(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 10 December 2009 (10.12.2009) WO 2009/147400 Al

(51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, C12N 5/00 (2006.0 1) C12N 5/06 (2006.0 1) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, PCT/GB2009/001404 KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (22) International Filing Date: ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, 5 June 2009 (05.06.2009) NO, NZ, OM, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, TZ, (25) Filing Language: English UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 0810304.6 5 June 2008 (05.06.2008) GB GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, 0821 363.9 2 1 November 2008 (21 . 1 1.2008) GB ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, (71) Applicant (for all designated States except US): ITI ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, SCOTLAND LIMITED [GB/GB]; 180 St. Vincent MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, TR), Street, Glasgow, Strathclyde G2 5SG (GB). OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). (72) Inventor; and (75) Inventor/Applicant (for US only): MCRAE, Robert, Published: Scott [GB/GB]; ITI Scotland Limited, 180 St. Vincent — with international search report (Art. 21(3)) Street, Glasgow, Strathclyde G2 5SG (GB). — before the expiration of the time limit for amending the (74) Agent: GOODFELLOW, Hugh, Robin; Carpmaels & claims and to be republished in the event of receipt of Ransford, 43-45 Bloomsbury Square, London WClA amendments (Rule 48.2(h)) 2RA (GB). (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM,

(54) Title: STEM CELL CULTURE MEDIA AND METHODS

FIG. 1 Influence of FXR agonists on hES cell proliferation

Rel

CA DCA TDCA LCA Z-G control

(57) Abstract: The invention provides new culture media and methods for pluripotent stem cells, in particular human embryonic stem (hES) cells, which provide significant advantages over known culture media and methods. The culture media of the inven tion comprise, amongst other ingredients, a nuclear hormone receptor agonist, in particular a farnesoid X receptor (FXR) agonist, a retinoid X receptor (RXR) or retinoic acid receptor (RAR) agonist, a peroxisome proliferator-activated receptor (PPAR) agonist, and/or a thyroid hormone receptor (THR) agonist. The invention also provides related culture medium supplements, compositions and uses. STEM CELL CULTURE MEDIA AND METHODS

All documents cited herein are incorporated by reference in their entirety.

TECHNICAL FIELD

The invention is in the field of stem cell culture media and methods, in particular culture media and methods for expanding populations of pluripotent stem cells, e.g. human embryonic stem (liES) cells.

BACKGROUND

There is great interest in culture media and methods for expanding populations of pluripotent stem cells, particularly hES cells which are especially difficult to culture. Clinical and research applications of pluripotent stem cells require reproducible cell culture methods to provide adequate numbers of cells 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. (1998) Science 282:1 145-1147). 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 cells (the stem cells 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 cells 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 cell pluripotency and self-renewal, and some feeder cell-free hES cell 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 cell line is sub- optimal or inappropriate for other hES cell lines (e.g. see Amit et al. (2004) Biology of Reproduction 70:837-845; Yao et al (2006) PNAS 103(18):6907-6912; Lu et al (2006) PNAS 103(15):5688-5693; and Mannello et al. (2007) Stem Cells 25(7):1603-1609).

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 hES cell culture media and methods.

THE INVENTION

The invention provides new culture media and methods for pluripotent stem cells, which provide significant advantages over known culture media and methods. The invention also provides related culture medium supplements, compositions and uses.

An advantage of the culture media of the invention is that they can 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. Another advantage of the culture media of the invention is that they can be used to rapidly expand a population of pluripotent stem cells, i.e. they allow large numbers of cells to be produced in a relatively short time. A further advantage of the culture media of the invention is that they can be used to expand several different types of pluripotent stem cell lines, i.e. different types of pluripotent stem cell can be cultured using a single culture medium. Another advantage of the culture media of the invention is that they can 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. The culture media of the invention therefore provide significant advantages over known culture media in terms of the scalability, reproducibility and robustness of pluripotent stem cell culture.

The culture media of the invention comprise, amongst other ingredients, a farnesoid X receptor (FXR) agonist, a retinoid X receptor (RXR) or retinoic acid receptor (RAR) agonist, a peroxisome proliferator-activated receptor (PPAR) agonist, and/or a thyroid hormone receptor (THR) agonist. The FXR, RXR, RAR, PPAR and THR are all members of the nuclear hormone receptor superfamily, which means they are all ligand- activated transcription factors, and they are functionally inter-linked by virtue of their hetero-dimerisation upon activation by ligands {e.g. see Aranda & Pascual (2001) Physiol Rev 81:1269-1304). The FXR, RXR, RAR, PPAR and THR work together to control transcription signalling pathways. Without wishing to be bound by the theory, the inventor believes that agonists to these types of nuclear hormone receptors, and particularly combinations of agonists as disclosed herein, may activate transcription signalling pathways that inhibit differentiation of stem cells and allow them to rapidly proliferate whilst maintaining their pluripotency.

Accordingly, the invention provides a culture medium for expanding a population of pluripotent stem cells, which comprises a farnesoid X receptor (FXR) agonist, a retinoid X receptor (RXR) or retinoic acid receptor (RAR) agonist, a peroxisome proliferator- activated receptor (PPAR) agonist, and/or a thyroid hormone receptor (THR) agonist. In some embodiments, the culture medium may comprise an FXR agonist, an RXR or RAR agonist and a THR agonist. In other embodiments, the culture medium may comprise an FXR agonist, an RXR or RAR agonist, a PPAR agonist and a THR agonist.

The invention also provides a culture medium supplement that comprises an FXR agonist, an RXR or RAR agonist, a PPAR agonist and/or a THR agonist.

The invention also provides a hermetically-sealed vessel containing a culture medium or culture medium supplement of the invention.

The invention also provides a method for preparing a culture medium as disclosed herein, comprising the steps of: (a) obtaining a culture medium; and (b) adding an FXR agonist, an RXR or RAR agonist, a PPAR agonist and/or a THR agonist to the culture medium.

The invention also provides a composition comprising: (a) a culture medium according to the invention; and (b) stem cells.

The invention also provides a composition containing: (a) a culture medium according to the invention; and (b) an extracellular matrix material.

The invention also provides the use of a culture medium of the invention for expanding a population of pluripotent stem cells.

The invention also provides a method for expanding a population of pluripotent stem cells, comprising: (a) providing a population of pluripotent stem cells; (b) providing a culture medium of the invention; (c) contacting the stem cells with the culture medium; and (d) culturing the cells under appropriate conditions.

The invention also provides the use of an FXR agonist, an RXR or RAR agonist, a PPAR agonist and/or a THR agonist for pluripotent stem cell culture.

The specific ingredients of the culture media, supplements and compositions of the invention can vary according to particular needs and applications. Likewise, the precise steps of the methods of the invention 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 pluripotent stem cell expansion, variables such as the amount of each ingredient in the culture medium or supplement, seeding densities, culture conditions, culture periods, etc. can be altered in further experiments. The amount of each of the ingredients described herein can be optimised independently of the other ingredients by routine optimisation or one or more ingredients can be added or removed. A culture medium can be tested for its ability to support expansion of pluripotent stem cells by testing it alongside or in place of a known culture medium or method.

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

Numerous textbooks are available that provide guidance on mammalian cell 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. M. Walker (1997), 'Mammalian Cell Culture: Essential 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. Helgason and C. L. Miller (2005), 'Stem Cells: From Bench to Bedside' by A. Bongso (2005) and 'Human Embryonic Stem Cell Protocols' by K. Turksen (2006). Pluripotent stem cells and cell culture reagents and apparatus for use in the invention are available commercially, e.g. from Cellartis AB (Gδteborg, Sweden), VitroLife AB (Kungsbacka, Sweden), GIBCO® (Invitrogen), Millipore Corporation (Billerica, Massachusetts), Sigma® (St. Louis, Missouri) and Biomol International L.P. (Exeter, UK).

Farnesoid X receptor fFXR) agonists

The inventor has discovered that the use of an FXR agonist improves pluripotent stem cell culture. Accordingly, the culture media of the invention may comprise an FXR agonist, and the invention provides the use of an FXR agonist for pluripotent stem cell culture.

The FXR (also known as NRH 14) is a member of the nuclear hormone receptor superfamily (i.e. it is a ligand-activated transcription factor) and is involved in the control of multiple cellular pathways. The FXR is expressed from a single gene in humans (NRHl4), although four isoforms are generated by alternative splicing.

An 'FXR agonist' is an agent that binds to and activates at least one FXR isoform. Various methods for determining if a given substance is an FXR agonist are known and might be used in conjunction with this invention. For example, determining whether a given substance is an FXR agonist may involve analysing whether the substance induces or represses the expression of FXR target genes, e.g. whether it induces expression of short heterodimer partner (SHP), phospholipid transport protein (PLTP), canalicular bile salt export pump (BSEP) and/or ileum bile acid binding protein (IBABP), or whether it represses expression of CYP7A1, CYP27A1 and/or CYP8B1 (see Claudel et al (2005) Arterioscler. Thromb. Vase. Biol. 25:2020-2030; Chiang (2002) Endocrine Reviews 23(4):443-463). Alternatively, or in addition, determining whether a given substance is an FXR agonist may involve testing the substance using a cell-based luciferase assay or using a surface plasmon resonance (SPR) coactivator association assay (e.g. as described in Fujino et al. (2004) Journal of Lipid Research 45:132-138).

Bile acids, bile acid intermediates and polyunsaturated fatty acids (PUFA) are all known to be FXR agonists in vitro, and several synthetic FXR agonists have been generated (see Claudel et al. (2005) Arterioscler. Thromb. Vase. Biol. 25:2020-2030; Chiang (2002) Endocrine Reviews 23(4):443-463; Houten et al. (2006) EMBO Journal 25:1419- 1425; and Soisson et al. (2008) PNAS vol. 105 no. 14:5337-5342).

An FXR agonist in a culture medium of the invention may be a cholesterol metabolite, such as a bile acid or a bile acid intermediate. Bile acids are steroid carboxylic acids derived from cholesterol. All bile acids comprise two connecting units, a rigid steroid nucleus and a short aliphatic side chain. The steroid nucleus of bile acids has the saturated tetra-cyclic hydrocarbon perhydrocyclo-pentanophenanthrene, containing three six-member rings and a five-member ring. In addition, there are angular methyl groups at positions C-18 and C-1 9. Bile acids are facially amphipathic molecules which contain a side chain that terminates in a carboxyl group (see Mukhopdhyay & Maitra (2004) Current Science 87(12):1666-1683). The principle bile acids in humans are the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA), and the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA). Accordingly, in some embodiments the FXR agonist is cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) or lithocholic acid (LCA).

Bile acids are often conjugated to glycine or taurine (e.g. to form glycocholic acid or taurocholic acid for CA), and these conjugates may also be used in the culture media of the invention. For example, glycocholic acid, taurocholic acid, glycodeoxycholic acid or taurodeoxycholic acid may be used.

Where a bile acid or a bile acid intermediate is to be used, it may be provided in the form of a bile salt, preferably in the form of a sodium salt.

An FXR agonist in a culture medium of the invention may be a polyunsaturated fatty acid (PUFA), such as an arachidonic acid, linolenic acid or docosahexaenoic acid. In some embodiments, an FXR agonist may be α-linolenic acid (ALA), γ-linolenic acid (GLA) or di-homo-γ-linolenic acid (DGLA, also sometimes referred to as DiHLA). The inventor's data show that DGLA provides improved stem cell expansion relative to other linolenic acids. The invention therefore also provides the use of DGLA for pluripotent stem cell culture, and a culture medium for expanding pluripotent stem cells that comprises DGLA. An FXR agonist in a culture medium of the invention may be a synthetic FXR agonist, such as MFA-I (e.g. see Soisson et al. (2008) PNAS), GW4064 {e.g. see Maloney et al (2000) J. Med. Chem. 43:2971-2974) or 6α-ethyl-CDCA (6-ECDCA) (e.g. see Houten et al. (2006) EMBO Journal 25:1419-1425).

Farnesol, forskolin, androsterone or etiocholanolone might also be useful as FXR agonists in the culture media of the invention (e.g. see Howard et al. (2000) Toxicol Appl Pharmacol. 163(2):195-202).

Retinoid X receptor (RXR) or retinoic acid receptor (RAR) agonists

The inventor has discovered that the use of an RXR or RAR agonist improves pluripotent stem cell culture. Accordingly, the culture media of the invention may comprise an RXR or RAR agonist, and the invention provides the use of an RXR or RAR agonist for pluripotent stem cell culture.

The retinoid receptors are members of the nuclear hormone receptor superfamily (i. e. they are ligand-activated transcription factors) and are involved in the control of multiple cellular pathways. The two families of retinoid receptors (RARs and RXRs) each consist of three receptor types (α, β, γ) encoded by different genes, and multiple isoforms are generated by alternative splicing. RXRs and RARs are functionally inter-linked, because RARs function as heterodimers with RXRs. RXRs can also form homodimers, as well as heterodimers with other nuclear hormone receptors such as PPAR (see Aranda & Pascual (2001) Physiol Rev 81:1269-1304; Szanto et al. (2004) Cell Death and Differentiation 11:S126-S143; Germain et al. (2006) Pharmacological Reviews 58:712- 725; and Germain et al. (2006) Pharmacological Reviews 58:760-772).

An 'RXR or RAR agonist' is an agent that binds to and activates at least one RXR or RAR receptor type (α, β, γ) or isoform (splice variant). Various methods for determining if a given substance is an RXR or RAR agonist are known and might be used in conjunction with this invention. For example, determining whether a given substance is an RXR or RAR agonist may involve analysing whether the substance induces or represses the expression of RXR target genes. Alternatively, or in addition, determining whether a given substance is an RXR or RAR agonist may involve testing the substance using a reporter gene assay system (e.g. see the methods of Perlmann et al. and Luria et al referenced in Szanto et al. (2004) Cell Death and Differentiation 11:Sl 26-S143; Germain et al. (2006) Pharmacological Reviews 58:712-725; and Germain et al. (2006) Pharmacological Reviews 58:760-772).

Natural and synthetic RAR and RXR agonists are known. Known RXR agonists include

9-cis-retinoic acid (9cRA), methoprene acid (MPA) 5 methoprenic acid, docosahexaenoic acid, phytanic acid, UAB20, UAB30, 4-methyl-UAB30, UAB76, UABl 12, CD3254, LG100268, LGD1069 (targretin), AGN194204 and SRl 1237 (see e.g. Germain et al (2006) Pharmacological Reviews 58:760-772). Known RAR agonists include 9-cis- retinoic acid (9cRA), AGN195183, Am580, TTNPB, Am80, CO666, BMS270394, BMS961, BMS641 and BMS753 (see e.g. Germain et al. (2006) Pharmacological Reviews 58:712-725). It is envisaged that any of these natural and synthetic RAR and RXR agonists may be useful in the culture media of the invention.

An RXR or RAR agonist in a culture medium of the invention may be a retinoid. The term 'retinoid' is commonly used to refer to dietary Vitamin A (retinol), retinol metabolites, and synthetic analogues thereof. Retinoids are typically compounds that comprise four isoprenoid units joined in a head-to-tail manner. An RXR or RAR agonist used in the invention may be a first generation retinoid (e.g. retinol, retinal, tretinoin, isotretinoin or alitretinoin), a second generation retinoid (e.g. etretinate or its metabolite acitretin) or a third generation retinoid (e.g. tazarotene, bexarotene or adapalene).

An RXR or RAR agonist in a culture medium of the invention may be a rexinoid. The term 'rexinoid' is commonly used to refer to synthetic RXR-specific agonists. Known rexinoids include SRl 1237, LG100268 and LGD 1069 (see Germain et al. (2006) Pharmacological Reviews 58:760-772).

In some embodiments, the RXR or RAR agonist is a retinol, retinal or retinoic acid. For example, the RXR or RAR agonist may be all-trans-retinol (ATR)5 13-cis-retinol (13cROL), 11-cis-retmal, retinyl acetate (RETACT) 4-hydroxy-retinoic acid (4HRA), 13-cis-retinoic acid (13cRA) or 9-cis-retinoic acid (9cRA).

Peroxisome proliferator-activated receptor (TPAR) agonists

The inventor has discovered that the use of a PPAR agonist improves pluripotent stem cell culture. Accordingly, the culture media of the invention may comprise a PPAR agonist, and the invention provides the use of a PPAR agonist for pluripotent stem cell culture.

The peroxisome proliferator-activated receptors are members of the nuclear hormone receptor superfamily (i.e. they are ligand-activated transcription factors) and are involved in the control of multiple cellular pathways. Three different PPAR types exist in humans: PPARα (NRlCl) PPARβ (NR1C2) and PPARγ (NR1C3). The PPARs exhibit broad, isotype-specific, tissue expression patterns. All of the PPARs function via heterodimerisation with the retinoid X receptor (RXR).

A 'PPAR agonist' is an agent that binds to and activates at least one PPAR type (i.e. at least one of PPARα, PPARβ and PPARγ). Various methods for determining if a given substance is a PPAR agonist are known and might be used in conjunction with this invention. For example, determining if a given substance is a PPAR agonist may involve analysing whether the substance induces or represses the expression of PPAR target genes, e.g. whether is affects expression of a gene encoding acyl-coenzyme A synthetase (ACS), a gene encoding muscle-type carnitine palmitoyltransferase- 1 (MCPT-I) and/or a gene encoding long-chain acyl-coenzyme A dehydrogenase (LCAD) (e.g. see the methods described in Gilde et al. (2003) Circulation Research 92(5):5 18-524). Alternatively, or in addition, determining if a given substance is a PPAR agonist may involve analysing whether the substance induces or represses the expression of a reporter gene under the control of a PPAR response element, or may involve using a Scintillation Proximity Assay (SCA), a Differential Protease Sensitivity Assay (DPSA), a Ligand Induced Complex (LIC) assay and/or a Coactivator-Dependent Receptor Ligand Assay (CARLA) (e.g. see the methods described in Desvergne & Wahli (1999) Endocrine Reviews 20(5):649-688).

A large number of natural and synthetic PPAR agonists are known (e.g. see Michalik et al. (2006) Pharmacological Reviews 58:726-725; Gilde et al. (2003) Circulation Research 92(5):5 18-524; Peraza et al. (2005) Toxicological Sciences 90(2):269-295; and Desvergne & Wahli (1999) Endocrine Reviews 20(5):649-688). Some of these known agonists are specific for a single PPAR isotype, whilst others target multiple PPAR subtypes. The inventor's data suggest that many of the known natural and synthetic PPAR agonists will be of use in the culture media of the invention. Natural PPAR agonists include unsaturated fatty acids such as ω3-PUFAs (e.g. α- linolenic acid, γ-linolenic acid, eicosapentaenoic acid and docohexaenoic acid), ω6- PUFAs (e.g. linolenic acid, dihomo-γ-linoleic acid and arachidonic acid), ω9-mUFAs (e.g. palmitoleic acid, oleic acid, elaidic acid, erucic acid and nervonic acid) and ω2- mUFAs (petroselinic acid). Natural PPAR ligands also include saturated fatty acids (such as capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid and behenic acid), dicarboxylic fatty acids (such as dodecanedoic acid) and eicosanoids (such as 8-HEPE, 8-HETE, 8S-HETE, 8R-HETE, 12-HETE, Leukotriene B4, 9-HODE and 13-HODE). Synthetic PPAR ligands include prostaglandin 12 analogs (such as carbaprostacyclin, iloprost and cicaprost), leukotriene B4 analogs (such as trifluoromethyl leukotriene B4, ZK 151657 and ZK 158252), leukotriene D4 antagonists (such as Ly 171883), hypolipidemic agents (such as clofibric acid, ciprofibric acid, bezafibric acid, fenofibric acid, pirixinic acid (Wy-14643), GW 2331, GW 2433 and eicosatetraynoic acid), hypoglycemic agents (thiazolidinediones, such as rosiglitazone (BRL 49653), AD-5075, troglitazone (CD-045), pioglitazone, and ciglitazone), hypolipidemic and hypoglycaemic agents (non-thiazolidinediones, such as L-165041, L- 165461, L-783483 and L-796449), nonsteroidal anti-inflammatory drugs (such as indomethacin, diclofenac, flufenamic acid, fenoprofen and ibuprofen), carnitine palmitoyl transferase I (CPTl) inhibitors (such as LY-171883, 2-bromopalmitate and tetradecylglycidic acid) and fatty acyl-CoA dehydrogenase inhibitors (such as ortylthiopropionic acid, tetradecylpropionic acid, nonylthioacetic acid and tetradecylthioacetic acid). It is envisaged that any of these natural and synthetic PPAR agonists may be useful in the culture media of the invention.

In some embodiments, the PPAR agonist may be a glitazone. Glitazones are synthetic PPAR agonists that activate PPARα, and include rosiglitazone, troglitazone, pioglitazone, and ciglitazone. In some embodiments, the PPAR agonist may be a glitazar. Glitazars are synthetic PPAR agonists that activate both PPARα and PPARγ, and include muraglitazar, tesaglitazar, ragaglitazar, reglitazar and farglitazar. In some embodiments, the PPAR agonist may be a fibric acid derivative (fibrate), such as clofibrate (or clofibric acid), bezafibrate (or bezafibric acid), ciprofibrate (or ciprofibric acid), fenofibrate (or fenofibric acid), etofibrate, or gemfibrozil. Known PPAR agonists also include plialate monoesters {e.g. monoethylhexyl phthalate), trichloroacetic acid, dichloroacetic acid, perfluorooctanoic acid (PFOA), perfluoroctanesulfonic acid (PFOS), oleylethanolamide, pristanic acid, pterostilbene, retinoic acid, 5-ASA, AD-5061, BADGE, BVT0.13, COOH, CDDO, DRF2519, FMOC- L-leucine, GW409544, GW7647, GW9578, GW501516, GW0742, GW2433, GW0072, GW2331, GWl 929, GW7845, LGl 0074, LTB4, LY-5 18674, LY-5 10929, LY-465608, L-764406, MCC555, NS-220, nTzDpa, PAT5A, KRP-297/MK-0767, SB-219993, SB- 219994, TAK-559 and TZD18 {e.g. see Michalik et al. (2006) Pharmacological Reviews 58:726-725; Gilde et al. (2003) Circulation Research 92(5):518-524; Peraza et al. (2005) Toxicological Sciences 90(2):269-295; and Desvergne & Wahli (1999) Endocrine Reviews 20(5):649-688). It is envisaged that any of these natural and synthetic PPAR agonists may be useful in the culture media of the invention.

Thyroid hormone receptor (THR) agonists

The inventor has discovered that the use of a THR agonist improves pluripotent stem cell culture. Accordingly, the culture media of the invention may comprise a THR agonist. The invention also provides the use of a THR agonist for pluripotent stem cell culture.

The THR is a member of the nuclear hormone receptor superfamily {i.e. it is a ligand- activated transcription factor) that is activated in vivo via binding to the tyrosine-based thyroid hormones tri-iodothyronine (T3) and thyroxine (T4) produced by the thyroid gland. Activation of the THR has multiple effects in vivo (see Yen (2001) Physiol. Rev. 81:1097-1 142; Goglia (2005) Biochemistry (Moscow) 70(2):203-213; Pinna et al. (2002) Endocrinology 143(5):1789-1800; Lombardi et al. (2000) 141(5):1729~1734). There are at least four types of THR, termed THR-αl , THR-α2, THR-βl and THR-β2.

An 'THR agonist' is an agent that binds to and activates at least one THR type {i.e. at least one of THR-αl , THR-α2 THR- βl and THR-β2). Various methods for determining if a given substance is a THR agonist are known and might be used in conjunction with this invention. For example, determining if a given substance is a THR agonist may involve analysing whether the substance induces or represses the expression of endogenous THR target genes. Alternatively, or in addition, determining if a given substance is a THR agonist may involve analysing whether the substance induces or represses the expression of a reporter gene under the control of a thyroid hormone response element (TRE) (e.g. see the methods cross-referenced in Yen (2001) Physiol. Rev. 81:1097-1 142).

A THR agonist in a culture medium of the invention may be an iodothyronine (i. e. an iodinated derivative of thyronine). A THR agonist may be a di-iodothyronine, a tri- iodothyronine or a tetra-iodothyronine. For example, a THR agonist may be 3,5-di- iodothyronine (3,5-T2) 3,5-diiodo-L-tyrosine dihydrate (DLTdH), 3,3'-di-iodothyronine

(3,3'-T 2), 3,3'-T 2 sulphate (3,3-T2S), 3,5,3'-tri-iodothyronine (T3), 3,3',5'-tri- iodothyronine (reverse T3), 3,3',5-T sulphate (3,3',5-T 3S), 3,5-diiodo-4- hydroxyphenylpropionic acid (DIHPA, also sometimes referred to as DHPPA), 3,5,3',5'- tetra-iodothyronine (T4), 3,5,3',5'-tetraiodo-L-thyronine or 3,3',5-triiodo-thyroacetic acid (TRIAC). In some embodiments, the THR agonist is 3,5-diiodo-L-thyronine (3,5-DLT).

The THR agonist may be a synthetic THR agonist, e.g. the heterocyclic thyromimetic GC-I (e.g. see Manzano et al. (2003) Endocrinology 144(12):5480-5487 and Flamant et al. (2006) Pharmacological Reviews 58:705-711).

Culture Media

The culture media of the invention contain a nuclear hormone receptor (NHR) agonist. The culture media of the invention may comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or twelve or more, different NHR agonists.

The culture media of the invention comprise an FXR agonist, an RXR or RAR agonist, a PPAR agonist and/or a THR agonist. The culture media may comprise two or more, three or more, four or more, or five or more, different FXR agonists. The culture media of the invention may comprise two or more, three or more, four or more, or five or more, different RXR or RAR agonists. The culture media of the invention may comprise two or more, three or more, four or more, or five or more, different PPAR agonists. The culture media of the invention may comprise two or more, three or more, four or more, or five or more, different THR agonists. In some embodiments, a culture medium of the invention comprises an FXR agonist, an RXR or RAR agonist and a THR agonist. In other embodiments, a culture medium of the invention comprises an FXR agonist, an RXR or RAR agonist, a PPAR agonist and a THR agonist. A culture medium of the invention may comprise between about lOpM and about 10OmM of an FXR agonist, between about lOpM and about 10OmM of an RXR or RAR agonist, between about lOpM and about 10OmM of a PPAR agonist, and/or between about lOpM and about 10OmM of a THR agonist. A culture medium of the invention may comprise at least about lOpM of an FXR agonist, at least about lOpM of an RXR or RAR agonist, at least about lOpM of a PPAR agonist, and/or at least about lOpM of a THR agonist.

In some embodiments, a culture medium of the invention may comprise between about 10OnM and about ImM of an FXR agonist. For example, a culture medium may comprise between about 250 and about 75OnM of an FXR agonist, between about 300 and about 70OnM of an FXR agonist, between about 350 and about 65OnM of an FXR agonist, between about 400 and about 60OnM of an FXR agonist, between about 450 and about 55OnM of an FXR agonist, or about 50OnM of an FXR agonist. For example, a culture medium may comprise between about 250 and about 75OnM of cholic acid, between about 300 and about 70OnM of cholic acid, between about 350 and about 65OnM of cholic acid, between about 400 and about 60OnM of cholic acid, between about 450 and about 55OnM of cholic acid, or about 50OnM of cholic acid.

In some embodiments, a culture medium of the invention may comprise between about InM and about ImM of an RXR or RAR agonist. For example, a culture medium may comprise between about 10 and about 50OnM of an RXR or RAR agonist, between about 20 and about 40OnM of an RXR or RAR agonist, between about 25 and about 35OnM of an RXR or RAR agonist, or between about 30 and about 30OnM of an RXR or RAR agonist. For example, a culture medium may comprise between about 10 and about 50OnM of all trans retinol, between about 20 and about 40OnM of all trans retinol, between about 25 and about 35OnM of all trans retinol, or between about 30 and about 30OnM of all trans retinol.

In some embodiments, a culture medium of the invention may comprise between about InM and about ImM of a PPAR agonist. For example, a culture medium may comprise between about 1OnM and about 750µM of a PPAR agonist, between about 25nM and about 500µM of a PPAR agonist, between about 5OnM and about 250µM of a PPAR agonist, or between about 75nM and about lOOµM of a PPAR agonist. For example, a culture medium may comprise about l OµM of a PPAR agonist.

In some embodiments, a culture medium of the invention may comprise between about InM and about 10OmM of a THR agonist. For example, a culture medium may comprise between about 25 and about 75nM of a THR agonist, between about 30 and about 7OnM of a THR agonist, between about 35 and about 65nM of a THR agonist, between about 40 and about 6OnM of a THR agonist, between about 45 and about 55nM of a THR agonist, or about 5OnM of a THR agonist. For example, a culture medium may comprise between about 25 and about 75nM of 3,5-diiodo-L-thyronine (3,5-DLT), between about 30 and about 7OnM of 3,5-DLT, between about 35 and about 65nM of 3,5-DLT, between about 40 and about 6OnM of 3,5-DLT, between about 45 and about 55nM of 3,5-DLT, or about 5OnM of 3,5-DLT.

Cell culture media typically contain a large number of ingredients, which are necessary to support maintenance of the cultured cells. A culture medium of the invention will therefore normally contain many other ingredients in addition to an FXR agonist, an RXR or RAR agonist, a PPAR agonist and/or a THR agonist. Suitable combinations of ingredients can readily be formulated by the skilled person, taking into account the following disclosure. A culture medium 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.

A culture medium according to the invention may be generated by modification of an existing cell culture medium. The skilled person understands the types of culture media that might be used for pluripotent stem cell culture. Potentially suitable cell culture media are available commercially, and include Dulbecco's Modified Eagle Media (DMEM), Minimal Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimal Essential Medium (G-MEM), Basal Medium Eagle (BME), DMEM/Ham's F12, Advanced DMEM/Ham's F12, Iscove's Modified Dulbecco's Media and Minimal Essential Media (MEM).

A culture medium for use in the invention may comprise one or more amino acids. The skilled 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-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and combinations thereof. Some culture media will contain all 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-glutamine 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.

A culture medium for use in 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 Bl), riboflavin (vitamin B2), niacin (vitamin B3), D-calcium pantothenate (vitamin B5), pyridoxal/pyridoxamine/pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL- alpha tocopherol (vitamin E), biotin (vitamin H) and menadione (vitamin K).

A culture medium for use in 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 CaCl2, CuSO4-5H2O, Fe(NO 3)-9H2O, FeSO4-7H2O, MgCl, MgSO4, KCl,

NaHCO 3, NaCl, Na2HPO4, Na2HPO4-H2O and ZnSO4-7H2O.

The osmolarity of the medium may be in the range from about 200 to about 400 mθ sm/kg, in the range from about 290 to about 350 mθ sm/kg, or in the range from about 280 to about 310 mθ sm/kg. The osmolarity of the medium may be less than about 300 mθ sm/kg (e.g. about 280 mθ sm/kg).

A culture medium for use in 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 cell culture media. Sugars which may be present include glucose, galactose, maltose and fructose. The sugar is preferably glucose, particularly D-glucose (dextrose). A carbon energy source will normally be present at between about 1 and about 10 g/L.

A culture medium for use in 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. NaHCO 3), chlorides (e.g. CaCl2), sulphates (e.g. MgSO4) and phosphates (e.g.

NaH 2PO ). These buffers are generally used at about 50 to about 500 mg/1. Other buffers such as N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesul-phonic acid] (HEPES) and 3-[N- morpholinoj-propanesulfonic acid (MOPS) may also be used, normally at around 1000 to around 10,000 mg/1.

A culture medium of the invention may contain serum. Serum obtained from any appropriate source may be used, including fetal 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, a culture medium of 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, a culture medium of 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.

A culture medium 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 cholesterol or cholesterol substitute can readily be determined from the literature or by routine experimentation. A synthetic cholesterol substitute may be used rather than cholesterol derived from an animal source. For example, Synthecol™ (Sigma S5442) may be used in accordance with the manufacturer's instructions.

The culture medium 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. In some embodiments, a culture medium of the invention may comprise transferrin at about 5.5µg/ml.

The culture medium may further comprise albumin or an albumin substitute, such as bovine serum albumin (BSA), human serum albumin (HSA), a plant hydrolysate (e.g. a rice or soy hydrolysate), Albumax® I or Albumax® II. The optimal amount of albumin or albumin substitute can readily be determined from the literature or by routine experimentation. In some embodiments, a culture medium of the invention may comprise albumin at about 0.5µg/ml.

The culture medium 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 of the invention may comprise insulin at about 1Oµg/ml.

The culture medium 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.

A culture medium of the invention may comprise one or more additional nutrients or growth factors that have previously been reported to benefit pluripotent stem cell culture. For example, a culture medium may comprise fibroblast growth factor (FGF), transforming growth factor beta 1 (TGFp 1), leukemia inhibitor factor (LIF), ciliary neurotrophic factor (CNTF), interleukin 6 (IL-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 homologs or analogs thereof. In some embodiments, bFGF is used. bFGF may be used at from about 1 ng/ml to about 50 ug/ml, e.g. at about 5 ng/ml, at about 10 ng/ml, or at about 40 ng/ml.

A culture medium for use in the invention may comprise one or more trace elements, such as ions of barium, bromium, cobalt, iodine, manganese, cliromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc and/or aluminium.

A culture medium may further 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/litre).

The medium may comprise a reducing agent, such as β-mercaptoethanol at a concentration of about 0.1 mM.

Preferred culture media are described in the Examples herein. In one embodiment, a culture medium of the invention comprises a bile acid, a retinol and a diiodothyronine. For example, a culture medium may comprise cholic acid, all-trans-retinol (ATR) and 3,5-diiodo-L-thyronine. In another embodiment, a culture medium may comprise cholic acid, all-trans-retinol, 3,5-diiodo-L-thyronine, cholesterol, transferrin, L-glutamine, progesterone, putrescine, insulin, selenite and DL-alpha-tocopherol (vitamin E).

In one embodiment, a culture medium may comprise transferrin, insulin, progesterone, putrescine, and sodium selenite.

'N2 Supplement' (available from Invitrogen, Carlsbad, CA; www.invitiOgen.com ; catalog no. 17502-048; and from PAA Laboratories GmbH, Pasching, Austria; www.paa.com ; catalog no. F005-004; Bottenstein & Sato, PNAS, 76(1):514-517, 1979) may be used to formulate a culture medium that comprises contains transferrin, insulin, progesterone, putrescine, and sodium selenite. N2 Supplement is supplied by PAA

Laboratories GmbH as a 10Ox liquid concentrate, containing 500µg/ml human transferrin, 500µg/ml bovine insulin, 0.63 µg/ml progesterone, 161 1µg/ml putrescine, and 0.52 µg/ml sodium selenite. N2 Supplement may be added to a culture medium as a concentrate or diluted before addition to a culture medium. It may be used at a Ix final concentration or at other final concentrations. Use of N2 Supplement is a convenient way to incorporate transferrin, insulin, progesterone, putrescine and sodium selenite into a culture medium of the invention.

Accordingly, the invention provides a culture medium as described herein for expanding a population of pluripotent stem cells, which comprises transferrin, insulin, progesterone, putrescine, and sodium selenite (optionally comprising N2 Supplement). The invention also provides a method as described herein for preparing a culture medium, which comprises adding transferrin, insulin, progesterone, putrescine, and sodium selenite to a culture medium (optionally by supplementing the medium with N2 Supplement).

In one embodiment, a culture medium may comprise biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri- iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin.

'B27 Supplement' (available from Invitrogen, Carlsbad, CA; www.invitrogen.com ; currently catalog no. 17504-044; and from PAA Laboratories GmbH, Pascbing, Austria; www.paa.com ; catalog no. F01-002; Brewer et al, J Neurosci Res., 35(5):567-76, 1993) may be used to formulate a culture medium that comprises biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri¬ iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin. B27 Supplement is supplied by PAA Laboratories GmbH as a liquid 5Ox concentrate, containing amongst other ingredients biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin. Of these ingredients at least linolenic acid, retinol, retinyl acetate and tri-iodothyronine (T3) are nuclear hormone receptor agonists as described elsewhere herein. B27 Supplement may be added to a culture medium as a concentrate or diluted before addition to a culture medium. It may be used at a Ix final concentration or at other final concentrations. Use of B27 Supplement is a convenient way to incorporate biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri- iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin into a culture medium of the invention.

Accordingly, the invention provides a culture medium as described herein for expanding a population of pluripotent stem cells, which comprises biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri¬ iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin (optionally comprising B27 Supplement). The invention also provides a method as described herein for preparing a culture medium, which comprises adding biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin to a culture medium (optionally by supplementing the medium with B27 Supplement).

N2 Supplement and B27 Supplement may be used in combination in a culture medium of the invention (see Example 5 herein). The invention therefore also provides a culture medium as described herein for expanding a population of pluripotent stem cells, which has been supplemented with N2 Supplement and B27 Supplement. The invention also provides a method as described herein for preparing a culture medium, which comprises adding N2 Supplement and B27 Supplement to a culture medium.

A culture medium of the invention will normally be formulated in deionized, distilled water. A culture medium of the invention 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 -200C or -800C) for storage or transport. The medium may contain one or more antibiotics to prevent contamination. The medium may have an endotoxin content of less that 0.1 endotoxin units per ml, or may have an endotoxin content less than 0.05 endotoxin units per ml. Methods for determining the endotoxin content of culture media are known in the art.

In some embodiments, the culture medium is a conditioned medium. Conditioned medium is produced by culturing a population (typically of non-pluripotent) cells 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 cells that support growth of stem cells. In some embodiments, the medium comprises conditioned VitroHES (VitroLife AB, Sweden).

Where a conditioned medium is used, the medium may be conditioned on mammalian cells, e.g. mouse cells 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 cells and human fallopian epithelial cells. Preferably, mEF cells are used. Conditioned medium may be prepared by well known methods, e.g. by culturing mEFs and harvesting the culture medium after an appropriate time {e.g. ~ 1 day at 37°C). 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 culturing 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 pluripotent stem cell 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 370C.

The period over which a conditioned medium can support pluripotent stem cell 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 cell growth and differentiation. Conditioned medium is typically used to support cell 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, the culture medium is a fresh culture medium. 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).

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. By 'different types' is meant that the combination of ingredients in the conditioned medium prior to conditioning is different to the combination of ingredients in the fresh medium (e.g. the conditioned medium may be conditioned VitroHES and the fresh medium may be DMEM/F12). In other words, the mixed culture medium is not one that would be obtained by merely diluting a concentrated medium with an non- concentrated or diluted form of the same medium, nor is it one that would be obtained by adding to a conditioned medium more fresh medium of the same type.

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. Accordingly, in one aspect the invention provides a method for preparing a mixed culture medium for expanding a population of pluripotent stem cells, comprising: (a) providing a conditioned medium; (b) providing a fresh medium; and (c) mixing at least part of the conditioned medium with at least part of the fresh medium, thereby forming a mixed culture medium, wherein the conditioned medium and the fresh medium are of different types. The invention also provides methods, compositions and uses as described herein involving such mixed culture media.

In a preferred embodiment, the culture medium of the invention is a mixture of a conditioned medium and a fresh medium of different types, which comprises a bile acid, a retinol and a diiodothyronine. In another preferred embodiment, the culture medium of the invention is a mixture of a conditioned medium and a fresh medium of different types, which comprises cholic acid, all-trans-retinol and 3,5-diiodo-L-thyronine.

In a preferred embodiment, a culture medium of the invention is a mixture of a conditioned medium and a fresh medium, which comprises biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri- iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin. B27 Supplement (available from Invitrogen, Carlsbad, CA; www.invitrogen.com ; currently catalog no. 17504-044; and from PAA Laboratories GmbH, Pasching, Austria; www.paa.com : catalog no. F01-002; Brewer et al, J Neurosci Res., 35(5):567-76, 1993) and/or N2 Supplement (available from Invitrogen, Carlsbad, CA; www.invitrogen.com catalog no. 17502-048; and from PAA Laboratories GmbH, Pasching, Austria; www.paa.com ; catalog no. F005-004; Bottenstein & Sato, PNAS, 76(1):514-517, 1979) may be used to formulate such a culture medium. As noted elsewhere herein, use of B27 Supplement is a convenient way to incorporate biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin into a culture medium of the invention. As noted elsewhere herein, use of N2 Supplement is a convenient way to incorporate transferrin, insulin, progesterone, putrescine and sodium selenite into a culture medium of the invention.

In one embodiment, a culture medium of the invention is a mixture of a conditioned medium and a fresh medium, which has been supplemented with B27 Supplement and N2 Supplement.

In one embodiment, a culture medium of the invention is a mixture of a conditioned medium and a fresh medium of different types, which comprises biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin.

In one embodiment, a culture medium of the invention is a mixture of a conditioned medium and a fresh medium of different types, which has been supplemented with B27 Supplement and N2 Supplement. In this embodiment, the fresh medium may be DMEM/F12 (Invitrogen). The conditioned medium may be VitrohES (Vitrolife AB) conditioned on mouse embryonic fibroblast cells. For example, the invention provides a culture medium comprising a mixture of mEF-conditioned VitrohES and fresh DMEM/F12, which has been supplemented with B27 Supplement and N2 Supplement.

If a conditioned medium is mixed with a fresh medium, the conditioned medium and fresh medium may be mixed to form a mixed culture medium that comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%, by volume (or by dry weight) conditioned medium.

If a conditioned medium is mixed with a fresh medium, the conditioned medium and fresh medium may be mixed to form a mixed culture medium that comprises at least 5%, at least 10%, at least 15%, at least 20%5.at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%, by volume (or by dry weight) fresh medium.

A culture medium may be a Ix formulation or a concentrated formulation, e.g. a 2x to 25Ox concentrated medium formulation. In a Ix 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 water (preferably deionized and distilled) or any appropriate solution, e.g. an aqueous saline solution, an aqueous buffer or a culture medium.

As noted elsewhere herein, the inventor believes that agonists to FXR, RXR, RAR, PPAR and THR, and particularly combinations of agonists as disclosed herein, may activate complex nuclear signalling pathways that inhibit differentiation of stem cells and allow them to rapidly proliferate whilst maintaining their pluripotency.

A culture medium as disclosed herein may be capable of expanding a population of stem cells in a pluripotent, undifferentiated and proliferative state for at least 3 passages under appropriate conditions. Stem cells 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 cell culture. Preferably, a culture medium is capable of expanding a population of stem cells 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 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 cells in a pluripotent, undifferentiated and proliferative state for more than 3 passages, more than 4 passages, more than 5 passages, more than 10 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 of the invention, the stem cells are 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 60, at least 70, at least 80, at least 90 or at least 100, passages under appropriate conditions.

A culture medium as disclosed herein may be capable of expanding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different pluripotent stem cell 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 SA121, SA181 and SA461 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 10, passages under appropriate conditions.

As noted elsewhere herein, the invention also provides a hermetically-sealed vessel containing a culture medium of the invention. 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, ajar, a vial or a bag.

As noted elsewhere herein, the invention also provides a method for preparing a culture medium, comprising the steps of: (a) obtaining a culture medium; and (b) adding a farnesoid X receptor (FXR) agonist, a retinoid X receptor (RXR) or retinoic acid receptor (RAR) agonist, a peroxisome proliferator-activated receptor (PPAR) agonist, and/or a thyroid hormone receptor (THR) agonist to the culture medium. Various different methods for preparing culture media are envisaged, depending on the specific ingredients to be included in the culture medium. For example, a method for preparing a culture medium may comprise the steps of: (a) obtaining a culture medium; and (b) adding a bile acid, a retinol and a diiodothyronine to the culture medium. In one embodiment, a method for preparing a culture medium may comprise the steps of: (a) obtaining a culture medium; and (b) adding cholic acid, all-trans-retinol and 3,5-diiodo- L-thyronine to the culture medium. In some of these embodiments, the culture medium may be a conditioned medium, or a mixture of a conditioned medium and a fresh medium of different types, as described elsewhere herein.

Cell Culture Methods and Uses of the Culture Media

The culture media of the invention can be used to expand a population of pluripotent stem cells. Accordingly, the invention provides the use of any culture medium as disclosed herein for expanding a population of pluripotent stem cells.

The invention also provides a method for expanding a population of pluripotent stem cells, comprising: (a) providing a population of pluripotent stem cells; (b) providing a culture medium as disclosed herein; (c) contacting the stem cells with the culture medium; and (d) culturing the stem cells under appropriate conditions.

A method for 'expanding' a population of cells is one that involves increasing the number of stem cells in an initial population to generate an expanded population, whilst maintaining pluripotency and without significant differentiation, i.e. one that involves growth and division of stem cells, but not their differentiation.

The methods of the invention may comprise culturing the cells in contact with an extracellular matrix material as described elsewhere herein. For example, the invention provides a method for expanding a population of pluripotent stem cells, comprising: (a) providing a population of pluripotent stem cells; (b) providing a culture medium as disclosed herein; (c) contacting the stem cells with the culture medium; and (d) culturing the cells under appropriate conditions and in contact with an extracellular matrix material. The invention also provides the use of a culture medium as disclosed herein and an extracellular matrix material to expand a population of pluripotent stem cells.

The methods of the invention may comprise a step of passaging stem cells into a culture medium as disclosed herein. For example, the invention provides a method for expanding a population of pluripotent stem cells, comprising: (a) providing a population of pluripotent stem cells; (b) providing a culture medium as disclosed herein; (c) contacting the stem cells with the culture medium; (d) culturing the cells under appropriate conditions; (e) passaging the cells into a culture medium as disclosed herein; and (f) further culturing the cells under appropriate conditions.

It Will be appreciated that the steps of the methods disclosed herein can 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 pluripotent stem cells may be performed before, after or at the same time as, the step of providing a culture medium.

Cells may be passaged in the methods of the invention using known methods, e.g. by incubating the cells 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. Collagenase, 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 of the invention do not comprise any step of manually selecting undifferentiated cells when the cells are passaged. In some embodiments, the cell culture methods of the invention comprise automated passaging of the stem cells, i.e. without manipulation by a laboratory worker.

The pluripotent stem cells will be seeded onto a support at a density that promotes cell proliferation but which limits differentiation. Typically, a plating density of at least 15,000 cells/cm 2 is used. A plating density of between about 15,000 cells/cm 2 and about 200,000 cells/cm 2 may be used. Single-cell suspensions or small cluster of cells will normally be seeded, rather than large clusters of cells, as in known in the art.

The environment used to culture the stem cells may be sterile and temperature stable.

The inventor has found that the culture media disclosed herein are particularly advantageous because they can be used to expand pluripotent stem cells without the need to adapt the cells to the culture medium, as is commonly required when transferring stem cells into a new culture medium. Various different methods for adapting cell cultures to new media are known in the art. Accordingly, in some embodiments the methods of the invention do not include any step of adapting a population of stem cells to a new culture medium, e.g. by gradually changing the components of the medium. The invention therefore provides a method for expanding a population of pluripotent stem cells, comprising: (a) providing a population of pluripotent stem cells; (b) providing a first culture medium; (c) culturing the cells in the first culture medium under appropriate conditions; (d) providing a second culture medium, which is a culture medium as disclosed herein, and which is different to the first culture medium; (e) replacing the first culture medium with the second culture medium, exchanging the first culture medium with the second culture medium or passaging the cells from the first culture medium into the second culture medium; and (f) further culturing the cells in the second culture medium under appropriate conditions, wherein the method does not comprise any step of adapting the population of stem cells to the second culture medium.

The methods and uses of the invention may involve any culture medium or supplement as described herein. Accordingly, in some embodiments the methods of the invention may be serum and/or serum replacement-free methods. In some embodiments, the methods of the invention may be used to culture cells in the absence of contact with a layer of feeder cells.

The methods of the invention may be performed using any suitable cell culture vessel as a support. Cell culture vessels of various shapes and sizes {e.g. flasks, single or multiwell plates, single or multiwell dishes, bottles, jars, vials, bags, bioreactors) and constructed from various different materials (e.g. plastic, glass) are known in the art. A suitable cell culture vessel can readily be selected by the skilled person.

Culture Medium Supplements

The invention also provides a culture medium supplement that can be used to produce a culture medium as disclosed herein. A "culture medium supplement' is a mixture of ingredients that cannot itself support pluripotent stem cells, but which enables or improves pluripotent stem cell culture when combined with other cell culture ingredients. The supplement can therefore be used to produce a functional cell culture medium of the invention by combining it with other cell culture ingredients to produce an appropriate medium formulation. The use of culture medium supplements is well known in the art. The invention provides a culture medium supplement that comprises a farnesoid X receptor (FXR) agonist, a retinoid X receptor (RXR) or retinoic acid receptor (RAR) agonist, a peroxisome proliferator-activated receptor (PPAR) agonist, and/or a thyroid hormone receptor (THR) agonist. The supplement may contain any agonist (or combination of agonists) disclosed herein. The supplement may also contain one or more additional cell culture ingredients as disclosed herein, e.g. 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 25Ox concentrated liquid supplement) or may be a dry supplement. Both liquid and dry supplements are well known in the art. A supplement may be lyophilised.

A supplement of the invention 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.

As noted elsewhere herein, the invention also provides a hermetically- sealed vessel containing a culture medium supplement of the invention. Hermetically-sealed vessels may be preferred for transport or storage of the culture media supplements disclosed herein, to prevent contamination. The vessel may be any suitable vessel, such as a flask, a plate, a bottle, ajar, a vial or a bag.

Compositions and Cell Culture Systems

The cell culture media and cell culture supplements disclosed herein can be used to expand a population of pluripotent stem cells. Accordingly, the invention provides compositions arising during use of the cell culture media and cell culture supplements of the invention. For example, the invention provides a composition comprising: (a) a culture medium according to the invention; and (b) 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 cells that is co-cultured with, and which provides a surface suitable for growth of, the pluripotent cells of interest. The feeder cell layer provides an environment in which the cells of interest can grow. Feeder cells are often mitotically inactivated (e.g. by irradiation or treatment with mitomycin C) to prevent their proliferation. As noted elsewhere herein, the culture media of the invention are particularly advantageous because they can be used to culture cells without feeder cell contact, i. e. the methods of the invention do not require a layer of feeder cells to support the stem cells.

Accordingly, the compositions of the invention may be feeder cell-free compositions. A composition is conventionally considered to be feeder cell-free if the pluripotent stem cells in the composition have been cultured for at least one passage in the absence of a feeder cell layer. A feeder cell-free composition of the invention 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 cells in the composition).

An extracellular matrix material may be used to substitute the supportive function of a feeder cell layer. The invention therefore also provides a composition containing: (a) a culture medium according to the invention; and (b) an extracellular matrix material. These compositions may further comprise pluripotent stem cells.

A variety of substances have been used as extracellular matrix materials for pluripotent stem cell culture, and an appropriate material can readily be selected by the skilled person. An extracellular matrix material may comprise fϊ bronectin, vitronectin, laminin, collagen (particularly collagen II, collagen III or collagen IV), thrombospondin, osteonectin, secreted phosphoprotein I heparan sulphate, dermatan sulphate, gelatine, merosin, tenasin, decorin, entactin or a basement membrane preparation from Engelbreth-Ho Im-Swarm (EHS) mouse sarcoma cells (e.g. Matrigel®; Becton Dickenson). A synthetic extracellular matrix material, such as ProNectin (Sigma Z378666) may be used. Mixtures of extracellular matrix materials may be used, if desired.

Preferably, the extracellular matrix material comprises fibronectin. Bovine fibronectin, recombinant bovine fibronectin, human fibronectin, recombinant human fibronectin, mouse fibronectin, recombinant mouse fibronectin or synthetic fibronectin may be used.

The extracellular matrix material will normally be coated onto a cell culture vessel, but may (in addition or alternatively) be supplied in solution. A fibronectin solution of about lmg/ml may be used to coat a cell culture vessel. In some embodiments, a cell culture vessel is coated with fibronectin at about 1 µg/cm2 to about 250 µg/cm2, or at about 1 µg/cm to about 150 µg/cm . In ome embodiments, a cell culture vessel is coated with fibronectin at 8 µg/cm2 or 125 µg/cm2.

The invention also provides the use of (a) an extracellular matrix material; and (b) a famesoid X receptor (FXR) agonist, a retinoid X receptor (RXR) or retinoic acid receptor (RAR) agonist, a peroxisome proliferator-activated receptor (PPAR) agonist, and/or a thyroid hormone receptor (THR) agonist, for expanding a population of pluripotent stem cells. For example, the invention provides the use of fibronectin, cholic acid, all-trans-retinol and 3,5-diiodo-L-thyronine for expanding a population of pluripotent stem cells.

The invention also provides a composition or cell culture vessel comprising (a) an extracellular matrix material; and (b) a farnesoid X receptor (FXR) agonist, a retinoid X receptor (RXR) or retinoic acid receptor (RAR) agonist, a peroxisome proliferator- activated receptor (PPAR) agonist, and/or a thyroid hormone receptor (THR) agonist. For example, the invention provides a composition or cell culture vessel comprising fibronectin, cholic acid, all-trans-retinol and 3,5-diiodo-L-thyronine.

The compositions of the invention may comprise serum, or may be serum-free and/or serum-replacement free, as described elsewhere herein.

The invention also provides a composition or cell culture vessel comprising (a) an extracellular matrix material; and (b) a culture medium which comprises biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin.

The invention also provides a composition or cell culture vessel comprising (a) an extracellular matrix material; and (b) a culture medium which has been supplemented with B27 Supplement and N2 Supplement.

The invention also provides a composition or cell culture vessel comprising (a) an extracellular matrix material; and (b) a culture medium which is a mixture of a conditioned medium and a fresh medium, and which comprises biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin.

The invention also provides a composition or cell culture vessel comprising (a) an extracellular matrix material; and (b) a culture medium which is a mixture of a conditioned medium and a fresh medium, and which has been supplemented with B27 Supplement and N2 Supplement.

The invention also provides a composition or cell culture vessel comprising (a) an extracellular matrix material; and (b) a culture medium which is a mixture of a conditioned medium and a fresh medium of different types, and which comprises biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin.

The invention also provides a composition or cell culture vessel comprising (a) an extracellular matrix material; and (b) a culture medium which is a mixture of a conditioned medium and a fresh medium of different types, and which has been supplemented with B27 Supplement and N2 Supplement. For example, the invention provides a composition or cell culture vessel comprising (a) an extracellular matrix material; and (b) a culture medium comprising a mixture of mEF-conditioned VitrohES and fresh DMEM/F12, which has been supplemented with B27 Supplement and N2 Supplement.

Pluripotent stem cells

The culture media and methods disclosed herein are useful for expanding a population of pluripotent stem cells, 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 cells of all three germ layers (endoderm, mesoderm and ectoderm) under appropriate conditions.

Pluripotent stem cells are not totipotent, i. e. they cannot form an entire organism, such as a foetus. Pluripotent stem cells for use in the invention can be obtained using well- known methods (see below). It is envisaged that various types of pluripotent stem cells may be used in conjunction with the invention, whether obtained from embryonic, foetal or adult tissue. Stem cells may be cloned directly from an organism for use in the invention, but established stem cell lines will typically be used. Accordingly, in some embodiments the initial population of stem cells are the progeny of previously-isolated stem cells or are the progeny of an established stem cell line, such that the invention does not involve any use of a tissue sample.

The culture media disclosed herein may be used to culture mammalian stem cells, particularly primate embryonic stem cells. Primate embryonic stem cells 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 cells are prepared from the inner cell mass (ICM) of a mammalian blastocyst using known techniques. For example, human ES cells can be obtained using the methods described in Thomson et al. (1998) Science 282:1 145-1147, Thomson et al. (1998) Curr. Top. Dev. Biol. 38:133 and US patent 5,843,780. In some embodiments where hES cells are used, the initial population of cells are the progeny of previously-isolated hES cells or are the progeny of an established line of hES cells, such that the invention does not involve any use of a human embryo. In other embodiments, the initial population of hES cells are the progeny of cells or a cell line obtained using a method that did not involve any use of a human embryo.

Commercially available hES cell lines can be used. Examples of commercially available stem cell lines that might be used in the invention include the SA461, SAl 2 1 and SAl 8 1 hES cell lines (Cellartis AB, Gδteborg, Sweden).

The inventor has already successfully tested the culture media and methods of the invention with the SA461 hES cell line (see the Examples herein), and preliminary results suggest that the same culture media and methods can be used with the SAl 2 1 and

SAl 8 1 hES cell lines. Accordingly, any of these cell lines can be used in the invention.

A culture media as disclosed herein may also be used to culture embryonic germ (EG) cells, particularly human EG cells. EG cells are prepared from primordial germ cells using known techniques. For example, human EG cells can be obtained using the method described in Shamblott et al. (1998) PNAS 95:13726 and US patent 6,090,622.

A culture media as disclosed herein may also be used to culture genetically modified pluripotent stem cells. Genetically modified cells include those that have been transiently or stably modified by transformation, transfection, transduction, etc.. For example, the culture media may be used to culture GFP reporter lines, or disease models (e.g. cells containing mutations or deletions in one or more genes).

The culture media disclosed herein may also be used to culture cells that have been induced or transformed to form cells with ES cell-like properties. These pluripotent stem cells may be genetically modified cells, such as mouse or human 'induced pluripotent stem' (iPS) cells. iPS cells are typically derived from adult somatic cells by transducing them with 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 al. (2007) Cell 13 1(5):861-72; Yu et al (2007) Science 318(5858):1917-1920; and Yamanaka S. (2008) Cell Prolif. 4 1 Suppl 1:51-6).

When a culture medium of the invention is used to expand a population of pluripotent stem cells, the total number of undifferentiated, pluripotent stem cells in the population will preferably increase at least 1.5 fold, at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold or at least 50 fold, between the time when a medium of the invention is applied to an initial cell population and the end of the culture period. It will be appreciated that the cells 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 cells are cultured in different cell culture vessels after passaging, or if cells are discarded during passaging, this can be taken into account when calculating the fold difference in cell numbers obtained during a known culture period.

A 'population' of cells is any number of cells greater than 1, but is preferably at least IxIO3 cells, at least IxIO4 cells, at least IxIO5 cells, at least IxIO6 cells, at least IxIO7 cells, at least IxIO8 cells, or at least IxIO9 cells. In preferred embodiments of the invention, 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 stem cells (% by cell number) in an initial cell population will be undifferentiated, pluripotent and proliferative cells.

In preferred embodiments of the invention, 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 stem cells (% by cell number) in an expanded population (z. e. the population after expansion of the initial population using a culture medium or method as disclosed herein) will be undifferentiated, pluripotent and proliferative cells.

Methods for identifying undifferentiated, pluripotent and proliferative stem cells, and for identifying the % of such cells in a population, are known and suitable methods for use with this invention can be selected by the skilled person depending on the stem cell type that is used.

Pluripotent 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 can be allowed to form embryoid bodies in vitro, then the embryoid bodies studied to identify cells of all three germ layers. Alternatively, stem cells can be allowed to form teratomas in vivo {e.g. in SCID mice), then the teratomas studied to identify cells of all three germ layers. Accordingly, in preferred 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 stem cells in an expanded population (or in an initial population) are capable of differentiating into cells of all three germ layers in vitro or in vivo.

The genomic integrity of stem cells can be confirmed by karyotype analysis. Stem sells can be karyotyped using known methods. A normal karyotype is where all chromosomes are present {i.e. euploidy) with no noticeable alterations. Accordingly, in preferred embodiments of the invention 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 stem cells in an expanded population (or in an initial population) exhibit normal karyotypes.

Pluripotent 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 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). Accordingly, in preferred 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 stem cells in an expanded population (or in an initial population) express OCT-4, SSEA-3, SSEA-4, TRA- 1-60 and/or TRA-I -81 at levels appropriate for hES cells.

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 cells in an expanded population (or in an initial population) will (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, SSEA-3, SSEA-4, TRA- 1-60 and TRA- 1-81 at levels appropriate for hES cells.

Undifferentiated, pluripotent and proliferative stem cells may also be identified by their morphological characteristics. Undifferentiated, pluripotent and proliferative stem cells are readily recognisable by those of skill in the art. For example, in a normal microscope image hES cells typically have high nuclear/cytoplasmic ratios, prominent nucleoli and compact colony formation with poorly discernable cell junctions. hES cells may also be identified by determining their alkaline phosphatase activity. hES cells have alkaline phosphatase activity, which can be detected by known methods.

General

References in the singular (e.g. to "a cell" and equivalent references) encompass the plural (e.g. "cells") unless the context requires otherwise. The term "comprising" encompasses "including" as well as "consisting of e.g. a culure 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" can be omitted.

Various aspects and embodiments of the invention will now be described in more detail by way of example, with reference to specific culture media and methods. It will be appreciated that modification of detail may be made without departing from the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 illustrates the effects of some FXR agonists and antagonists on the proliferation of SA461 hES cells after 24 and 48 hours.

Figure 2 illustrates the effect of dihomo-γ-linolenic acid on the proliferation of SA461 hES cells after 24 hours.

Figure 3 illustrates the effects of some THR agonists on the proliferation of SA461 hES cells after 24 and 48 hours.

Figure 4 illustrates the effects of some PPAR agonists on the proliferation of SA461 hES cells after 24 and 48 hours.

Figure 5 illustrates the effects of some RXR/RAR agonists on the proliferation of SA461 hES cells after 24 and 48 hours.

Figures 6-8 illustrate the typical morphology of SA461 hES cells following 6 passages in a culture system of the invention.

Figure 9 illustrates the normal karyotype of SA461 hES cells following 6 passages in a culture medium according to this invention.

Figure 10 illustrates a high alkaline phosphatase activity in SA461 hES cells following 6 passages in a culture medium according to the invention. Figures 11-20 illustrate immunostaining of pluripotency markers in SA461 hES cells following 6 passages in a culture medium of the invention.

Figure 11 shows the results of OCT4 immunostaining on SA461 hES cells following 6 passages in a culture medium of the invention. Figure 12 shows the results of DAPI staining of the SA461 hES cells of Figure 11.

Figure 13 shows the results of SSEA3 immunostaining on SA461 hES cells following 6 passages in a culture medium of the invention. Figure 14 shows the results of DAPI staining of the SA461 hES cells of Figure 13.

Figure 15 shows the results of SSEA4 immunostaining on SA461 hES cells following 6 passages in a culture medium of the invention. Figure 16 shows the results of DAPI staining of the SA461 hES cells of Figure 15.

Figure 17 shows the results of TRAl 60 immunostaining on SA461 hES cells following 6 passages in a culture medium of the invention. Figure 18 shows the results of DAPI staining of the SA461 hES cells of Figure 17.

Figure 19 shows the results of TRAl 8 1 immunostaining on SA461 hES cells following 6 passages in a culture medium of the invention. Figure 20 shows the results of DAPI staining of the SA461 hES cells of Figure 19.

Figure 2 1 shows an example of an expression profile of various markers of hES cell pluripotency and differentiation as measured by QRT PCR in SA461 hES cells following 6 passages in a culture medium according to this invention.

Figure 22 shows an example of the morphology of SA461 hES cells cultured for 4 passages in a culture medium containing dihomo-γ-linolenic acid and 3,5-diiodo-L- thyronine (3,5-DLT).

Figure 23 shows an example of the morphology of SA461 hES cells cultured for 4 passages in media containing dihomo-γ-linolenic acid, 3,5-diiodo-L-thyronine (3,5- DLT), all-trans-retinol and retinyl acetate.

Figure 24 shows an example of the morphology of SA461 hES cells cultured for 4 passages in media without exogenous lipid or NHR agonists. Figure 25 shows an example of the morphology of SA461 hES cells cultured for 4 passages in media containing 3,5-diiodo-L-thyronine (3,5-DLT) and all-trans-retinol.

Figure 26 shows an example of the morphology of SA461 hES cells cultured for 4 passages in media containing all-trans-retinol, 3,5-diiodo-L-thyronine (3,5-DLT) and cholic acid (CA).

Figure 27 shows the results of OCT4 immunostaining on SA461 hES cells following 3 enzymatic passages in a culture medium as described in Example 5. Figure 28 shows the results of DAPI staining of the SA461 hES cells of Figure 27.

Figure 29 illustrates the typical morphology of SA461 hES cells following 3 enzymatic passages in a culture system as described in Example 5.

Figure 30 shows the results of smooth muscle actin immunostaining on differentiated cells derived from SA461 hES cells following 3 enzymatic passages in a culture medium as described in Example 5. Figure 3 1 shows the results of DAPI staining of the cells of Figure 30.

Figure 32 shows the results of beta tubulin immunostaining on differentiated cells derived from SA461 hES cells following 3 enzymatic passages in a culture medium as described in Example 5. Figure 33 shows the results of DAPI staining of the cells of Figure 32.

Figure 34 shows the results of HNF3β immunostaining on differentiated cells derived from SA461 hES cells following 3 enzymatic passages in a culture medium as described in Example 5. Figure 35 shows the results of DAPI staining of the cells of Figure 34.

Figure 36 shows the results of in vivo differentiation of SA121 hES cells to tissue representative of all three germ layers following 23 passages in a culture medium as described in Example 5.

Figure 37 shows the results of in vivo differentiation of SA461 hES cells to tissue representative of all three germ layers following 17 passages in a culture medium as described in Example 5. The following examples are provided to illustrate the invention, and do not limit the scope of the preceding description or the following claims. The culture media and methods described in the examples herein were designed primarily for hES cell expansion, and may be adapted for use with other types of pluripotent stem cells by routine optimisation without undue experimentation.

EXAMPLES

Example 1: Investigation of the influence of NHR agonists on hES cell proliferation

This example identifies the presence of factors that either induce or support hES cell proliferation in the absence of conditioned medium and peptide growth factors. It is known that lipids and lipophilic agents bound by albumin act to support hES cell self renewal (Garcia-Gonzalo and Belmonte, 2008). Nuclear hormone receptors (NHRs) are transcription factors that regulate gene expression in response to lipophilic ligands. The inventor postulated that the efficacious agents may be NHR agonists. NHRs are known to influence cell growth and differentiation in other cell types, have widely varying effects dependant on different patterns of receptor dimerisation and are present in serum. Agonists that have been investigated include agonists for the FXR, the RXR and RAR, the PPAR, and the THR.

Materials and Methods

The hES cells used in these experiments were line SA461 from human blastocysts established by Cellartis AB (Goteborg, Sweden). For these assays this cell line was dissociated to single cells and plated in a 96 well format before culture in a basal medium to determine the activity of the various ligands without the interference or masking effect of exogenous growth factors or conditioned medium. The proliferation inducing ability was established by comparison with control wells using a proliferation assay based on the activity of cellular mitochondrial dehydrogenases, a measurment directly proportional to cell number. The lack of washing or harvesting steps in this assay removes the possibility of cell loss during processing.

SA461 stem cells at passage 74 were treated with Y27632 (Calbiochem) for 4 hours then trypsinised and seeded as a single cell suspension in VitrohES (Vitrolife AB) with 1OµM Y27632 in 96 well plate format on a fibronectin coated surface (16.6µg/cm2) at a density of 10,000 cells/well. To study the effects of nuclear hormone receptor agonists and antagonists, the cell culture medium was changed 24 hours after attachment to Advanced DMEM/F12 (Invitrogen) and agonist or antagonist added. Each compound was resuspended in DMSO as a 1OmM stock solution prior to addition to the culture medium.

The following FXR agonists and antagonists were tested: cholic acid (CA), deoxycholic acid (DCA), taurodeoxycholic acid (TDCA), lithocholic acid (LCA) and Z- Guggulsterone (Z-G) (all Biomol). The effect of dihomo- γ-linolenic acid (DGLA or DiHLA) (Biomol), a polyunsaturated fatty acid FXR agonist, was also tested.

The following THR agonists were tested: 3,5-diiodo-L-thyronine (3,5-DLT), 3,5-diiodo- L-tyrosine dihydrate (DLTdH) and 3,5-diiodo-4-hydroxyphenylpropionic acid (DIHPA, also sometimes referred to as DHPPA) (all Biomol).

The following PPAR agonists and antagonists were tested: 5,8,1 1,14-eicosatetraynoic acid, bezafibrate, clofibric acid, gemfibrozil, WYl 4643 and tetradecylthio acetic acid (all Biomol).

The following RXR and RAR agonists were tested: 13-cis retinoic acid (13cRA), 9-cis retinoic acid (9cRA), methoprene acid (MPA), 13-cis retinol (13cROL), retinyl acetate (RETACT), acitretin (ACT), 4-hydroxyretinoic acid (4HRA) and all trans retinol (ATR) (all Biomol) resuspended in DMSO.

Cell number was assayed after a further 24 hours (and in some experiments after a further 48 hours) using the Rapid Cell Proliferation Kit (Calbiochem) according to the manufacturers instructions with a 1 hour incubation and absorbance read at 450nm on a microplate reader.

Results

The results of these experiments are shown in Figures 1-5. Figures 1 and 2 show that in the case of the FXR both bile acids (cholesterol metabolites) and dihomo-γ-linolenic acid (a polyunsaturated fatty acid) were able to markedly increase cell growth rates over a 48 hour timescale. The importance of FXR activity in liES cell self-renewal is further demonstrated by the rapid and effective suppression of cell growth produced by exposure to the known FXR antagonist Z-Guggulsterone (Z-G) (see Figure 1). Figure 3 shows that the three different THR agonists tested were all able to markedly increase cell growth rates over a 48 hour timescale. Figure 4 shows that the six different PPAR agonists tested were also all able to markedly increase cell growth rates over a 48 hour timescale. Figure 5 illustrates that the RXR/RAR agonists tested produced two distinct groups of influence. In the first group were those agonists found to induce proliferation such as 13-cis-retinoic acid, 9-cis-retinoic acid and 4-hydroxyretinoic acid. In the second group were those agonists found to suppress proliferation including all-trans-retinol, retinyl acetate and 13-cis-retinol. Without wishing to be bound by the theory, the inventor believes that NHR agonists which appear to reduce hES cell proliferation in an assay of the type described in this example may be useful in hES cell culture media, as well as those which are found to increase hES cell proliferation in such an assay, because of the complex nature of NHR signalling pathways, particularly when combinations of agonists are used. For example, the difference in the results in Figure 5 may be the result of the recruitment of different binding partners after RXR and/or RAR activation by the different RXR and RAR agonists tested. The inventor's results suggest opposing ligand- specific signalling functions, but confirm that RXR and RAR agonists play a role in hES cell proliferation and self-renewal. For example, although all-trans-retinol appears to reduce hES cell proliferation relative to the control in Figure 5, it has been shown to significantly improve hES cell culture when incorporated into a culture medium of the invention (see Examples 2 and 3).

In summary, significant changes in hES cell proliferation were observed in response to different families of nuclear hormone receptor agonists, as well as to a variety of different individual agonists. These results show that all the nuclear hormone receptor families tested contain multiple different agonists capable of modulating hES cell proliferation when added to a basal culture medium. These results demonstrate that hES cells are sensitive to a wide range of nuclear hormone receptor agonists, and open the way for identification of further related agonists useful in stem cell culture. In light of the disclosure herein, a skilled person will readily be able to identify further agonists to the nuclear hormone receptor families tested for use in the culture media and methods of the invention. Example 2: Further investigations into using NHR agonists in hES cell culture

In light of the results of the experiments described above, the inventor further tested the effects of incorporating nuclear hormone receptor agonists into stem cell culture media. The inventor hypothesised that introduction of the agonists into a culture medium prior to conditioning might result in the utilisation, degradation or metabolism of agonists by the fibroblast feeder cell layer. Accordingly, in these experiments the agonists were used as a supplement to a culture medium which has already undergone a conditioning step.

The SA461 hES cell line was provided by Cellartis AB (Gδteborg, Sweden) and initially expanded, as instructed by the provider, on mitotically inactivated mouse embryonic fibroblasts (MEFs). The MEFs used in this case were isolated from the dl3 foetuses of pregnant ICR strain mice. A conditioned medium was produced by conditioning VitrohES (Vitrolife AB) media containing 4ng/ml bFGF on mitotically inactivated MEFs seeded at 65,000 cells/cm2 on a gelatinised culture surface at a ratio of 0.14ml/cm 2 for 24 hours before filter sterilising and storage at -800C. A fresh medium was also produced, consisting of DMEM/F12 (Invitrogen) containing Synthechol™ supplement (Invitrogen), transferrin, insulin and selenite, progesterone and putrescine (Invitrogen),

L-glutamine lOOµM (Invitrogen) and DL-alpha tocopherol 30OnM (Sigma) to replace routine culture components which may be degraded or exhausted in the conditioned media by fibroblast exposure. The inclusion of dihomo-γ-linolenic acid (lOµM), all- trans-retinol (3OnM), cholic acid (l µM), 3,5-diiodo-L-thyronine (5OnM), and retinyl acetate (1OnM) was assessed for effect on the morphology of hES cells over prolonged culture. The conditioned and fresh media were combined in equal ratio then supplemented with 10ng/ml bFGF to produce a complete culture medium. This complete medium was mixed immediately prior to use.

Cells were passaged manually from the feeder based colonies onto IVF dishes pre-coated with fibronectin matrix at passage 51, incubated with the complete medium mentioned 0 above and cultured in a 5% CO2 incubator at 37 C. Medium changes took place every 48 hours. As described in more detail below, images taken at passage 4 demonstrate that some combinations of agonists produced expansive regions of hES like morphology, and one combination did not contain evidence of differentiating cells (Figures 22-26). A combination of an FXR agonist (.dihomo-γ-linolenic acid) and a THR agonist (3,5- - diiodo-L-thyronine) alone produced uneven fissured colonies with peripheral differentiation, although the majority of the cells did still exhibit the stem cell like morphology of small round cells with a high nuclear to cytoplasic ratio (Figure 22).

A combination of an FXR agonist (dihomo-γ-linolenic acid), a THR agonist (3,5-diiodo- L-thyronine) and two RXR/RAR agonists (all-trans-retinol and retinyl acetate) resulted in both peripheral and central differentiation (Figure 23).

A media without any nuclear receptor agonists, and in the absence of cholesterol to prevent endogenous production of bile acids, produced relatively homogenous colonies with areas of peripheral differentiation and a fragile morphology which rendered them difficult to passage (Figure 24).

A combination of an RXR/RAR agonist (all-trans-retinol) and a THR agonist (3,5- diiodo-L-thyronine) produced expansive areas of morphologically acceptable hES-like cells with both peripheral and central regions of differentiation (Figure 25).

A combination of an FXR agonist (cholic acid), a THR agonist (3,5-diiodo-L-thyronine) and a RXR/RAR agonist (all-trans-retinol) allowed the growth of cell colonies exhibiting typical hES-like morphology without visible indication of differentiated cell types. (Figure 26). This particular cell culture medium was continued in culture and the cells characterised at passage 6 (see Example 3).

These results clearly demonstrate that the supplementation of cell culture media for hES cell expansion with nuclear hormone receptor agonists can assist in the maintenance of the typical morphology of an undifferentiated embryonic stem cell over multiple passages in a feeder-free culture system.

Example 3: Human ES cells cultured with a combination of NHR agonists retain an undifferentiated morphology and phenotype

A conditioned medium was produced as in Example 2. A fresh medium was also produced, consisting of DMEM/F12 (Invitrogen) containing Synthechol™ supplement (Invitrogen), transferrin, insulin, selenite, progesterone and putrescine (Invitrogen), L- glutamine lOOµM (Invitrogen), DL-alpha tocopherol 30OnM (Sigma), the THR agonist 3,5-diiodo-L-thyronine (5OnM) (Biomol), the RXR/RAR agonist all-trans-retinol (3OnM) (Biomol), and the FXR agonist cholic acid (l µM) (Biomol). 3,5-diiodo-L- thyronine, all-trans-retinol and cholic acid were resuspended in DMSO as 1OmM stock solutions before final dilution in the complete culture media. The hES cells were cultured continuously as described in Example 2 for 6 passages with a passage interval of between 4-7 days to fresh fibronectin coated IVF dishes. The hES cells rapidly adopted a consistent morphology and at 6 passages they still retained a characteristic morphology, forming dense colonies with small rounded cells and a high nucleus to cytoplasm ratio with multiple visible nucleoli (Figures 6-8).

Immunostaining experiments were performed using antibodies against the pluripotency markers OCT4, SSEA 3 and SSEA 4, TRA 181 and TRA 160 with associated nuclear DAPI staining (DAPI: Final con.: 0.5ug/ml; Oct-4: (1:500), 2ary: Goat-a-mouse-IgG- FITC (1:150); SSEA-I: (1:200), 2ary: Goat-a-Mouse-IgM-FITC (1:150); SSEA-3: (1:200), 2ary: Goat-a-Rat-IgM-Cy3 (1:150); SSEA-4: (1:200), 2ary: Goat-a-Mouse-IgG- FITC (1:150); TRA-1-60: (1:200), 2ary: Goat-a-Mouse-IgM-FITC (1:150); TRA-1-81: (1:200), 2ary: Goat-a-Mouse-IgM-FITC (1:150)). Primary antibodies were incubated overnight at 4 C and secondary at room temperature for 2h. The entire visible population stained positive for the undifferentiated markers OCT4, SSEA3, SSEA4, TRA 160 and TRAl 81, with no loss of expression around the margin of the colonies (Figures 11-20).

Karyotpye anatysis was also performed. Briefly, cells were incubated in culture with O.l µg/ml of colcemid, trypsinised, resuspended in 0.075M KCl then fixed in 3:1 methanol/acetic acid. After staining karotypes were examined and band analysis performed at Cellartis AB (Gδteborg, Sweden). The cells, which were established from a male embryo, possessed a normal 46XY karyotype (Figure 9).

QT-PCR analysis of marker expression profiles was also performed. RNA was extracted using the RNEasy™ kit (Qiagen, Valencia, CA) according to manufacturers instructions, genomic DNA eliminated by DNase 1 digestion using the Turbo™ DNase (Austin, Texas), reverse transcription performed using Superscript II (Invitrogen, Carlsbad, CA) and QRT PCR using Taqman Low Density Arrays (Applied Biosystems, Foster City, CA), all according to manufacturers instructions. This QRT-PCR analysis demonstrated a high level of expression of the pluripotency associated genes LIN28, DNMT3B, GAL, ZFP42, SFRP2, PODXL, POU5F1, SOX2, NANOG and TDGFl, with low or no expression of the differentiation markers AFP, SERPINAl, FGF5, PAX4, TAT, SST, INS, IPFl, T, COL2A1, PECAMl, MYF5, CDH5, MYODl, DES, RUNX2, EOMES, CGB GCMl, GFAP, OLIG2, SYP, NES, PAX6 and TH (Figure 21).

The alkaline phosphatase activity of the cells was also analysed (Figure 10). Cells were PBS rinsed then fixed in citrate-acetone-formaldehyde solution prior to staining with FRV-alkaline/NaptholAS-BI Alkaline solution and final slide preparation with DAKO mounting media.

This example confirms that hES cells expanded in a culture medium as disclosed herein retain the unique morphology and molecular phenotype characteristic of undifferentiated hES cells, whilst retaining a stable and normal karyotype after prolonged culture.

Summary of Examples 1-3

The inventor's experiments show that an advantage of the culture media of the invention is that they can 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. The inventor's experiments show that another advantage of the culture media of the invention is that they can be used to rapidly expand a population of pluripotent stem cells, i.e. they allow large numbers of cells to be produced in a relatively short time. The inventor's experiments show that a further advantage of the culture media of the invention is that they can be used to expand several different types of pluripotent stem cell lines, i. e. different types of pluripotent stem cell can be cultured using a single culture medium. The inventor has already successfully tested the culture media and methods of the invention with the SA461 hES cell line (as described above), and preliminary results suggest that the same culture media and methods can be used with other hES cell lines. The inventor's experiments also show that the culture media of the invention can 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. The inventor's experimental data therefore suggest that the culture media of the invention benefit from significant advantages over known culture media in terms of the scalability, reproducibility and robustness of pluripotent stem cell culture. Example 4: Human ES cells cultured with a combination of NHR agonists retain an undifferentiated morphology and phenotype - further experiments

Further experiments were performed to confirm the ability of RXR/RAR agonists, in particular retinoids, and FXR agonists to improve pluripotent stem cell culture. These experiments used the same SA461 hES cell line and the same medium composition as in Example 3, except the all-trans-retinol (ATR) was substituted with either 13-cis retinol (13cROL) or retinyl acetate (RETACT). The results of these experiments (not shown) confirm that 13-cis retinol (13cROL) and retinyl acetate (RETACT) provide a similar effect to all-trans-retinol (ATR) in assisting the maintenance of the typical morphology of an undifferentiated embryonic stem cell over multiple passages in a feeder-free culture system.

In other experiments, it was found that removal of the all-trans-retinol (ATR) from the culture medium described in Example 3 resulted in reduced robustness of hES cell culture. It was also found that removal of both the all-trans-retinol (ATR) and the cliolic acid from the culture medium described in Example 3 resulted in widespread differentiation of the hES cells.

Further experiments were performed to confirm the ability of FXR and PPAR agonists to improve pluripotent stem cell culture. These experiments used the same SA461 hES cell line and the same medium composition as in Example 3, except that (i) progesterone and putrescine were omitted from the fresh medium, (ii) the FXR agonist di-homo-γ- linolenic acid (10 µM) and the PPAR agonist clofibric acid (250 ng/ml) were added to the fresh medium; and (iii) human serum albumin (500 mg/L) was added to the fresh medium. The results of these experiments (not shown) indicate that this culture medium composition can assist in the maintenance of the typical morphology of an undifferentiated embryonic stem cell over multiple passages in a feeder-free culture system. This culture medium composition was found to be particularly advantageous in improving pluripotent stem cell culture, in that it allows (to some extent) enzymatic passaging of cells in feeder-free culture.

Further experiments (data not shown) have confirmed that SA461 hES cells expanded in a culture medium as described in Example 3 retain the unique morphology and molecular phenotype characteristic of undifferentiated hES cells, whilst retaining a stable and normal karyotype, even after several months in feeder-free culture.

Example 5: Human ES cells cultured with a combination of NHR agonists retain an undifferentiated morphology and phenotype —further experiments

A conditioned medium was produced as in Example 2. A fresh medium was also produced using DMEM/F12 (Invitrogen) and transferrin, insulin, selenite, progesterone and putrescine (as N2 Supplement; Invitrogen), L-glutamine (Invitrogen) and B27 Supplement (Invitrogen).

The SA461 and SAl 2 1 hES cell lines (Cellartis AB, Gδteborg, Sweden) were cultured continuously using enzymatic passage with trypsin for between 3 and 23 passages with a passage interval of 4-7 days to fresh fibronectin coated culture vessels. Immunostaining, karyotype analysis and QRTPCR were performed as described in Example 3.

In vitro differentiation was performed by transferring cells back to feeder based culture for one passage then manually cutting segments for embryoid body formation in suspension based culture in DMEM containing 10% FBS (both Invitrogen).

In vivo differentiation was performed both directly from cells cultured in the medium described and indirectly using cells expanded in the medium described then passaged back to feeder culture for one passage before undergoing teratoma formation. For teratoma formation cells were enzymatically dissociated to clusters then implanted under the kidney capsule of SCID mice. Teratomas were harvested and sectioned for histological examination after 8 weeks. All three germ layers were detectable in teratomas derived from both lines SA461 and SAl 2 1 after 17 and 23 passages in this culture medium, respectively.

Some of the results of these experiments are shown in Figures 27-37. The results of these experiments demonstrate that hES cells expanded in this culture medium retain the expression of markers of pluripotency and typical morphology of hES cells. They also demonstrate through in vitro and in vivo differentiation after prolonged culture that the cells retain their ability to differentiate into cells of all three germ layers. The culture medium described in this example has also been used successfully with the

SAl 8 1 hES cell line (Cellartis AB, Gδteborg, Sweden).

The culture medium composition described in this example has been found to be particularly advantageous in improving pluripotent stem cell culture. The use of this culture medium composition provides an especially robust culture system. It has been found to support proliferation and enzymatic passaging of multiple hES cell lines in prolonged feeder-free culture (at least the SA461, SA121 and SAl 8 1 hES cell lines).

The additional experiments described in Examples 4 and 5 confirm that the culture media of the invention benefit from significant advantages over known culture media in terms of the scalability, reproducibility and robustness of pluripotent stem cell culture.

It will be understood that the invention has been described by way of example only and modification of detail may be made without departing from the spirit and scope of the invention. CLAIMS

1. A culture medium for expanding a population of pluripotent stem cells, which is a mixture of a conditioned medium and a fresh medium, and which comprises biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, triiodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin.

2. A culture medium according to claim 1, which has been supplemented with B27 Supplement and N2 Supplement.

3. A culture medium according to claim 1 or claim 2, wherein the culture medium is a mixture of a conditioned medium and a fresh medium of different types.

4. A composition or cell culture vessel comprising (a) an extracellular matrix material; and (b) a culture medium which is a mixture of a conditioned medium and a fresh medium, and which comprises biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin.

5. The composition or cell culture vessel according to claim 4, wherein the culture medium has been supplemented with B27 Supplement and N2 Supplement.

6. A composition or cell culture vessel according to claim 4 or claim 5, comprising a culture medium which is a mixture of a conditioned medium and a fresh medium of different types.

7. Use of a culture medium according to any of claims 1-3 for expanding a population of pluripotent stem cells.

8. A method for expanding a population of pluripotent stem cells, comprising: (a) providing a population of pluripotent stem cells; (b) providing a culture medium according to any of claims 1-3; (c) contacting the stem cells with the culture medium; and (d) culturing the stem cells under appropriate conditions. 9. A culture medium for expanding a population of pluripotent stem cells, which comprises a retinoid X receptor (RXR) or retinoic acid receptor (RAR) agonist, a farnesoid X receptor (FXR) agonist, a peroxisome proliferator-activated receptor (PPAR) agonist, and/or a thyroid hormone receptor (THR) agonist.

10. A culture medium according to claim 9, wherein the RXR or RAR agonist is a retinoid.

11. A culture medium according to claim 10, wherein the retinoid is a retinol, a retinal or a retinoic acid.

12. A culture medium according to claim 10, wherein the retinoid is all-trans-retinol (ATR), 13-cis retinoic acid (13cRA), 9-cis retinoic acid (9cRA), methoprene acid (MPA), 13-cis retinol (13cROL), retinyl acetate (RETACT), acitretin (ACT) or 4-hydroxyretinoic acid (4HRA).

13. A culture medium according to any preceding claim, wherein the FXR agonist is a cholesterol metabolite.

14. A culture medium according to claim 13, wherein the cholesterol metabolite is a bile acid.

15. A culture medium according to claim 14, wherein the bile acid is cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) or lithocholic acid (LCA), or a glycine or taurine conjugate thereof.

16. A culture medium according to any of claims 9-12, wherein the FXR agonist is an arachidonic acid, a linolenic acid or a docosahexaenoic acid.

17. A culture medium according to claim 16, wherein the FXR agonist is α-linolenic acid (ALA), γ-linolenic acid (GLA) or di-homo-γ-linolenic acid (DGLA).

18. A culture medium according to claim 17, wherein the FXR agonist is di-homo-γ- linolenic acid (DGLA).

19. A culture medium according to any preceding claim, wherein the PPAR agonist is an unsaturated fatty acid, a saturated fatty acid, a dicarboxylic fatty acid, an eicosanoid, a prostaglandin 12 analog, a leukotriene B4 analog, a leukotriene D4 antagonist, a hypolipidemic agent, a hypoglycemic agent, a hypolipidemic and hypoglycaemic agent, a nonsteroidal anti-inflammatory drug, a carnitine palmitoyl transferase I (CPTl) inhibitor, or a fatty acyl-CoA dehydrogenase inhibitor.

20. A culture medium according to claim 19, wherein the PPAR agonist is 5,8,1 1,14- eicosatetraynoic acid, bezafibrate, clofibric acid, gemfibrozil, WYl 4643 or tetradecylthioacetic acid.

21. A culture medium according to any preceding claim, wherein the THR agonist is an iodothyronine.

22. A culture medium according to claim 21, wherein the iodothyronine is a di- iodothyronine, tri-iodothyronine or tetra-iodothyronine.

23. A culture medium according to claim 22, wherein the iodothyronine is 3,5-

diiodothyronine (3,5-T ), 3,3'-diiodothyronine (3,3-T2), 3,3'-T sulphate (3,3-

T2S), 3,5-diiodo-L-tyiOsine dihydrate (DLTdH), 3,5,3'-triiodo-L-thyronine (T3),

3,3',5-T 3 sulphate (3,3',5-T 3S), 3,5,3',5'-tetra-iodothyronine (T4), 3,5,3',5'- tetraiodo-L-thyronine or 3,5-diiodo-4-hydroxyphenylpropionic acid (DIHPA).

24. A culture medium according to claim 23, wherein the iodothyronine is 3,5- diiodo-L-thyronine.

25. A culture medium according to any preceding claim, which comprises between about lOpM and about 10OmM of an FXR agonist, between about lOpM and about 10OmM of an RXR or RAR agonist, between about lOpM and about 10OmM of a PPAR agonist, and/or between about lOpM and about 10OmM of a THR agonist.

26. A culture medium according to any preceding claim, which comprises at least about lOpM of an FXR agonist, at least about lOpM of an RXR or RAR agonist, at least about lOpM of a PPAR agonist, and/or at least about lOpM of a THR agonist. 27. A culture medium according to any of claims 1-26, which comprises (a) an FXR agonist and (b) a RXR or RAR agonist, a PPAR agonist, or a THR agonist.

28. A culture medium according to any of claims 1-26, which comprises (a) a RXR or RAR agonist and (b) an FXR agonist, a PPAR agonist, or a THR agonist.

29. A culture medium according to any of claims 1-26, which comprises (a) a PPAR agonist and (b) an FXR agonist, a RXR or RAR agonist, or a THR agonist.

30. A culture medium according to any of claims 1-26, which comprises (a) a THR agonist and (b) an FXR agonist, a RXR or RAR agonist, or a PPAR agonist.

31. A culture medium according to any of claims 1-26, which comprises an FXR agonist, an RXR or RAR agonist and a THR agonist.

32. A culture medium according to any of claims 1-26, which comprises an FXR agonist, an RXR or RAR agonist, a PPAR agonist and a THR agonist.

33. A culture medium according to any preceding claim, which comprises cholesterol or a cholesterol substitute.

34. A culture medium according to any preceding claim, which comprises transferrin or a transferrin substitute.

35. A culture medium according to any preceding claim, which comprises albumin or an albumin substitute.

36. A culture medium according to any preceding claim, which comprises L- glutamine, progesterone, putrescine, insulin or an insulin substitute, selenite and DL-alpha tocopherol (Vitamin E).

37. A culture medium for expanding a population of pluripotent stem cells, which comprises a bile acid, a retinol and a diiodothyronine.

38. A culture medium according to claim 37, which comprises cholic acid, all-trans- retinol and 3,5-diiodo-L-thyronine. 39. A culture medium according to claim 38, which comprises cholic acid, all-trans- retinol, 3,5-diiodo-L-thyronine, cholesterol, transferrin, L-glutamine, progesterone, putrescine, insulin, selenite and DL-alpha-tocopherol (Vitamin E).

40. A culture medium according to any preceding claim, which comprises serum or a serum replacement.

41. A culture medium according to any preceding claim, wherein the medium is a conditioned medium, or is a mixture of conditioned and fresh medium.

42. A culture medium according to claim 41, wherein the conditioned medium has been conditioned on mammalian cells.

43. A culture medium according to claim 42, wherein the conditioned medium has been conditioned on murine embryonic fibroblast (mEF) cells.

44. A culture medium according to any of claims 1-43, wherein the medium is a fresh medium.

45. A culture medium according to any of claims 1-44, which comprises transferrin, insulin, progesterone, putrescine, and sodium selenite.

46. A culture medium according to claim 45, which has been supplemented with N2 Supplement.

47. A culture medium according to any of claims 1-46, which comprises biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin.

48. A culture medium according to claim 47, which has been supplemented with B27 Supplement.

49. A culture medium according to any of claims 1-48, which has been supplemented with N2 Supplement and B27 Supplement.

50. A culture medium according to any of claims 1-49, which is a mixture of a conditioned medium and a fresh medium, and which comprises biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin.

51. A culture medium according to claim 50, which is a mixture of a conditioned medium and a fresh medium, and which has been supplemented with B27 Supplement and N2 Supplement.

52. A culture medium according to claim 51, wherein the culture medium is a mixture of a conditioned medium and a fresh medium of different types.

53. A method for preparing a culture medium according any of the preceding claims, comprising the steps of: (a) obtaining a culture medium; and (b) adding a retinoid X receptor (RXR) or retinoic acid receptor (RAR) agonist, a farnesoid X receptor (FXR) agonist, a peroxisome proliferator-activated receptor (PPAR) agonist, and/or a thyroid hormone receptor (THR) agonist to the culture medium.

54. A culture medium supplement that comprises a retinoid X receptor (RXR) or retinoic acid receptor (RAR) agonist, a farnesoid X receptor (FXR) agonist, a peroxisome proliferator-activated receptor (PPAR) agonist, and/or a thyroid hormone receptor (THR) agonist.

55. A culture medium supplement according to claim 54, wherein the supplement is a concentrated liquid supplement.

56. A culture medium supplement according to claim 54, wherein the supplement is a dry supplement.

57. A hermetically-sealed vessel containing a culture medium according to any of claims 1-52 or a culture medium supplement according to any of claims 54-56.

58. A composition comprising: (a) a culture medium according to any of claims 1- 52; and (b) pluripotent stem cells.

59. A composition containing: (a) a culture medium according to any of claims 1-52; and (b) an extracellular matrix material. 60. A composition according to claim 59, wherein the extracellular matrix material is fibronectin.

61. A composition according to claim 59 or 60, further comprising stem cells.

62. A composition according to any of claims 58-61, which is a feeder cell-free composition.

63. The use of (a) an extracellular matrix material; and (b) a retinoid X receptor (RXR) or retinoic acid receptor (RAR) agonist, a farnesoid X receptor (FXR) agonist, a peroxisome proliferator-activated receptor (PPAR) agonist, and/or a thyroid hormone receptor (THR) agonist, for expanding a population of pluripotent stem cells.

64. The use of fibronectin, cholic acid, all-trans-retinol and 3,5-diiodo-L-thyronine for expanding a population of pluripotent stem cells.

65. A composition or cell culture vessel comprising (a) an extracellular matrix material; and (b) a retinoid X receptor (RXR) or retinoic acid receptor (RAR) agonist, a farnesoid X receptor (FXR) agonist, a peroxisome proliferator- activated receptor (PPAR) agonist, and/or a thyroid hormone receptor (THR) agonist.

66. A composition or cell culture vessel according to claim 65, comprising (a) an extracellular matrix material; and (b) biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri¬ iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin.

67. A composition or cell culture vessel according to claim 65, comprising (a) an extracellular matrix material; and (b) a culture medium which has been supplemented with B27 Supplement and N2 Supplement.

68. A composition or cell culture vessel according to claim 65, comprising (a) an extracellular matrix material; and (b) a culture medium which is a mixture of a conditioned medium and a fresh medium, and which has been supplemented with B27 Supplement and N2 Supplement. 69. A composition or cell culture vessel according to claim 68, comprising (a) an extracellular matrix material; and (b) a culture medium which is a mixture of a conditioned medium and a fresh medium of different types, and which has been supplemented with B27 Supplement and N2 Supplement.

70. A composition or cell culture vessel comprising fibronectin, cholic acid, all- trans-retinol and 3,5-diiodo-L-thyronine.

71. Use of a culture medium according to any of claims 1-52 for expanding a population of pluripotent stem cells.

72. A method for expanding a population of pluripotent stem cells, comprising: (a) providing a population of pluripotent stem cells; (b) providing a culture medium according to any of claims 1-52; (c) contacting the stem cells with the culture medium; and (d) culturing the stem cells under appropriate conditions.

73. Use of a retinoid X receptor (RXR) or retinoic acid receptor (RAR) agonist for pluripotent stem cell culture.

74. The use of claim 72, wherein the RXR or RAR agonist is a retinoid.

75. The use of claim 73, wherein the retinoid is a retinol, a retinal or a retinoic acid.

76. The use of claim 73, wherein the retinoid is all-trans-retinol (ATR), 13-cis retinoic acid (13cRA), 9-cis retinoic acid (9cRA), methoprene acid (MPA), 13- cis retinol (13cROL) retinyl acetate (RETACT), acitretin (ACT) or 4- hydroxyretinoic acid (4HRA).

77. Use of a farnesoid X receptor (FXR) agonist for pluripotent stem cell culture.

78. The use of claim 76, wherein the FXR agonist is a bile acid.

79. The use of claim 77, wherein the bile acid is cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) or lithocholic acid (LCA), or a glycine or taurine conjugate thereof.

80. The use of claim 76, wherein the FXR agonist is an arachidonic acid, a linolenic acid or a docosahexaenoic acid. 81. The use of claim 79, wherein the FXR agonist is α-linolenic acid (ALA), γ-linolenic acid (GLA) or di-homo- γ-linolenic acid (DGLA).

82. The use of claim 80, wherein the FXR agonist is di-homo- γ-linolenic acid (DGLA).

83. Use of a peroxisome proliferator-activated receptor (PPAR) agonist for pluripotent stem cell culture.

84. The use of claim 82, wherein the PPAR agonist is an unsaturated fatty acid, a saturated fatty acid, a dicarboxylic fatty acid, an eicosanoid, a prostaglandin 12 analog, a leukotriene B4 analog, a leukotriene D4 antagonist, a hypolipidemic agent, a hypoglycemic agent, a hypolipidemic and hypoglycaemic agent, a nonsteroidal anti-inflammatory drug, a carnitine palmitoyl transferase I (CPTl) inhibitor, or a fatty acyl-CoA dehydrogenase inhibitor.

85. The use of claim 83, wherein the PPAR agonist is 5,8,1 1,14-eicosatetraynoic acid, bezafibrate, clofibric acid, gemfibrozil, WYl 4643 or tetradecylthioacetic acid.

86. Use of a thyroid hormone receptor (THR) agonist for pluripotent stem cell culture.

87. The use of claim 85, wherein the THR agonist is a di-iodothyronine, tri¬ iodothyronine or tetra-iodothyronine.

88. The use of claim 86, wherein the THR agonist is 3,5-diiodothyronine (3,5-T 2),

3,3'-diiodothyronine (3,3-T ), 3,3'-T sulphate (3,3-T 2S), 3,5-diiodo-L-tyrosine

dihydrate (DLTdH), 3,5,3'-triiodo-L-thyronine (T3), 3,3',5-T 3 sulphate (3,3 ',5-

T3S), 3,5,3',5'-tetra-iodothyronine (T4), 3,5,3',5'-tetraiodo-L-thyronine or 3,5- diiodo-4-hydroxyphenylpropionic acid (DIHPA).

89. The use of claim 87, wherein the THR agonist is 3,5-diiodo-L-thyronine.

90. A culture medium, method, supplement, vessel, composition or use according to any preceding claim, wherein the pluripotent stem cells are human cells. 91. A culture medium, method, supplement, vessel, composition or use according to any preceding claim, wherein the pluripotent stem cells are embryonic stem cells.

92. A culture medium, method, supplement, vessel, composition or use according to claim 90, wherein the stem cells are human embryonic stem (hES) cells.

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

94. A culture medium, method, supplement, vessel, composition or use according to any preceding claim, wherein the pluripotent stem cells are cells that have been induced or transformed to form cells with ES cell-like properties.

95. A culture medium, method, supplement, vessel, composition or use according to any preceding claim, wherein the pluripotent stem cells are SA461, SA121, or SAl 8 1 hES cells (Cellartis AB, Gδteborg, Sweden).

INTERNATIONAL EAR H REP RT nterna ona app ca on o PCT/GB2009/001404

A CLASSIFICATION OF SUBJECT MATTER , INV. C12N5/00 C12N5/06

According to International Patent Classification (IPC) orto both national classification and IPC B FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) C12N

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and where practical search terms used) EPO-Internal , BIOSIS, EMBASE, MEDLINE, WPI Data

C DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document with indication where appropriate of the relevant passages Relevant to claim No

ALEXANIAN A R ET AL: "Quiescent neural 1,3,4,6, cells regain multi potent stem cell 33-36, characteristics influenced by adult neural 41,42, stem cells in co-culture" 44,45, EXPERIMENTAL NEUROLOGY, ACADEMIC PRESS, 47,50, NEW YORK, NY, US, 57,59,62 vol. 191, no. 1 , 1 January 2005 (2005-01-01), pages 193-197, XP004676600 ISSN: 0014-4886 page 195, coTumn 2 , paragraph 1 - page 40,43, 196, column 1 , paragraph 1 48,53, 57,60

-/--

Further documents are listed in the continuation of Box C See patent family annex

* Special categories of cited documents "T" later document published after the international filing date π π 1 or p o ty date and not in conflict with the application but A" document defining the general state of the art which is not cited to understand the principle or theory underlying the considered to be of particular relevance invention 1 E' earlier document but published on or after the international "X" document of particular relevance the claimed invention filing date cannot be considered novel or cannot be considered to 1L1 document which may throw doubts on pπoπty claιm(s) or involve an inventive step when the document is taken alone which is cited to establish the publication date of another "Y" document of particular relevance, the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the "O" document referring to an oral disclosure use exhibition or document is combined with one or more other such docu¬ other means ments such combination being obvious to a person skilled in the art 1P" document published prior to the international filing date but later than the prionty date claimed "&" document member of the same patent family

Date of the actual completion of the international search Date of mailing of the international search report

6 October 2009 20/10/2009

Name and mailing address of the ISA/ Authorized officer European Patent Office P B 5818 Patentlaan 2 NL - 2280 HV Rijswijk TeI (+31-70) 340-2040, Fax (+31-70) 340-3016 Friedrich, Christof INTERNATIONAL SEARCH REPORT n e a o PCT/GB2009/001404

C(Continuatlon). DOCUMENTS CONSIDERED TO BE RELEVANT

Category" Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

X US 2004/102375 Al (LANIER LORENE M [US] ET 1,3,4,6, AL) 27 May 2004 (2004-05-27) 33-36, 41 ,42, 44,45, 47,50, 57,59,62 Y paragraph [0091] 40,43, 48,53, 57,60

X WO 2004/099395 A (CELLARTIS AB [SE]; 1-6, ERIKSSON PETER [SE]; ZETTERSTROEM MATHILDA 33-36, [SE] ; EN) 18 November 2004 (2004-11-18) 4 1 ,42, 44-47, 49-52, 57,59,62 Y page 8 - page 9 ; c l aims 18-20 40,43, 48,53, 57,60

X WO 2005/103232 A ( INST NAT SANTE RECH MED 1, 3,4,6, [FR] ; BALSE ELISE [FR] ; TESSIER LUC-HENRI 33-36, [FR]) 3 November 2005 (2005-11-03) 41,42, 44,45, 47,50, 57,59,62 Y c l aims 15,24 40,43, 48 S53, 57,60

Y GUNILLA CAISANDER ET AL: "Chromosomal 7,8,58, integrity maintained i n five human 6 1 ,71 , embryonic stem cel l l ines after prolonged 72,90-95 i n vitro cul ture" CHROMOSOME RESEARCH, KLUWER ACADEMIC PUBLISHERS, DO, vol . 14, no . 2 , 1 March 2006 (2006-03-01) , pages 131-137, XP019234937 ISSN: 1573-6849 the whole document INTERNATIONAL SEARCH REPORT PCT/GB2009/001404

C(Continuatlon). DOCUMENTS CONSIDERED TO BE RELEVANT

Category * Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

Y MANNELLO F ET AL: "Conci se review: No 7,8,58, breakthroughs for human mesenchymal and 61 ,71, embryonic stem cel l culture: Conditi oned 72,90-95 medium, feeder l ayer , or feeder-free; medium with fetal cal f serum, human serum, or enriched p l asma; serum-free, serum repl acement nonconditioned medium, or ad hoc formul a? Al l that g l itters i s not go" STEM CELLS, ALPHAMED PRESS, DAYTON, OH, us, vol '. 25, no. 7 , 1 January 2007 (2007-01-01) , pages 1603-1609 , XP002466979 ISSN: 1066-5099 the whole document

Y PROwSE ANDREW B J ET AL: " A proteome 7,8,58, analysis of .conditioned medi a from human 61 ,71 , neonatal fibrobl asts used i n the 72,90-95 maintenance of human embryonic stem cel l s " PROTEOMICS, " vol . 5 , no. 4 , March 2005 (2005-03) , pages 978-989, XP002538300 ISSN: 1615-9853 the whole document

Y CHIN ET AL: "Identi fication of proteins 7,8,58, from feeder conditioned medium that 61 ,71, support human embryonic stem cel l 's " 72,90-95 JOURNAL OF BIOTECHNOLOGY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol . 130 , no . 3 , 19 June 2007 (2007-06-19) , pages 320-328, XP022119696 ISSN: 0168-1656 the whole document

Y PROWSE ANDREW B J ET AL: "Identificati on 7,8,58, of potential p i uri potency determinants for 61 ,71, human embryonic stem cel l s following 72,90-95 proteomic analysi s of human and mouse fibrobl ast conditioned medi a" JOURNAL OF PROTEOME RESEARCH, vol . 6 , no. 9 , September 2007 (2007-09) , pages 3796-3807, XP002538301 ISSN: 1535-3893 the whole document _ / INTERNATIONAL SEARCH REPORT PCT/GB2009/001404

C(Contlnuatlon). DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

LIU Y ET AL: "A novel chemical -defined 7,8,58, medium with bFGF and N2B27 supplements 61,71, supports undifferentiated growth in human 72,90-95 embryonic stem cells" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 346, no. 1 , 2 1 July 2006 (2006-07-21), pages 131-139, XP024925297 ISSN: 0006-291X [retrieved on 2006-07-21] table 1

GARCIA-GONZALO FRANCESC R ET AL: 7,8,58, "Albumin-Associ ated Lipids Regul ate Human 61,71, Embryonic Stem Cel l Sel f-Renewal " 72,90-95 PLOS ONE , vol. 3 , no. 1 , January 2008 (2008-01), page Article No.: el384, XP002548861 ISSN: 1932-6203 page 6 , column 1 , paragraph 2 - column 2 , paragraph 1 International application No. INTERNATIONAL SEARCH REPORT PCT/GB2009/001404

Box No. Il Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)

This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons:

Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely:

2. Claims Nos.: because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically;

3. Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Box No. Ill Observations where unity of invention is lacking (Continuation of item 3 of first sheet)

This International Searching Authority found multiple inventions in this international application, as follows:

see additi onal sheet

As all required additional search fees were timely paid by the applicant, this international search report covers allsearchable claims.

2. As all searchable claims could be searched without effort justifying an additional feeS this Authority did not invite payment of additional fees.

3. A s only some of the- required additional search fees were timely paid by the applicant, this international search reportcovers only those claims for which fees were paid, specifically claims Nos.: l-8,33-36(parts) ,40-53(parts ) ,57-62(parts) ,71-72(parts ) ,90-95(parts)

4. No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.:

Remark on Protest The additional search fees were accompanied by the applicant's protest and, where applicable, the payment of a protest fee. The additional search fees were accompanied by the applicant's protest but the applicable protest fee was not paid within the time limit specified in the invitation.

A No protest accompanied the payment of additional search fees. International Application No. PCT/GB2009 /001404

FURTHER INFORMATION CONTINUED FROM PCT/ISA/ 210

This International Searching Authority found multiple (groups of) inventi ons i n thi s i nternational appl icati on , as fol l ows :

1. claims : l,3(parts) ,4,6-8(parts) ,33-36(parts) , 40-45(parts) ,47-48(parts) ,50(p arts) ,53(parts ) ,57-62(parts) , 71-72(parts) ,90-95(parts)

Cu lture medium which is a mixture of a conditioned medium and a fresh medium which comprises B27 and use thereof.

2 . claims: 2,3(parts),5,6-8(parts),33-36(parts), 40-52(parts ),57-62(parts ),71-7 2(parts ),9Q-95(parts )

Culture medium which is a mixture of a conditioned medium and a fresh medium which comprises B27 and N2 and use thereof.

3 , claims: 9-32 ,33-36(parts ),40-52(parts ), 53(parts),54 1 56,57-62(parts),63,65-6 9 ,71-72(parts ),73-89 ,90-95(parts )

Culture media comprising RXR, RAR, FXR, PPAR, or THR. Should prior art documents disclosing culture media comprising nuclerar hormone receptor agonists be retrieved in a further search, this group of invention shall be devided into further individual inventions.

4 . cl aims : 37-39 ,40-52(parts ) ,57-62(parts) , 71-72(parts),90-95(parts)

Culture medium compri sing bi l e acid, retinol , and di iodothyronine.

5. cl aims : 64, 70,90-95(parts )

Use of fibronecti n, chol i c acid, al l-trans-reti nol and 3,5-di i odo-L-thronine for expanding pi uri potent stem cel l s International application No Information on patent family members PCT/GB2009/001404

Patent document Publication Patent family Publication cited in search report date member(s) date

US 2004102375 A l 27-05-2004 NONE

WO 2004099395 A 18-11-2004 US 2007020608 A l 25-01-

WO 2005103232 A 03-11-2005 EP 1589097 A l 26-10- US 2008280318 A l 13-11-