected from the group consisting of OCT-4, Sox2, Klf4, c-MYC, Nanog, Lin28, alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Exemplary pluripotent stem cells can be generated using methods known in the art. "Induced pluripotent stem cells" (iPS cells or iPSCs) can be generated by protein introduction of reprogramming factors into somatic cells.

[0047] Pluripotent stem cells according to the present invention can be derived from any species. Embryonic stem cells have been successfully derived in, for example, mice, several species of non-human primates, and humans, and embryonic stem-like cells have been generated from numerous additional species. Thus, those skilled in the art can generate embryonic stem cells and embryo-derived stem cells from any species, including, but not limited to, humans, non-human primates, rodents (mice, rats), ungulates (such as cows and sheep), dogs (domestic and wild), cats (domestic and wild cats such as lions, tigers, and cheetahs), rabbits, hamsters, gerbils, squirrels, guinea pigs, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, and marine mammals (such as dolphins and whales).

[0048] Similarly, iPS cells can be derived from any species. These iPS cells have been successfully generated using mouse and human cells. Furthermore, iPS cells have been successfully generated using embryonic, fetal, neonatal, and adult tissues. Therefore, iPS cells can be readily generated using donor cells from any species. Thus, iPS cells can be generated from any species, including, but not limited to, humans, non-human primates, rodents (mice, rats), ungulates (e.g., cows, sheep), dogs (domestic and wild), cats (domestic and wild, such as lions, tigers, and cheetahs), rabbits, hamsters, goats, elephants, pandas (including giant pandas), pigs, raccoons, horses, zebras, and marine mammals (e.g., dolphins and whales).

[0049] In certain embodiments, the pluripotent stem cells according to the invention or for use in the invention are animal cells, hi certain embodiments, the pluripotent stem cells according to the invention or for use in the invention are derived from food animal species.

[0050] Preferably, the pluripotent stem cells according to or for use in the present invention are derived from livestock or poultry animals, including but not limited to domestic cattle, pigs, sheep, goats, lambs, camels, buffalo, and rabbits.

[0051] Preferably, the pluripotent stem cells according to the present invention or for use in the present invention are porcine or bovine pluripotent stem cells. Most preferably, they are porcine pluripotent stem cells. In a specific embodiment, the stem cells according to the present invention are porcine epiblast stem cells (pEpiSCs).

[0052] Poultry species include, but are not limited to, domestic chickens, turkeys, ducks, geese, and pigeons. In certain embodiments, the cells are derived from common game birds such as wild deer, domestic chickens, waterfowl, and hares. Preferably, the pluripotent stem cells according to or for use in the present invention are not human cells.

[0053] Transcriptional repressor proteins bind to response elements and prevent gene expression.

[0054] Transcriptional regulatory proteins can be activated or inactivated by several mechanisms, including binding of substances, interaction with other transcription factors (e.g., homodimerization or heterodimerization) or coregulatory proteins, phosphorylation, and / or methylation. Transcriptional regulatory factors can be controlled by activation or inactivation.

[0055] When the transcriptional regulatory protein is a transcriptional activator protein, it is preferred that the transcriptional activator protein requires activation. This activation can be achieved by any suitable means, but it is preferred that the transcriptional regulatory protein is activated by adding an exogenous substance to the cell. The supply of the exogenous substance to the cell can be controlled, and thus the activation of the transcriptional regulatory protein can be controlled. Alternatively, the exogenous substance can be supplied to inactivate the transcriptional regulatory protein, and then the supply can be stopped to activate the transcriptional regulatory protein.

[0056] When the transcriptional regulatory protein is a transcriptional repressor protein, it is preferred that the transcriptional repressor protein requires inactivation, and therefore a substance is provided to prevent the transcriptional repressor protein from repressing transcription, thus allowing transcription to occur.

[0057] Any suitable transcriptional regulatory protein can be used, preferably an activatable or inactivatable transcriptional regulatory protein. Preferably, the transcriptional regulatory protein is obtained by supplying an exogenous substance to control the transcriptional regulatory protein. Such transcriptional regulatory proteins are also called inducible transcriptional regulatory proteins.

[0058] Thus, in certain embodiments, pluripotent stem cells according to the present invention are regulated by exogenously supplied substances.

[0059] In certain embodiments, the exogenously supplied substance is selected from the group consisting of a peptide (e.g., those described in Klotzsche, et al., Journal of Biological Chemistry 280.26 (2005): 24591-24599 or Schlicht et al., Applied and environmental microbiology 72.8 (2006): 5637-5642, or the inducer described in Goeke, et al., Journal of molecular biology 416.1 (2012): 33-45, which are incorporated herein by reference), an aptamer (e.g., the RNA aptamer described in Hunsicker et al., "Chemistry & biology" 16.2 (2009): 173-180, which are incorporated herein by reference), tetracycline, and anhydroteracycline or a derivative thereof. Preferably, the exogenously supplied substance is doxycycline.

[0060] In certain embodiments, the transcriptional regulatory proteins described herein are selected from the group consisting of tetracycline-responsive transcriptional activator protein (rtTa), tetracycline repressor (TetR), VgEcR synthetic receptor, or hybrid transcriptional regulatory proteins comprising a DNA-binding domain from yeast GAL4 protein, a truncated ligand-binding domain from the human progesterone receptor, or an activation domain from human NF-kB.

[0061] Tetracycline-controlled transcriptional activation is a method of inducible gene expression well known in the art, in which transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g., the more stable doxycycline). In this system, the transcriptional activator protein is the tetracycline-responsive transcriptional activator protein (rtTA) or its derivatives. The rtTA protein can bind to DNA at specific TetO operator sequences. Several repeats of such TetO sequences are placed upstream of a minimal promoter (such as the CMV promoter), and together they form a tetracycline response element (TRE). This system has two forms, depending on whether the addition of tetracycline or a derivative activates (Tet-On) or inactivates (Tet-Off) the rTA protein.

[0062] In the Tet-Off system, tetracycline or its derivatives bind to rTA, inactivating it and rendering it unable to bind to the TRE sequence, thereby preventing transcription of the TRE-controlled gene. The Tet-On system consists of two components: (1) a constitutively expressed tetracycline-responsive transcription activator protein (rtTa) and an rtTa-sensitive inducible promoter (Tet Responsive Element, TRE). This can be bound by tetracycline or its more stable derivatives (including doxycycline (dox)), causing activation of rtTa, allowing it to bind to the TRE sequence and inducing expression of the TRE-controlled gene. In a preferred embodiment of the present invention, the transcriptional regulatory protein is rtTA.

[0063] When the transcriptional regulatory protein is rtTA, the inducible promoter inserted into at least one additional GSH site contains a tetracycline-responsive element (TRE) rather than the first GSH site. Thus, in certain embodiments, the inducible promoter contains a Tet-Responsive Element (TRE).

[0064] In some embodiments, the transcriptional regulatory protein is rtTA and, when it contains a TRE, the exogenously supplied substance is the antibiotic tetracycline or one of its derivatives.

[0065] In certain embodiments of the invention, the expression construct inserted into the second genetic safe harbor site is a fusion protein encoding both a PPAR-γ protein and a CEBPα protein as described herein. In certain embodiments, the expression construct inserted into the second genetic safe harbor site encodes a PPAR-γ protein, a linker, and a CEBPα protein, and in preferred embodiments, the construct comprises or consists of SEQ ID NO:4.

[0066] In certain embodiments, the linker sequence may be a cleavable linker. That is, the linker sequence may include a sequence of amino acids that can be cleaved. For example, the linker sequence may include a sequence that can act as a substrate for an enzyme capable of cleaving a peptide bond, i.e., a cleavage site. Many such cleavage sites are known to and available to those skilled in the art of molecular biology. In some embodiments, the cleavable linker may include a self-cleavage site. A self-cleavage site is automatically cleaved without the need for enzymatic treatment. For example, a family of 2A self-cleaving peptides, or 2A peptides, has been described, including the 2A peptides P2A, E2A, F2A, and T2A. F2A is derived from foot-and-mouth disease virus; E2A is derived from equine rhinitis A virus; P2A is derived from porcine teschovirus-1 2A; and T2A is derived from thosea asigna virus 2A. In certain embodiments, the cleavable linker is therefore selected from the group consisting of P2A, E2A, F2A, and T2A.

[0067] 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.

[0068] In certain embodiments, the expression construct inserted into the second genetic safe harbor site encoding the PPAR-γ protein, the linker, and the CEBPα protein described herein comprises or consists of the sequence of SEQ ID NO:4.

[0069] 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 chain or light chain, etc.

[0070] In a particular embodiment, the inducible promoter used in the present invention is the tetOn promoter. The third generation TetOn promoter is preferred.

[0071] [Culture method] The inventors of the present application have surprisingly found that the differentiation time required to obtain adipocytes can be dramatically reduced by using the pluripotent cells described herein. Thus, in a further aspect, the present invention relates to a method for producing adipocytes, the method comprising: a) culturing the pluripotent stem cells described herein in a growth medium; followed by b) Inducing adipocyte differentiation by adding an exogenous substance as described herein.

[0072] In certain embodiments, the methods of the present invention are ex vivo methods.

[0073] In certain embodiments, the method is for the production of mature adipocytes, defined herein as adipocytes that exhibit lipid accumulation and / or express detectable levels of PPARy, FABP4, PLIN1, and adiponectin.

[0074] In a specific embodiment, the method of the present invention relates to a method for producing white adipocytes. In a specific embodiment, the proliferation and / or differentiation medium does not contain or is substantially free of 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 is substantially free of insulin.

[0075] The insulin-free or substantially insulin-free growth and / or differentiation medium may optionally contain IGF-1 and / or LR3.

[0076] The proliferation and / or differentiation medium may comprise up to about 20 μg / mL insulin, such as up to about 10 μg / mL insulin, such as about 5 μg / mL insulin, for example 1 μg / mL insulin.

[0077] The inventors have surprisingly found that the use of the pluripotent cells described herein obviates the need to culture cells through a commitment induction step. Typically, when culturing adipocytes, several culture stages can be distinguished. The commitment or decision phase involves the formation of preadipocytes, which have lost the ability to differentiate into other cell types. Differentiation of preadipocytes into adipocytes is driven by a highly regulated network of transcription factors whose sequential expression promotes morphological and biochemical characteristics of adipocytes, such as insulin responsiveness, lipid transport and synthesis, and secretory capacity. Differentiation stages are 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 differentiation of pluripotent stem cells into mature adipocytes without the need for a commitment induction step and without forcing overexpression. The ability of cells to skip this commitment induction step is particularly advantageous because it reduces the amount of compounds and small molecules that would normally need to be present in the differentiation medium. For example, it has been found that the cells described herein can differentiate in the absence of BMP-4, activin A, and FGF2, which are normally required for full differentiation. These compounds and small molecules can be omitted from the differentiation medium, reducing the cost of the medium and facilitating regulatory acceptance. The terms differentiation phase and differentiation stage are used interchangeably herein.

[0078] Thus, in certain embodiments, the methods described herein do not include an additional participation induction step.

[0079] The methods described herein dramatically reduce the time to differentiate the pluripotent cells described herein into mature adipocytes. In certain embodiments, the time to generate mature adipocytes using the claimed methods is up to 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, or 2 days. Using the pluripotent cells described in the described methods, the inventors observed a conversion rate of at least 95% by the fourth day of culture, meaning that at least 95% of the cells are mature after four days of culture. Thus, in certain embodiments, the time to generate at least 95% mature adipocytes is up to four days.

[0080] In a further aspect, the present invention provides an adipocyte, preferably a mature adipocyte, obtainable by the methods described herein.

[0081] The cell culture described herein can be performed under so-called 2D culture conditions, which are considered the conventional approach to culturing cells. However, the described methods can also be easily adapted to allow culturing under 3D conditions, as shown in the examples below.

[0082] 3D cell culture is an artificially created environment that allows cells to grow and interact with their surroundings in three dimensions. In such cultures, cells typically form 3D colonies, sometimes called "spheroids." 3D culture approaches can more accurately model in vivo cell growth and behavior. Those skilled in the art can easily perform 3D cell culture, for example, by utilizing any of several commercially available culture tools. For example, 3D culture can be performed using scaffolding or scaffold-free techniques. Scaffold-based techniques utilize supports such as solid scaffolds and hydrogels to culture cells in 3D. Such scaffolds can be designed to mimic the natural extracellular matrix (ECM) present in vivo. Scaffold-free techniques do not require the use of a scaffold for cell growth. Instead, 3D spheroids can be established using, for example, low-attachment plates, hanging drop plates, micropatterned surfaces, rotating bioreactors, magnetic levitation, and magnetic 3D bioprinting.

[0083] Cells transduced with lentiviral vectors are considered unsafe for food use and unsafe for human and non-human nutrition. The pluripotent cells described herein of the methods described herein obviate the need to use lentivirally transduced cells. Thus, in certain embodiments, adipocytes produced according to the methods disclosed herein are for human and non-human nutrition. In certain embodiments, the produced adipocytes can be used in the production of cultivated meat for human consumption.

[0084] 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, hi certain embodiments, the methods described herein are for ex vivo or in vivo tissue engineering.

[0085] In a particular aspect, the use is for the production of cultivated meat, i.e., the present invention provides the use of pluripotent stem cells as described herein, or adipocytes obtained by the methods described herein, in the production of cultivated meat.

[0086] In yet a further aspect, the present invention provides food products (also referred to as "foodstuffs") comprising the pluripotent stem cells described herein or adipocytes generated and / or obtained by the methods described. In certain embodiments, the food product is or further comprises an edible composition for human or non-human consumption. For example, an edible composition for human or non-human consumption comprises at least one of muscle cells, mature muscle cells, minerals, synthetic substances, flavoring substances (e.g., herbs and spices), plant-based proteins, or proteins of microbial origin, such as yeast proteins. Plant-based proteins and yeast proteins suitable for use in foods are known to those of skill in the art. In certain embodiments, the food product is cultured meat or a cultured meat product.

[0087] In yet a further aspect, the invention provides a method of producing a food product, the method comprising combining the pluripotent stem cells described or produced herein and / or the resulting adipocytes with an edible composition for human or non-human consumption described herein. In certain embodiments, the food product is cultured meat.

[0088] [Array Description]

[0089] [Table 1]

[0090] [Example] This invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

[0091] Materials and Methods [Differentiation of porcine epiblast-derived stem cells (pEpISCs) into adipocytes] Undifferentiated pEpISCs (Opti-Ox PPARγ and Opti-Ox CEBPα-PPARγ) were cultured in N2B27 growth medium (50% DMEM Ham's) on hESC-qualified geltrex (A1413301, Thermo Scientific)-coated plates. Cells were grown in F-12 (L0093-500, Biowest), 50% Neurobasal medium (21103049, Thermofisher), B27 supplement (17504044, Thermofisher), N2 supplement (17502001, Thermofisher), glutamax (35050061, Thermofisher), 10 mM 2-mercaptoethanol (31350010, Thermofisher), 0.02 μg / mL activin A (QK001, Q-kine), 0.10 μg / mL FGF2 (QK002, Q-kine), and 0.625 μg / mL XAV939 (X3004, Sigma-Aldrich). For 2D adipocyte differentiation, gentle cell dissociation reagent (07174, STEMCELL After treatment with the EpISC technology, single cells were obtained and assessed for cell number and viability. Single pEpISC cells were cultured at 50,000–150,000 cells / cm. 2The cells were seeded onto corresponding cell culture plates at a density of 10 μM. After overnight incubation in growth medium containing 10 μM Rockland-related 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 the stem cell commitment to the adipocyte lineage. On day 2, the medium was replaced with adipocyte maturation medium consisting of DMEM Ham's F-12 (L0093-500, Biowest) containing 15% knockout serum replacement (10828-028, Thermofisher), 1 μg / mL insulin (12585014, Thermofisher), and 1 μM dexamethasone (D1756, Sigma-Aldrich). pEpISCs were differentiated for the indicated days. 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.

[0092] For spheroid or aggregate differentiation experiments, undifferentiated single pEpISCs (Opti-Ox PPARγ and Opti-Ox CEBPα-PPARγ) were cultured in 25 mL of growth medium (50% DMEM Ham's F-12 (L0093-500, Biowest), 50% Neurobasal medium (21103049, Thermofisher), B27 supplement (17504044, Thermofisher), N2 supplement (17502001, Thermofisher), glutamax (35050061, Thermofisher), 10 mM MgCl ). Cells were seeded at 3 million cells / mL in a 250 mL shake flask containing 2-mercaptoethanol (31350010, Thermofisher), 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, Thermofisher), and 10 ng / mL X FGF2. The next day, once small spheroids had formed, the medium was replaced with supplemented StemPro-34 SFM. Similarly, maturation medium was added on day 2 of differentiation and refreshed every 2 days until the end of the experiment.

[0093] [Oil Red O staining] Undifferentiated and differentiated pEpISCs Opti-Ox PPARγ and pEpISCs Opti-Ox CEBPα-PPARγ were stained with Oil Red O (O0625, Sigma) as previously described. Briefly, cells in culture or in aggregates were washed with phosphate-buffered saline (PBS) and fixed with paraformaldehyde (PFA) for 40 minutes at room temperature. After three washes with PBS and permeabilization with 60% isopropanol, cells were stained with Oil Red O (0.5% in isopropanol) diluted to 40% in water for 30 minutes at room temperature. pEpISCs were then washed several times with deionized water until only lipid droplets were properly stained with the dye.

[0094] Undifferentiated and differentiated cells were photographed under an inverted bright-field microscope (Oxion Inverso, Euromex) or a fluorescence microscope (EVOS M7000, Thermo Fisher Scientific). The triglyceride content of pEpISCs Opti-Ox PPARγ and pEpISCs Opti-Ox CEBPα-PPARγ was estimated at various checkpoints during adipogenesis by extracting Oil Red O dye from cells with 100% isopropanol and measuring the resulting solution spectrophotometrically at 540 mM (Glomax Discover, Promega). Oil Red O data were adjusted by estimating cell number measured spectrophotometrically with HOESCH 33342 staining (H1399, Thermo Fisher Scientific) according to the manufacturer's instructions.

[0095] pEpISCs Opti-Ox PPARγ and pEpISCs Opti-Ox CEBPα-PPARγ differentiated in aggregates or spheroids were stained according to the same protocol described above. Image acquisition followed two different approaches. To quickly confirm the quality of adipocytes, stained aggregates were transferred to slides. After removing excess buffer, the slides were coverslipped using hard set mounting medium (P36984, Thermo Fisher Scientific). This process flattens the spheroid aggregates, providing an overview of the presence of lipids and an estimate of their distribution. For more precise analysis, unstained and fixed cell aggregates were cryopreserved in sucrose solution. The frozen 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 lipids).

[0096] [Triglyceride content] Intracellular triglyceride content was quantified from undifferentiated and differentiated cultures / aggregates of pEpISCs Opti-Ox PPARγ and pEpISCs Opti-Ox CEBPα-PPARγ using a triglyceride quantification colorimetric / fluorimetric assay kit (MAK266-1KT, Sigma-Aldrich) according to the manufacturer's instructions. Briefly, total lipids were extracted with a 5% Nonidet P40 substitute (11754599001, Sigma-Aldrich) solution in deionized water under heating. The addition of lipase liberated glycerol from triglycerides, which subsequently reacted to produce a color that could be measured spectrophotometrically at 570 nm (Glomax Discover, Promega). TG concentrations were calculated based on a standard curve constructed from TG standards and normalized to total cellular protein content.

[0097] [Real-time quantitative PCR analysis (RT-qPCR)] Total RNA from undifferentiated and differentiated pEpISCs Opti-Ox PPARγ and pEpISCs Opti-Ox CEBPα-PPARγ was extracted using the Reliaprep Cell Miniprep System (Z6012, Promega) according to the manufacturer's instructions. RNA concentration and quality were measured using a microspectrophotometer DS-11 (DeNovix). Five hundred nanograms of purified total RNA from all samples was first treated with DNase I to eliminate potential genomic DNA contamination and then reverse-transcribed into cDNA using the iScript gDNA Clear cDNA Synthesis Kit (1725035BUN, BioRad). Specific primers for porcine pluripotent and mature adipocyte markers were designed for real-time quantitative PCR analysis (Table I). At least three samples from two independent experiments were amplified in triplicate using the PowerTrack SYBR Green Master Mix (A46112, Thermofisher) in a thermocycler system according to the manufacturer's guidelines. The RT-qPCR conditions were 95°C for 30 seconds, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The YWHAZ gene was used as a housekeeping gene to normalize the expression levels of the target genes. Relative gene quantification was calculated by the 2-ΔΔCt method.

[0098] [result] Example 1: Development of an inducible transgene overexpression method by dual GSH targeting in animal cells. To explore the potential of OPTi-OX for forward programming of porcine pPSCs (pPSCs), we generated PPARG OPTi-OX pPSCs. We sequentially targeted an rtTA cassette to the porcine ROSA26 GSH under the control of the CAG promoter, followed by a PPARG transgene to the porcine AAVS1 GSH under the control of a doxycycline-inducible element, and observed robust and homogeneous inducible transgene expression. Induction of PPARG expression after doxycycline treatment resulted in lipid accumulation in the cells. These results demonstrated that overexpression of PPARG alone was sufficient to promote adipogenesis in pPSCs. However, the PPARG lipid accumulation and cell morphology of pEpiSCs were far from comparable to those of porcine mature adipocytes, and some specific late adipogenic markers, such as PLIN1 or adiponectin, were not expressed even after 21 days of differentiation.

[0099] After systematic screening for adipogenic factors by regulating key signaling cascades involved in adipogenesis, we selected PPARg and CEBPa for use in a combinatorial cell reprogramming strategy. To simultaneously express PPARg and CEBPa upon doxycycline induction, we designed a knock-in containing PPARg, a P2A "self-cleaving" peptide linker, and CEBPa all in a single open reading frame. The P2A peptide linker is a well-characterized short peptide linker of 18-22 amino acids that results in two separate gene products expressed from a single open reading frame by ribosomal skipping during translation. Stable knock-in of AAVS1 GSH was selected by the integration of a puromycin resistance cassette and the addition of puromycin to the cell culture medium. After selection, single pEpiSC cells were plated, and clonal cell lines were isolated for expansion and analysis. Doxycycline-inducible PPARg-P2A-CEBPa incorporation was subsequently confirmed using PCR genomic analysis, Sanger sequencing, and RT-qPCR. Using a dual GSH targeting approach, we selected clonal lines carrying two copies of each transgene and observed that homozygous targeting of both elements allowed for inducible overexpression (data not shown). Importantly, the dual GSH targeting approach did not affect SC self-renewal or differentiation as determined by RT-qPCR (data not shown).

[0100] Example 2: 2D short-term differentiation of EpiSCs-PPARγ-CEBPα into adipocytes pEpISCs Opti-Ox PPARγ and pEpISCs Opti-Ox PPARγ-CEBPα were differentiated with doxycycline for up to 8 days. Photographs were taken using a bright-field microscope at undifferentiated cells (day 0) and during differentiation (days 2, 4, 6, and 8). As can be seen in Figure 1, pEpISCs containing Opti-Ox-PPARγ-CEBPα generated larger lipid droplets during differentiation than pEpISCs containing Opti-Ox-PPARγ-CEBPα, indicating greater cellular maturity and lipid accumulation. This difference was already apparent as early as day 4 after the initiation of differentiation, indicating significantly faster differentiation of pEpISCs containing Opti-Ox PPARγ-CEBPα.

[0101] Using RT-qPCR, we evaluated the relative mRNA gene expression of porcine endogenous PPARγ, CEBPα, adiponectin, LPL, perilipin-1, FABP4, CD36, and CEBPδ. The results are shown in Figure 2. As expected, pEpiSC PPARγ-CEBPα cells expressed Opti-Ox CEBPα and Opti-Ox PPARγ, confirming the stably transgene-expressing ability of the Opti-Ox system even after 2 days of differentiation, confirming the usefulness of the cell model. pEpiSC PPARγ-CEBPα cells also showed higher expression of several adipocyte markers (LPL and CD36, perilipin-1 (indicating lipid accumulation)), CEBPδ and ZBTB16 (late adipogenesis markers), adiponectin, and FABP4 (a marker of terminal cell differentiation). Adiponectin and perilipin-1 were not expressed in pEpiSC PPARγ. pEpiSC PPARγ-CEBPα demonstrates a higher differentiation potential. Additionally, we observed that Oct4 expression was induced in pEpiSC PPARγ-CEBPα on day 2, an effect not observed in pEpiSC PPARγ. Oct4 is known to be a key regulator of cell commitment to the adipocyte lineage, indicating that pEpiSC PPARγ-CEBPα does not require a commitment step during differentiation, allowing for more rapid differentiation.

[0102] Next, we quantified the total triglyceride content from undifferentiated and differentiated cells (Figure 3) using Oil Red O staining. Oil Red O is a lipophilic dye that stains neutral triglycerides and lipids. These results indicate that pEpiSC PPARγ-CEBPα had significantly higher triglyceride content and differentiated more rapidly and efficiently than pEpiSC PPARγ.

[0103] Example 3: 2D long-term differentiation of EpiSCs-PPARγ-CEBPα into adipocytes The experiment described in Example 2 was repeated, but here, pEpISCs Opti-Ox PPARγ and pEpISCs Opti-Ox PPARγ-CEBPα cells were differentiated for up to 20 days, and differentiated cells were harvested on days 7, 14, and 21 for Oil Red O staining (Figure 4). pEpiSC-CEBPα-PPARγ cells accumulate larger lipid droplets than pEpiSC-PPARγ cells during adipogenesis. This difference was detectable already at day 7, with pEpISCs Opti-Ox PPARγ-CEBPα having a well-defined adipocyte-like morphology and homogenous lipid accumulation. In addition, pEpiSC-PPARγ-CEBPα cells did not need to reach confluence before initiating differentiation (data not shown), indicating that pEpiSC-PPARγ-CEBPα is less dependent on external adipogenic factors (ECM, secreted factors).

[0104] Example 4: EpiSCs-PPARγ-CEBPα skips a committed step during differentiation. To confirm that the commitment differentiation step was unnecessary, EpiSCs-PPARγ-CEBPα were differentiated in 2D using two differentiation protocols: Protocol D (including commitment + maturation steps) and Protocol F (maturation step only). Cells were harvested during differentiation on days 4, 12, and 20 and analyzed under a bright-field microscope (Figure 6A). Neutral lipid content was estimated by Oil Red O, adjusted for cell number measured by the HOESCH method. Both dyes were quantified spectrophotometrically (Figure 6B).

[0105] Although an intervention step was required to improve the differentiation capacity of pEpiSC PPARγ (data not shown), the adipogenic potential of EpiSCs-PPARγ-CEBPα was comparable between protocols D and F (Figure 6A). Indeed, because neutral lipid accumulation per cell was higher in EpiSCs-PPARγ-CEBPα differentiated without the mesoderm step (Figure 6B), this model is more promising in terms of cost savings (process and media) and because some small molecules can be excluded for regulatory acceptance of cultivated meat.

[0106] Example 5: 2D differentiation of EpiSCs-PPARγ-CEBPα: Differentiation using Protocol D and Protocol F with KSR + insulin + dexamethasone, KSR + dexamethasone (without additional insulin), and KSR + insulin (without dexamethasone). To investigate whether EpiSCs-PPARγ-CEBPα require additional insulin and dexamethasone during the maturation step of differentiation, EpiSCs-PPARγ-CEBPα were differentiated in 2D using differentiation protocols D and F in the presence or absence of additional insulin and / or dexamethasone. Cells were harvested during differentiation on days 2, 7, and 13 and analyzed under a bright-field microscope (Figure 7A). Adipogenic potential was comparable between protocols D and F with insulin and dexamethasone and those without insulin and / or dexamethasone. Because insulin and dexamethasone can be omitted, this model is more promising in terms of cost savings (process and media) and because some small molecules can be omitted for regulatory approval of cultivated meat.

[0107] Example 6: 3D culture of EpiSCs-PPARγ-CEBPα 3D suspension culture of cells is a necessary step for large-scale scale-up to produce the quantities of adipocytes required for cultivated meat products at a cost-competitive price (e.g., in shakers and / or bioreactors). While 3D suspension culture of cells allows cells to grow in an environment that closely resembles the cells' physiological in vivo environment, the transition from 2D to 3D culture must be performed appropriately, and differentiation data obtained in 2D experiments must be validated in 3D.

[0108] For this purpose, EpiSCs-PPARγ-CEBPα were adapted for 3D suspension cell growth and propagated as aggregates; EpiSCs-PPARγ-CEBPα grown in adherent 6-well cell plates were dissociated into single cells using Accumax (00-4666-56, ThermoFisher Scientific) according to the manufacturer's instructions, transferred to 150 mL shaker flasks containing 12.5 mL of medium and a RHO / ROCK pathway inhibitor, and then expanded for at least three cycles. The medium during 3D growth was refreshed daily. The 3D-adapted EpiSCs-PPARγ-CEBPα were then used in shaker and bioreactor experiments.

[0109] To examine adipogenic potential in 3D, 3 million cells / mL were seeded into 250 mL shaker flasks containing 25 mL of medium and EpiSCs-PPARγ-CEBPα were differentiated using differentiation protocol D in the presence of additional insulin and dexamethasone. The medium was changed every two days. Cells were harvested during differentiation day 6, stained with Oil Red O and DAPI, and visualized by fluorescence microscopy using the pancake method described previously (Figure 5). The relative gene expression of porcine PPARγ and CEBPα Opti-Ox-derived mRNA, as well as porcine endogenous PPARγ, CEBPα, adiponectin, LPL, perilipin-1, FABP4, CD36, and CEBPβ ​​mRNA, was analyzed as previously described herein. The relative gene expression was not significantly different from that observed in cells grown in 2D culture (data not shown), confirming that adipocytes are well adapted for proliferation and differentiation in 3D culture.

[0110] Example 7: 3D differentiation of EpiSCs-PPARγ-CEBPα in suspension: Protocol optimization: Differentiation using Protocol D and Protocol F with KSR + insulin + dexamethasone, KSR + dexamethasone (without additional insulin), and KSR + insulin (without dexamethasone). To confirm that additional insulin and dexamethasone could be omitted in 3D, EpiSCs-PPARγ-CEBPα were differentiated on a shaker and the procedure described in Example 5 was repeated in 3D. pEpiSC-PPARγ-CEBPα differentiated in 3D in suspension under both Protocol D (mesoderm step + maturation step) and Protocol F (maturation step only) (Figure 8). Intracellular triglycerides were also detectable in the center of the aggregates, indicating sufficient perfusion of nutrients and factors within the 3D structures (including the large aggregates). Thus, pEpiSC-PPARγ-CEBPα can also be differentiated into adipocytes without the addition of insulin or dexamethasone to the maturation medium, again confirming that this model is more promising in terms of cost reduction (process and medium) and because some small molecules can be omitted for regulatory approval of cultivated meat.

[0111] Example 8: 3D differentiation of EpiSCs-PPARγ-CEBPα in suspension in a bioreactor: To investigate whether pEpiSC PPARγ-CEBPα can be expanded and differentiated in a bioreactor, a fed-batch procedure was designed using the Ambr250 modular bioreactor system (Sartorius) to grow and differentiate 6 × 10 61 / mL of undifferentiated cells were seeded into 120 mL of DMEMF / 12 medium (21041-025, Gibco) supplemented with KSR, BMP4, FGF2, activin A, GlutaMAX, and ascorbic acid at the same concentrations described in the previous experiment. The glucose concentration was maintained above 7 mM, the temperature was kept at physiological levels, the agitation speed was sufficient to prevent aggregate size from exceeding the limits of nutrient perfusion, 7.5% sodium bicarbonate was added as needed to maintain pH at 7.4 ± 0.5, and the dissolved oxygen setpoint was set at 30–70%. Additional insulin and doxycycline were added on days 0, 2, 4, and 6, and dexamethasone was added only once on day 2. Aggregates were harvested on days 0, 3, 5, and 7 of differentiation for further analysis. The concentrations of medium components were monitored every 24 h to identify potential limitations (data not shown). Samples for neutral lipid quantification and gene expression analysis were collected on days 0, 3, 5, and 7. Aggregate size was measured on days 0 and 6.

[0112] Intracellular triglycerides were already detected in pEpiSC PPARγ-CEBPα after 3 days of differentiation, whereas lipid accumulation constantly increased 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 and highlight the potential use of this model for scale-up processes.

[0113] Example 9: Differentiation of EpiSCs-PPARγ-CEBPα in the absence of insulin PPARγ-CEBPα pEpISCs were seeded at 125,000 cells / cm2 in growth medium plus Fasudil at 3.5 μM in Geltrex-precoated 6-well plates. Cells were evenly distributed across the wells and incubated overnight at 38.5°C and 5% CO2 to ensure adherence. Adipogenic differentiation was induced the following day by refreshing the medium with maturation medium (Protocol F) containing 20 μg / mL insulin (control, condition Figure 10A), 10 μg / mL insulin (condition Figure 10B), 5 μg / mL insulin (condition Figure 10C), or no insulin (condition Figure 10D), supplemented with 0.5 μg / mL doxycycline. The insulin-free maturation medium was also supplemented with 0.05 μg / mL IGF-1 (condition Figure 10E) and 0.05 μg / mL IGF-1 / LR3 (condition Figure 10F). Maturation medium was changed every 2 days and cells were incubated at 38.5°C and 5% CO2 during differentiation. Brightfield images were taken on day 8 of differentiation. These results confirm that insulin is not required for efficient differentiation.

[0114] Descriptions (features) and embodiments of the methods and compositions disclosed herein are set forth below. Each description and embodiment disclosed by the invention as so defined may be combined with any other description and / or embodiment, unless expressly indicated otherwise. In particular, any feature indicated as being preferred or advantageous may be combined with one or more other features indicated as being preferred or advantageous.

[0115] [Embodiment] The present invention provides at least the following numbered statements / embodiments: 1.i) an expression construct for expression of a transcriptional regulatory protein inserted into a first genetic safe harbor site; ii) an expression construct for the expression of a PPAR-γ protein, wherein the coding sequence for the PPAR-γ protein is operably linked to an inducible promoter; and iii) an expression construct for the expression of a CEBPα protein, wherein the coding sequence for the CEBPα protein is operably linked to an inducible promoter; A pluripotent stem cell comprising: 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; A pluripotent stem cell in which an inducible promoter is regulated by a transcriptional regulatory protein. 2. The pluripotent stem cell of embodiment 1, wherein both of the expression constructs of ii) and iii) are inserted into a second genetic safe harbor site that is different from the first genetic safe harbor site. 3. The pluripotent stem cell of embodiment 1 or 2, wherein the cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines. 4. The pluripotent stem cells according to any one of embodiments 1 to 3, which are of a livestock or poultry species. 5. The pluripotent stem cells according to embodiment 4, wherein the livestock species is porcine or bovine, preferably porcine. 6. The pluripotent stem cell according to any one of embodiments 2 to 5, wherein the expression construct inserted into the second genetic safe harbor site encodes a PPAR-γ protein, a linker, and a CEBPα protein, preferably, the linker is P2A, more preferably, the linker comprises the sequence of SEQ ID NO: 3. 7. The pluripotent stem cell of embodiment 6, wherein the construct comprises the sequence of SEQ ID NO: 4. 8. The pluripotent stem cell according to any one of embodiments 1 to 7, 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 embodiments 1 to 8, wherein the transcriptional regulatory protein is selected from the group consisting of tetracycline-responsive transcriptional activator protein (rtTa), tetracycline repressor (TetR), VgEcR synthetic receptor, or a hybrid transcriptional regulatory protein, wherein the hybrid transcriptional regulatory protein comprises a DNA-binding domain derived from yeast GAL4 protein, a truncated ligand-binding domain derived from human progesterone receptor, and an activation domain derived from human NF-kB, and preferably, the transcriptional regulatory protein is rtTA. 10. The pluripotent stem cell of any one of embodiments 1 to 9, wherein the inducible promoter comprises a Tet-responsive element (TRE). 11. The pluripotent stem cell according to any one of embodiments 1 to 10, wherein the inducible promoter is the tetON promoter. 12. The pluripotent stem cell according to any one of embodiments 1 to 11, wherein the first and further genetic safe harbor sites are selected from any two of the hROSA26 locus, the AAVS1 locus, the CLYBL gene, or the CCR5 gene, preferably the genetic safe harbor sites are the hROSA26 locus and the AAVS1 locus. 13. A method for producing adipocytes, preferably white adipocytes, comprising: a) culturing the pluripotent stem cells according to any one of the preceding claims in a growth medium; followed by b) inducing adipocyte differentiation by adding an exogenous substance according to claim 8, preferably wherein the proliferation and / or differentiation medium does not contain insulin and / or dexamethasone. 14. The method of embodiment 13, wherein the differentiation stage is at most 10 days, at most 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, or 3 days. 15. The method of embodiment 13 or 14, wherein the adipocytes produced are for human and non-human nutrition. 16. Use of the pluripotent stem cells according to any one of embodiments 1 to 12, or the method for producing adipocytes according to any one of embodiments 13 to 15, for tissue engineering, and optionally for the production of cultivated meat. 17. A food product comprising pluripotent stem cells according to any one of embodiments 1 to 12 or adipocytes obtained by the method according to any one of claims 13 to 15. 18. The food product of embodiment 17, wherein the food product is cultured meat.

Claims

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

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

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

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

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

6. The pluripotent stem cell of any one of claims 2 to 5, wherein the expression construct inserted into the second genetic safe harbor site encodes a 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.

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

4.

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

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

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

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

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

13. A method for producing adipocytes, preferably white adipocytes, comprising: (a) culturing the pluripotent stem cells according to any one of claims 1 to 12 in a growth medium, followed by (b) inducing adipocyte differentiation by adding an exogenous substance according to claim 8, wherein the proliferation and / or differentiation medium preferably does not contain insulin and / or dexamethasone.

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

15. 15. The method of claim 13 or 14, wherein the adipocytes produced are for human and non-human nutrition.

16. Use of the pluripotent stem cells of any one of claims 1 to 12, or the method for producing adipocytes of any one of claims 13 to 15, for tissue engineering, optionally for the production of cultivated meat.

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

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

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