WO2025141211A1

WO2025141211A1 - Cells for forward programming

Info

Publication number: WO2025141211A1
Application number: PCT/EP2024/088672
Authority: WO
Inventors: Angelica AGUILERA GOMEZ
Original assignee: Meatable BV
Current assignee: Meatable BV
Priority date: 2023-12-29
Filing date: 2024-12-30
Publication date: 2025-07-03
Anticipated expiration: 2026-06-29

Abstract

The present invention relates to a modified pluripotent cell and to a method for forward programming said cell. The invention further relates to use of said modified pluripotent stem cell for tissue engineering and to a food product comprising said modified pluripotent stem cell.

Description

Cells for forward programming

Field of the invention

The present invention relates to a modified pluripotent cell and to a method for forward programming said cell. The invention further relates to use of said modified pluripotent stem cell for tissue engineering and to a food product comprising said modified pluripotent stem cell

Background of the invention

Stem cell research holds great promise for research of human development, regenerative medicine, disease modelling, drug discovery, cell transplantation and also for use in the field of cultivated meat.

Moreover, stem cell-derived cells enable studying physiological and pathological responses of human and non-human animal cell populations that are not easily accessible. This often entails the study of genes (and other forms of regulatory mechanisms encoded in non-protein-coding RNAs - ncRNAs). Unfortunately, controllable transcription or expression of genetic information in human and non-human animal cells has been proven to be particularly difficult.

Moreover, for several key aspects of regenerative medicine, disease modelling, drug discovery, cell transplantation and cultivated meat production, manipulation and manufacture of mature, human and non-human animal cell types from easily accessible sources is required.

Controlling expression of transgenes in cells is the basis of biological research. However, this has proven to be difficult in human cells and even more so in non-human animal cells, for example livestock, which are relatively less well studied and understood.

Moreover, there is a real need for the in vitro derivation of many highly desirable cell types in a quantity and quality suitable for drug discovery, regenerative medicine and food production purposes.

Because directed differentiation of stem cells into desired cell types is often challenging, other approaches have emerged, including direct reprogramming of cells into the desired cell types. In particular, forward programming, as a method of directly converting pluripotent stem cells, including human and non-human animal PSCs, to mature cell types has been recognised as a powerful strategy for the derivation of cells. This reprogramming involves the forced expression of key lineage transcription factors (or non-coding RNAs, including IncRNA and microRNA) in order to convert the stem cell into a particular mature cell type.

Also in this context, controllable expression of genetic information in human and non-human animal cells has been challenging. Currently available forward programming protocols are largely based on lentiviral transduction of cells, which results in variegated expression or complete silencing of randomly inserted inducible cassettes. This results in additional purification steps in order to isolate a sub-population expressing the required transcription factors. Thus, further refinements of these methods are clearly required. Specifically in relation to non-human animal cells, and as consumers become more sensitive to environmental, ethical, animal welfare and health concerns, there is a growing interest in finding alternative protein sources which ideally will be sustainable and will contain the nutrients normally provided by meat in the human diet.

Cultivated meat has arisen as another alternative to traditional animal agriculture that aims to produce the muscle and adipose tissues that normally comprise animal meats, except using in vitro tissue and biological engineering techniques. Despite efforts to develop robust protocols for scalable generation of animal cell types from easily accessible and renewable sources, the differentiation of animal (pluripotent) stem cells into specific cell types often remains cumbersome, lengthy, and difficult to reproduce and/or has not been established yet.

Accordingly there remains a need in the art forthe production and culturing of mature animal cells that are suitable for human consumption and that can be produced in a scalable and cost effective manner.

Summary of the invention

The inventors have developed cells, in particular pluripotent stem cells, in which the stable introduction of an inducible cassette into the genome of a cell is possible, whilst at the same time being able to control the transcription of that inducible cassette. This allows the forward programming of such cells. Critically, transcription is controlled by molecules considered to be food safe which is especially important in the field of cultivated meat, i.e. abscisic acid or analogues thereof.

Specifically, the inventors have found that it is possible to insert an inducible cassette and control transcription of the genetic material within that inducible cassette by using a dual genomic safe harbour targeted system herein described which may be induced by abscisic acid or an analogue thereof.

Such a method is highly desirable, since there is reduced risk of epigenetic silencing of the inserted genetic material, and it is possible to obtain a homogenous population of cells transcribing the inducible cassette and the inducer of transcription is food safe. This has benefits in any cell type in which it is desired to introduce an inducible cassette and control transcription of the inserted genetic material, in particular in pluripotent stem cells and may be especially applicable in the context of the production of cultivated food products, such as cultivated meat products.

According to the invention, there is thus provided a cell, such as a pluripotent stem cell, comprising:

(i) an expression construct for expression of a transcriptional regulator protein inserted into a first genetic safe harbour site; and

(ii) an expression construct for expression of a protein, wherein the coding sequence for the protein is operably linked to an inducible promoter; wherein the expression construct of (ii) is inserted into a further genetic safe harbour site that is not the first genetic safe harbour site, wherein the inducible promotor is regulated by the transcriptional regulator protein, and wherein the activity of the transcription regulator protein is controlled by abscisic acid or an analogue thereof.

The cell of the invention, such as a pluripotent stem cell, may be one in which: the coding sequence in expression construct (ii) encodes a protein involved in cell differentiation; and/or the coding sequence in expression construct (ii) encodes a transcription factor; and/or the coding sequence in expression construct (ii) encodes a MYOD protein, a MYOG protein, a PAX7 protein, a CEBPa protein or a PPARy protein; and/or the activity of the transcriptional regulator protein is controlled by exogenously supplied abscisic acid or an analogue thereof; and/or the transcriptional regulator protein is a transcriptional regulator protein comprising a DNA binding domain from the yeast GAL4 protein; and/or the inducible promoter may include a GAL4 binding site; and/or the expression construct (i) is for the expression of at least two fusion proteins:

- a first fusion protein a comprises a VP16 protein and one of a PYL1 protein; and a PP2C protein; and

- a second fusion protein comprises a DNA binding domain from the yeast GAL4 protein and the other of a PYL1 protein and a PP2C protein; and/or

The first and further genomic safe harbour sites are selected from any two of the hROSA26 locus, the AAVS1 locus, the CLYBL gene or the CCR5 gene, preferably wherein the genetic safe harbour site are hROSA26 locus and the AAVS1 locus; and/or the cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines and/or the cell is from a livestock or poultry species, optionally wherein the livestock species is porcine or bovine.

The invention also relates to a method for the forward of a cell comprising: a ) culturing a cell, such as a pluripotent stem cell, of the invention in a culture medium; and b) inducing forward programming by culturing the cell in a culture medium in the presence of abscisic acid, optionally wherein the abscisic acid is exogenously added.

In such a method: the culture media in a) and b) may have the same composition; and/or the cell may be fully differentiated at most 10 days, at most 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days or 2 days; and/or the produced cells may be muscle cells or adipose cells for human and non-human dietary consumption.

The invention further relates to: use of a cell, such as a pluripotent stem cell, of the invention or use of a method of the invention for producing a cell for tissue engineering, optionally for the production of cultivated meat. In addition, the invention relates to a food product comprising a cell, such as a pluripotent stem cell of the invention, or a cell obtained by the method of the invention. Such as food product may be cultivated meat.

Description of the figures

Figure 1 a shows a schematic representation of a protein-protein interaction based system for inducible gene expression. The system is dependent on ABA induced interaction between PYL1 and ABI1 . In the presence of abscisic acid ABI and PYL1 are brought into close proximity resulting in a DNA binding domain (GAL4 DNA binding domain) and a transcriptional activator domain also being brought into close proximity such that they can drive promoter mediated expression of a gene of interest.

Figure 1 b shows two gene expression cassettes used to target two different safe harbours. The upper construct comprises a fragment of PYL1 (also referred to as "PYLcs") linked to a transcriptional activation domain (transcription activator domain of VP16) and a fragment of ABI1 , a PP2C member, (also referred to as "ABIcs") linked to a DNA binding domain (GAL4 DNA binding domain). These two elements are linked by an IRES. The second construct comprises binding sites for the DNA binding domain upstream of two transcription factors (TS). In the presence of abscisic acid, the PYL1 fragment and the ABI1 fragment are brought into close proximity such that the transcriptional activation domain and DNA binding domain can drive expression of the two TFs.

Figure 2a shows a map of the construct to target the Rosa locus comprising: PYLcs linked to the transactivation domain of VP16; and ABIcs linked to the GAL4 DNA binding domain.

Figure 2b shows a more detailed map of the VP16-PYLcs-HA-IRES-GAL4-ABI portion of the targeting construct in Figure 2a.

Figure 3a shows a map of the construct to target the AAVS1 locus comprising: 10 x UAS sequences upstream of the hsp70 promoter operably linked to the MYOD1 transcription factor.

Figure 3b shows a more detailed map of the 10 x UAS-HSP70 portion of the targeting construct in Figure 2a.

Figure 4 shows induction to differentiation of cells edited with Plant Based Switch-ABA, harboring the reprogramming transcription factors, MyoD1-MyoG. Immunofluorescent images (20X) stained with DAPI (blue), MyoD (green), and Heavy Chain (red) were analyzed. Pictures numbered 1 , 2, 3, 4, 5, and 6 serve as controls, treated with different concentrations of DMSO (the ABA solvent). Pictures numbered 7, 8, 9, 10, and 11 contain ABA at concentrations of 100, 200, 300, 400, and 500 pM, respectively. Picture numbered 12 shows unedited cells treated with 500 pM ABA. Notably, from 100 pM to 500 pM, the induction of reprogramming factors is observed in a positive dose-dependent manner, as evidenced by the increased intensity of the Heavy Chain marker.

Figure 5 shows induction to differentiation of cells edited with Plant Based Switch-ABA, harboring the reprogramming transcription factors, MyoD1-MyoG. Immunofluorescent images (20X) stained with DAPI (blue), MyoD (green), and Heavy Chain (red) were analyzed. Edited cells exhibit clear reprogramming to muscle, as evidenced by the expression of MyoD1 (green) and Heavy Chain (red), when treated daily with 500 uM of ABA.

Figure 6 shows comparison Induction-Potency to differentiation of cells edited with Plant Based Switch-ABA or edited with doxycycline based switch both harboring the reprogramming transcription factors, MyoD1-MyoG. Figures a, b, c, and d depict immunofluorescent images (20X) stained with DAPI (blue) and TITIN (red). All cells shown here exhibit clear reprogramming towards muscle, as indicated by the expression of TITIN (red), when treated every 2 days with either doxycycline or ABA over a period of 6 days.

Figure 7 shows qPCR Comparison of Induction Potency for Differentiation of Cells Edited with the Plant-Based Switch (ABA) or Doxycycline-Based Switch, Both Harboring the Reprogramming Transcription Factors MyoD1 and MyoG. Figures a, b, and c show the expression levels of the markers Desmin, Oct4, and Sox2. All cells depicted here exhibit clear reprogramming towards muscle, as evidenced by the expression of TITIN (red), when treated every 2 days with either doxycycline or ABA over a period of 6 days.

Figure 8 shows induction to differentiation of cells edited with Plant Based Switch-ABA, harboring the reprogramming transcription factors, PPARg-CEBPa. Immunofluorescent images stained with Phalloidin (green) and Lipitox (red) were analyzed. Edited cells demonstrate clear reprogramming to fat-producing cells after 7 days, as indicated by the detection of Lipitox (red). These cells were treated daily with varying concentrations of ABA. Lipid droplets are observed at all ABA concentrations.

Figure 9 shows induction to differentiation of cells edited with Plant Based Switch-ABA, harboring the reprogramming transcription factors, PPARg-CEBPa. Immunofluorescent images (20X) stained with Phalloidin (green) and Lipitox (red) are presented, with unmerged channels (red and white) highlighting lipid droplet production. Edited cells exhibit clear reprogramming to fatproducing cells after 7 days of daily treatment, as indicated by the detection of Lipitox (red or white). These cells were treated daily with varying concentrations of ABA. Lipid droplets are observed at all ABA concentrations tested, in a dose-dependent manner.

Description of the Sequence Listing

Description of the invention

Definitions Unless defined otherwise, 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. 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 in no way limited to the method. In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but 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 more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

As used herein, the term "and/or" indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases. As used herein, with "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, 1 1 , 12, 13, 14, 15, ... ,etc.

The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1 % of the value.

The term "heterologous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced, but has been obtained from another cell or synthetically or recombinantly produced. Generally, though 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 for proteins 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. 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 herein encompassed 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 where at least two of the combined sequences are foreign with respect to each other.

The terms "expression vector" or “expression construct" refer to nucleotide sequences that are capable of effecting expression of a gene in host cells or host organisms compatible with such sequences. These expression vectors typically include at least suitable transcription regulatory sequences and optionally, 3' transcription termination signals. Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements

As used herein, the term “operably linked” refers to a 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 instance, a transcription regulatory sequence is operably linked to a coding sequence if it 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 encoding regions, contiguous and in reading frame, inducible promoter

As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill 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, e.g. by the application of a chemical inducer. In the case of the present invention, the control is effected by the transcriptional regulator protein.

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

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

The inventors have surprisingly found that controlled gene expression for the differentiation of, for example pluripotent cells into mature cells, can be induced by abscisic acid or an analog thereof.

A specific example of this approach is set out in Figure 1 . Figure 1 a is a of representation of a protein-protein interaction based system for inducible gene expression. The system is dependent on abscisic acid (ABA) induced interaction between PYL1 and ABI1 . In the presence of abscisic acid, the ABI and PYL1 proteins are brought into close proximity resulting in a DNA binding domain (GAL4 DNA binding domain) and a transcriptional activator domain also being brought into close proximity such that they can drive promoter mediated expression of a gene of interest.

Figure 1 b shows two gene expression cassettes used to target two different safe harbours. The upper construct comprises a fragment of PYL1 (also referred to as "PYLcs") linked to a transcriptional activation domain (transcription activator domain of VP16) and a fragment of ABI1 , a PP2C member, (also referred to as "ABIcs") linked to a DNA binding domain (GAL4 DNA binding domain). These two elements are linked by an IRES. The second construct comprises binding sites for the DNA binding domain upstream of two transcription factors (TS). In the presence of abscisic acid, the PYL1 fragment and the ABI1 fragment are brought into close proximity such that the transcriptional activation domain and DNA binding domain can drive expression of the two TFs. Critically in the invention, the two expression cassettes are targeted to two different genetic safe harbours.

The cell of the invention may be particularly applicable in the reliable and scalable production of cultivated meat production where it is important that molecules used are food safe.

Accordingly, in a first aspect the invention relates to a pluripotent stem cell comprising:

(ii) an expression construct for expression of a protein inserted into a second genetic safe harbour site, wherein the coding sequence for the protein is operably linked to an inducible promoter, wherein the first and second genetic safe harbour sites are different genetic safe harbour sites, wherein the inducible promotor is regulated by the transcriptional regulator protein, and wherein the activity of the transcription regulator protein is controlled by abscisic acid or an analogue thereof. The coding sequence in expression construct (ii) may encodes a protein involved in cell differentiation. The coding sequence in expression construct (ii) may encode a transcription factor. The coding sequence in expression construct (ii) may encode two or more such proteins.

Thus, a pluripotent stem cell of the invention may encode a MYOD protein, a MYOG protein, a PAX7 protein, a CEBPa protein or a PPARy protein.

Furthermore, a pluripotent stem cell of the invention may encode: a MYOD protein and a MYOG protein; a MOYD protein and a PAX7 protein; or a CEBPa protein or a PPARy protein.

The protein combinations are described in detail in International patent publication numbers WO2024/170696 (describing a combination of a MYOD protein and a MYOG protein) and WO2024/170702 (describing a combination of a MOYD protein and a PAX7 protein) and in International patent publication number W02024/084082 (describing a combination of a CEBPa protein and a PPARy protein).

In a pluripotent stem cell of the invention, the transcriptional regulator protein may be expressed as a single protein, but more typically as separate DNA binding and activation domains. In the latter case, the DNA binding and activation domains must brought into proximity with each other in order that transcription may take place (of the protein encoded in construct (ii)).

Thus, a transcriptional regulator protein may comprise a DNA binding domain, such as that from the yeast GAL4 protein. Such binding domains are well known to those skilled in the art. A coding sequence for a suitable DNA binding domain comprises the sequence set out in SEQ ID NO: 3. Variants of this sequence may also be suitable for use in the invention.

The transcriptional regulator protein may also comprise an activation domain, for example a transactivation domain, such as a VP16 transactivation domain (also referred to herein as the VP16 protein and the like). Again, such binding domains are well known to those skilled in the art. A coding sequence for a suitable VP16 transactivation domain comprises the sequence set out in SEQ ID NO: 1 . Variants of this sequence may also be suitable for use in the invention.

Where the transcriptional activator comprises a GAL4 DNA binding domain, the inducible promoter in expression construct (ii) typically includes a GAL4 binding site, preferably multiple such sites. An example of such a GAL4 binding site comprises the sequence set out in SEQ ID NO: 6. Alternative GAL4 binding sites are well known to those skilled in the art.

In a pluripotent stem cell according to the invention, expression construct (i) may allow for the expression of at least two fusion proteins (or chimeric proteins):

- a first fusion protein may comprise an activation domain, such as a VP16 protein, and one of a PYL1 protein; and a PP2C protein; and

- a second fusion protein may comprise a DNA binding domain, such as a DNA binding domain from the yeast GAL4 protein, and the other of a PYL1 protein and a PP2C protein.

That is to say, the pluripotent stem cell according to the invention may be one wherein wherein the expression construct (i) is for the expression of at least two fusion proteins selected from: - a first fusion protein which may comprise an activation domain, such as a VP16 protein, and one of a PYL1 protein; and a PP2C protein; and

- a second fusion protein which may comprise a DNA binding domain, such as a DNA binding domain from the yeast GAL4 protein, and one of a PYL1 protein and a PP2C protein; or

- a first fusion protein which may comprise an activation domain, such as a VP16 protein, and a PP2C protein; and

- a second fusion protein which may comprise a DNA binding domain, such as a DNA binding domain from the yeast GAL4 protein, and a PYL1 protein; or

- a first fusion protein which may comprise an activation domain, such as a VP16 protein, and a PYL1 protein; and

- a second fusion protein which may comprise a DNA binding domain, such as a DNA binding domain from the yeast GAL4 protein, and a PP2C protein.

When these two fusion proteins are expressed in the presence of abscisic acid or an analog thereof, the PYL1 protein and PP2C protein are brought into proximity with each other which then brings the activation domain, such as a VP16 protein, and the DNA binding domain, such as a DNA binding domain from the yeast GAL4 protein, into proximity with each other thus constituting a functional transcriptional regulator protein.

That functional transcriptional regulator protein can then drive expression form the inducible promoter in expression construct (ii).

Any suitable PYL1 protein may be used in a cell of the invention. The PYR/PYL/RCAR- like family belongs to the SRPBCC (START/RHO_alpha_C/PITP/Bet_v1/CoxG/CalC) domain superfamily of proteins that bind hydrophobic ligands. SRPBCC domains have a deep hydrophobic ligand-binding pocket. PYR/PYL/RCAR plant proteins are receptors involved in signal transduction. They bind abscisic acid (ABA) and mediate its signaling. A preferred PLY1 protein is PYLcs (complementary surface of PYL1) fragment. (PYLcs, amino acids 33 to 209). Such proteins are well known to those skilled in the art. A coding sequence for a suitable PYLcs domain comprises the sequence set out in SEQ ID NO: 2. Variants of this sequence may also be suitable for use in the invention.

Any suitable PP2C protein may be used in a cell of the invention. PP2C proteins are protein phosphatase type 2C proteins. Abscisic acid produces its effects by binding to the pyrabactin resistance (PYR)/PYR1-like (PYL)Zregulatory component of abscisic acid receptor (RCAR) family of intracellular receptors, and the resulting complexes inhibit the activity of protein phosphatase type 2Cs (PP2Cs). A specific PP2C protein suitable for use in the invention is ABI1 , more particularly a fragment comprising the interacting complementary surfaces (CSs) of ABI1 (ABIcs, amino acids 126 to 423). Such proteins are well known to those skilled in the art. A coding sequence for a suitable ABIcs domain comprises the sequence set out in SEQ ID NO: 4. Variants of this sequence may also be suitable for use in the invention. The fusion proteins described above are examples of how an abscisic acid inducible system for forward programming may be implemented. The PYL and PP2C proteins may be fused to alternative proteins (i.e. other than the VP16 transactivation domain and the GAL4 DNA binding domain) which, when the PYL and PP2C proteins are in close proximity (i.e. in the presence of abscisic acid) allow a functional transcriptional regulator to be activated other than that based on GAL4 DNA binding domain and VP16 proteins. That is to say other combinations of a DNA binding domain and a transcriptional activation domain may be used.

For all variants of proteins mentioned herein, a variant may have at least about 99%, at least about 98%, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75% or at least about 70% sequence identity to the protein in question.

In a pluripotent stem cell according to the invention, the activity of the transcriptional regulator protein is controlled by exogenously supplied abscisic acid or an analogue thereof. That is to say, the pluripotent stem cell is not typically one which is capable of producing endogenous abscisic acid.

In chemistry, an analogue (or analog) is a compound in which one or more individual atoms have been replaced, either with a different atom, or with a different functional group. Another use of the term in chemistry refers to a substance which is similar in structure to another substance. Analogues to abscisic acid suitable for use in the invention will thus differ from abscisic acid chemically or structurally, but critically in the context of the invention will retain ability to control activity of the transcriptional regulator protein. Any such substance is suitable for use in in the invention as an analog of abscisic acid.

In one embodiment the method as disclosed herein comprises a transcriptional regulator protein, wherein the transcriptional regulator protein is abscisic acid, or an analogue thereof. In a preferred embodiment, abscisic acid, or an analogue thereof, is provided up to at least 100 pM, at least 150 pM, at least 200 pM, at least 250 pM, at least 300 pM, at least 350 pM, at least 400 pM, at least 450 pM, at least 500 pM. In a more preferred embodiment, abscisic acid, or an analogue thereof, is provided at 500 pM.

In certain embodiments, the nucleic acid molecules encoding the proteins according to the invention are codon-optimized for expression in mammalian cells. Methods of codon-optimization are known and have been described previously (e.g. WO 96/09378 for mammalian cells). A sequence is considered codon-optimized if at least one non-preferred codon as compared to a wildtype sequence is replaced by a codon that is more preferred. Herein, a non-preferred codon is a codon that is used less frequently in an organism than another codon coding for the same amino acid, and a codon that is more preferred is a codon that is used more frequently in an organism than a non-preferred codon. The frequency of codon usage for a specific organism can be found in codon frequency tables, such as in http://www.kazusa.or.jp/codon. Preferably more than one nonpreferred codon, preferably most or all non-preferred codons, are replaced by codons that are more preferred. Preferably the most frequently used codons in an organism are used in a codon- optimized sequence. Replacement by preferred codons generally leads to higher expression. A transcriptional regulator protein is a protein that bind to DNA, preferably sequence- specifically to a DNA site located in or near a promoter, and either facilitating the binding of the transcription machinery to the promoter, and thus transcription of the DNA sequence (a transcriptional activator) or blocks this process (a transcriptional repressor). Such entities are also known as transcription factors.

The DNA sequence that a transcriptional regulator protein binds to is called a transcription factor-binding site or response element, and these are found in or nearthe promoter of the regulated DNA sequence.

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

The method of the invention envisages a split transcriptional regulator protein which is only active in the presence of abscisic acid (when the PYL and PP2C proteins are brought into close proximity).

A genetic safe harbour (GSH) site is a locus within the genome wherein a gene or other genetic material may be inserted without any deleterious effects on the cell or on the inserted genetic material. Most beneficial is a GSH site in which expression of the inserted gene sequence is not perturbed by any read-through expression from neighboring genes and expression of the inducible cassette minimizes interference with the endogenous transcription program. More formal criteria have been proposed that assist in the determination of whether a particular locus is a GSH site in future (Papapetrou et al, 201 1 , Nature Biotechnology, 29(1), 73-8. doi: 1 0. 1 038/nbt. 1 71 7.) These criteria include a site that is (i) 50 kb or more from the 5’ end of any gene, (ii) 300 kb or more from any gene related to cancer, (iii) 300 kb or more from any microRNA(miRNA), (iv) located outside a transcription unit and (v) located outside ultra-conserved regions (UCR). It may not be necessary to satisfy all of these proposed criteria, since GSH already identified do not fulfil all of the criteria. It is thought that a suitable GSH will satisfy at least 2, 3, 4 or all of these criteria.

In certain embodiments of the invention, the first and further genomic safe harbour 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 further genomic safe harbour sites are located on 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.

In certain embodiments of the invention, the first and further genomic safe harbour sites are selected from any two of the safe harbour sites ROSA26, AAVS1 , the CLYBL gene and the CCR5 gene. The pluripotent stem cell according to any one of the preceding claims, wherein said first and further genomic safe harbour sites are selected from any two of the hROSA26 locus, the AAVS1 locus, the CLYBL gene or the CCR5 gene, preferably wherein the genetic safe harbour site are ROSA26 locus and the AAVS1 locus.

Preferably, the genetic safe harbour sites are ROSA26 locus and the AAVS1 locus.

As used herein, the term "pluripotent stem cells" includes embryonic stem cells, embryo- derived stem cells, epliblast-derived stem cells (EpiSCs), induced pluripotent stem cells and somatic cells, regardless of the method by which the pluripotent stem cells are derived. Accordingly, in certain embodiments the pluripotent stem cell is 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, 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 iPSC) can be produced by protein transduction of reprogramming factors in a somatic cell.

The pluripotent stem cell according to the invention can be from any species. Embryonic stem cells have been successfully derived in, for example, mice, multiple species of non-human primates, and humans, and embryonic stem-like cells have been generated from numerous additional species. Thus, one of skill in the art can generate embryonic stem cells and embryo- derived stem cells from any species, including but not limited to, human, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, gerbils, squirrel, guinea pig, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like.

Similarly, iPS cells can be 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, newborn, and adult tissue. Accordingly, one can readily generate iPS cells using a donor cell from any species. Thus, one can generate iPS cells from any species, including but not limited to, human, non-human primates, rodents (mice, rats), ungulates (cows, sheep, etc.), dogs (domestic and wild dogs), cats (domestic and wild cats such as lions, tigers, cheetahs), rabbits, hamsters, goats, elephants, panda (including giant panda), pigs, raccoon, horse, zebra, marine mammals (dolphin, whales, etc.) and the like.

In certain embodiments, the pluripotent stem cell according to the invention, or for use in the invention is a human or animal cell. In certain embodiments the pluripotent stem cell according to the invention, or for use in the invention if from an edible animal species.

Preferably, the pluripotent stem cell according to the invention, or for use in the invention is from a livestock or poultry animal. Livestock species include but are not limited to domestic cattle, pigs, sheep, goats, lamb, camels, water buffalo and rabbits.

Preferably, the pluripotent stem cell according to the invention, or for use in the invention is a bovine pluripotent stem cells or a porcine pluripotent stem cell. In certain embodiments, the stem cell according to the invention is a bovine epiblast stem cell (pEpiSC) or a porcine epiblast stem cell (bEpiSC).

Poultry species include but are not limited to domestic chicken, turkeys, ducks, geese and pigeons. In certain embodiments, the cells originate from common game species such as wild deer, gallinaceous fowl, waterfowl and hare. In certain embodiments, the inducible promotor that is operably linked to the one or mor proteins (such as transcription factors) in expression construct (ii) is HSP 70, CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc. Each protein to be expressed may be operably linked to the same or different promoters.

A pluripotent stem cell of the invention may be especially useful in forward programming as applied to the generation of cultivated meat. Accordingly, in a further aspect, the invention relates to a method for the production of cells such as adipocytes and muscle cells (in particular skeletal muscle cells), comprising a ) culturing the pluripotent stem cell as described herein in a proliferation medium: followed by b) inducing differentiation by adding exogenous abscisic acid as described herein.

In certain embodiments, the method of the invention is an ex vivo or an in vitro method.

In certain embodiments, the method is for the production of mature adipocytes or mature muscle cells, such as skeletal muscle cells. Mature adipocytes are herein defined as adipocytes which show lipid accumulation and/or express detectable levels of PPARy FABP4, PLIN1 and adiponectin. In certain embodiments, the method of the invention relates to a method for production of white adipocytes.

In certain embodiments the method as described herein does not comprise an additional commitment phase induction step.

The method as described herein may reduce the differentiation time of the pluripotent cells as described herein to mature adipocytes or skeletal muscle cells dramatically. In certain embodiments, the time to produce mature adipocytes using the method as claimed 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 as described in the method as described, is may be possible to achieve a conversion rate of at least 95% by day 4 of culture, meaning that at least 95% of the cells are mature after 4 days of culture. Accordingly, in certain embodiments the time to produce at least 95% mature adipocytes is at most 4 days.

In a further aspect, the invention provides for adipocytes, preferably mature adipocytes, and myocytes, preferably skeletal muscle cells, as obtained by the method as described herein.

Culturing the cells as described herein can be performed under so called 2D culturing conditions, which is considered the conventional approach to culturing cells. However, the method as described can also easily be adapted to allow culturing under 3D conditions.

3D cell culture is an artificially-created environment which enables cells to grow or interact with their surroundings in three dimensions. In such culture, cells typically form 3D colonies, which may be referred to as "spheroids". The 3D culture approach may more accurately model the cells' in vivo growth and behaviour. The skilled person is readily able to carry out 3D cell culture, for example by taking advantage of any of a number of commercially-available culturing tools. For example, the 3D culture may be carried out using scaffold or scaffold-free techniques. Scaffoldbased techniques make use of supports such as solid scaffolds and hydrogels to enable the cells to form a 3D culture. Such scaffolds may aim to mimic the natural extracellular matrix (ECM), which is present in vivo. Scaffold-free techniques dispense with the use of the scaffold on which to grow the cells. Instead, 3D spheroids may be established through the use of, for example, low-adhesion plates, hanging-drop plates, micro-patterned surfaces, rotating bioreactors, magnetic levitation and magnetic 3D bioprinting.

Cells that have been transduced with lentiviral vectors are not considered food safe or not safe for human and non-human dietary consumption. The pluripotent cells as described herein of the method as described herein obviates the need to use lentivirally transduced cells. Accordingly, in certain embodiments, the adipocytes that are produced according to the method as disclosed herein are for human and non-human dietary consumption. In certain embodiments, the produced adipocytes can be used in the production of cultured meat for human consumption.

In a further aspect, the invention provides for a use of a pluripotent stem cell as described herein or the cells obtained by the method as described herein for forward programming of cells, for example in tissue engineering. The cells obtained may be suitable for use in a method of treatment and in research. In certain aspects, the use is for the production of cultured meat. That is to say, the invention provides for a use of a pluripotent stem cell as described herein or the cells obtained by the method as described herein for the production of cultured meat.

In yet a further aspect, the invention provides for a food product (also referred to as “foodstuff’) comprising the pluripotent stem cells as described herein or the adipocytes produced and/or obtained by the method as described. In certain embodiments, the food product is or further comprises an edible composition for human or non-human consumption. The edible composition for human or non-human consumption for example comprises at least one of adipocytes, myocytes, mature muscle cells, minerals, synthetic substances, flavoring substances (such as for examples herbs and spices), plant based proteins or proteins from microbial origin such as yeast proteins. Plant based proteins and yeast proteins suitable forthe use in food products are known to the skilled person in the art. In certain embodiments, the food product is cultured meat or a cultured meat product.

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

Examples

The present invention is further illustrated by the following Examples which should not be construed as limiting the scope of the invention.

Materials and Methods

Epiblast-derived Stem Cells (EplSCs) differentiation to skeletal muscle cells. Undifferentiated bEplSCs (capable of expressing MyoD1 and MYOG) may be grown as set out in International patent publication nos. WO2024/170696 and WO2024/170702.

Immunofluorescence Imaging

Immunofluorescent images (20X) stained with DAPI, MyoD, Heavy Chain, TITIN, Phalloidin, Lipitox were prepared and analyzed following the protocol described in International patent publication nos. WO2024/170696 and WO2024/170702.

Real-time quantitative PCR analysis (RT-qPCR)

Total RNA from undifferentiated and differentiated cells is extracted using Reliaprep Cell Miniprep System (Z6012, Promega) according to the manufacturer's instructions. RNA concentration and quality is determined with a Microvolume Spectrophotometer DS-11 (DeNovix). Five hundred nanograms of purified total RNA from every sample are first treated with DNase I to remove possible genomic DNA contamination and are subsequently reverse transcribed into cDNA using iScript gDNA Clear cDNA Synthesis Kit (1725035BUN, BioRad). Specific primers for bovine pluripotency and mature adipocyte markers are designed to perform real-time quantitative PCR analysis (table I). At least three samples from two independent experiments are amplified in triplicates in an (thermocycler) system using PowerTrack SYBR Green Master Mix (A46112, Thermofisher) according to manufacturer’s guidelines. RT-qPCR conditions were 95 °C for 30 seconds, followed by 40 cycles of of 15 s at 95°C and 1 min at 60°C. The YWHAZ gene is used as a housekeeping gene to normalize the target gene expression levels. Relative gene quantification is calculated by the 2-AACt method.

Results

Example 1: Development of an abscicic acid inducible transgene overexpression method by dual genomic safe harbor (GSH) targeting in animal Cells

To explore the myogenic reprogramming potential of a dual safe harbour expression system inducible by abscisic acid for bovine pluripotent stem cells (bPSCs), bPSCs capable of expressing MYOD1 and MYOG are generated.

A first SV40 NLS-VP1 -PYLcs -HA-IRES-GAL4 DNA binding domain-ABIcs construct (see Figure 2a and b) and a second 10 x UAS-HSP70 promoter-MYOD1-MYOG construct (see Figure 3a and b (for targeting to AAVS1 locus) are sequentially targeted to the Rosa GSH and the AAVS1 GSH. In the presence of abscisic acid, MYOD1 and MYOG are expressed.

Inducible transgene expression is determined to show that the system is functioning correctly. Further, we observed that MYOD reprogramming of pPSCs directly generates myotubes after 8 days of expression (Figure 1 , left). This finding demonstrated that MYOD can directly reprogram pPSCs into myotubes in 2D cultures.

Example 2: EpiSCs-MYOD-MYOG 2D differentiation into multinucleated skeletal muscle cells To further determine xthat the system is working, bEplSCs capable of expressing MYOD- MYOG are expanded in 3D suspension culture, single celled and seeded in coated plastic wells with 200k cells/cm². One day after attachment, abscisic acid is added to medium to start the differentiation. Cells are differentiated for 8 days and the extent of differentiation may be determined by the extent of formation of titin positive skeletal muscle cells.

Example 3: 3D culture of EpiSCs-MYOD-MYOG

For the expansion (proliferation) phase of cell culture in the production of cultivated meat, shakers and/or bioreactors are often used to scale up to large volumes and generate the amounts of cell mass needed for a cultivated meat product at a cost competitive price. Commonly, primary cells are used derived from muscle biopsies. However, primary cultures have limited self-renewal, lose their capacity to differentiate during expansion and are variable in quality between biopsies.

Other sources such as ESCs and iPSCs have high expansion capacity but are in an undifferentiated state and differentiation must be induced.

For meat production, the differentiation of cells into myotubes and subsequently into myofibers is an integral part of the process, which usually occurs in a subsequent separate step. Differentiation of skeletal muscle cells requires often distinct conditions in terms of nutrients and physical environment. The necessary nutrients can be provided by switching from a nutrient rich proliferation medium to a differentiation medium consisting of low serum, but providing the physical environment that the cells require in order to differentiate is more challenging. In addition, the substrate requirements for the proliferation and differentiation phases are typically different in terms of surface chemistry and topography. A particular challenge for skeletal muscle cells in suspension is that skeletal muscle cells need attachment sides to poles, a scaffold, or a surface to form elongated stretched multinucleated myotubes.

In this Example, bEpiSCs-MYOD-MYOG (see Example 1) are adapted to 3D suspension cell growth and grown as aggregates; bEpiSCs expanded in an adherent 6-well cell plate are single celled and transferred to a 150mL shaker flask in 12,5mL media and a RHO/ROCK pathway inhibitor and thereafter expanded for at least 3 cycles. Media during expansion in 3D is refreshed daily. 3D adapted EpiSCs are subsequently used in shaker and bioreactor experiments.

To explore skeletal muscle differentiation in 3D suspension culture, 0.5 million cells/mL are inoculated in a 150 mL shaker flask containing 12,5 mL of media and bEpiSCs-MYOD-MYOG are differentiated using a differentiation protocol. Media is changed every second day. After a set number of days of differentiation of EpiSCs-MYOD-MYOG, the extent of multinucleated skeletal muscle cells formed with elongated morphology is determined and the amount of titin protein determined.

In this simplified bioprocess, where the same culture system can be used for both proliferation and differentiation phases via a medium change or by only adding the abscisic acid inducer of expression, the amount and quality of differentiation into skeletal muscle cells can be used to determine the effectiveness of the use of abscisic acid as an induction molecule. Example 4: Plant Based-Abscisic Acid (ABA) switch for both species Bovine-Porcine

Current technology is based on a doxycycline-inducible system for controlling the protein abundance of reprogramming transcription factors, thereby inducing a desired phenotype. However, the use of doxycycline is limited by its slow clearance and regulatory concerns, additionally a final product free of antibiotics is favorable. To address this issue, we innovatively modified our current antibiotic-based inducible system by integrating proteins from the plant abscisic acid (ABA) stress response pathway, enabling the induction of the desired phenotype through the application of exogenous ABA.

According to the literature, ABA's stability and ability to induce transgene expression last for a maximum of 24 hours and are dose-dependent up to an upper limit of 100 pM (Liang FS, Ho WQ, Crabtree GR. Engineering the ABA plant stress pathway for regulation of induced proximity. Sci Signal. 2011 Mar 15;4(164):rs2). Here, we demonstrate that the expression of transgenes in bovine cells induced for muscle differentiation is maintained over time with a concentration of 500 pM ABA. In comparison, a concentration of 100 pM results in limited and results in weak transgene expression (Figures 4 and 5).

Remarkably, the potency of the ABA-inducible system is comparable to the doxycycline- based system for reprogramming bovine EpiSCs to muscle at a concentration of 500 pM ABA. This is true even when ABA is replenished every 48 hours, contrasting with the described stability and inducibility of ABA over 24 hours at concentrations up to 100 pM (Figures 6 and 7). Additionally, cells reprogrammed to muscle by ABA exhibit unexpectedly better striation, as evidenced by the orientation of TITIN and nuclear reorganization (Figure 6).

A key innovative step involves our ability to sustain effective transgene expression well beyond the known limits of ABA stability and inducibility. This extends the ABA's functional timeframe to 48 hours and increases its effective concentration to 500 pM, a significant deviation from the existing knowledge that suggested a maximum of 24 hours and 100 pM.

ABA has already been shown in mammals to control adipose tissue at nanomolar concentrations by increasing energy expenditure, thus reducing fat accumulation in brown and white adipose tissues (Magnone, M.; Sturla, L.; Guida, L.; Spinelli, S.; Begani, G.; Bruzzone, S.; Fresia, C.; Zocchi, E. Abscisic Acid: A Conserved Hormone in Plants and Humans and a Promising Aid to Combat Prediabetes and the Metabolic Syndrome. Nutrients 2020, 12, 1724). In several studies, ABA treatment of adipocytes compared with insulin resulted in lower triglyceride accumulation (Sturla, L.; Mannino, E., et al., Abscisic acid enhances glucose disposal and induces brown fat activity in adipocytes in vitro and in vivo, Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 2017, 1862(2), 131-144). Contrary to our expectations, we successfully induced the accumulation of lipid droplets in porcine EpiSC cells harboring the reprogramming transcription factors PPARG-CEBPA, which are typically used to reprogram cells toward adipocytes. Furthermore, the accumulation of lipid droplets in ABA-treated cells (harboring PPARg- CEBPA) increased strikingly in a dose-dependent manner (Figures 8 and 9). This counterintuitive result underscores another innovative aspect of our approach: despite ABA's known role as an adipocyte-repressing component, we achieved efficient lipid droplet accumulation, highlighting the versatility and robustness of our designed ABA-switch system. This finding not only challenges the existing paradigm but also opens new avenues for further research and application in antibiotic-free metabolic reprogramming.

Overall, optimizations in the machinery and induction protocols have enabled us to replace the doxycycline-based system, known as the most potent inducible system, with an optimized and equally potent plant-based system. This innovation addresses the issue of antibiotic usage in products intended for human consumption and any Meatable final product. The new system has improved muscle formation and overall product quality by employing the ABA-switch for muscle reprogramming. Furthermore, we were able to counterintuitively induce the accumulation of lipid droplets in cells reprogrammed to adipocytes using the ABA-switch, despite ABA being a known adipocyte-repressing component.

Claims

1 . A pluripotent stem cell comprising:

2. The pluripotent stem cell according to claim 1 , wherein the coding sequence in expression construct (ii) encodes a protein involved in cell differentiation.

3. The pluripotent stem cell according to claim 1 or 2, wherein the coding sequence in expression construct (ii) encodes a transcription factor.

4. The pluripotent stem cell according to any one of the preceding claims, wherein the coding sequence in expression construct (ii) encodes a MYOD protein, a MYOG protein, a PAX7 protein, a CEBPa protein or a PPARy protein.

5. The pluripotent stem cell according to any one of the preceding claims, wherein the activity of the transcriptional regulator protein is controlled by exogenously supplied abscisic acid or an analogue thereof, up to at least about 500pl.

6. The pluripotent stem cell according to any one of the preceding claims, wherein the transcriptional regulator protein is a transcriptional regulator protein comprising a DNA binding domain from the yeast GAL4 protein.

7. The pluripotent stem cell according to any one of the preceding claims, wherein the inducible promoter includes a GAL4 binding site.

8. The pluripotent stem cell according to any one of the preceding claims, wherein the expression construct (i) is for the expression of at least two fusion proteins selected from: - a first fusion protein a comprising a transcriptional activation domain and one of a PYL1 protein; and a PP2C protein; and

- a second fusion protein comprising a DNA binding domain and the other one of a PYL1 protein and a PP2C protein; or

- a first fusion protein comprising a transcriptional activation domain and a PP2C protein; and

- a second fusion protein comprising a DNA binding domain and a PYL1 protein; or

- a first fusion protein comprising a transcriptional activation domain and a PYL1 protein; and

- a second fusion protein comprising a DNA binding domain and a PP2C protein.

9. The pluripotent stem cell according to any one of the preceding claims, wherein the expression construct (i) is for the expression of at least two fusion proteins selected from:

- a first fusion protein comprising a VP16 protein and one of a PYL1 protein and a PP2C protein; and

- a second fusion protein comprising a DNA binding domain from the yeast GAL4 protein and one of a PYL1 protein and a PP2C protein; or

- a first fusion protein comprising a VP16 protein and a PP2C protein; and

- a second fusion protein comprising a DNA binding domain from the yeast GAL4 protein and a PYL1 protein; or

- a first fusion protein comprising a VP16 protein and a PYL1 protein; and

- a second fusion protein comprising a DNA binding domain from the yeast GAL4 protein and a PP2C protein.

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

11 . The pluripotent stem cell according to any one of the preceding claims, wherein the cell is selected from the group consisting of embryonic stem cells, induced pluripotent stem cells, embryonic cell lines, and somatic cell lines.

12. The pluripotent stem cell according to any one of the preceding claims, wherein the pluripotent stem cells are of a livestock or poultry species.

13. The pluripotent stem cell according to claim 4, wherein the livestock species is porcine or bovine.

14. A method for the forward programming of cells comprising: a) culturing a pluripotent stem cell according to any one of the preceding claims in a culture medium; and b) inducing forward programming by culturing the cells in a culture medium in the presence of abscisic acid, optionally wherein the abscisic acid is exogenously added.

15. The method according to claim 14 for the forward programming of cells, wherein the media in a) and b) have the same composition.

16. The method according to claim 14 or 15, wherein the differentiation stage is at most 10 days, at most 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days or 2 days.

17. The method according to any one of claims 14 to 16 wherein the produced cells are muscle cells or adipose cells for human and non-human dietary consumption.

18. Use of a pluripotent stem cell according to any one of claims 1 to 13 or use of the method for producing a cell according to any one of claims 14 to 17 fortissue engineering, optionally for the production of cultivated meat.

19. A food product comprising the pluripotent stem cell according to any one of claims 1 to 13 or a cell obtained by the method according to any one of claims 14 to 17.

20. The food product according to claim 19, wherein the food product is cultivated meat.