AU2003229140B2 - Methods of regulating differentiation in stem cells - Google Patents

Description

WO 03/104442 PCT/AU03/00713 METHODS OF REGULATING DIFFERENTIATION IN STEM CELLS Technical Field The present invention relates to methods for inhibiting spontaneous 5 differentiation of stem cells. The invention also relates to media useful in propagating stem cells in an undifferentiated state, methods for identifying agents useful for inhibiting stem cell differentiation, and methods of treating stem cell related disorders. 10 Background Art In general, stem cells are undifferentiated cells which can give rise to a succession of mature functional cells. For example, a haematopoietic stem cell may give rise to any of the different types of terminally differentiated blood cells. Embryonic stem (ES) cells are derived from the embryo and are 15 pluripotent, thus possessing the capability of developing into any organ, cell type or tissue type or, at least potentially, into a complete embryo. ES cells may be derived from the inner cell mass of the blastocyst, which have the ability to differentiate into tissues representative of the three embryonic germ layers (mesoderm, ectoderm, endoderm), and into the extra-embryonic 20 tissues that support development. Human embryonic stem cells (hES cells) are pluripotent cell lines derived from the inner cell mass of the blastocyst. These cells have the ability to differentiate into functional tissues representative of the three embryonic germ layers (mesoderm, ectoderm, endoderm), and into extra-embryonic 25 tissues that support development. Because of their ability to generate these different cellular fates, hES cells are considered to be of great potential for future therapies. However, during routine culture in vitro, established hES cell lines have a tendency to spontaneously differentiate. Because the pluripotency of 30 these cells is associated with their undifferentiated state, it is desirable to find a way to limit this spontaneous differentiation. Contrary to what is seen in mouse embryonic stem cells, leukemia inhibitory factor (LIF) does not prevent the spontaneous differentiation of hES cells [1]. Thus, a common WO 03/104442 PCT/AU03/00713 2 way to grow and then to maintain hES cells in an optimum state is to cultivate them on feeder layers, which are constituted by primary mouse embryonic fibroblasts (MEF), in media supplemented with high doses of foetal calf serum. 5 However, serum contains a wide variety of biologically active compounds that might have the potential to adversely affect hES cell growth and differentiation. Furthermore, there is a biosafety issue if cells cultured in animal serum are subsequently used for implantation in a human or for the production of a biological therapeutic. 10 With regard to these issues and in order to establish a serum-free culture system to grow hES cells, it is of great importance to identify the specific factors in serum that are responsible for its beneficial effect on the growth of hES cells. Thus, alternative approaches to traditional culture systems are desirable, such as the use of a serum replacement medium 15 such as Knockout Serum Replacement [2, 3]. Sphingosine-1 -phosphate (S1 P) and lysophosphatidic acid (LPA) are two small bioactive lysophospholipids, present in serum (at concentration of up to 1 and 5 pM respectively) {4], released by activated platelets, which act on a wide range of cell types derived from the three developmental germ 20 layers. Most of the effects of these lysophospholipids seem to be mediated by specific lysophospholipid G-protein coupled receptors (LPL receptors) previously named endothelial differentiation gene (Edg) receptors. Up to now, eight distinct mammalian LPL/Edg receptors have been identified: S1 P 1 I/Edg-1, SI P 2 /Edg-5, S1 P 3 /Edg-3, S1 P4/Edg-6 and SI P 5 /Edg 25 8 are specific for SIP while LPAI/Edg-2, LPA 2 /Edg-4 and LPA 3 /Edg-7 are specific for LPA (for reviews see [5, 6]). Each of these receptors is coupled to at least one G protein and can activate or inhibit specific signalling pathways. For instance, all these receptors are coupled to Gv/o proteins (for review see [5, 6]). 30 By activating notably these Gvo 0 proteins, SIP and LPA can stimulate the extracellular-signal-regulated kinases I and 2 (ERKI/2), which are members of the mitogen-activated protein (MAP) kinase family, and thus are involved in regulation of major cellular events, such as cell proliferation or WO 03/104442 PCT/AU03/00713 3 differentiation. SIP and LPA are potent biological agents involved in numerous cell events, such as proliferation, differentiation, death or migration (for review see [5]) since the very early stages of development. SIP stimulates mammalian angiogenesis, at least via SIP 1 and SIP 2 5 [7-10]. Thus, SIP 1 knockout mice show impaired blood vessel maturation. Moreover, in the zebrafish, SIP is required for normal heart development [11]. Thus, in these animals, the mutation of the gene ml! that encodes the SIP receptor Mil (very similar to the mammalian SIP 2 receptor) impairs migration of cardiac progenitor cells [11]. 10 On the other hand, LPA seems to be mainly involved in neurogenesis [121. For instance, LPA, probably via LPA 1 , stimulates cell cycle morphological changes and cell migration of cultured cortical neuroblasts. Moreover, LPA, probably via LPA 2 , regulates the migration of post-mitotic neurons to their final destination. Last but not least, LPA 1 knockout mice 15 present abnormal cerebral cortices and olfactory bulbs, probably due to impaired development, demonstrating LPA 1 is essential for a normal brain development [13]. Within serum, Platelet-Derived Growth Factor (PDGF) is a major protein growth factor that has been widely described as a potent mitogen of 20 numerous kinds of cells. PDGF has also been shown to induce chemotaxis, actin re-organization, and to prevent apoptosis. This growth factor belongs to a family of dimeric isoforms of polypeptide chains, A, B, C and D that act through different tyrosine kinase receptors: PDGFR-a and PDGFR-p. SIP and PDGF have additional effects that induce biological 25 responses. Thus SIP and PDGF are able to regulate smooth muscle cell migration, proliferation and vascular maturation. Moreover, Hobson et aL. (2001), and Rosenfeld et a. (2001) demonstrated that PDGF-stimulated cell motility is SIPr-dependent in HEK 293 cells and MEF [14, 15] while Kluk et a/. (2003) showed that this effect was independent of SIP 1 in vascular 30 smooth muscles and MEF [16]. Last but not least, it is now proposed that PDGF is able to stimulate the enzyme sphingosine kinase, which leads to an increase in SIP intracellular concentration [17], an effect that would be 4 responsible for PDGF-induced proliferation in Swiss 3T3 cells [17] and vascular smooth muscle cells [18]. The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a 5 context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application. 10 Summary of the Invention In one aspect the present invention provides a method for modulating spontaneous differentiation of a stem cell, which method comprises incubating the stem cell in the presence of an agonist of a LPL receptor and/or a ligand of a class Ill tyrosine kinase receptor, wherein the agonist is 15 selected from the group consisting of S1P, dihydro S1P, LPA, PAF and SPC or functional equivalents thereof. In another aspect the present invention provides a serum free or substantially serum free medium useful for modulating spontaneous differentiation of a stem cell, comprising an agonist of a LPL receptor and/or 20 a ligand of a class IlIl tyrosine kinase receptor, wherein the agonist is selected from the group consisting of S1P, dihydro S1P, LPA, PAF and SPC or functional equivalents thereof. Also described is a method of treating or preventing a disorder of stem cell differentiation comprising administering to an animal in need thereof a 25 composition containing an agonist of a LPL receptor and/or a ligand of a class Ill tyrosine kinase receptor, wherein the agonist is selected from the group consisting of S1P, dihydro S1P, LPA, PAF and SPC or functional equivalents thereof. Also described is a pharmaceutical composition comprising a class Ill 30 tyrosine kinase receptor ligand and/or a LPL receptor agonist, wherein the agonist is selected from the group consisting of S1 P, dihydro S1 P, LPA, PAF and SPC or functional equivalents thereof.

5 In a further aspect the present invention provides a method of producing a population of proliferating undifferentiated stem cells from a stem cell which method comprises incubating the stem cell in the presence of an agonist of the LPL receptor and/or a ligand of a class IlIl tyrosine kinase 5 receptor, wherein the agonist is selected from the group consisting of S1P, dihydro S1 P, LPA, PAF and SPC or functional equivalents thereof. In another aspect the present invention provides a method of producing a population of proliferating undifferentiated stem cells from a stem cell which method comprises incubating the stem cell in the presence of an 10 agonist of the LPL receptor and/or a ligand of a class Ill tyrosine kinase receptor, wherein the agonist is selected from the group consisting of S1 P, dihydro S1 P, LPA, PAF and SPC or functional equivalents thereof. Description of the Invention 15 The present inventors investigated the role of the LPL receptor agonists S1P, dihydro S1P and LPA, and the ligand of a class Ill tyrosine kinase receptor, PDGF, in modulating the fate of hES cells in culture. The present inventors have established that hES cells are target cells for S1P, dihydro SIP, LPA and PDGF, through expression of the LPL 20 receptors, PDGFR-a and PDGFR-p and through stimulation of ERKs by these agonists. Moreover the present inventors have found that S1P and PDGF slightly inhibit the spontaneous differentiation of hES cells while co incubation with both S1P and PDGF strongly reduces the spontaneous differentiation of hES cells. These findings provide a basis for the 25 establishment of a serum-free culture medium for stem cells and in particular hES cells. Throughout the description and claims of this specification, the word "comprise" and variations of that word, such as "comprising" and "comprises" are not intended to exclude other additives, steps or integers. 30 In a first aspect the present invention provides a method for modulating spontaneous differentiation of a stem cell, which method comprises incubating the stem cell in the presence of an agonist of a LPL receptor, wherein the agonist is selected from the group consisting of SlP, dihydro S1 P, LPA, PAF and SPC or functional equivalents thereof.

6 In a second aspect the present invention provides a method for modulating spontaneous differentiation of a stem cell, which method comprises incubating the stem cell in the presence of a ligand of a class Ill tyrosine kinase receptor. 5 In a third aspect the present invention provides a method for modulating spontaneous differentiation of a stem cell, which method comprises incubating the stem cell in the presence of an agonist of a LPL receptor and a ligand of a class Ill tyrosine kinase receptor, wherein the agonist is selected from the group consisting of S1P, dihydro S1 P, LPA, PAF 10 and SPC or functional equivalents thereof. Sphingosine-1 -phosphate (S1P), an agonist of the LPL receptors has the ability to at least partially inhibit the spontaneous loss of stem cell phenotype in cell culture. It has also been found that the method does not affect the ability of stem cells to proliferate. 15 Preferably, the LPL receptor is selected from the group consisting of S1P1, S1P2 and S1P3. As used herein the term "modulating the differentiation of a stem cell" includes the inhibition or enhancement of cellular differentiation. The term also includes partial inhibition or enhancement of cellular differentiation. In a 20 preferred form of the method, the modulation is inhibition of differentiation. Typically the agonist is a phospholipid. As used herein, the term "phospholipid" refers to a molecule that includes a backbone attached to two fatty acid moieties and a phosphate group. The backbone on which the fatty acid molecules are attached is 25 variable and may be based on glycerol or sphingosine for example. A diagram of a generic phospholipid is shown below. y Fatty c acid e r o Phosphate Aco l The term "lysophospholipid" refers to a phospholipid molecule where one of the fatty acid chains has been removed. The removal of a fatty acid 6a chain may be accomplished by treatment of the phospholipid with an enzyme such as phospholipidase A2. The phospholipid or lysophoholipid may have a sphingosine 5 backbone, and particularly, the lysophospholipid may be a phosphorlyated amino alcohol. The agonist is selected from the group consisting of S1P, dihydro S1 P, LPA, PAF and SPC or functional equivalents thereof. In a highly preferred form of the invention the lysophospholipid is sphingosine-1 -phosphate (S1P) or a functional equivalent thereof. S1P is a 10 small bioactive phospholipid, present in serum, released by activated platelets, which has the following structure: WO 03/104442 PCT/AU03/00713 7 CH 0- o 'CHI OA O +U The skilled person will understand that bioactive molecules such as 5 phospholipids and lysophospholipids may be altered in a number of ways and still retain biological activity. Accordingly, the scope of the present invention includes altered forms of phospholipids and lysopholipids that retain their LPL receptor agonist activity. The scope of the present invention also includes synthetic peptidic agonists of the LPL receptors. 10 The skilled person will be familiar with methods which can be applied to testing phospholipids or lysophospholipids for the ability to modulate the ability of a stem cell to differentiate. Suitable methods are found herein, and include reactivity with antibodies such as GCTM-2 which are directed to stem cell specific markers, and simple morphological evaluation of cells by light 15 microscopy. For example, the effect of the agonist on the differentiation of stem cells into neuronal or endodermal lineages may be studies by analysis of marker expression as shown in PCT/ AU01/00278 and PCT/AUOI/00735. The phospholipid or lysophospholipid may be extracted from a 20 biological source such as serum. In addition, mast cells and monocytes are able to produce SIP while adipocytes produce LPA, however the main source of LPA and S1P is activated platelets. Alternatively, the phospholipid may be synthesised by procedures well known in the field of organic chemistry. 25 Preferably, cells that have been exposed to a LPL receptor agonist are not substantially negatively affected in their ability to proliferate. Therefore, an advantage of the methods and compositions described herein is that it is possible to expand a population of hES cells without leading to a loss in pluripotency. Methods for determining the proliferative capability of a hES 8 cell will be known by the skilled person and include detection of the cell proliferation marker PCNA as described herein. Typically the ligand is a PDGF or functional equivalent thereof. The tyrosine kinase receptor may be PDGFR-a or PDGFR-p. 5 In a preferred embodiment the PDGF is PDGFaa, PDGFab or PDGFbb which bind to the two types of receptors. The method may also include use of TNF alpha, NGF (nerve growth factor), muscarinic acetylcholine agonists, serum or phorbol esters - which again are compounds that have additive or synergistic effects with S1P in 10 other cell types. The stem cell may be derived from foetal tissue or adult tissue. The stem cell is typically an ES cell. Preferably the stem cell is a hES cell. As used herein the term "embryonic stem cell" means a cultured cell line derived from preimplantation stages of development capable of 15 differentiation into tissues representative of all three embryonic germ layers. Theses cells: - express SSEA-3, SSEA-4, TRA 1-60, GCTM-2, alkaline phosphatase and Oct-4 - Grow as flat colonies with distinct cell borders 20 - Differentiate into derivatives of all three embryonic germ layers - Are feeder cell dependent (feeder cell effect on growth not reconstituted by conditioned medium from feeder cells or by feeder cell extracellular matrix) - Are highly sensitive to dissociation to single cells and show 25 poor cloning efficiency even on a feeder cell layer - Do not respond to Leukemia Inhibitory Factor In a fourth aspect the present invention provides a serum free medium useful for modulating spontaneous differentiation of a stem cell having a LPL receptor, comprising an agonist of the LPL receptor and a ligand of a class III 30 tyrosine kinase receptor, wherein the agonist is selected from the group consisting of S1 P, dihydro S1P, LPA, PAF and SPC or functional equivalents thereof.

9 In a fifth aspect the present invention provides a serum free medium useful for modulating spontaneous differentiation of a stem cell, comprising a ligand of a class Ill tyrosine kinase receptor. The medium is useful in propagating stem cells such as human 5 embryonic stem cells in an undifferentiated state. Typically the ligand is a PDGF or functional equivalent thereof. The tyrosine kinase receptor may be PDGFR-a or PDGFR-p. In a preferred embodiment the PDGF is PDGFaa, PDGFab or PDGFbb. 10 The medium may also include TNF alpha, NGF (nerve growth factor), muscarinic acetylcholine agonists, serum or phorbol esters - which again are compounds that have additive or synergistic effects with S1 P. Typically the agonist is a phospholipid. The agonist is selected from the group consisting of S1P, LPA, PAF, 15 dihydro S1 P and SPC or functional equivalents thereof. The stem cells may be derived from foetal tissue or adult tissue. The stem cells are typically embryonic stem cells. Preferably the stem cells are from embryonic tissue. Typically the stem cells are of human origin. 20 The base medium is typically a standard serum free medium that is supplemented with phospholipids and ligand as well as a buffering agent. A suitable buffering agent is 25mM Hepes. The medium is of use in inhibiting the differentiation of pluripotent stem cells. 25 The cell culture medium may be based on any of the base media known in the art useful for the growth and/or maintenance of stem cells such as hES cells. Such media include but are not limited to Dulbecco's Modified Eagles Medium (DMEM), KNOCKOUT-DMEM or hES medium. In a preferred form of the invention the medium is based on DMEM supplemented 30 with insulin, transferrin and selenium. The optimal concentration of LPL agonist in the medium may be determined by routine experimentation. However, in a preferred form of the invention the 9a agonist is present in the medium at a concentration of from 0.1 pM to 10pM where the agonist is S1P. In a highly preferred form of the invention the agonist is present in the medium at a concentration of about 10pM. It would be expected that the optimum concentration will vary WO 03/104442 PCT/AU03/00713 10 according to a number of parameters including the species of agonist, the line of stem cells being cultured, the base medium used, and other culture conditions such as temperature, carbon dioxide concentration, and humidity. The optimal concentration of ligand in the medium may be determined 5 by routine experimentation. However, in a preferred form of the invention the ligand is present in the medium at a concentration of from I ng/ml to 20ng/ml where the ligand is PDGFaa, PDGFab or PDGFbb. In a highly preferred form of the invention the ligand is present in the medium at a concentration of 20 ng/ml. Again, it would be expected that the optimum concentration will 10 vary according to a number of parameters including the species of agonist, the line of stem cells being cultured, the base medium used, and other culture conditions such as temperature, carbon dioxide concentration, and humidity. The skilled person understands that it is often necessary to culture 15 hES cells on feeder cells, and the present invention contemplates methods including the use of such feeder cells. The concentration of agonist may also need to be optimised according to the feeder cell line used. In a fifth aspect the present invention provides a stem cell grown and/or maintained in a cell culture medium of the invention. 20 Cells of the present invention will find many uses in biology and medicine. The properties of pluripotentiality and immortality are unique to ES cells and enable investigators to approach many issues in human biology and medicine for the first time. ES cells potentially can address the shortage of donor tissue for use in transplantation procedures, particularly where no 25 alternative culture system can support growth of the required committed stem cell. However, it must be noted that almost all of the wide ranging potential applications of ES cell technology in human medicine-basic embryological research, functional genomics, growth factor and drug discovery, toxicology, and cell transplantation are based on the assumption that it will be possible 30 to increase the proliferation and therefore grow ES cells on a large scale, to introduce genetic modifications into them, and to direct their differentiation. The present invention provides a method of producing a population of proliferating undifferentiated stem cells from a stem cell which method 11 comprises incubating the stem cell in the presence of an agonist of the LPL receptor and a ligand of a class III tyrosine kinase receptor. The present invention also provides a method of producing a population of proliferating undifferentiated stem cells from a stem cell which 5 method comprises incubating the stem cell in the presence of a ligand of a class Ill tyrosine kinase receptor. The present invention further provides a method of producing a population of proliferating undifferentiated stem cells from a stem cell which method comprises incubating the stem cell in the presence of an agonist of 10 the LPL receptor, wherein the agonist is selected from the group consisting of S1 P, dihydro S1P, LPA, PAF and SPC or functional equivalents thereof. These methods therefore provide for the expansion of stem cell populations. The invention also provides a population of undifferentiated stem cells 15 produced by at least one of these methods. Preferably, the LPL receptor is selected from the group consisting of S1P1, S1P2 and S1P3. Typically the agonist is a phospholipid. The agonist is selected from the group consisting of S1P, dihydro 20 S1 P, LPA, PAF and SPC or functional equivalents thereof. In a highly preferred form of the invention the lysophospholipid is sphingosine-1 -phosphate (S1 P) or a functional equivalent thereof. Typically the ligand is a PDGF or functional equivalent thereof. The tyrosine kinase receptor may be PDGFR-a or PDGFR-p. 25 In a preferred embodiment the PDGF is PDGFaa, PDGFab or PDGFbb which bind to the two types of receptors. The ligand may also be TNF alpha, NGF (nerve growth factor), muscarinic acetylcholine agonists, serum or phorbol esters. The stem cell may be derived from foetal tissue or adult tissue. 30 The stem cell is typically an ES cell. Preferably the stem cell is a hES cell. Also described is a method of treating or preventing a disorder of stem cell differentiation comprising administering to an animal in need thereof a composition containing an agonist of a LPL 12 receptor. Methods for the preparation of pharmaceutical compositions are well known in the art, as set out in textbooks such as Remington's Pharmaceutical Sciences, 18 th Edition, Mack Publishing Company, Easton, Pennsylvania, USA, the contents of which is incorporated herein. 5 The present invention also provides a method of treating or preventing a disorder of stem cell differentiation comprising administering to an animal in need thereof a composition containing an agonist of a LPL recepetor. The present invention also provides a method of treating or preventing a disorder of stem cell differentiation comprising administering to an animal in 10 need thereof a composition containing a ligand of a class IlIl tyrosine kinase receptor. Also described is a method of treating or preventing a disorder of stem cell differentiation comprising administering to an animal in need thereof a composition containing an agonist of a LPL receptor and a ligand of a class 15 1il tyrosine kinase receptor. The present invention also provides a method of treating or preventing a disorder of stem cell differentiation comprising administering a stem cell as described herein. Disorders of stem cell differentiation are well known to those skilled in the art, and include, but are not limited to the following: 20 Acute Leukemias Acute Lymphoblastic Leukemia (ALL) Acute Myelogenous Leukemia (AML) Acute Biphenotypic Leukemia 25 Acute Undifferentiated Leukemia Chronic Leukemias Chronic Myelogenous Leukemia (CML) Chronic Lymphocytic Leukemia (CLL) 30 Juvenile Chronic Myelogenous Leukemia (JCML) Juvenile Myelomonocytic Leukemia (JMML) Myelodysplastic Syndromes Refractory Anemia (RA) WO 03/104442 PCT/AU03/00713 13 Refractory Anemia with Ringed Sideroblasts (RARS) Refractory Anemia with Excess Blasts (RAEB) Refractory Anemia with Excess Blasts in Transformation (RAEB-T) Chronic Myelomonocytic Leukemia (CMML) Stem Cell Disorders Aplastic Anemia (Severe) Fanconi Anemia Paroxysmal Nocturnal Hemoglobinuria (PNH) Pure Red Cell Aplasia Myeloproliferative Disorders Acute Myelofibrosis Agnogenic Myeloid Metaplasia (myelofibrosis) Polycythemia Vera Essential Thrombocythemia Lymphoproliferative Disorders Non-Hodgkin's Lymphoma Hodgkin's Disease Phagocyte Disorders Chediak-Higashi Syndrome Chronic Granulomatous Disease Neutrophil Actin Deficiency Reticular Dysgenesis Inherited Metabolic Disorders Mucopolysaccharidoses (MPS) Hurler's Syndrome (MPS-IH) Scheie Syndrome (MPS-IS) Hunter's Syndrome (MPS-II) Sanfilippo Syndrome (MPS-Ill) Morquio Syndrome (MPS-IV) Maroteaux-Lamy Syndrome (MPS-VI) Sly Syndrome, Beta-Glucuronidase Deficiency (MPS-VII) Adrenoleukodystrophy Mucolipidosis 11 (1-cell Disease) Krabbe Disease WO 03/104442 PCT/AU03/00713 14 Gaucher's Disease Niemann-Pick Disease Wolman Disease Metachromatic Leukodystrophy Histiocytic Disorders Familial Erythrophagocytic Lymphohistiocytosis Histiocytosis-X Hemophagocytosis Inherited Erythrocyte Abnormalities Beta Thalassemia Major Sickle Cell Disease Inherited Immune System Disorders Ataxia-Telangiectasia Kostmann Syndrome Leukocyte Adhesion Deficiency DiGeorge Syndrome Bare Lymphocyte Syndrome Omenn's Syndrome Severe Combined Immunodeficiency (SCID) SCID with Adenosine Deaminase Deficiency Absence of T & B Cells SCID Absence of T Cells, Normal B Cell SCID Common Variable Immunodeficiency Wiskott-Aldrich Syndrome X-Linked Lymphoproliferative Disorder Other Inherited Disorders Lesch-Nyhan Syndrome Cartilage-Hair Hypoplasia Glanzmann Thrombasthenia Osteopetrosis Inherited Platelet Abnormalities Amegakaryocytosis / Congenital Thrombocytopenia Plasma Cell Disorders WO 03/104442 PCT/AU03/00713 15 Multiple Myeloma Plasma Cell Leukemia Waldenstrom's Macroglobulinemia Other Malignancies Breast Cancer Ewing Sarcoma Neuroblastoma Renal Cell Carcinoma Thus, the present invention may be used to treat a patient having a stem cell related disease by administration of a composition described herein, or by administering populations of stem cells produced by a method 5 described herein The agonist is typically a phospholipid. The phospholipid may be a lysophospholipid and may have a sphingosine backbone. Preferably the agonist is selected from the group consisting of S1P, dihydro SIP, LPA, PAF and SPC or functional equivalents thereof. SIP and dihydro SIP are 10 lysophospholipids with a sphingosine backbone, as is SPC, while LPA is a lysophosphospholipid with a glycerol backbone, and PAF is a phospholipid with a glycerol backbone. The tyrosine kinase receptor may be PDGFR-a or PDGFR-p and the ligand a PDGF or functional equivalent thereof. 15 In a preferred embodiment the PDGF is PDGFaa, PDGFab or PDGFbb. The method may also include use of TNF alpha, NGF (nerve growth factor), muscarinic acetylcholine agonists, serum or phorbol esters - which again are compounds that have additive or synergistic effects with SIP in 20 other cell types. Also provided is a pharmaceutical composition comprising a class Ill tyrosine kinase receptor ligand and a LPL receptor agonist. The composition may also include use of TNF alpha, NGF (nerve growth factor), muscarinic acetylcholine agonists, serum or phorbol esters - which again are compounds 25 that have additive or synergistic effects with S1 P in other cell types.

WO 03/104442 PCT/AU03/00713 16 A skilled person will be able to provide formulations and dosage forms of the agonist. Furthermore, the optimum dosage for a given clinical situation could be determined by routine experimentation. The compositions may be administered parenterally. For parenteral 5 administration, the agonist and/or ligand may be combined with sterile aqueous or organic media to form injectable solutions or suspensions. The injectable solutions prepared in this. manner may then be administered intravenously, intraperitoneally, subcutaneously, or intramuscularly. Additional methods of administration may include, but are not limited to, 10 topical, sublingual, anal and vaginal methods of administration according to methods which are commonly known by those skilled in the art. The amount of agonists or ligand used for preparation of a pharmaceutical composition should be varied according to principles well known in the art taking into account the severity of the condition being treated 15 and the route of administration. In general, such a pharmaceutical composition would be administered to a warm blooded animal, preferably a mammal and most preferably a human, so that an effective dose, usually a daily dose administered in unitary or divided portions, is received. Dosages depend upon a number of factors, including the condition or disease being 20 treated, characteristics of the subject and the type of pharmaceutical form or formulation used. Such deviations are within the scope of this invention. Suitable pharmaceutically acceptable carriers for preparing a pharmaceutical composition include inert solid fillers or diluents and sterile aqueous or organic solutions. The antagonist and/or ligand are present in 25 such pharmaceutical compositions in amounts sufficient to provide the desired dosage according to the range described above. Thus, for oral administration the agonist and/or ligand may be combined with a suitable solid or liquid carrier or diluent to form capsules, tablets, powders, syrups, solutions, suspensions and the like. The pharmaceutical compositions may, if 30 desired, contain additional components such as flavorants, sweeteners, excipients and the like. Controlled release, sustained release, and delayed release oral or parenteral compositions may be used.

WO 03/104442 PCT/AU03/00713 17 The tablets, pills, capsules, and the like may also contain one or more binders such as gum tragacanth, acacia, corn starch or gelatin; one or more excipients such as dicalcium phosphate; one or more disintegrating agents such as corn starch, potato starch, alginic acid; one or more lubricants such 5 as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin. When a dosage unit form is a capsule, for example a gel capsule, it may contain, in addition to or instead of materials of the above type, a liquid carrier such as a fatty glyceride or mixtures of fatty glycerides. Dosage forms may also include oral suspensions. 10 Various other materials may be present as coatings or to modify the physical form of a dosage unit. For instance, tablets may be coated with shellac, sugar or both. A syrup or elixer may contain, in addition to the active ingredient(s), sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry or orange flavor. 15 The pharmaceutical forms suitable for injectable use include sterile solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sufficiently fluid to enable incorporation into a syringe and injection therefrom and must be substantially stable under the conditions of 20 manufacture and storage. In addition, the form must be substantially sterile and must be preserved against contamination of microorganisms such as bacteria and fungi. Sterilization may be achieved by filtration through microorganism retaining filters, by incorporating sterilizing agents into the compositions, or by irradiating or heating the compositions wherein such 25 irradiation or heating is both appropriate and compatible with the applicable formulation. Additional pharmaceutical forms may include suppositories, sublingual tablets, topical dosage forms and the like, and these may be prepared according to methods which are commonly known by those skilled in the art. 30 The present invention provides use of an agonist of the LPL receptors and a ligand of a class IlIl tyrosine kinase receptor for modulating spontaneous differentiation of a stem cell having a lysophospholipid (LPL) receptor and PDGF receptors.

18 The present invention also provides use of a ligand of a class Ill tyrosine kinase receptor in modulating spontaneous differentiation of a stem cell. The present invention further provides use of an agonist of the LPL 5 receptor for modulating spontaneous differentiation of a stem cell having a lysophospholipid (LPL) receptor, wherein the agonist is selected from the group consisting of S1P, dihydro S1P, LPA, PAF and SPC or functional equivalents thereof. Preferably, the LPL receptor is selected from the group consisting of 10 S1P1, S1P2 and S1P3. Typically the agonist is a phospholipid. The phospholipid or lysophoholipid may have a sphingosine backbone, and particularly, the lysophospholipid may be a phosphorlyated amino alcohol. 15 In a highly preferred form of the invention the lysophospholipid is sphingosine-1 -phosphate (S1 P) or a functional equivalent thereof. Typically the ligand is a PDGF or functional equivalent thereof. The tyrosine kinase receptor may be PDGFR-a or PDGFR-p. In a preferred embodiment the PDGF is PDGFaa, PDGFab or 20 PDGFbb which bind to the two types of receptors. TNF alpha, NGF (nerve growth factor), muscarinic acetylcholine agonists, serum or phorbol esters may also be used as compounds that have additive or synergistic effects with S1 P in other cell types. The stem cell may be derived from foetal tissue or adult tissue. 25 The stem cell is typically an ES cell. Preferably the stem cell is a hES cell. The present invention provides use of an agonist of the LPL receptor and a ligand of a class IlIl tyrosine kinase receptor in producing a population of proliferating undifferentiated stem cells from a stem cell. 30 The present invention also provides use of a ligand of a class IlIl tyrosine kinase receptor in producing a population of proliferating undifferentiated stem cells from a stem cell. The present invention further provides use of a method of an agonist of the LPL receptor in producing a population of proliferating undifferentiated 19 stem cells from a stem cell, wherein the agonist is selected from the group consisting of S1 P, dihydro S1P, LPA, PAF and SPC or functional equivalents thereof. Preferably, the LPL receptor is selected from the group consisting of 5 S1P1,S1P2and S1P3. Typically the agonist is a phospholipid. In a highly preferred form of the invention the lysophospholipid is sphingosine-1 -phosphate (S1 P) or a functional equivalent thereof. Typically the ligand is a PDGF or functional equivalent thereof. 10 The tyrosine kinase receptor may be PDGFR-a or PDGFR-p. In a preferred embodiment the PDGF is PDGFaa, PDGFab or PDGFbb which bind to the two types of receptors. The ligand may also be TNF alpha, NGF (nerve growth factor), muscarinic acetylcholine agonists, serum or phorbol esters. 15 The stem cell may be derived from foetal tissue or adult tissue. The stem cell is typically an ES cell. Preferably the stem cell is a hES cell. Also provided is the use of a composition containing an agonist of a LPL receptor and a ligand of a class Ill tyrosine kinase receptor in a method 20 of treating or preventing a disorder of stem cell differentiation. The present invention also provides use of a composition containing a ligand of a class IlIl tyrosine kinase receptor in a method of treating or preventing a disorder of stem cell differentiation. The agonist is typically a phospholipid. The phospholipid may be a 25 lysophospholipid and may have a sphingosine backbone. The agonist is selected from the group consisting of S1 P, dihydro S1 P, LPA, PAF and SPC. S1 P and dihydro SIP are lysophospholipids with a sphingosine backbone, as is SPC, while LPA is a lysophosphospholipid with a glycerol backbone, and PAF is a phospholipid with a glycerol backbone.

WO 03/104442 PCT/AU03/00713 20 The tyrosine kinase receptor may be PDGFR-a or PDGFR-p and the ligand a PDGF or functional equivalent thereof. In a preferred embodiment the PDGF is PDGFaa, PDGFab or PDGFbb. 5 The method may also include use of TNF alpha, NGF (nerve growth factor), muscarinic acetylcholine agonists, serum or phorbol esters - which again are compounds that have additive or synergistic effects with SIP in other cell types. 10 Abbreviations dH-SIP: dihydro-sphingosine-1 -phosphate; EDG: endothelial differentiation gene; ERK: extracellular signal-regulated kinase; MAP kinase: mitogen-activated protein kinase; MEF: mouse embryonic fibroblasts; hES cells: human embryonic stem cells; LPA: lysophosphatidic acid; LPL: 15 lysophospholipid; PAF: platelet-activated factor; PCNA: proliferating cell nuclear antigen; PDGF: platelet-derived growth factor; PDGFR: platelet derived growth factor receptor; SIP: sphingosine-1-phosphate; SPC: sphingosylphosphorylcholine; SPK: sphingosine kinase. 20 Brief Description of the Accompanying Figures FIGURE 1:shows hES cells are target of S1 P, LPA and PDGF. RT-PCR for LPL receptors (A, B), PDGFR-a (alpha) and PDGFR-p (beta) (C), SPK-I and SPK-2 (D), with (+) or without (-) RT. Immunostaining of hES cells with Hoechst 33342 (E, H), PDGFR-a (F) or PDGFR-p (I) and GCTM-2 (G, J) 25 antibodies. SIP, LPA and PDGF stimulate ERKs phosphorylation in hES cells. (K) Western blots experiment were performed using protein lysate from hES cells. Cells were pre-treated or not with U0126 (30 pM, 1 hr) and incubated for 5 min in the absence (C, control) or presence of SIP (S,10 pM), LPA (L, 50 M) or PDGF (P, 20 ng/ml). The phosphorylation of Erk1 and 30 Erk2 (P-ErkI and P-Erk2) was assessed by immunoblotting with a polyclonal anti-active MAP kinase as described in Materials and Methods. After a WO 03/104442 PCT/AU03/00713 21 stripping procedure, the same blots were reprobed with a monoclonal anti MAP kinase, allowed the detection of Erk1 and Erk2. These data are representative of results from at least 3 independent experiments. FIGURE 2 shows S1P and PDGF inhibit the spontaneous differentiation 5 of hES cells. (A) hES cells grown with MEF, before the depletion of serum from the medium. (B, C, D, E) hES cells grown without serum after 8 days, in the absence (B) or in the presence of SIP (10 pM) (C), PDGF (20 ng/ml) (D), SIP (10 pM) plus PDGF (20 ng/mI) (E). (F) hES cells grown without serum, in the presence or in the absence (control) of SIP (10 pM), PDGF (20 ng/ml), 10 SIP (10 pM) plus PDGF (20 ng/ml). In A-E, data are representative of at least 3 independent experiments. In F, data expressed as percentages of alkaline phosphatase activity in absence of serum for eight days (% of control), are the means ± SEM of at least 2 independent experiments, each run in triplicate. 15 FIGURE 3 shows SIP and PDGF inhibit the spontaneous differentiation of hES cells independently of MEF. hES cells mechanically dissociated and cultivated for 4 days in the absence (C, control) or presence of S1 P (10 pM) or/and PDGF (20 ng/ml) in a media depleted in serum. (A) Quantification of the number of GCTM2+ cells. (B) Quantification of the number of 20 PCNA+/GCTM2+ cells. These data are the mean ± SEM of results obtained in at least 3 independent experiments. FIGURE 4 shows hES cells are target of SIP, LPA and PDGF. RT-PCR for LPL receptors (A, B), PDGFR-a (alpha) and PDGFR-p (beta) (C), SPK-I and SPK-2 (D), with (+) or without (-) RT. Immunostaining of hES cells with 25 Hoechst 33342 (E, H), PDGFR-a (F) or PDGFR-p (I) and GCTM-2 (G, J) antibodies. S1 P, LPA and PDGF stimulate ERKs phosphorylation in hES cells. FIGURE 5 shows S1 P and PDGF inhibit the spontaneous differentiation of hES cells in the absence of serum. (A-C) hES cells with or without (control) WO 03/104442 PCT/AU03/00713 22 the indicated agonists. Dihydro-S1P: DHS1P. (D) Sphingosine kinase activity measurement following incubation of hES cells with PDGF. FIGURE 6 shows characterization of hES cells. (A) hES cells grown in the presence of SIP + PDGF, passage 14. (B) RT-PCR using mRNA from 5 hES cells grown in the presence of SIP and PDGF using specific primers for Oct-4, cripto, SPKI and SPK2, with (+) or without (-) RT, passage 7. Immunostaining of hES cells grown in the presence of SIP + PDGF with GCTM-2 (C), Oct-4 (D), TG-30 (E) or TRA-1-60 (F), passage 13. (G) Karyotyping of hES cells grown in the presence of S1P + PDGF, passage 8. 10 (H) Neuronal differentiation into neurospheres. (I) stubulin immunostaining FIGURE 7 shows Edg receptor mRNAs are expressed in hES cells. RT-PCR experiments were performed using mRNA isolated from hES cells using specific primers for human Edg receptors. In each case, experiments were conducted either in the presence (+) or absence (-) of reverse 15 transcriptase. The RT-PCR products were separated by electrophoresis on 1.5% agarose gel and revealed by ethidium bromide fluorescence. Molecular sizes (in bp) of the products were calculated using I kB plus DNA ladder markers (M). These data are representative of at least 6 independent experiments, each carried out on mRNAs prepared from different cultures of 20 hES cells. FIGURE 8 shows SIP inhibits the spontaneous differentiation of hES cells. (A) hES cells grown with feeder, before the depletion in serum. (B) hES cells grown without serum-after 8 days (B, C) and 12 days (D, E), in absence (B, D) or presence of SIP (C, E, 10 piM). These data are 25 representative of at least 3 independent experiments. FIGURE 9 shows SIP inhibits the spontaneous differentiation of hES cells. Double staining experiments were performed using antibodies for PCNA and GCTM-2. These data are representative of at least 3 independent experiments.

WO 03/104442 PCT/AU03/00713 23 FIGURE 10 shows SIP stimulates ERKs phosphorylation in hES cells. Western blots experiments were performed using protein lysate from hES cells. (A) Cells were pre-treated or not with U0126 (30 pM, I hr) and incubated for 5 min in the absence (C, control) or presence of S1P (10 pM). 5 (B) Cells were incubated for different time periods in the absence or presence of SIP (10 piM). (C) Cells were incubated for 5 min with various concentrations of S1 P. The phosphorylation of Erk1 and Erk2 (P-Erk1 and P Erk2) was assessed by immunoblotting with a polyclonal anti-active MAP kinase as described in Materials and Methods. After a stripping procedure, 10 the same blots reprobed with a monoclonal anti-MAP kinase, allowed the detection of Erk1 and Erk2. These data are representative of at least 3 independent experiments. The invention will now be more fully described with reference to the following non-limiting Examples. 15 Best Method and Other Methods of Carrying out the Present Invention EXAMPLE 1 20 Cell culture hES-3 cells were cultured as previously described 1. The serum-free culture medium consisted of DMEM (without sodium pyruvate, glucose 4500 mg/I, Invitrogen, Mount Waverley, VIC, Australia) supplemented with insulin/transferrin/selenium 1%, p-mercaptoethanol 0.1 mM, NEAA 1 %, 25 glutamine 2 mM, Hepes 25 mM, penicillin 50 U/ml and streptomycin 50 mg/ml (all from Invitrogen). Medium was changed every 2 days and cells were passaged every week. SIP and dihydro-S1P were obtained from Biomol (Plymouth Meeting, PA, USA) and were dissolved in methanol. LPA was obtained from Sigma (Castle Hill, NSW, Australia) and was dissolved in 30 ethanol. Extemporaneous dilutions of all lipids were made in water containing WO 03/104442 PCT/AU03/00713 24 0.1 % fatty acid-free bovine serum albumin (BSA) (Sigma). Human PDGF AB, PDGF-AA, PDGF-BB were from PreproTech Inc. (Rocky Hill, NJ, USA). RT-PCR experiments. 5 Total RNA was extracted from hES cells and reverse transcribed (RT) as previously described '.The cDNA samples were amplified by PCR with sense and antisense primers (Sigma) designed for the specific detection of mouse (data not shown) or human DNA target sequences (Table 1) using Taq DNA polymerase (Biotech International Ltd, Perth, WA, Australia) as 10 previously described 18. The specific amplified DNA fragments were sized by electrophoresis on 1.5 % (w/v) agarose gel and stained with ethidium. Molecular sizes (bp) were calculated using 1 kb plus DNA ladder markers (M). The amplicons were purified and sequenced. Experiments were performed on hES-2 and hES-3. 15 Table I Sense and antisense primers Gene sense and antisense primers Size Annealing References (bp) temp ( 0 C) S1 P 1 CCACAACGGGAGCAATAACT 480 52 2 GTAAATGATGGGGTTGGTGC S1P 2 CCAATACCTTGCTCTCTCTGGC 502 52 2 CAGAAGGAGGATGCTGAAGG

SIP

3 TCAGGGAGGGCAGTATGTTC 505 52 2 CTGAGCCTTGAAGAGGATGG

SIP

4 CGGCTCATTGTTCTGCACTA 701 52 2 WO 03/104442 PCT/AU03/00713 25 GATCATCAGCACCGTCTTCA

SIP

5 TTCTGATACCAGAGTCCGGG 460 52 2 CAAGGCCTACGTGCTCTTCT LPA, GCTCCACACACGGATGAGCAACC 621 56 3 GTGGTCATTGCTGTGAACTCCAGC

LPA

2 AGCTGCACAGCCGCCTGCCCCGT 775 56 TGCTGTGCCATGCCAGACCTTGTC

LPA

3 CCATAGCAACCTGACCAAAAAGAG 482 56 TCCTTGTAGGAGTAGATGATGGGG PDGFRa ATCAATCAGCCCAGATGGAC 891 58 TTCACGGGCAGAAAGGTACT PDGFRp AATGTCTCCAGCACCTTCGT 698 58 AGCGGATGTGGTAAGGCATA Crypto CAGAACCTGCTGCCTGAATG 185 55 GTAGAAATGCCTGAGGAAACG SPK-1 ACCCATGAACCTGCTGTCTC 227 55 CAGGTGTCTTGGAACCCACT SPK-2 TGGCAGTGGTGTAAGAACC 200 55 CAGTCAGGGCGATCTAGGA Oct-4 CGTTCTCTTTGGAAAGGTGTTC 320 55

ACACTCGGACCACGTCTTTC

WO 03/104442 PCT/AU03/00713 26 Immunofluorescence. In some experiments, hES-3 cells plated onto 8-well chamber slides, with or without MEF, were fixed in ethanol or paraformaldehyde (for PDGFR) the day after plating. In others, hES-3 cells were mechanically dissociated, in 5 order to obtain a monolayer culture and then plated onto 8-well chamber slides without MEF and were fixed in ethanol 4 days after the first treatment. Immunostaining was performed using the following antibodies: anti human PDGFR-a or PDGFR-p (R&D Systems Inc.), GCTM-2, and/or PCNA (Chemicon, Boronia, VIC, Australia), TRA-1-60, Oct-4. Nuclei were 10 evidenced by Hoechst-33342. Slides were mounted and observed by fluorescent microscopy with a Leica microscope at X1 0, X20 and X40. Specificity was verified by the absence of any staining in the negative controls. In some experiments, cells were counted to determine the ratio of GCTM-2 positive (GCTM2+), PCNA positive (PCNA+) and GCTM2+/PCNA+ 15 cells within the global population. GCTM-2 quantification. hES-3 cells plated with MEF, were fixed in ethanol and immunostained with GCTM-2 and then with an alkaline phosphatase-coupled secondary 20 antibody (Dako). The activity of alkaline phosphatase was detected by adding a solution of 4-nitrophenyl phosphate (Roche, Mannheim, Germany), followed by reading the optical density (OD) at 405 nm. In order to validate the technique as a relevant indicator of the proportion of GCTM-2 positive cells, standard curves were done with the teratocarcinoma cell line 25 GCT27C4, known to express GCTM-2. This showed a linear correlation between the number of cells and the OD read at 405 nm (data not shown). Western blot analysis. hES-3 cells plated without MEF for 24 hrs were depleted of serum for 30 a further 18 hrs. Cells pre-treated or not with U0126 (Sigma, 30 pM, 1 hr), were incubated in the presence of the different agents for 5 min and were lysed by removal of the supernatants and addition of a reducing loading buffer containing 1 mM sodium orthovanadate (Sigma). Protein lysates WO 03/104442 PCT/AU03/00713 27 (approx. 80 pg) were separated by SDS-polyacrylamide gel electrophoresis (10 % polyacrylamide, w/v), transferred to nitrocellulose (Hybond nitrocellulose, Amersham) and immunoblotting was carried out using rabbit polyclonal anti-active mitogen-activated protein (MAPK) antibodies raised 5 against a dually phosphorylated MAPK peptide (Promega, Madison, WI, USA). Peroxidase-coupled secondary antibody (Dako) was detected by exposure of autoradiographic films in the presence of a chemiluminescent detection reagent (ECL, Amersham). Stripping of antibodies was achieved and membranes were then reprobed with rabbit polyclonal anti-ERKI/2 10 antibodies (Promega), and then with peroxidase-coupled secondary antibodies (Dako). Membranes probed with either rabbit polyclonal anti active p38 (Promega) or rabbit polyclonal anti-active JNK (Promega) antibodies were also performed, using the same procedure as described above. 15 Protein quantification. hES-3 cells were lysed and the amount of proteins was determined using a colorimetric assay based on the Bradford dye-binding test (Bio-Rad Laboratories, Regents Park, NSW, Australia). 20 Statistical analysis. Each set of experiments was performed at least 3 times (n refers to number of independent experiments performed on different cell cultures). Data are expressed as the mean ± SEM. Significance of the differences was 25 evaluated by using the ANOVA followed by Student-Newman Keuls test. Values of P < 0.05 were considered significant and were respectively indicated by *. RESULTS 30 hES cells (Figure IA) expressed mRNA transcripts for three SIP receptors: S1P 1 , S1P 2 and S1P 3 and for each of LPA receptors: LPA 1 , LPA 2 and LPA 3 (Figure 1 B), while these cells did not express mRNA for S1 P 4 and S1P 5 (data not shown). hES cells also expressed mRNA transcripts for WO 03/104442 PCT/AU03/00713 28 PDGFR-a (Figure 1C) and PDGFR-p (Figure 1C) as well as the corresponding proteins, as revealed by immunostaining (Figure 1 E-J). MEF expressed SIP 1 , SIP 2 , SIP 3 , LPA 1 and LPA 2 , PDGFR-a and PDGFR-p but neither SIP 4 , S1P 5 nor LPA 3 (data not shown), as previously shown by 5 others ~6-. Because the MAP kinases ERKs are implicated in cell proliferation and differentiation, we examined the effects of SIP, LPA and PDGF-AB (PDGF) on their activation in hES cells. After 5 min, SIP, LPA and PDGF stimulated the phosphorylation of ERKs in hES cells (Figure 1K), an effect that was totally inhibited in presence of the MEK inhibitor U0126 (30 pM) 10 (Figure 1K). Next, it was examined whether SIP, LPA and PDGF could modulate the fate of hES cells. When hES cells were grown on MEF, in a serum-free culture media, they spontaneously differentiated. As shown in Figure 2, after 8 days in such conditions (control), the colonies were bigger than those 15 observed before the removal of serum (Figure 2A) and hES cells gave rise to different kinds of cells (Figure 2B). After 8 days, LPA (up to 50 pM) did not have an obvious effect on growth of the colonies, as ascertained by morphological (data not shown) whilst in the presence of either SIP (10 pM) or PDGF (20 ng/ml), the colonies appeared flatter and less differentiated as 20 compared to the control condition (Figure 2C, 2D). Thus, after 8 days of treatment, when GCTM-2 levels of cells were quantified by measuring the activity of alkaline phosphatase, cells treated with SIP or PDGF were respectively 16.6 ± 4.1 % (n=7) and 16.6 ± 7.0 % (n=7) more GCMT2+ than the control cells (Figure 2F). Strikingly, the co-incubation of both S1P (10 pM) 25 and PDGF (20 ng/ml) induced a strong inhibition of spontaneous differentiation, not observed in the presence of one or the other agent (Figure 2E) with a higher percentage of GCTM2+ cells of 40.1 ± 7.5 % (n=7) than in the control cells (Figure 2F). As GCTM-2 is a stem cell marker, these results suggest that the combination of PDGF and SIP in a serum-free culture 30 media strongly prevents the spontaneous differentiation of hES cells.

WO 03/104442 PCT/AU03/00713 29 In order to identify the effects of SIP and PDGF on hES cells, we carried out experiments in which we forced the cells to differentiate, by 1) mechanically dissociating them before plating and 2) growing them in the absence of MEF and serum. SIP or/and PDGF were added to the culture 5 medium and their effects on differentiation and proliferation were quantified by immunostaining the cells with PCNA, a marker of proliferation, and GCTM-2 (Figure 3). After 4 days in medium without serum, most of the control cells were differentiated, with only 30.8 ± 7.7 % (n=13) of GCTM2+ cells (Figure 3A). By contrast, when either SIP (10 pM) or PDGF (10 ng/ml) 10 was added to the medium, 47.9 ± 3.8 % (n=13) or 53.7 ± 13.2 % (n=3) of the cells respectively were GCTM2+, and 53.7 ± 3.5 % (n=3) of the cells were GCTM2+ in presence of both S1P and PDGF. Within the hES cell population, a large proportion expressed PCNA, showing that the majority of these stem cells still proliferated (Figure 3B). However, there was no statistically 15 significant difference in the proliferating rate of hES cells between the control cells and the ones treated with either SIP or/and PDGF (Figure 3B). Altogether, these data suggest that SIP and PDGF mostly act on the differentiation of hES cells grown in the absence of serum rather then on the proliferating state of hES cells. Moreover, because the hES cells were 20 cultivated in absence of MEF, these experiments clearly show that S1 P and PDGF are able to directly target the hES cells. We next investigated the effect of dihydrosphingosine-1-phosphate (dihydro-SIP, 10 pM), an SIP analogue that can only mimics the receptor dependent effects of SIP. By measuring the GCTM2 levels of the cells, we 25 showed that the effect seen in presence of S1 P and PDGF was mimicked by dihydro-S1P and PDGF (125.7 ± 9.7 % of control (n=3)), demonstrating that SIP's effect is receptor-dependent (Figure 2F). We then investigated which WO 03/104442 PCT/AU03/00713 30 isoform of PDGF was the most potent in inhibiting the spontaneous differentiation of hES cells. When added with SIP, the isoform BB was the most potent (182.0 ± 26.0 % of control (n=2)), followed by AB (125.7 ± 9.7 % of control (n=3)), while AA elicited little effect (120.5 ± 4.5 % of control (n=2)) 5 (Figure 2F). Passaging The hES cells have successfully been passaged through at least 18 passages in PDGF and SIP, with no serum. After passage 13 the cells have stained positive for the stem cell markers GCTM-2, Oct-4 and TG30. After 10 passage 7 the cells expressed mRNA for SPKI and SPK2 showing the probable expression of the enzymes as well as the stem cell markers Oct-4, and Crypto. After passage 8: karyotyping of hES cells - is being carried out to show that these cells when cultured in serum free conditions with PDGF and S1 P have maintained a normal karyotype. 15 DISCUSSION Since hES cells spontaneously differentiate in culture, a phenomenon that leads to a loss of their pluripotency, the identification of the compounds that are able to prevent this differentiation is of particular interest. In this study, we describe for the first time that hES cells are targets of S1 P, LPA 20 and PDGF. As revealed by RT-PCR analysis, these cells express the mRNA for the receptors SIP 1 , S1P 2 , S1P 3 , LPA 1 , LPA 2 and LPA 3 .. Referring to studies performed in rodent or in human, these receptors are widely expressed in the body (for reviews see 9'10). The absence of expression of S1P 4 and S1P 5 in 25 these cells is in accordance with the fact that these receptors seem to be mostly expressed in highly differentiated tissues, such as lymphoid tissue for S1P 4 and in brain's white matter for SIP 5 1. Moreover, hES cells express WO 03/104442 PCT/AU03/00713 31 the PDGF-receptors a and P, as revealed by RT-PCR and immunostaining. In hES cells, the addition of both PDGF and SIP inhibit very strongly the spontaneous differentiation, suggesting that these two molecules do cross talk. These combined effects could be attributed to the fact that 1) PDGF 5 stimulates the formation of intracellular SIP which would then act as a second messenger, for instance in the regulation of calcium homeostasis 13 and in the suppression of apoptosis, as shown in fibroblasts 4 and other cell types 15,16, but up to now the intracellular targets of SIP remain unclear; 2) SIP acts extracellularly through its receptors, and thus activates different 10 intracellular signalling pathways, such as the MAP kinases, involved in cell proliferation. The presence of both intracellular and extracellular SIP might then lead to a stronger inhibition of differentiation than the ones observed in presence of either S1P or PDGF. Also reported is a new cross link between PDGF and S1 P signals, in which both molecules need to be present. Such a 15 mechanism has recently been described for the first time by Katsuma et at (2002) 7 in mesangial cells. As shown by others, S1P, LPA and PDGF receptors are expressed in MEF 7 and these molecules are able to regulate multiple signalling pathways. Thus, Ishii et a! (2001) demonstrated that in these cells, SIP activates 20 phospholipase C, inhibits the production of cAMP and activates Rho 7. In MEF, PDGF stimulates migration. The effect observed in presence of PDGF and S1 P on hES cells might be in part due to an effect through the MEF. SIP, LPA and PDGF are all present in serum from different species, including bovine and human. However, the concentration of these molecules 25 varies from one species to another. Thus, it is believed that this could explain the commonly observed phenomenon with current cell culturing techniques where there is not only species dependant variation in the performance of WO 03/104442 PCT/AU03/00713 32 serum used to supplement cell culture systems but also intra-species batch to batch variations as well. Altogether, these data suggest that within the lipids and the proteins present into the serum, both SIP and PDGF are key elements in the 5 regulation of spontaneous differentiation of hES cells. Identification of compounds having an ability to inhibit differentiation. allows the design of simple culture media more suitable for hES cell propagation. Moreover, in a therapeutic view, it is important to determine compounds that allow cultivation of hES cells in a serum-free environment. 10 EXAMPLE 2 Cell culture. hES-3 cells were cultured as previously described 1 The serum-free culture medium consisted of DMEM (without sodium pyruvate, glucose 4500 mg/I, Invitrogen, Mount Waverley, VIC, Australia) supplemented with 15 insulin/transferrin/selenium 1%, p-mercaptoethanol 0.1 mM, NEAA I %, glutamine 2 mM, Hepes 25 mM, penicillin 50 U/ml and streptomycin 50 mg/ml (all from Invitrogen). Media was changed every 2 days and cells were passaged every week. SIP and dihydro-S1P were obtained from Biomol (Plymouth Meeting, PA, USA). LPA was obtained from Sigma (Castle Hill, 20 NSW, Australia). Extemporaneous dilutions of all lipids were made in water containing 0.1 % fatty acid-free bovine serum albumin (BSA) (Sigma). SIP and dihydro-S1P were used at 10 mM. Human PDGF-AB, PDGF-AA, PDGF BB were from PreproTech Inc. (Rocky Hill, NJ, USA) and were used at 20 ng/ml. 25 RT-PCR experiments.

WO 03/104442 PCT/AU03/00713 33 Total RNA was extracted from hES cells and reverse transcribed (RT) as previously described '.The cDNA samples were amplified by PCR with sense and antisense primers (Sigma) designed for the specific detection of mouse (data not shown) or human DNA target sequences (Table 1) using 5 Taq DNA polymerase (Biotech International Ltd, Perth, WA, Australia) as previously described 18. The specific amplified DNA fragments were sized by electrophoresis on 1.5 % (w/v) agarose gel and stained with ethidium. Molecular sizes (bp) were calculated using 1 kb plus DNA ladder markers (M). The amplicons were purified and sequenced. Experiments were 10 performed on hES-2 and hES-3. Immunofluorescence. Cells were fixed in paraformaldehyde 4% (for PDGFR staining) or 100 % ethanol and immunostained as previously described 1 using the following antibodies: anti-human PDGFR-a or PDGFR-P (R&D Systems Inc., 15 Minneapolis, MN, USA), GCTM-2 (this laboratory), TRA-1-60 (gift from P. Andrews, University of Sheffield), Oct-4 (Santa Cruz, CA, USA), TG-30 (this laboratory). Nuclei were counter-stained with Hoechst-33342 (Chemicon). Specificity was verified by the absence of any staining in the negative controls. 20 Sphingosine kinase activity. hES-3 cells plated without MEF for 24 hr and depleted of serum for a further 18 hr were incubated in the presence of PDGF (20 ng/ml) for various time periods and were harvested and lysed by sonication (2 W for 30 s at 40C) in lysis buffer containing 50 mM Tris/HCI (pH 7.4), 10% glycerol, 0.05% 25 Triton X-100, 150 mM NaCl, 1 mM dithiothreitol, 2 mM Na 3

VO

4 , 10 mM NaF, 1 mM EDTA and protease inhibitors (Complete

TM

, Roche, Mannheim, WO 03/104442 PCT/AU03/00713 34 Germany). SPK activity was determined using D-erythro-sphingosine and [a 32 P]ATP as substrates, as previously described 19. Protein concentrations in cell homogenates were determined with Coomassie Brilliant Blue reagent (Bio-Rad, Regent Park, NSW, Australia) using bovine serum albumin as 5 standard. GCTM-2 quantification. Cells were fixed in 100 % ethanol and immunostained with GCTM-2 followed by alkaline phosphatase-coupled secondary antibodies (Dako). Alkaline phosphatase activity was detected by adding a solution of 4 10 nitrophenyl phosphate (Roche), and the concentration of the reaction product was determined by reading the optical density (OD) at 405 nm. In order to validate the technique as an accurate indicator of the proportion of GCTM-2 positive cells, standard curves were carried out with the embryonal carcinoma cell line GCT27C4, known to express GCTM-2 20. This showed a 15 linear correlation between the number of cells and the OD read at 405 nm (data not shown). Neuronal induction of hES cells. hES-3 cells (passages 11, 13-15) were differentiated into noggin cells by a noggin treatment then into neurospheres and last into neurons as 20 previously described in PCT/AU01/00735. Statistical analysis. All experiments were performed at least 3 times. Data are expressed as the mean ± SEM of at least 3 independent experiments. Significance of the differences was evaluated using an ANOVA followed by Student 25 Newman Keuls test. Values of P < 0.05 were considered significant (*).

WO 03/104442 PCT/AU03/00713 35 Results hES cells expressed mRNA transcripts for three SIP receptors: SIP 1 , S1P 2 and S1P 3 and for each of the LPA receptors (Fig. 4A-B). However these cells did not express mRNA for SIP 4 and SIP 5 . Contrary to mouse 5 embryonic stem cells, hES cells expressed mRNA transcripts for PDGFR-a and PDGFR-P (Fig. 4C) as well as the corresponding proteins, as revealed by immunostaining (Fig. 4E-J). As previously shown by others 6-8,18,19 we show that MEF expressed S1P 1 , S1P 2 , S1P 3 , LPA 1 , LPA 2 , PDGFR-a and PDGFR-p. Thus in a co-culture system, S1P, LPA and PDGF could be active 10 on either cell type. We next examined whether S1 P, LPA and PDGF could affect growth or differentiation of hES cells. When hES cells were grown on MEF in a serum free culture medium, they spontaneously differentiated into different kinds of cells. After 2 weeks in a serum-free media, LPA (up to 50 pM) had no 15 obvious effect on size or morphology of hES cell colonies whilst in the presence of either S1P (10 pM) or PDGF-AB (PDGF, 20 ng/ml), the colonies appeared flatter and less differentiated as compared to the controls. Moreover, the co-incubation of S1 P and PDGF induced a strong inhibition of spontaneous differentiation. To quantify these effects, we used an ELISA 20 based assay to measure expression of the stem cell surface antigen GCMT-2 (GCTM2+) in cells treated for 2 weeks with different agonists. Thus, cells treated with S1P or PDGF were respectively 18.0 ± 17.0 % (n=3) and 50.3 + 18.4 % (n=3) more GCMT2+ than the controls and the ones treated with both S1P and PDGF were 152.7 ± 54.9 % (n=3) more GCTM2+ than the controls 25 (Fig. 5A). Using the same technique, we showed that cells treated with S1P and either PDGF-AA or PDGF-BB showed a GCTM2 expression similar to the one observed with S1P and PDGF (PDGF-BB: 294.3 ± 77.3 % of control, n=3, PDGF-AA: 220.3 ± 49.0 % of control, n=3; Fig. 5A). Moreover, the effect WO 03/104442 PCT/AU03/00713 36 of SIP in combination with PDGF was mimicked by the use of dihydrosphingosine-1-phosphate (dihydro-SIP, 10 mM), a S1P analogue that mimics its receptor-dependent effects, in combination with PDGF (227.0 ± 59.9 % of control (n=3), Fig. 5A). Furthermore, dihydro-SI P on its own had a 5 more potent effect on hES cells than SIP (223.0 ± 27.0 % of control, n=3; Fig. 5A). Together, these results suggest that the combination of PDGF and S1P in a serum-free culture medium prevents the spontaneous differentiation of hES cells. This effect is dependent upon SIP's receptors and both PDGFRs, as PDGF-AA only binds to PDGFR-a while PDGF-AB and PDGF 10 BB bind to both receptors. Moreover, treatment of hES cells with the MAP kinase kinase inhibitor U0126 (Promega, 10 mM) for 7 days, totally inhibited the effect of PDGF and SIP on GCTM2 expression (Fig. 5B), strongly suggesting that the activation of the extracellular signal-regulated kinases is required to maintain hES cells undifferentiated. As SPK is a key molecule in 15 PDGF signalling pathways, we verified the presence of SPK transcripts in hES cells and showed expression of both SPK-1 and SPK-2 mRNA (Fig.4D). We next investigated if PDGF modulates SPK activity in hES cells (Fig. 5D). PDGF (20 ng/ml) enhanced in a time-dependent manner the SPK activity in hES cells (Fig. 5D). This effect lasted for at least 60 min and SPK activity 20 reached 1.6 fold the basal values (75.3 ± 3.92 nmol/min/mg, n=3) after 30 min of incubation (Fig. 5D). In contrast, PDGF (20 ng/ml) did not induce a significant statistical activation of SPK in MEF. Moreover, treatment of hES cells with dimethylsphingosine (DMS, 3 pLM, Fig. 5C), a non-specific inhibitor of SPK, for 7 days, inhibited the effect of PDGF and SIP, suggesting an 25 involvement of SPK in the maintenance of hES in an undifferentiated state. To date, hES cells have been grown in a serum-free medium supplemented with S1P (10 pM) and PDGF (20 ng/ml) for 19 passages. As WO 03/104442 PCT/AU03/00713 37 these cells still express SPK-I and SPK-2 mRNA (Fig. 6B), we can expect the PDGF-activation of SPK to be involved in the propagation of hES cells. RT-PCR studies showed that hES cells expressed the mRNA for Oct-4 and cripto (Fig. 6B), and immunostaining showed immunoreactivity to the stem 5 cell markers GCTM-2, Oct-4, TG-30 and Tra-1-60 (Fig. 6C-F). These hES cells retained a normal karyotype (Fig. 6G). Moreover, these HES cells still responded to noggin treatment and were able to form neurospheres (Fig. 6H) and neuronal cells as ascertained by immunostaining for ptubulin (Fig. 61), Map2, nestin, synaptophysin, N-cam and NF200 (Pera et al submitted). 10 Altogether, these data demonstrate that HES cells grown in the presence of SIP and PDGF retain the characteristics of HES cells propagated in normal serum conditions. Discussion In this study, we show for the first time that hES cells are targets of 15 SIP, LPA and PDGF and we also show an interaction between SIP and PDGF signal, in that extracellular SIP and PDGF need to be present together to exert a potent biological effect. Katsuma et al. (2002) 17 reported a similar mechanism in mesangial cells where application of S1P and PDGF increases proliferation. In hES cells the addition of both SIP and PDGF 20 maintains these cells in the undifferentiated state, and still allows them to follow differentiation. These combined effects could be attributed to the fact that (i) SIP acts extracellularly through its receptors to modulate intracellular signalling pathways; (ii) and that PDGF stimulates the formation of intracellular SIP which would either be secreted or act as an intracellular 25 messenger, for instance in the regulation of calcium homeostasis 13 and in the suppression of apoptosis, as shown in fibroblasts 14 and other cell types 15,16. Whether SIP is secreted or acts as a second messenger needs to be further investigated. However, because the maintenance of hES cells in an WO 03/104442 PCT/AU03/00713 38 undifferentiated state only occurs in the presence of both PDGF and SI P, we could expect that intracellular SIP, produced in response to PDGF, acts within the cells, as its cell-surface receptors are likely to have already been engaged by SIP previously added to the culture media. To our knowledge, 5 this study is the first one to report a cross-talk involving SIP and two isoforms of PDGFR, instead of only PDGFR-p. These data demonstrate that SIP and PDGF are key elements in the regulation of spontaneous differentiation of hES cells. Their identification as compounds having an ability to inhibit differentiation allows the design of a simple serum-free 10 culture medium more suitable for hES cell propagation. The following materials and methods relate to Examples 3 to 5. Reagents SIP AND LPA were obtained from Biomol (Plymouth Meeting, PA, USA) and were dissolved in methanol. Freshly prepared dilutions of agonists 15 were made in water containing 0.1% fatty acid-free bovine serum albumin (BSA) (Sigma). Protease, sodium orthovanadate and U0126 were from Sigma. was from Calbiochem (San Diego, CA, USA). Pertussis Toxin (PTX) was from List Biological Laboratories (Campbell, CA, USA). GCTM-2, Oct-4, PCNA, Hoechst-33342 20 Cell culture hES-3 cells were cultured as previously described [1]. Human stem cells were grown on MMC treated fibroblasts' feeder layer. Fibroblasts were plated on gelatine treated dishes. A combination of human and mouse 25 derived fibroblasts were used at a density of approximately 25,000 and 70,000 cells per cm2 respectively. The fibroblasts were plated up to 48 hours before culture of the stem cells. Mouse fibroblasts only could also support the growth of the stem cells. However, while human fibroblasts could also support stem cells, they created an uneven and unstable feeder layer. 30 Therefore, the human fibroblasts were combined with the mouse fibroblasts WO 03/104442 PCT/AU03/00713 39 to augment and achieve better support of growth and prevention of differentiation. The medium that was used for the growth of human stem was DMEM 5 (GIBCO, without sodium pyruvate, with glucose 4500mg/L) supplemented with 20% FBS (Hyclone, Utah) (2-mercaptoethanol - 0.1mM (GIBCO), Non Essential Amino Acids - NEAA 1% (GIBCO), glutamine 2mM.(GIBCO), penicillin 50u/ml, and streptomycin 50mg/ml (GIBCO) 10 For direct observation, hES-3 cells were coated into 12-well plates (3 colonies per well), with or without mouse embryonic feeders (MEFs). The day following the plating, cells were incubated with the different agents in serum free medium containing insulin, transferring and selenium. Media was changed the 2 nd day and then every 2 days. 15 For immunostaining, hES-3 cells were coated on chamber slides after mechanical dissociation, in order to obtain a monolayer culture. The day following the plating, cells were incubated with the different agents in a media depleted in serum. Media was changed the 2 nd day and the cells were 20 fixed 4 days after the first treatment. For immunoblot analysis, cells were transferred into 24 well plates (8 colonies per well) without MEFs, and 24 hr later, were grown in the absence of serum for 18 hrs. 25 In some experiments, cells were pre-treated for 1 hr with U0126 (30 tM) or for 18 hrs with PTX (100 pg/ml). RT-PCR experiments 30 Cells were washed with PBS and hES colonies were removed by treatment with protease. Purified mRNA was extracted from hES cultures using Dynabeads* Oligo (dT) 2 5 (Dynal, Oslo, Norway), according to the WO 03/104442 PCT/AU03/00713 40 supplier's instruction. RT was performed using superscript TM 11 Rnase H Reverse Transcriptase (Invitrogen, Life technologies), according to the supplier's protocol. After cooling on ice, the cDNA samples were amplified by PCR with sense and antigens primers (synthesis performed by Sigma 5 Genosys, Castle Hill, Australia) designed for the specific detection of human Edg-1, Edg-2, Edg-3, Edg-4, Edg-5, Edg-6, Edg-7 and Edg-8 DNA target sequences. The primers used for Edg-1, Edg-3, Edg-5, Edg-6 and Edg-8 were previously designed by Hornu et al. (2001) [1]. These primer pairs were 10 5'-CCACAACGGGAGCAATAACT-3' (sense) and 5'-GTAAATGATGGGGTTGGTGC-3' (antigens) (expected PCR product: 480 bp) for Edg-1 ; 5'-TCAGGGAGGGCAGTATGTTC-3' (sense) and 15 5'-CTGAGCCTTGAAGAGGATGG-3' (antisense) (505 bp) for Edg-3; 5'-CCAATACCTTGCTCTCTCTGGC-3' (sense) and 5'-CAGAAGGAGGATGCTGAAGG-3' (antisense) (502 bp) for Edg-5; 20 5'-CGGCTCATTGTTCTGCACTA-3' (sense) and 5'-GATCATCAGCACCGTCTTCA-3' (antisense) (701 bp) for Edg-6; 5'-TTCTGATACCAGAGTCCGGG-3' (sense) and 5'-CAAGGCCTACGTGCTCTTCT-3' (antisense) (460 bp) for Edg-8. 25 For Edg-2 and Edg-4, the primer pairs designed by Goetzl et al. (1999) were used: 5'-GCTCCACACACGGATGAGCAACC-3' (sense) and 5'-GTGGTCATTGCTGTGAACTCCAGC-3' (antisense) (621 bp) for Edg 30 2, 5'-AGCTGCACAGCCGCCTGCCCCGT-3' (sense) and 5'-TGCTGTGCCATGCCAGACCTTGTC-3' (antisense) (775 bp) for Edg-4.

WO 03/104442 PCT/AU03/00713 41 For Edg-7, the primer pairs designed by Goetlz et al. (2000) were used: 5'-CCATAGCAACCTGACCAAAAAGAG-3' (sense) and 5'-TCCTTGTAGGAGTAGATGATGGGG-3' (antisense) (482 bp). 5 PCR experiments were performed in a mixture (25 pl) containing 0.25 units of Taq DNA polymerase (Biotech International Ltd, Perth, WA, Australia) and 2 pM of each primer in a buffer including 67 mM Tris-HCI, pH 8.8, 1.5 mM MgCl2, 16.6 mM [NH 4

]

2

SO

4 , 0.45% Triton X-100, 0.25 mM of 10 each dATP, dGTP, dCTP, dTTP. Absence of contaminating genomic DNA was confirmed by control reactions with mRNA that had not been treated with reverse transcriptase. PCR experiments were run with the following steps: initial denaturation at 94*C for 5 min, 35 cycles of denaturation at 94"C for 30 sec, annealing at 520C (Edg-1, Edg-3, Edg-5, Edg-6, Edg-8) or 560C 15 (Edg-2, Edg-4, Edg-7) for 2 min, extension at 740C for 2 min, and a final extension at 740C for 7 min. The specific amplified DNA fragments were purified by electrophoresis on 1.5 % (w/v) agarose gel, stained with ethidium bromide and photographed. The amplicons were purified and sequenced. 20 Immunofluorescence Cells were washed in PBS, fixed with MeOH, and immunostaining was performed, using the specific stem cell marker antibody GCTM-2, and the specific cell proliferation marker PCNA. Cells were then washed and the nucleuses were stained with Hoechst-33342 (1 pg/ml). Slides were mounted 25 and then observed by fluorescent microscopy. Cells were then counted in order to determine the ratio of proliferating stem cells within the overall population. Western blot analysis 30 hES3 cells were lysed following removal of the supernatants by addition of a reducing loading buffer (2% SDS, 62.5 mM Tris pH 6.8, 0.1 M DTT, 0.01% bromophenol blue) containing 1 mM sodium orthovanadate.

WO 03/104442 PCT/AU03/00713 42 Samples were boiled for 10 min and centrifuged at 13000g for 15 min, and protein lysates (approx. 80 pg) were separated by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide, w/v). Proteins were transferred to nitrocellulose (Hybond-ECL, Amersham) and immunoblotting was carried out 5 with rabbit polyclonal anti-active mitogen-activated protein (MAPK) antibodies raised against a dually phosphorylated MAPK peptide (Promega, Madison, WI, USA). Peroxidase-coupled secondary antibody (Dako) was detected by exposure of autoradiographic films in the presence of a chemiluminescent detection reagent (ECL, Amersham). Stripping of 10 antibodies was achieved by incubating the membrane during 30 min at 500C in a buffer containing 100 mM mercaptoethanol, 2% SDS, 62.5 mM Tris-HCI pH 6.7. The membrane was then reprobing with rabbit polyclonal anti ERKI/2 antibodies (Promega), and then with peroxidase-coupled secondary antibodies (Dako). 15 Blots probed with either rabbit polyclonal anti-active p38 (Promega) or rabbit polyclonal anti-active JNK (Promega) or mouse polyclonal GCTM-2 antibodies were also performed, using the same procedure as described above. 20 Protein quantification hES3 cells were lysed and their quantity was determined by using a colorimetric assay based on the Bradford dye-binding test (Bio-Rad Laboratories, Regents Park, NSW, Australia). Each set of experiments was performed at least 3 times (n refers to 25 number of independent experiments performed on different cell cultures). EXAMPLE 3 The results presented in Figure 7A indicate that hES cells expressed mRNA transcripts for the three SIP receptors : Edg-1, Edg-3 and Edg-5 30 while these cells do not seem to express mRNA for Edg-6 and Edg-8 (data not shown). Moreover, hES cells express mRNA transcripts for each of LPA receptors : Edg-2, Edg-4 and Edg-7 (Figure 7B). The nucleotide sequences WO 03/104442 PCT/AU03/00713 43 of all purified PCR products were analysed and revealed to be identical to the corresponding regions in the human receptor genes. EXAMPLE 4 5 Applicants next determined whether SIP could modulate the fate of hES cells. When hES cells were grown on MEFs, in a culture media depleted in serum, they spontaneously differentiated. As shown in Figure 8, after 8 days in such conditions, hES cells colonies contained enlarged flattened cells which formed cystic structures (Figure 2A, 2B). Even after 12 days, LPA (up 10 to 50 pIM) did not seem to affect the growth of the colonies (data not shown). In presence of SIP (10 pM, 8 days), the colonies were more compact and less differentiated than in the control condition (Figure 8C). This effect of S1 P was more obvious after 12 days of treatment (Figure 8D, 8E). The inhibitory effect of SIP on cell differentiation and the lack of effect of LPA were also 15 observed when hES cells were grown without MEFs, suggesting that S1P did not directly act on the feeder cells (n=3, data not shown). In order to understand and quantify the effect of SIP on the spontaneous differentiation of hES cells, double immunostaining experiments were carried out. For that purpose, Applicants used two specific antibodies, 20 one as a stem cell marker, GCTM-2, and one for proliferation, PCNA, a marker that is only expressed during the S phase of the cell cycle, in order to determine the ratio of proliferating stem cells (Figure 9). After 4 days in a media without serum, most of the control cells were differentiated (Figures 9A, 9C and 9E), as revealed by the fact that only 16 % of the cells still 25 expressed GCTM-2 (Figure 10A). By contrast, when SIP (10 pM) was added to the media, 68 % of the cells were GCTM-2 positive, suggesting that most of the cells remained stem cells (Figures 9B, 9D, 9F and 10B). Within these cell populations, a large part expressed PCNA, suggesting that most of these stem cells still proliferated (Figures 9G and 9H). However, no marked 30 difference in the proliferating rate of hES cells between the control cells and the ones treated with SI P were observed (Figure 10). Altogether, these data WO 03/104442 PCT/AU03/00713 44 suggest that SIP mostly acts on the differentiation of hES cells observed in absence of serum rather then acts on the proliferating state of hES cells. EXAMPLE 5 5 Because the MAP kinases ERKs have often been implicated in cell proliferation and differentiation, the effects of SIP on the activation of the ERKs were then investigated. After 5 min, SIP stimulated the phosphorylation of ERKs in hES cells (Figure 10), an effect that was totally inhibited in presence of the MEK inhibitor U0126 (30 pM) (Figure 10A). SIP 10 stimulated ERKs for at least 60 min and in a concentration dependant manner (Figure 1OB, 10C). These results show clearly that treatment of human ES cells with SI P results in inhibition of spontaneous differentiation. S1P is a major component of serum, and is therefore likely to account for much of the beneficial effect of 15 calf serum in human ES cultures. Although human ES cells express receptors for both SIP and LPA, the latter lysophospholipid is inactive on human ES cells. This suggests that particular members of the Edg receptor family have distinct effects on human ES cell behaviour.

WO 03/104442 PCT/AU03/00713 45 REFERENCES 1. Reubinoff, B. E., Pera, M. F., Fong, C. Y., Trounson, A. & Bongso, A. Embryonic stem cell lines from human blastocysts: somatic 5 differentiation in vitro. Nat Biotechnol 18, 399-404 (2000). 2. Hornuss, C., Hammermann, R., Fuhrmann, M., Juergens, U. R. & Racke, K. Human and rat alveolar macrophages express multiple EDG receptors. Eur J Pharmacol 429, 303-8 (2001). 3. Goetzl, E. J., Dolezalova, H., Kong, Y. & Zeng, L. Dual mechanisms 10 for lysophospholipid induction of proliferation of human breast carcinoma cells. Cancer Res 59, 4732-7 (1999). 4. Basciani, S. et al. Expression of platelet-derived growth factor-A (PDGF-A), PDGF-B, and PDGF receptor-alpha and -beta during human testicular development and disease. J Clin Endocrinol Metab 15 87, 2310-9 (2002). 5. van Eijk, M. J. et al. Molecular cloning, genetic mapping, and developmental expression of bovine POU5F1. Biol Reprod 60, 1093 103 (1999). 6. Rosenfeldt, H. M., Hobson, J. P., Milstien, S. & Spiegel, S. The 20 sphingosine-1-phosphate receptor EDG-1 is essential for platelet derived growth factor-induced cell motility. Biochem Soc Trans 29, 836-9 (2001). 7. Ishii, I. et al. Selective loss of sphingosine 1-phosphate signaling with no obvious phenotypic abnormality in mice lacking its G protein 25 coupled receptor, LP(B3)/EDG-3. J Biol Chem 276, 33697-704 (2001). 8. Heldin, C. H. & Westermark, B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev 79, 1283-316 (1999). 9. Takuwa, Y., Takuwa, N. & Sugimoto, N. The edg family g protein coupled receptors for lysophospholipids: their signaling properties and 30 biological activities. J Biochem (Tokyo) 131, 767-71 (2002). 10. Chun, J. et al. International Union of Pharmacology. XXXIV. Lysophospholipid Receptor Nomenclature. Pharmacol Rev 54, 265-9 (2002). 11. Graler, M. H., Bernhardt, G. & Lipp, M. EDG6, a novel G-protein 35 coupled receptor related to receptors for bioactive lysophospholipids, WO 03/104442 PCT/AU03/00713 46 is specifically expressed in lymphoid tissue. Genomics 53, 164-9 (1998). 12. Im, D. S. et al. Characterization of a novel sphingosine 1-phosphate receptor, Edg-8. J Biol Chem 275, 14281-6 (2000). 5 13. Mattie, M., Brooker, G. & Spiegel, S. Sphingosine-1-phosphate, a putative second messenger, mobilizes calcium from internal stores via an inositol trisphosphate-independent pathway. J Biol Chem 269, 3181-8 (1994). 14. Cuvillier, 0. et al. Suppression of ceramide-mediated programmed cell 10 death by sphingosine-1 -phosphate. Nature 381, 800-3 (1996). 15. Van Brocklyn, J. R. et al. Dual actions of sphingosine-1-phosphate: extracellular through the Gi-coupled receptor Edg-1 and intracellular to regulate proliferation and survival. J Cell Biol 142, 229-40 (1998). 16. Olivera, A. et al. Sphingosine kinase expression increases intracellular 15 sphingosine-1-phosphate and promotes cell growth and survival. J Cell Biol 147, 545-58 (1999). 17. Katsuma, S. et al. Signalling mechanisms in sphingosine 1-phosphate promoted mesangial cell proliferation. Genes Cells 7, 1217-30 (2002). 18. Pebay, A. et al. Sphingosine-1-phosphate induces proliferation of 20 astrocytes: regulation by intracellular signalling cascades. Eur J Neurosci13, 2067-76 (2001). 19. Pitson, S. M. et al. Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes. 25 Biochem J 350 Pt 2, 429-41 (2000). 20. Andrews, P. W. et al. Comparative analysis of cell surface antigens expressed by cell lines derived from human germ cell tumours. Int J Cancer 66, 806-16 (1996). 30 21. Baron, V. & Schwartz, M. Cell adhesion regulates ubiquitin-mediated degradation of the platelet-derived growth factor receptor beta. J Biol Chem 275, 39318-23 (2000). 22. Kluk, M. J., Colmont, C., Wu, M. T. & Hla, T. Platelet-derived growth factor (PDGF)-induced chemotaxis does not require the G protein 35 coupled receptor SI P(1) in murine embryonic fibroblasts and vascular smooth muscle cells. FEBS Lett 533, 25-8 (2003).

Claims (24)

1. A method for modulating spontaneous differentiation of an embryonic (ES) stem cell, which method comprises incubating the ES cell in the 5 presence of an agonist of a LPL receptor, wherein the agonist is selected from the group consisting of SIP, dihydro S1P, LPA, PAF and SPC or functional equivalents thereof.

2. A method for modulating spontaneous differentiation of an ES cell, which method comprises incubating the ES cell in the presence of an agonist 10 of the LPL receptor and a ligand of a class Ill tyrosine kinase receptor, wherein the agonist is selected from the group consisting of S1P, dihydro S1 P, LPA, PAF and SPC or functional equivalents thereof.

3. A method according to claim 1 or 2 wherein the modulation is inhibition of differentiation. 15

4. A method according to any one of claims 1 to 3 wherein the LPL receptor is selected from the group consisting of S1 P1, S1 P2, and S1 P3.

5. A method according to any one of claims 2 to 4 wherein the tyrosine kinase receptor is PDGFR-a or PDGFR-p.

6. A method according to any one of claims 2 to 5 wherein the ligand is a 20 PDGF or functional equivalent thereof and wherein the PDGF is selected from the group comprising PDGFaa, PDGFab or PDGFbb.

7. A method according to any one of claims 1 to 6 comprising use of TNF alpha, NGF (nerve growth factor), a muscarinic acetylcholine agonist, or a serum or phorbol ester. 25

8. A method according to claims 1 to 7 wherein the ES cell is a hES cell.

9. A serum free or substantially serum free media for modulating spontaneous differentiation of an ES cell, comprising an agonist of a LPL receptor and a ligand of a class Ill tyrosine kinase receptor, wherein the agonist is selected from the group consisting of S1 P, dihydro S1 P, LPA, PAF 30 and SPC or functional equivalents thereof.

10. A media according to claim 9 wherein the modulation is inhibition of differentiation. 48

11. A media according to claim 9 or 10 wherein the LPL receptor is selected from the group consisting of S1 P1, S1 P2, and S1 P3.

12. A media according to any one of claims 9 to 11 wherein the tyrosine kinase receptor is PDGFR-a or PDGFR-p. 5

13. A media according to any one of claims 9 to 12 wherein the ligand is a PDGF or functional equivalent thereof and wherein the PDGF is selected from the group comprising PDGFaa, PDGFab or PDGFbb.

14. A media according to any one of claims 9 to 13 comprising TNF alpha, NGF (nerve growth factor), a muscarinic acetylcholine agonist, or a serum or 10 phorbol ester.

15. A media according to any one of claims 9 to 14 wherein the ES cell is a hES cell.

16. A media according to claim 9 to 15 wherein the media is based on DMEM supplemented with insulin, transferrin and selenium. 15

17. A media according to any one of claims 9 to 16 wherein the agonist is S1P and is present in the media at a concentration of from 0.1 pM to 10pM.

18. A media according to any one of claims 9 to 17 wherein the ligand is present in the media at a concentration of from 1 ng/ml to 20ng/ml where the ligand is either PDGFaa, PDGFab or PDGFbb. 20

19. A serum free or substantially serum free media when used for modulating spontaneous differentiation of an ES cell, comprising an agonist of the LPL receptor, wherein the agonist is selected from the group consisting of PAF and SPC.

20. A method of producing a population of proliferating undifferentiated ES 25 cells from an ES cell which method comprises incubating the ES cell in the presence of an agonist of the LPL receptor, wherein the agonist is selected from the group consisting of S1P, dihydro S1P, LPA, PAF and SPC or functional equivalents thereof.

21. A method of producing a population of proliferating undifferentiated ES 30 cells from an ES cell which method comprises incubating the ES cell in the presence of an agonist of a LPL receptor and a ligand of a class Ill tyrosine kinase receptor, wherein the agonist is selected from the group consisting of S1 P, dihydro S1P, LPA, PAF and SPC or functional equivalents thereof. 49

22. A method according to claim 1 or 2 substantially as hereinbefore described with reference to the examples.

23. A media according to claim 9 or 19 substantially as hereinbefore described with reference to the examples. 5

24. A method according to claim 20 to 21 substantially as hereinbefore described with reference to the examples.