WO2013054112A1 - Culture media for pluripotent stem cells - Google Patents

Abstract

This invention relates to growth media for the culture of pluripotent stem cells, in particular embryonic stem (ES) cells, which comprise a β-catenin/P300 antagonist and an Activin/TGFβ ligand. The invention also provides related culture medium supplements, compositions, methods and uses.

Description

Culture Media for Pluripotent Stem Cells

Field of Invention

The invention relates to stem ceil culture media and methods, in particular culture media and methods for expanding populations of pluripotent stem cells, e.g. human embryonic stem (hES) ceils.

Background of invention

There is great interest in culture media and methods for expanding populations of pluripotent stem ceils, particularly hES cells which are especially difficult to culture. Clinical and research applications of pluripotent stem cells require reproducible ceil culture methods to provide adequate numbers of ceils of suitable quality. Numerous different culture media and methods have been tested for pluripotent stem cells, with varying degrees of success. hES cells were originally derived using mouse embryonic fibroblasts (mEFs) as feeder cells (Thomson et al. (1 998) Science 282:1 145-1 147). hES cells are still commonly maintained using human or murine embryonic fibroblasts as feeder cells, or as a source of conditioned medium (or both). The use of feeder cells is

undesirable, because it complicates passaging of the stem ceils (the stem ceils must be separated from the feeder cells at each passage, and new feeder cells are required at each passage). The use of feeder cells can also lead to contamination of the stem ceils with the feeder cells, complicating analysis of the results of any experiments performed on the stem cells. Efforts have been made to identify the extrinsic factors provided by feeder cell layers that are necessary for maintaining hES ceil piuripotency and self-renewal, and some feeder ceil-free hES ceil culture methods have been reported. However, feeder cell-free methods for hES cell culture currently remain of limited application and there remains much uncertainty regarding the necessary extrinsic factors. A problem that often occurs in hES cell culture is that the optimal culture medium for one hES ceil line is sub-optimal or inappropriate for other hES ceil lines (e.g. see Amit et ai. (2004) Biology of Reproduction 70:837-845; Yao et ai. (2006) PNAS 103(18) :69Q7-691 2; Lu et al. (2006) PNAS 1 03(1 5):5688- 5693; and Manneilo et ai. (2007) Stem Cells 25{7) :1603-1 609). Although signalling from the TGFp superfamiiy has been identified as an agent for the maintenance of human p!uripotent ceils, all commercially available iterations have been dependant on interaction with exogenous FGF signalling. It is widely recognised that there remains a great need for further improvements to pluripotent stem cell culture media and methods, in particular for improvements to h ES ceil culture media and methods.

Summary of Invention

The present inventor has found that p-catenin/pSOO antagonists improve pluripotent stem cell culture when used in combination with Activin/TGFp !igands. For example, differentiation of the pluripotent stem cells may be inhibited and the cells allowed to proliferate rapidly whilst maintaining their pluripotency, An aspect of the invention provides a cuiture medium for pluripotent stem cells which comprises a p-caienin/P30G antagonist and an Activin/TGFp iigand.

Another aspect of the invention provides the use of a combination of an β- catenin/p300 antagonist and an Activin/TGFp Iigand in the culture of pluripotent stem ceils.

Another aspect of the invention provides a method for cuituring or expanding pluripotent stem ceils, comprising cuituring a population of pluripotent stem cells in a culture medium which comprises a p-catenin/P30Q antagonist and an Activin/TG Fp Iigand.

Suitable p-catenin/PSGQ antagonists include I D-8 and pharmaceutical equivalents analogs derivatives, salts or prodrugs of I D-8. Suitable Activin/TG Fp iigands include TG Fp superfamiiy ligands, such as Activin A, Activin B, Activin AB, TG FJ31 , TG Fp2, TGFp3, nodal, and GDF 1 -1 5.

Any combination of the p-catenin/P30Q antagonists and Activin/TG Fp ligands iisted above may be employed. In some embodiments, a culture medium of the invention may further comprise one or more additional active ingredients, such as an FGFR agonist, an EGFR agonist, an IGFR agonist, a BMP antagonist or a Wnt agonist, either singly or in combination.

In some embodiments, a culture medium of the invention may further comprise a BMP antagonist.

Suitable BMP antagonists include smail molecules, such as dorsomorphin (CAS: 868405-64-3; 6-[4-[2-(1 -Pipendinyi)ethoxy]phenyi]-3-(4-pyridinyi)-pyrazolo[1 ,5- ajpyrimidine dihydrochloride, as well as noggin, foilistatin, dan, chordin, and gremlin.

Another aspect of the invention provides a cell culture comprising a culture medium according to any of the aspects or embodiments described above and a population of pluripotent stem cells.

Another aspect of the invention provides a cell culture composition containing: (a) a culture medium according to any of the aspects or embodiments described above and an extracellular matrix material. Another aspect of the invention provides a method for producing a culture medium for pluripotent stem ceils comprising;

(a) obtaining a culture medium ; and,

(b) introducing an Activin/TGFp ligand and a p-catenin/P300 antagonist to the culture medium,

thereby producing a culture medium according to any of the aspects or embodiments described above.

A method may further comprise introducing an extracellular matrix material and/or a population of pluripotent stem cells to the medium.

Other aspects as described herein provide culture medium supplements,

compositions and methods of use of a culture medium according to any of the aspects or embodiments described above. Brief Description Of Drawings

Figure 1 shows the morphological appearance of NOTT2 hES cells cultured in medium containing I D-8, Activin A and ΤΘΡβΙ at low density post passage (60,000 ceils/cm²), images at 4x magnification.

Figure 2 shows the morphological appearance of NOTT2 hES ceils cultured in medium containing I D-8, Activin A and TGFpl at packed confluence, images at 4x magnification.

Figure 3 shows the flow cytometry showing percentage of OCT4 expressing cells.

Figure 4 shows the immunocytochemistry showing expression of OCT4, NANOG, SOX2, TRA160, TRA181 and absence of expression of SSEA1 in NOTT2 hES cells cultured in medium containing I D-8, Activin A and TGFpi .

Figure 5 shows the immunocytochemistry showing expression of, AS A (top) β- tubuiin (middle) and SOX17 (bottom) in cells differentiated from NOTT2 hES cells cultured in medium containing ID-8, Activin A and TGFpl using embryoid body formation.

Figure 6 shows the morphological appearance of NOTT2 hES cells cultured in medium containing ! D~S, Activin A, Noggin and bFGF at packed confluence, passage 20. images at 4x magnification.

Figure 7 shows the morphological appearance of NCL5 hES cells cultured in medium containing I D-8, Activin A, Noggin and bFGF at packed confluence, passage 20. images at 4x magnification. Figures 8A to 8F show immunocytochemistry showing expression of OCT4 (A), NANOG (B), SOX2 (G), TRA160 (D), TRA181 (E) and absence of expression of SSEA1 (F) in NOTT2 hES cells cultured in medium containing ID-8, Activin A, Noggin and bFGF for 20 passages. Images at 1 0x magnification. Figure 9 shows the morphological appearance of NOTT2 hES cells cultured in medium containing I D-8, Activin A, Noggin and Wnt3a at packed confluence at passage 13. Images at 10x magnification. Figure 10 shows the morphological appearance of NCL5 hES ceils cultured in medium containing I D-8, Activin A, Noggin and Wnt3a at packed confluence. Images at 10 x magnification.

Figure 1 1 shows the immunocytochernistry showing expression of QCT4, NANOG, SOX2, and absence of expression of SSEA1 in NOTT2 hES ceils cultured in medium containing ID-8, Activin A, Noggin and Wnt3a.

Figure 12 shows the real time PGR expression profiling of NOTT2 hES cells cultured in medium containing ID-8, Activin A, Noggin and Wnt3a and of embryoid bodies differentiated from those ceils.

Figure 13 shows immunocytochernistry showing expression of OCT4, NANOG, SOX2, SSEA4 and TRA1 -80 and absence of expression of SSEA1 in hiPS cells cultured in medium containing I D-8, Activin A and TGFpL Figure 14 shows immunocytochernistry showing expression of β-tubulin, AS A and Foxa2 in ceils differentiated from hi PS cells cultured in medium containing ID-8, Activin A and TGFpl using embryoid body formation.

Detailed Description of Invention

This invention relates to culture media for pluripotent stem ceils and methods for culturing and expanding pluripotent stem cells, as well as related culture medium supplements, cell cultures and compositions and uses thereof.

The culture media for pluripotent stem cells described herein may offer advantages over known culture media in terms of the scalability, reproducibility and/or

robustness of pluripotent stem cell culture. Culture media described herein may be useful in culturing piuripotent stem cells without feeder ceil contact, i.e. they can be used to culture piuripotent stem cells in the absence of a layer of feeder ceils. Culture media described herein may be used to rapidly expand a population of piuripotent stem cells, i.e. they may allow large numbers of cells to be produced in a relatively short time relative to other culture media.

Culture media described herein may be useful in expanding several different types of piuripotent stem cell lines, i.e. different types of piuripotent stem ceil can be cultured using the culture media described herein.

Culture media described herein may be useful in culturing piuripotent stem cells without a step of adapting ceils to the culture medium, as is commonly required when transferring piuripotent stem ceils into known culture media.

As described above, culture media of the invention comprise a beta-catenin/P300 antagonist and an Aciivin/TGFp ligand. -catenin/p300 antagonists reduce, inhibit or block the interaction of β-catenin and P300 in mammalian cells and prevent or inhibit β-catenin from switching its cofactor from CBP to P300. A -catenin/p3G0 antagonist may enhance or promote the β- catenin/CBP interaction. For example, ID-8 acts as an enhancer of the

Wnt/CBP/catenin expression cassette which is associated with the maintenance of pluripotency. Preferred -catenin/p300 antagonists show little or no activity in reducing, inhibiting or blocking the interaction of β-catenin and CBP.

Suitable p-catenin/p300 antagonists include IQ-1 (CAS: 331001 -62-8; Pubchem CID 1 1 152734; 2-[(4-acety!phenyl)diazenyl]-2-(3,3-dimethyl-2

4-dihydro-1 H-isoquinolin-1 -yl)acetamide; Miyabayashi, T., et al. 2007. Proc. Nati.

Acad. Sci. U.S.A. 104: 5668-5673), I D-8 (CAS: 147591 -46-6; 1 -(4- ethoxyphenyl)-2- methyl-3-nitro-1 H-indoi-6-ol ; chemical formula C 6H₁₄N₂04) and pharmaceutical equivalents analogs derivatives, salts or prodrugs of IQ-1 and I D-8. Preferably, culture media of the invention may comprise at least 1 nM, at least 10nM, at least 50nM or at least 100nM beta-catenin/P300 antagonist. Culture media of the invention may comprise up to 1 μ , up to 10ΟμΜ, up to 1 mM or up to 1 M beta- catenin/P300 antagonist. Concentration ranges with any combination of these upper and lower concentrations of beta-catenin/P300 antagonist may be employed in the culture media described herein. For example, 1 nM to 50n , l OnIVS to 50nM 10nM to 500nM, or 50nM to 1 μΜ of beta-catenin/P300 antagonist may be employed in the culture media described herein, in some preferred examples, about 250nM of beta- catenin/P300 antagonist may be employed. In other preferred examples, about 25nM of beta-catenin/P3GQ antagonist may be employed.

Αο.ϊνϊη/ΤΘΡβ ligands stimulate Activin/TGP signalling in mammalian ceils through SMAD2 and/or SIV1AD3 intracellular signalling pathways. Suitable Αοίίνιη/ΤΘΡβ ligands include ligands of the ΤΘΡβ superfami!y, such as Activins, Growth and differentiation factors (GDFs), Anti-muiierian hormone (AMH), Activins, Nodals and TGFps.

Activins bind to Activin Type I I receptors, such as ACVR2A and ACVR2B and include Activin A, Activin B and Activin AB.

Activins are dimers of two peptides, Activin A is a homodimer of inhibin beta A, Activin B is a homodimer of inhibin beta B and Activin AB is a heterodimer of inhibin beta A and inhibin beta B. The database entry for the amino acid sequence of human inhibin beta A (INHBA) Gene I D NO: 3624) is NP_002183.1 Gl :4504699 and the database entry for the amino acid sequence of human inhibin beta B (INHBB; Gene I D NO:3825) is NP_002184.2 Gl :154813204. The sequences of other mammalian activin sequences are available on public databases (e.g. NCBi

Genbank or E BL-EBI Uniprot).

Suitable activins may be obtained as recombinant proteins from commercial sources (for example, R&D Systems, N USA; Sigma-AIdrich Co LLC, MO US) Growth and differentiation factors (GDFs) may include GDF 1 -15. The sequences of mammalian GDF sequences are well known in the art and are available on public databases (e.g. NCBi Genbank or E BL-EBI Uniprot). Suitable GDFs may be obtained as recombinant proteins from commercial sources (for example, R&D Systems, N USA; Sigma-A!dhch Co LLC, MO US)

Nodal binds to activin A receptor, type I IB ACVR2B. Nodal may then either form a receptor complex with activin A receptor, type IB (ACVR1 B) or with activin A receptor, type !C (ACVR1 C). The database entry for the amino acid sequence of human Nodal (Gene ID NO: 4838) is NP Q8Q525.3 GL222352098. The sequences of other mammalian Noda! sequences are available on public databases (e.g. NCBi Genbank or EMBL-EBI Uniprot). Suitable Nodal may be obtained as a recombinant protein from commercial sources (for example, R&D Systems, MN USA; Sigma-Aldrich Co LLC, MO US).

TGF s bind to TGF-beta receptor type-2 (TGFBR2) and may include TGF 1 , TGF 2, and TGF 3. The database entry for the amino acid sequence of human TGFpi (Gene ID NO: 7040) is NP_000651 .3 Gl : 63025222; the database entry for the amino acid sequence of human ΤΘΡβ2 (Gene I D NO: 7042) is N P_001 129071 .1 Gi : 208022853 and the database entry for the amino acid sequence of human TGFp3 (Gene ID NO:7043) is NP_003230.1 Gl :4507465. The sequences of other TGFP sequences are available on public databases (e.g. NCBI Genbank or EMBL- EBi Uniprot).

Suitable TGF s may be obtained as a recombinant protein from commercial sources (for example, R&D Systems, MN USA; Sigma-Aldrich Co LLC, MO US Preferably, the Activin/TGFP ligand is Activin A.

In some embodiments, culture media as described herein may comprise two or more Activin/TGFp ligands, such as Activin and ΤΟΡβΙ . Culture media of the invention comprise at least 0.01 ng/ml, at least 0.1 ng/mi or at least 1 ng/ml of an Activin/TGF ligand, such as Activin or TGFp. Culture media of the invention comprise up to 100 ng/ml, up to 500 ng/ml or up to 1 mg./ml of an Activin/TGF ligand, such as Activin or TGF .

Concentration ranges with any combination of these upper and lower concentrations of Activin/TGF ligand may be employed in the culture media described herein. For example, 0.1 ng/ml to 1 mg/mi or 1 ng/ml to 1 00 ng/ml of activin, preferably about 10ng/ml activin may be employed in the culture media described herein. 0.02 ng/ml to 200 ng/ml or 0.2ng/ml to 20 ng/ml of TGFp, preferably about 2ng/ml may be employed in the culture media described herein.

Optionally, culture media of the invention may further comprise other active factors, such as agonists of IGF, EGF, FGF, or Wnt signalling pathways or antagonists of the BMP pathway. Suitable factors are described in more detail below.

A culture media as described herein may further comprise a BMP antagonist.

Suitable BMP antagonists include noggin, foliistatin, gremlin, dan, and chordin, and small molecules, such as dorsomorphin.

BMP antagonists are well known in the art and available from commercial sources (for example, R&D Systems, MN USA; Sigma-Aldrich Co LLC, MO US)

Cell culture media typically contain a large number of ingredients, which are necessary to support maintenance of cultured ceils. Culture media as described herein will therefore normally contain many other ingredients in addition to the Activin/TGF ligand and p-catenin/P300 antagonist. Suitable combinations of ingredients may readily be formulated by the skilled person, taking into account the disclosure herein.

Culture media according to the invention will generally be a nutrient solution comprising standard cell culture ingredients, such as amino acids, vitamins, inorganic salts, a carbon energy source, and a buffer, as described in more detail below. Culture media according to the invention may comprise one or more amino acids. The skiiied person understands the appropriate types and amounts of amino acids for use in stem cell culture media, Amino acids which may be present include L- alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-pro!ine, L~serine, L-Ehreonine, L-tryptophan, L-tyrosine, L-vaiine and combinations thereof. Some culture media will contain ail of these amino acids. Generally, each amino acid when present is present at about 0.001 to about 1 g/L of medium (usually at about 0.01 to about 0.15 g/L), except for L-g!utamine which is present at about 0.05 to about 1 g/L (usually about 0.1 to about 0.75 g/L). The amino acids may be of synthetic origin.

Culture media according to the invention may comprise one or more vitamins. The skilled person understands the appropriate types and amounts of vitamins for use in stem cell culture media. Vitamins which may be present include thiamine (vitamin B1 ), riboflavin (vitamin B2), niacin (vitamin B3), D-calcium pantothenate (vitamin B5), pyridoxai/pyridoxamine/pyridoxine (vitamin B6), folic acid (vitamin B9),

cyanocobaiamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL- alpha tocopherol (vitamin E), biotin (vitamin H) and menadione (vitamin K).

Culture media according to the invention may comprise one or more inorganic salts. The skilled person understands the appropriate types and amounts of inorganic salts for use in stem cell culture media. Inorganic salts are typically included in culture media to aid maintenance of the osmotic balance of the cells and to help regulate membrane potential, Inorganic salts which may be present include salts of calcium, copper, iron, magnesium, potassium, sodium, zinc. The salts are normally used in the form of chlorides, phosphates, sulphates, nitrates and bicarbonates. Specific salts that may be used include CaCI₂, CuS0₄-5H₂0, Fe(N0₃)-9H₂0, FeS0₄-7H₂0, gCi, MgSCvi, KCi, NaHC0₃, NaCi, Na₂HP0 , Na₂HP0₄-H₂0 and ZnS0₄-7H₂0. The osmoiarity of the medium may be in the range from about 200 to about 400 mOsm/kg, or in the range from about 250 to about 350 mOsm/kg. Culture media according to the invention may comprise a carbon energy source, in the form of one or more sugars. The skilled person understands the appropriate types and amounts of sugars to use in stem ceil culture media. Sugars which may be present include glucose, galactose, maltose and fructose. The sugar is preferably glucose, particularly D-giucose (dextrose). A carbon energy source will normally be present at between about 1 and about 10 g/L

Culture media according to the invention may comprise a buffer. A suitable buffer can readily be selected by the skilled person. The buffer may be capable of maintaining the pH of the culture medium in the range about 6.5 to about 7.5 during normal culturing conditions, most preferably around pH 7.0. Buffers that may be used include carbonates (e.g. NaHC0₃), chlorides (e.g. CaC!₂), sulphates (e.g.

gS0₄) and phosphates (e.g. NaH₂P0₄). These buffers are generally used at about 50 to about 500 mg/i. Other buffers such as N~[2-hydroxyethy!]~piperazine~N'-[2- ethanesul-phonic acid] (HEPES) and 3-[N-morpho!ino]-propanesulfonic acid (MOPS) may aiso be used, normally at around 1000 to around 10,000 mg/l.

In some embodiments, culture media according to the invention may contain serum. Serum obtained from any appropriate source may be used, including foetal bovine serum (FBS), goat serum or human serum. Preferably, human serum is used. Serum may be used at between about 1 % and about 30% by volume of the medium, according to conventional techniques.

In other embodiments, culture media according to the invention may contain a serum replacement. Various different serum replacement formulations are commercially available and are known to the skilled person. Where a serum replacement is used, it may be used at between about 1 % and about 30% by volume of the medium, according to conventional techniques. in other embodiments, culture media according to the invention may be serum-free and/or serum replacement-free. A serum-free medium is one that contains no animal serum of any type. Serum-free media may be preferred to avoid possible xeno- contamination of the stem cells. A serum replacement-free medium is one that has not been supplemented with any commercial serum replacement formulation. Culture media according to the invention may comprise cholesterol or a cholesterol substitute. Cholesterol may be provided in the form of the HDL or LDL extract of serum. Where the HDL or LDL extract of serum is used, it is preferably the extract of human serum. The optimal amount of choiestero! or cholesterol substitute can readily be determined from the literature or by routine experimentation.

Culture media according to the invention may further comprise transferrin or a transferrin substitute. Transferrin may be provided in the form of recombinant transferrin or in the form of an extract from serum. Preferably, recombinant human transferrin or an extract of human serum is used. An iron chelate compound may be used as a transferrin substitute. Suitable iron chelate compounds are known to those of skill in the art, and include ferric citrate chelates and ferric sulphate chelates. The optimal amount of transferrin or transferrin substitute can readily be determined from the literature or by routine experimentation.

Culture media according to the invention may further comprise albumin or an albumin substitute, such as bovine serum albumin (BSA), human serum albumin (HSA), recombinant albumin (RA), a plant hydroiysate (e.g. a rice or soy

hydroiysate). The optimal amount of albumin or albumin substitute can readily be determined from the literature or by routine experimentation.

Culture media according to the invention may further comprise insulin or an insulin substitute. Natural or recombinant insulin may be used. A zinc-containing compound may be used as an insulin substitute, e.g. zinc chloride, zinc nitrate, zinc bromide or zinc sulphate. The optimal amount of insulin or insulin substitute can readily be determined from the literature or by routine experimentation. In some embodiments, a culture medium as described herein may comprise insulin at about 10pg/mL Culture media according to the invention may comprise progesterone, putrescine, and/or selenite. if selenite is present, it is preferably in the form of sodium selenite. The optimal amount of these ingredients can readily be determined from the literature or by routine experimentation. Culture media according to the invention may comprise one or more additional nutrients or growth factors that have previously been reported to benefit p!uripotent stem cell culture. For example, a culture medium may comprise fibroblast growth factor (FGF), leukemia inhibitor factor (UF), epidermal growth factor (EGF), insulin growth factor (IGF), platelet derived growth factor (PDGF) connective tissue growth factor (GTGF) ciliary neurotrophic factor (CNTF), interleukin 6 (!L-6) or stem cell factor (SCF). Antibodies or other ligands that bind to the receptors for such substances may also be used. Any form of FGF suitable for pluripotent stem cell culture may be used, e.g. basic FGF (bFGF; FGF-2), FGF-4, or homoiogs or analogs thereof.

Suitable concentrations of nutrients or growth factors are well known in the art and may be optimised for particular culture conditions or cell types. Typically, 50pg/ml to 100Mg/m! may be employed.

Some examples of the culture media described herein may lack factors or

ingredients which stimulate the Wnt and/or FGF signalling cascades. For example, some examples of culture media as described herein may lack FGF and/or Wnt3a. Other examples of the culture media described herein may include Wnt and/or FGF in addition to the p-catenin/p300 antagonist and the Activin/TGFp ligand.

Culture media according to the invention may comprise one or more trace elements, such as ions of barium, bromium, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc and/or aluminium.

Culture media according to the invention may comprise phenol red as a pH indicator, to enable the status of the medium to be easily monitored {e.g. at about 5 to about 50 mg/iitre).

Culture media according to the invention may comprise a reducing agent, such as β- mercaptoethanol, for example at a concentration of about 0.1 mM. Suitable concentrations of the cuiture media components described above are well known in the art and may be optimised for particular cell types using standard laboratory procedures. Culture media according to the invention may be generated by modification of existing cell culture media. Suitable culture media that may be used for piuripotent stem cell culture are well known in the art. Potentially suitable ceil cuiture media are available commercially, and include Dulbecco's Modified Eagle Media (D EM), Minimal Essential Medium (MEM), Knockout-DMEM (KG-DMEM), Glasgow Minima! Essential Medium (G-MEM), Basal Medium Eagle (BME), DMEM/Ham's F12, Advanced DMEM/Ham's F1 2, Iscove's Modified Dulbecco's Media and Minimal Essential Media (MEM) ,

Preferred culture media are described in the Examples herein.

The specific ingredients of the culture media, supplements and compositions as described herein can vary according to particular needs and applications. Likewise, the precise steps of the methods as described herein can vary according to particular needs and applications. The culture media, supplements, methods, compositions and uses according to this invention may be optimised by routine experimentation. For example, if a culture medium, supplement or composition fails to give the desired level of piuripotent stem ceil expansion, variables such as the amount of each ingredient in the cuiture medium or supplement, seeding densities, cuiture conditions, cuiture periods, etc. can be altered in further experiments.

The amount of each of the ingredients described herein may be optimised independently of the other ingredients by routine optimisation or one or more ingredients can be added or removed. A culture medium may be tested for its ability to support expansion of piuripotent stem ceils by testing it alongside or in place of a known culture medium or method.

The cuiture media, supplements, methods, compositions and uses as described herein are described in more detail below. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of ceii culture, molecular biology and microbiology, which are within the skill of those working in the art.

Numerous textbooks are available that provide guidance on mammalian ceil culture media and methods, including textbooks dedicated to culture media and methods for hES cells. Such textbooks include 'Basic Cell Culture Protocols' by J. Pollard and J. . Walker (1997), 'Mammalian Cell Culture: Essentia! Techniques' by A. Doyle and J. B. Griffiths (1997), 'Human Embryonic Stem Cells' by A. Chiu and M. Rao (2003), 'Culture of Animal Cells: A Manual of Basic Technique' by R. I. Freshney (2005), 'Basic Cell Culture Protocols' by C. Heigason and C. L. Miller (2005), 'Stem Cells: From Bench to Bedside' by A. Bongso (2005) and 'Human Embryonic Stem Ceil Protocols' by K. Turksen (2006).

Pluripotent stem cells and cell culture reagents and apparatus suitable for use in the invention are available commercially, e.g. from Cell Guidance Systems Ltd

(Cambridge, UK), , GIBCO™ (Invitrogen), Miilipore Corporation (Bilierica,

Massachusetts), Sigma™ (St. Louis, Missouri), Merck (Nottingham, UK) and Enzo Life Sciences (Exeter, UK). Cell culture media as described herein may be formulated in deionized, distilled water. A culture medium as described herein will typically be sterilized prior to use to prevent contamination, e.g. by ultraviolet light, heating, irradiation or filtration. The culture medium may be frozen {e.g. at -20 °C or -80 °C) for storage or transport. The medium may contain one or more antibiotics to prevent contamination. in some embodiments, cell culture media of the invention may be conditioned media. A conditioned medium is produced by culturing a population of ceils in a culture medium for a time sufficient to condition the medium, then harvesting the conditioned medium. Conditioned medium contains growth factors, cytokines and other nutrients secreted by the conditioning ceils that support growth of stem cells. Where a conditioned medium is used, the medium may be conditioned on mammalian cells, e.g. mouse ceils or human cells. Various different types of mammalian cells may be used to produce conditioned medium suitable for pluripotent stem cell culture, including mouse embryonic fibroblasts (mEF), human foreskin ceils and human fallopian epithelial cells. Preferably, mEF cells are used. Conditioned medium may be prepared by well known methods, e.g. by cuituring mEFs and harvesting the culture medium after an appropriate time (e.g. ~1 day at 37°G). The cells used to condition a medium may be irradiated or treated with a substance (e.g. mitomycin C) to prevent their proliferation.

An appropriate cuituring time to condition a medium may be estimated by the skilled person, based on known methods. Alternatively, the time required to condition the medium can be determined by assessing the effect of the conditioned medium on piuripotent stem ceil growth and differentiation. The conditioning time can be altered after assessing the effect of the conditioned medium on stem cell growth and differentiation. Typically, a medium will be conditioned for between about 1 and about 72 hours, such as between about 4 hours and about 48 hours, or between about 4 hours and about 24 hours, at 37 °C.

The period over which a conditioned medium can support piuripotent stem ceil expansion may likewise be estimated by the skilled person, based on known methods, or may be assessed experimentally. The period before replacement or exchange of conditioned medium can therefore be altered after assessing the effect of a conditioned medium on stem eel! growth and differentiation. Conditioned medium is typically used to support ceil growth for between about 6 hours and about 72 hours, such as between about 12 hours and about 56 hours, e.g. for about 24-36 hours or for about 24-48 hours, before replacement or exchange with a further batch of conditioned medium.

In other embodiments, cell culture media of the invention may be fresh culture media. A fresh medium is a medium that has not been conditioned. A fresh medium may be preferred, because such a medium may be chemically defined (i.e. all of the ingredients in the medium and their concentrations may be known), in contrast to a conditioned medium (which is not fully defined because the conditioning cells alter the composition of the medium, and because of batch-to-batch variations). For example, the ceil culture media may be chemically defined media. In other embodiments, the culture medium is a mixture of a fresh medium and a conditioned medium. When a conditioned medium and a fresh medium are mixed, the conditioned medium and the fresh medium may be of the same type or may be of different types. The use of a mixture of a conditioned medium and a fresh medium of different types may be preferred, as it may provide a more complex nutrient mixture that is of further benefit to pluripotent stem cells in culture.

A culture medium may be a 1 x formulation or a concentrated formulation, e.g. a 2x to 250x concentrated medium formulation, !n a 1 x formulation each ingredient in the medium is at the concentration intended for cell culture, in a concentrated formulation one or more of the ingredients is present at a higher concentration than intended for cell culture. Concentrated culture media is well known in the art. Culture media can be concentrated using known methods e.g. salt precipitation or selective filtration. A concentrated medium may be diluted for use with wafer (preferably deionized and distilled) or any appropriate solution, e.g. an aqueous saline solution, an aqueous buffer or a culture medium.

Cell culture media of the invention may be capable of expanding a population of pluripotent stem ceils in a pluripotent, undifferentiated and proliferative state for at least 3 passages under appropriate culture conditions. Stem ceils are considered to be in a pluripotent, undifferentiated and proliferative state if they exhibit certain characteristics as described in more detail elsewhere herein. Appropriate conditions can be selected by the skilled person from those normally used for pluripotent stem ceil culture.

Preferably, a culture medium may be capable of expanding a population of stem ceils in a pluripotent, undifferentiated and proliferative state for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 1 0, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 passages under appropriate conditions.

A culture medium may be capable of expanding a population of pluripotent stem ceils in a piuripotent, undifferentiated and proliferative state for more than 3 passages, more than 4 passages, more than 5 passages, more than 1 0 passages, more than 15 passages, more than 20 passages, more than 25 passages, more than 30 passages, more than 40 passages, more than 50 passages, or more than 100 passages. Accordingly, in some embodiments of the methods described herein, the stem celis may be cultured in a pluripotent, undifferentiated and proliferative state for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 80, at least 70, at least 80, at least 90 or at least 100 passages under appropriate conditions in a medium described herein.

A culture medium described herein may be capable of culturing or expanding at least 2, at least 3, at least 4, at least 5, at least 8, at least 7, at least 8, at least 9, or at least 10 different pluripotent stem ceil lines {e.g. different human ESC lines) in a pluripotent, undifferentiated and proliferative state for multiple passages under appropriate conditions. For example, a culture medium may be capable of expanding at least the NOTT2 and NCL5 hES cell lines (see elsewhere herein) in a pluripotent, undifferentiated and proliferative state for at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 1 0, passages under appropriate

conditions.

Another aspect of the invention provides a hermetically-sealed vessel containing a cuiture medium as described herein. Hermetically-sealed vessels may be preferred for transport or storage of the culture media, to prevent contamination. The vessel may be any suitable vessel, such as a flask, a plate, a bottle, a jar, a vial or a bag.

The cuiture media described herein can be used to expand a population of pluripotent stem ceils. Accordingly, other aspects of the invention relate to the use of the culture media described herein for expanding a population of pluripotent stem ceils.

Another aspect of the invention provides a method for culturing or expanding a population of pluripotent stem cells, comprising: culturing a population of pluripotent stem cells in a culture medium described herein.

Expansion of an initial population of pluripotent stem cells increases the number of stem cells in the population to generate an expanded population, whilst maintaining pluripotency (i.e. without significant differentiation of the cells in the population). In other words, expansion of a population comprises both growth and division of stem ceils, but not differentiation. A method for culturing or expanding a population of pluripotent stem cells may comprise;

culturing an initial population of pluripotent stem cells with a culture medium described herein to produce an expanded population of the pluripotent stem ceils.

A method described herein may comprise culturing pluripotent stem cells in contact with an extracellular matrix material as described elsewhere herein. For example, a method for culturing or expanding a population of pluripotent stem cells may comprise:

culturing population of pluripotent stem cells in a culture medium described herein, wherein the cells are in contact with an extracellular matrix material. Another aspect of the invention provides the use of a culture medium as disclosed herein and an extracellular matrix material to expand a population of pluripotent stem ceils.

A method described herein may comprise a step of passaging pluripotent stem cells into a culture medium as disclosed herein. For example, a method for culturing or expanding a population of pluripotent stem ceils may comprise:

(a) providing a population of pluripotent stem ceils and a culture medium as described above;

(b) culturing the population in a first portion of said culture medium;

(c) passaging the cells into a second portion of said culture medium; and

(d) further culturing the ceils in the second portion of said culture medium.

A method may further comprise:

(e) passaging the cells into a further portion of said culture medium ; and (!) further cuituring the cells in the further portion of said culture medium. Steps e) and f) may be repeated one, two, three or more times, as required.

It will be appreciated that the steps of the methods disclosed herein may be performed in any suitable order or at the same time, as appropriate, and need not be performed in the order in which they are listed. For example, in the above method the step of providing a population of p!uripotent stem cells may be performed before, after or at the same time as, the step of providing a culture medium. Techniques for passaging cells are well known in the art e.g. cells may be incubated with trypsin and EDTA for between 5 seconds and 15 minutes at 37°C. A trypsin substitute (e.g. TrypLE from invitrogen) may be used, if desired. Coilagenase, dispase, accutase or other known reagents may also be used to passage the cells. Passaging is typically required every 2-8 days, such as every 4-7 days, depending on the initial seeding density. In some embodiments, the cell culture methods as described herein do not comprise any step of manually selecting undifferentiated ceils when the cells are passaged, in some embodiments, the cell culture methods as described herein comprise automated passaging of the stem ceils, i.e. without manipulation by a laboratory worker, in some embodiments, the cell culture methods as described herein comprise cuituring the cells without contact to an adherent surface or use of a ceil culture substrate by continued maintenance in suspended state.

The pluripotent stem cells will be seeded onto a support at a density that promotes ceil proliferation but which limits differentiation. Typically, a plating density of at least 15,000 cells/cm² is used. A plating density of between about 1 5,000 cells/cm² and about 200,000 ceils/cm² may be used. Single-ceil suspensions or small cluster of ceils will normally be seeded, rather than large clusters of cells, as in known in the art.

The inventor has found that the culture media disclosed herein may be used to expand pluripotent stem cells without the need to adapt the cells to the culture medium, as is common!y required when transferring stem ceils into a new culture medium. Various different methods for adapting cell cultures to new media are known in the art.

Accordingly, in some embodiments, a method as described herein may not include any step of adapting a population of stem ceils to a new culture medium, e.g. by gradually changing the components of the medium. A method for culturing or expanding a population of pluripofent stem ceils as described herein may therefore comprise:

(a) providing a population of p!uripotent stem celis;

(b) providing a first cuiture medium;

(c) culturing the ceils in the first cuiture medium;

(d) providing a second culture medium,

(e) replacing or exchanging the first culture medium with the second cuiture medium; or passaging the celis from the first culture medium into the second cuiture medium; and

(f) further culturing the cells in the second cuiture medium.

The second medium is a culture medium as described herein and the method does not comprise any step of adapting the population of stem cells to the second culture medium.

The first medium may be a culture medium as described herein or a different culture medium. The methods and uses as described herein may involve any cuiture medium or supplement as described herein. In some embodiments, the methods as described herein may be serum-free and/or serum replacement-free methods. In some embodiments, pluripotent cells may be cultured in the absence of contact with a layer of feeder celis.

Any suitable cell culture vessel may be used as a support in the methods described herein. Suitable cell culture vessels of various shapes and sizes (e.g. flasks, single or multiwell plates, single or muitiwe!l dishes, bottles, jars, vials, bags, bioreactors) and constructed from various different materials {e.g. plastic, glass) are well-known in the art and may be readily selected by the skilled person.

Another aspect of the invention provides a culture medium supplement for use in producing a culture medium as disclosed herein.

A 'culture medium supplement' is a mixture of ingredients that cannot itself support pluripotent stem ceils, but which enables or improves p!uripotent stem ceil culture when combined with other cell culture ingredients. The supplement may therefore be combined with other ceil culture ingredients to produce a functional ceil culture medium as described herein. The use of culture medium supplements is well known in the art.

A culture medium supplement may comprise a p-catenin/P30G antagonist and an Activin/TGFP ligand. The supplement may comprise any combination of the β- catenin/P300 antagonists and Activin/TGF iigands which are disclosed herein. The supplement may also comprise one or more additional cell culture ingredients as disclosed herein. For example, the supplement may comprise one or more cell culture ingredients selected from the group consisting of amino acids, vitamins, inorganic salts, carbon energy sources and buffers.

A culture medium supplement may be a concentrated liquid supplement (e.g. a 2x to 250x concentrated liquid supplement) or may be a dry supplement. Both liquid and dry supplements are well known in the art.

A supplement may be iyophilised.

A supplement as described herein will typically be sterilized prior to use to prevent contamination, e.g. by ultraviolet light, heating, irradiation or filtration. A culture medium supplement may be frozen (e.g. at -20°C or -80°C) for storage or transport.

Cell culture media and supplements disclosed herein may be useful in expanding populations of pluripotent stem cells. Another aspect of the invention provides a cell culture comprising: (a) a culture medium as described above; and (b) a population of pluripotent stem cells.

Feeder cell layers are often used to support the culture of pluripotent stem cells, and to inhibit their differentiation. A feeder cell layer is generally a monolayer of ceils that is co-cultured with, and which provides a surface suitable for growth of, the pluripotent cells of interest. The feeder ceil layer provides an environment in which the ceils of interest can grow. Feeder cells may be mitoticaiiy inactivated {e.g. by irradiation or treatment with mitomycin C) to prevent their proliferation.

As noted elsewhere herein, the culture media as described herein are particularly advantageous because they can be used to culture cells without feeder cell contact, i.e. the methods as described herein do not require a layer of feeder cells to support the stem cells.

Accordingly, a cell culture comprising a culture medium and a population of pluripotent stem celis as described above may be feeder ceil-free (i.e. devoid of feeder cells). A composition is generally considered to be feeder ceil-free if the pluripotent stem ceils in the composition have been cultured for at least one passage in the absence of a feeder ceil layer.

A feeder cell-free composition as described herein will normally contain less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1 % feeder cells (expressed as a % of the total number of celis in the composition).

In some embodiments, an extracellular matrix material may be used to substitute for the supportive function of a feeder ceil layer. Another aspect of the invention therefore provides a cell culture composition containing: (a) a culture medium as described above; (b) an extracellular matrix material and optionally (c) a population of pluripotent stem ceils. Suitable extracellular matrix materials for pluripotent stem ceil culture are well-known in the art and may, for example, include laminin, fibronectin, vitronectin, collagen (particularly coiiagen Π, collagen 111 or collagen IV), thrombospondin, osteonectin, secreted phosphoprotein 1 , heparan sulphate, dermatan sulphate, gelatine, merosin, tenasin, decorin, entactin and a basement membrane preparation from Engelbreth- Holm-Swarm (EHS) mouse sarcoma ceils (e.g. atrigel®; Becton Dickenson).

Synthetic extracellular matrix materials, such as ProNectin (Sigma Z378666), may also be used. Mixtures of different extracellular matrix materials may be used, if desired.

The extracellular matrix material will normally be coated onto a ceil culture vessel, but may (in addition or alternatively) be supplied in solution. Suitable extracellular matrix material may comprise commercially available substrates such as Pluripro Matrix™ (Cell Guidance Systems, Cambridge UK) or CeiiStart™ (Invitrogen, Paisley, UK).

The compositions as described herein may comprise serum, or may be serum-free and/or serum-replacement free, as described elsewhere herein. The culture media and methods disclosed herein are useful for expanding a population of pluripotent stem ceils, whilst maintaining the pluripotency of the cells and without problematic differentiation of the cells.

'Pluripotent' stem cells are those that have the potential to differentiate into ceils of all three germ layers (endoderm, mesoderm and ectoderm) under appropriate conditions, but not extraembryonic tissue. Pluripotent stem ceils are not totipotent, i.e. they cannot form an entire organism, such as a foetus.

Pluripotent stem cells may be obtained using well-known methods (see below). Various types of pluripotent stem cells may be used as described herein, whether obtained from embryonic, foetal or adult tissue (see for example, Thomson et ai 1998 Science 282 1061 -62; Mali et a! 2008. Stem Cells 26 8 1998-2005; Jiang ef al 2002 Nature 418 (6893) 41 -49) . Pluripotent stem cells may be cloned directly from an organism for use pluripotent stem cells, but established pluripotent stem eel! lines will typically be used.

Accordingly, in some embodiments, an initial population of pluripotent stem ceil lines may be the progeny of previously-isolated pluripotent stem cell lines or may be the progeny of an established pluripotent stem ceil line, such that the use of tissue samples is avoided.

The culture media disclosed herein may be used to culture mammalian pluripotent stem ceils, particularly primate pluripotent stem ceils, such as primate embryonic stem cells. Primate pluripotent stem ceils that may be used in conjunction with the invention include human, Rhesus monkey and marmoset stem cells. Pluripotent ceils may include embryonic, foetal or adult stem cells and induced pluripotent stem (IPS) cells. In preferred embodiments, the pluripotent stem cells are human induced pluripotent stem (IPS) cells.

Primate embryonic stem ceils that may be used in conjunction with the invention include human, Rhesus monkey and marmoset embryonic stem cells. Mouse embryonic stem cells may also be used, in preferred embodiments, the embryonic stem cells are human embryonic stem (hES) cells.

ES ceils are prepared from the inner ceil mass (I CM) of a mammalian blastocyst using known techniques. For example, human ES cells can be obtained using the methods described in Thomson et a!. (1998) Science 282:1 145-1 147, Thomson et al. (1998) Curr. Top. Dev. Biol. 38:133 and US patent 5,843,780. in some embodiments, the initial population of hES cells may be the progeny or descendents of previously-isolated hES ceils or may be the progeny or descendents of an established line of hES cells. The initial population of hES ceils may be the progeny or descendents of cells or cell lines which were obtained using a method that did not involve human embryos.

Suitable hES lines include NOTT2 and NCL5 hES cell lines, which are described in more detail in the Examples. The culture media disclosed herein may aiso be used to culture pluripotent stem ceils that have been produced, reprogrammed or formed from non-p!uripotent cells. These pluripotent stem cells may be genetically modified cells, such as human induced pluripotent stem' (IPS) cells, as shown in Example 4. Alternatively methods of reprogramming have been described which may achieve p!uripotency without genetic modifications, for example by utilising modified RNA (Warren ef al (2010) Cell Stem Cell 7 5 618-630) or recombinant proteins (Zhou et al 2009 Ceil Stem Cell 4 (5) 381 -384). IPS ceils are typically derived from adult somatic ceils by introducing certain key pluripotency genes, in particular genes encoding transcription factors e.g. by transduction of mouse or human fibroblasts with four transcription factors: Oct- 3/4, Sox2, KLF4 and c-Myc (e.g. see Takahashi et ai, (2007) Cell 131 (5):861 -72; Yu et ai. (2007) Science 318(5858):1917-1920; and Yamanaka S. (2008) Cell Proiif. 41 Suppl 1 :51 -6). When a culture medium as described herein is used to expand a population of pluripotent stem cells, the total number of undifferentiated, pluripotent stem cells in the population may increase at least 1 .5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 1 0 fold, at least 20 fold, at least 30 fold, at least 40 fold or at least 50 fold, between the time when the initial cell population is exposed to the culture medium and the end of the culture period.

It will be appreciated that the pluripotent stem ceils may be passaged one or more times during the culture period, after which the cells may be cultured in different cell culture vessels or cells may be discarded, if pluripotent stem cells are cultured in different cell culture vessels after passaging, or if cells are discarded during passaging, this may be taken into account when calculating the fold difference in cell numbers obtained during a known culture period, as described above.

A 'population' of ceils may be any number of cells greater than 1 , but is preferably at least 1 xl 0 cells, least 1 x10² cells, at least 1 x10³ cells, at least 1 xl 0⁴ cells, at least 1 x10⁵ ceils, at least 1 x10⁶ ceils, at least 1 x10⁷ ceils, at least 1 x1 0^s cells, or at least 1 x10⁹ cells. In an initial population of plunpotent stem ceils, at least 50%, at least 55%, at ieast 60%, at least 70%, at Ieast 80%, at Ieast 85%, at Ieast 90%, at ieast 91 %, at Ieast 92%, at Ieast 93%, at Ieast 94% or at ieast 95%, of the stem cells {% by cell number) may be undifferentiated, plunpotent and proliferative cells.

In an expanded population of plunpotent stem cells (i.e. the population after expansion of the initial population using a culture medium or method as disclosed herein), at Ieast 50%, at Ieast 55%, at ieast 60%, at Ieast 70%, at Ieast 80%, at ieast 85%, at Ieast 90%, at Ieast 91 %, at ieast 92%, at Ieast 93%, at Ieast 94% or at Ieast 95%, of the stem cells (% by cell number) may be undifferentiated p!uripotent and proliferative ceils.

Methods for identifying undifferentiated, piuripotent and proliferative stem cells and for identifying the % of such ceils in a population are well-known in the art.

For example, piuripotent stem cells may be identified by their ability to differentiate into cells of all three germ layers e.g. by determining the ability of the cells to differentiate into cells showing detectable expression of markers specific for all three germ layers. Stem cells may be allowed to form embryoid bodies in vitro, and then the embryoid bodies studied to identify cells of all three germ layers. Alternatively, stem cells may be allowed to form teratomas in vivo (e.g. in 8CI D mice), then the teratomas studied to identify cells of ail three germ layers. Accordingly, in preferred embodiments at Ieast 50%, at ieast 55%, at least 60%, at least 70%, at least 80%, at Ieast 85%, at Ieast 90%, at ieast 91 %, at least 92%, at Ieast 93%, at Ieast 94% or at Ieast 95%, of the stem ceils in an expanded population (or in an initial population) may be capable of differentiating into cells of all three germ layers in vitro or in vivo. The genomic integrity of piuripotent stem ceils may be confirmed by karyotype analysis. Piuripotent stem ceils may be karyotyped using known methods. A norma! karyotype is where all chromosomes are present {i.e. eupioidy) with no noticeable alterations. For example, at Ieast 50%, at Ieast 55%, at least 60%, at Ieast 70%, at Ieast 80%, at Ieast 85%, at least 90%, at ieast 91 %, at Ieast 92%, at ieast 93%, at Ieast 94% or at Ieast 95%, of the piuripotent stem cells in a population may exhibit normal karyotypes. Piuripotent stem cells may be identified via phenotypic markers. Stem cell markers (both intracellular and extracellular) may be detected using known techniques, such as immunocytochemistry, flow cytometry (e.g. fluorescence-activated cell sorting) and reverse transcription-PCR (RT-PCR). For example, hES cells may be identified via detection of hES cell markers, such as OCT-4, stage-specific embryonic antigen 3 (SSEA-3), stage-specific embryonic antigen 4 (SSEA-4), tumour-rejecting antigen 1 -60 (TRA-1 -60) and tumour-rejecting antigen 1 -81 (TRA-1 -81 ). In some

embodiments, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94% or at least 95%, of the piuripotent stem cells in a population may express OCT-4, SSEA-3, SSEA-4, TRA-1 -60 and/or TRA-1 -81 at levels appropriate for hES cells.

For example, in a population, at least 50%, at least 55%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94% or at least 95%, of the piuripotent stem cells may (i) have the ability to differentiate into cells of all three germ layers in vitro or in vivo (ii) exhibit normal karyotypes; and/or (iii) express the markers OCT-4, NANOG, SOX2 TRA-1 -60 and TRA-1 -81 at levels appropriate for hES cells. Undifferentiated, piuripotent and proliferative stem cells may also be identified by their morphological characteristics. Undifferentiated, piuripotent and proliferative stem cells are readily recognisable by those of skill in the art. For example, in a normal microscope image, hPS ceils typically have high nuclear/cytoplasmic ratios and prominent nucleoli, when dense they pack together in a colony format or monolayer format as small oval entities without clear demarcation of the cell-cell boundary.

References in the singular (e.g. to "a cell" and equivalent references) encompass the plural (e.g. "ceils") unless the context requires otherwise.

The term "comprising" encompasses "including" as well as "consisting of" e.g. a culture medium or supplement "comprising" a specific ingredient may consist exclusively of that specific ingredient or may include one or more additional substances. The term "about" in relation to a numerical value x means, for example, x ±10% or x ±5%. Where necessary, the term "about" may be omitted. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to ail aspects and embodiments which are described. The above disclosure includes all combinations of the described features and culture media, supplements, methods and uses according to embodiments of the invention encompass ail of these combinations of features.

Ail documents mentioned in this specification are incorporated herein by reference in their entirety. Various aspects and embodiments as described herein will now be described in more detail by way of example, with reference to specific culture media and methods. The following examples are provided to illustrate the invention, and do not limit the scope of the preceding description or the following claims. EXAMPLES

The experimental data set out below shows that ! D-8 and Activin/TGFp may act in concert to improve the robustness, scalability, viability and ease of pluripotent stem ceil culture. Pluripotent stem cells may be transferred to the new culture system without a step of adaptation, as is often required. The experiments also demonstrate the ability to generate high quality of culture as evidenced by the high purity evidenced by OCT4 marker expression by flow cytometry. Multiple different formulations may be applied to culture pluripotent ceils when pairing the I D-8 and Activin/TGFp ligand combination with other agents or growth factors Example 1 : investigation of the utility of ID-8 in conjunction with Activin and TGFf31 for the maintenance of pluripotent stem ceil culture

This example identifies the combination of factors that either induce or support hES ceil proliferation in the absence of conditioned medium or feeder cells. Materials and Methods

The hES ceils used in these experiments were line NOTT2 from human blastocysts sourced from the UK Stem Cell Bank, For these assays this cell line was recovered from frozen vials and plated onto feeder based culture as previously described (ref). Once established the colonies were dissociated to single cells and plated in a 8 well plate format in, Pluripro Base Medium using Pluripro Matrix as a cell culture matrix (both Cell Guidance Systems, Cambridge, UK) according to manufacturer's protocol. This medium, with GABA (1 30ug/mi) and pipecolic acid (130ng/ml) was additionally supplemented with ID-8 (250nM), Activin A (10ng/ml) and ΤΟΡβΙ (2ng/mi) Control cultures included formulations without ID-8 and formulations with no Activin or TGFpl . The ability to maintain pluripotency was established by assaying for the expression of markers known to be associated with the pluripotent phenotype following extended passaging. NOTT2 stem cells at passage 40 were trypsinised and seeded as a single ceil suspension in the medium described above with 10μΜ Y27632 in a 6 well plate format at a density of 60,000 ceils/cm²

Results

The results of these experiments are shown in Figures 1 -5. Figures 1 and 2 show the maintenance of hES ceil morphology over sustained culture (9 passages at this time) by the absence of morphology associated with differentiated cells. Cultures consist of uniform ceil appearance and, once confluent, adopt the traditional tightly packed morphology with high nuc!ear/cytop!asmic ratio and prominent nucleoli. Figure 3 demonstrates by flow cytometry the high proportion of ceils expressing OCT4. Figure 4 demonstrates the expression profile of OCT4, TRA1 60, and SSEA4 as

representative of a pluripotent population. Figure 5 demonstrates immunostaining from embryoid body based spontaneous differentiation showing expression of genes, SOX1 7, ASMA, and β-tubuiin associated with each of the 3 germ layers. The control cultures with the absence of either I D-8 or of Activin/TGFp ligands did not sustain the undifferentiated phenotype, and failed to sustain cell culture beyond passage 4.

These results show that the combination of agonists which targets both β- catenin/P300 interaction and the Activin/TGFp pathway is capable of maintaining hES cell self-renewal, when added to a basal culture medium. Example 2: Further investigations into using lD-8 in hES cell culture: Human ES cells cultured with a combination I D-8, Activin/TGFB ligands and bFGF retain an undifferentiated phenptype

The effects of incorporating I D-8 and Activin into stem cell culture media were further tested.

Combinations of agonists were used as a supplement to other commonly recognised stem cell culture agents. The blastocyst derived cell lines NOTT2 and NCL5 were recovered from frozen vials and, once resuscitated, passaged into Pluripro Base Medium™ using Pluripro Matrix™ as a cell culture matrix (both Cell Guidance

Systems, Cambridge, UK) according to manufacturer's protocol. This medium, with GABA (1 30ug/ml), pipecolic acid (130ng/mi), was additionally supplemented with I D- 8 (250nM), Activin A (10ng/mi). and in this case IGF1 (25ng/ml), noggin (5ug/ml) and bFGF (20ng/mi). The ability to maintain piuripotency was established by assaying for the expression of markers known to be associated with the pluripotent phenotype following extended passaging.

NOTT2 stem cells at passage 25 were trypsinised and seeded as a single cell suspension in the medium described above with 10μΜ Y27832 in a 8 well plate format at a density of 60,000 cells/cm². Cells were grown to confluence and again passaged using the same protocol for repeated cycles, incubated with the complete medium described and cultured in a 5% C0₂ incubator at 37^eC. Medium changes took place every 24 hours. Data collected following extended passage demonstrates that these combinations of agents produced expansive regions of hES like morphology that did not contain evidence of differentiating cells.

Results

Figures 6 and 7 demonstrate the uniform morphology associated with

undifferentiated hES cells following 20 passages in culture. Figure 8 demonstrates the piuripotency associated expression profile by immunocytochemistry with positive expression of OCT4, NANOG, SOX2, TRA1 60, TRA181 and negative expression of S3 E / 1 , Example 3: Further investigations into using lD-8 in hES cell culture: Hum

cultured with a combination of I D-8, Activin/TGFB iigands and Wnt3a retain an undifferentiated phenptype

A further demonstration of the synergistic utility of the combination of iD-8 and Αοίίνίη/ΤΘΡβ Iigands with other agonists to enhance pluripotent cell culture was demonstrated using the supplementation with Wnt.

The blastocyst derived cell lines NOTT2 and NCL5 were recovered from frozen vials and, once resuscitated, passaged into Pluripro Base Medium ^IM using Piuripro Matrix ^{i M} as a cell culture matrix (both Ceil Guidance Systems, Cambridge, UK) according to manufacturer's protocol. This medium, with GABA (130ug/mi), pipecolic acid (130ng/mi), was additionally supplemented with ID-8 (250nM), Activin A

(10ng/mi) and, in this case, IGF1 (25ng/ml), noggin (5ug/ml) and nt3a (20ng/ml). The ability to maintain pluripotency was established by assaying for the expression of markers known to be associated with the pluripotent phenotype following extended passaging.

NOTT2 stem ceils at passage 25 were trypsinised and seeded as a single cell suspension in the medium described above with 10μΜ Y27832 in a 8 well plate format at a density of 60,000 cells/cm². Cells were grown to confluence and again passaged using the same protocol for repeated cycles, incubated with the complete medium described and cultured in a 5% C02 incubator at 37^eC. Medium changes took place every 24 hours. As before, the data collected following extended passage demonstrates that these combinations of agents produced expansive regions of hES like morphology that did not contain evidence of differentiating ceils.

Results

Figure 9 and 10 demonstrate the uniform morphology associated with

undifferentiated hES ceils following 13 passages in culture. Figure 1 1 demonstrates the pluripotency associated expression profile by immunocytochemistry with positive expression of OCT4, NANOG, SOX2, and negative expression of SSEA1 . Figure 10 shows expression of pluripotency and differentiation markers by QRTPCR in the undifferentiated and embryoid body differentiated populations. The above experiments show that the culture media comprising a p-catenin/P300 antagonist, such as ID-8, and an Acnvin/TGFB agonist may be used to culture pluripotent stem cells without feeder cell contact, i.e. they can be used to culture pluripotent stem cells in the absence of a layer of feeder cells. Populations of pluripotent stem cells cultured in the media display rapid expansion, i.e. the media allow large numbers of cells to be produced in a relatively short time. The culture media as described herein are also effective in expanding different pluripotent stem cell lines and may be used without a step of adapting cells to the culture medium, as is commonly required when transferring pluripotent stem cells into a new culture medium. Culture media comprising a p-catenin/P3GQ antagonist, such as ID-8, and an Activin/TGF agonist, may therefore offer advantages over known culture media in terms of the scalability, reproducibility and robustness of pluripotent stem cell culture. The above experiments also demonstrate the ability to enhance the quality of culture as evidenced by the high percentage of cells showing pluripotency marker expression under multiple different formulations when paring the ID-8 and

Activin/TGF ligand combination with other agents or growth factors already known in the art.

Example 4: Further investigation of the utility of I D-8 in conjunction with Activin and TGFB1 for the maintenance of pluripotent stem cell culture

A further demonstration of the synergistic utility of this combination of I D-8 and Activin/TGF ligands to enhance pluripotent ceil culture can be demonstrated by reducing the number of additional components. For these experiments a human iPSC line, purchased from System Biosciences and hereon referred to as hi PS cells, was used. This cell line was recovered from frozen vials, as described in for the hES lines in previous examples, and once resuscitated passaged into Pluripro Base™ Medium using Pluripro Matrix™ as a cell culture matrix (both Ceil Guidance

Systems, Cambridge, UK) according to manufacturer's protocol. This medium, with GABA (1 03ug/ml), pipecolic acid (130ng/mi), was additionally supplemented with I D- 8 (25nM), Activin A (10ng/ml) and ΤΟΡβΙ (2ng/ml). The ability to maintain

pluripotency was established by assaying for the expression of markers known to be associated with the pluripotent phenotype following extended passaging. hiPS cells at passage 16 were trypsinised and seeded as a single cell suspension in the medium described above with 10μΜ Y27632 in a 6 weii plate format at a density of 60,000 cells/cm2. Cells were grown to confluence and again passaged using the same protocol for repeated cycles, incubated with the complete medium described and cultured in a 5% C02 incubator at 37^SC. Medium changes took place every 24 hours, As before, the data collected following extended passage demonstrates that these combinations of agents produced expansive regions of hPSC like morphology that did not contain evidence of differentiating ceils.

Results

The uniform morphology demonstrated in the previous figures was again observed. Figure 13 demonstrates the pluripotency associated expression profile by

immunocytochemistry with positive expression of OCT4, NANOG, SOX2, SSEA4 and TRA1 -60 and negative expression of SSEA1 . Figure 14 demonstrates immunostaining from embryoid body based spontaneous differentiation showing expression of markers, Foxa2, ASMA, and β-tubulin associated with each of the 3 germ layers.

Claims

Claims:

1 . A culture medium for pluripotent stern cells which comprises;

a p-catenin/P300 antagonist and,

an Activin/TGFp !igand.

2. A culture medium according to claim 1 , wherein the p-catenin/P300 antagonist is ! D-8 or a pharmaceutical equivalent, analog, derivative, salt or prodrug thereof,

3. A culture medium according to claim 1 or claim 2 wherein the Activin/TGFp ligand is a member of the TGFp superfarnily.

4. A culture medium according to any one of the preceding claims wherein the Activin/TGFp ligand is selected from the group consisting of Activin A, Activin B, Activin AB, TGFp! , TGFp2, TGFp3, nodal, and GDF 1 -15.

5. A culture medium according to any one of the preceding claims wherein the medium comprises two or more Activin/TGFp !igands

6. A culture medium according to one of the preceding claims wherein the

Activin/TGFp ligand is Activin A and/or TGFpl ,

7. A culture medium according to one of the preceding claims which further

comprises a BMP antagonist.

8. A culture medium according to claim 7 wherein the BMP antagonist is

selected from the group noggin, foilistatin, dan, chordin and gremlin.

9. A culture medium according to any one of claims 1 to 8, wherein the BMP antagonist is a small molecule.

10. A culture medium according to one of the preceding claims comprising additionally an exogenous ligand selected from the group consisting of IGF, EGF, heregulin, FGF, Wnf, PDGF and homoiogs or analogs thereof.

1 1 . A culture medium according to any one of the preceding claims further comprising serum.

12. A culture medium according to any one of claims 1 to 10 further comprising serum replacement.

13. A culture medium according to any one of claims 1 to 10 which is serum free and serum replacement free.

14. A culture medium according to any one of the preceding claims further comprising cholesterol or a cholesterol substitute.

15. A culture medium according to any one of the preceding claims further comprising transferrin or a transferrin substitute.

16. A culture medium according to any one of the preceding claims further comprising albumin or an albumin substitute.

17. A culture medium according to any one of the preceding claims further comprising insulin or an insulin substitute.

18. A culture medium according to any one of the preceding claims further comprising progesterone, putrescine, and/or selenife.

19. A culture medium according to any one of the preceding claims wherein the medium is conditioned.

20. A culture medium according to any one of claims 1 to 19 wherein the medium is a fresh medium.

21 . A culture medium according to claim 20 wherein the medium is a chemicaily defined medium.

22. Use of a culture medium according to any one of claims 1 to 21 for the culture or expansion of p!uripotent stem cells.

23. A method for cuituring or expanding piuripotent stem ceils comprising

cu!turing a population of piuripotent stem ceils in a culture medium according to any one of claims 1 to 21 .

24. A method according to claim 23 comprising : (a) providing a population of piuripotent stem cells and a culture medium according to any one of claims 1 to 21 (b) cuituring the population in a first portion of said culture medium; (c) passaging the cells into a second portion of said culture medium ; and (d) further cuituring the cells in the second portion of said culture medium.

25. A method according to claim 24 comprising : (e) passaging the cells into a further portion of said culture medium; and (f) further cuituring the ceiis in the further portion of said culture medium.

28. A method according to claim 25 comprising repeating steps e) and f) one or more times.

27. A method for cuituring or expanding a population of piuripotent stem cells comprising (a) cuituring a population of piuripotent stem cells in a first culture medium; (b) replacing or exchanging the first culture medium with a second culture medium ; or passaging the ceils from the first culture medium into the second culture medium ; and (c) further cuituring the ceils in the second culture medium, wherein second medium is a culture medium according to any one of claims 1 to 21 and the population of stem ceils is not adapted to the second culture medium,

28. A ceil culture composition comprising a culture medium according to any one darns 1 to 21 and an extracellular matrix material.

29. A ceil culture comprising a culture medium according to any one claim of claims 1 to 21 or a cell culture composition according to claim 28 and a population of pluripotent stem cells.

30. A cell culture according to claim 29 which is feeder ceil free.

31 . A cell culture supplement comprising a β-θΒίβηίη/Ρ30υ antagonist and an Activin/TGF iigand, wherein the supplement is for use in the production of a cell culture medium according to any one of claims 1 to 21 .

32. A culture medium supplement according to claim 31 which is a concentrated liquid supplement.

33. A culture medium supplement according to claim 31 which is lyophiiised.

34. A culture medium, method, cell culture, supplement, composition or use according to any preceding claim, wherein the pluripotent stem cells are human ceils.

35. A culture medium, method, cell culture, supplement, composition or use according to any preceding claim, wherein the piuripotent stem cells are embryonic stem ceils.

36. A culture medium, method, ceil culture, supplement, composition or use according to any preceding claim, wherein the stem cells are human embryonic stem (hES) cells.

37. A culture medium, method, cell culture, supplement, composition or use according to any preceding claim, wherein the piuripotent stem cells are cells that have been genetically modified.

38. A culture medium, method, ceil culture, supplement, composition or use according to any preceding claim, wherein the piuripotent stem cells are iPS cells.

39. A method for producing a culture medium for piuripotent stem cells

comprising;

(a) obtaining a culture medium ; and,

(b) introducing an Activin/TGFp ligand and/or a -catenin P300 antagonist to the culture medium thereby producing a culture medium according to any of claims 1 to 21 .