WO2006084314A1 - Stem cell populations and classification system - Google Patents

Abstract

A method of identifying, isolating, and classifying closely related populations of stem cells grown under substantially identical conditions is disclosed. The methods utilize a binding agent such as an antibody which binds to cell surface markers such as GCTM-2 and CD9 which are quantitatively associated with the pluripotency of the cell. Based on these methods a classification system is disclosed whereby cells are ranked based on their ability to differentiate.

Description

STBM CELL POPULATIONS AND CLASSIFICATION SYSTEM FIELD OF THE INVENTION

This invention relates generally to methods of classifying cells and in particular human embryonic stem cells. Further, the invention relates to a classification system whereby cells are assigned α class based on the pluripotency of the cell.

BACKGROUND OF THE INVENTION

There are a large number of different types of cells. One general type is stem cells. However, there are several types of stem cells including mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs) and embryonic stem cells (ESCs). The ability to isolate specific sub-populations of celIs from cell suspensions is of critical importance to many applications in the biological sciences as well as to many therapies in clinical medicine. For example, the basis of many medical therapies for treating a variety of human diseases and for countering the effects of a variety of physiological injuries involves the isolation, manipulation, expansion, and/or alteration of specific biological cells. One particularly important example involves the reconstitution of the hematopoietic system via bone marrow or progenitor cell transplantation. More specific examples include: autologous, syngeneic, and allogenic stem cell transplants for immune system reconstilntion following (he myeloablalive effects of severe high dose chemotherapy or therapeutic irradiation: severe exposure to certain chemical agents; or severe exposure to environmental radiation, for example from nuclear weapons or accidents involving nuclear power generators,

Mesenchymal stem cells (MSCs) are the formative pluripotential blast cells found inter alia in bone marrow, blood, dermis and periosteum that are capable of differentiating into more than . one specific type of mesenchymal or connective tissue (i.e. the tissues of the body that support the specialized elements; e.g. adipose, osseous, stroma, cartilaginous, elastic and fibrous connective tissues) depending upon various influences from bioactive factors, such as cytokines. The potential to differentiate into cells such as osteoblasts and chondrocytes is retained after isolation and expansion in culture; differentiation occurs when the cells are induced in vitro under specific conditions or placed in vivo ar the site of damaged tissue.

Epitopes on the surface of the human mesenchymal stem cells (hMSCs) are reactive with certain monoclonal antibodies known as SH2, SH3 and SH4 described in U.S. Pat No. 5,486,359. These antibodies can be used as reagents to screen and capture the mesenchymal stem cell population from a heterogeneous cell population, such as exists, for example, in bone marrow.

Hematopoietic stem cells (HSCs) are the torraativti pluripotential blast cells found inter alia in bone marrow and peripheral blood that are capable of differentiating into any of the specific types of hematopoietic or blood cells, such as erythrocytes, lymphocytes, macrophages and megakaryocytes. The expression of a particular antigen or antigens on the cell surface or in the cytoplasm and the intensity of expression indicate the stage of maturation and lineage commitment, of the hematopoietic stem cell. Human hematopoietic stem cells (hIISCs) fare reactive with certain monoclonal antibodies, such as CD34, recognized as being specific for hematopoietic cells.

T hus, human hematopoietic stem cells and human mesenchymal stem cells have been readily distinguishable by their ϊmmunυspecific profiles.

These profiles are useful in both cell identification and isolation. At the present time it is not known how many of the markers associated with stern cells are- indicative of their potential to develop into various more mature cell types. One marker wliich has been indicated as present on stem cells, CD34, is also found on a significant number of lineage-committed progenitors. Another marker that provides for some emiehruent of progenitor activity is Class II HLA (particularly a conserved DR epitope recognized by a monoclonal antibody designated J I -43). However, these markers are found on numerous lineage-committed hematopoietic cells. Therefore, in vi<jw of the small proportion of the total number of cells in many cell populations which are stem cells, the uncertainly of the markers associated with stem cells, as distinct from more differentiated cells, and the general inability to conduct a definitive biological assay for human stem cells., the identification and purification of stem cells has been elusive.

The first six years of research on human embryonic stem cells (hESC) have led to significant advances in our ability to maintain and manipulate these fascinating cultured cell lines (Pcra, M.F., Reubinoff, B., and Trounson, A. (2000). Human embryonic stem cells. J Cell Sci 113 (Pt 1), 5-10; Pera, MF ., and Trounson, A.O- (2004). Human embryonic stem cells: prospects for development. Development 131, 5515-5525; Hoffman, L.M., and Carpenter, M.K. (2005). Characterization and culture of human embryonic stem cells. Nat Biotcchnol 23, 699-708). The initial reports of the derivation of pluripotent stem cells from the human blastocyst (Thomson, JA., Itskovilvs-EIdor, J,, Shapiro, S.S., Waknitz, MA., Swiergiel, JJ,, Marshall, V.S., and Jones, J.M. (1998). Embryonic stem cell lines derived from human blastocysts. Science 282, 1 145- 1 147; Reubinoff, B.E., Peru, M. R, Fong, C.Y., Trounson, A., and Bongso, A. (2000). Embryonic stem cell lines from human blastocysts; somatic differentiation in vitro. Nat Biotechnol 18, 399-404) have been abundantly confirmed, technology for the maintenance and manipulation of hI7.SC has been successfully disseminated around the world, there have been improvements to the culture system used in the first derivations, the differentiation in vitro of hESC into n variety of tissue types of enormous potential significance to research and medicine, including neural tissue, blood, cardiac muscle, and a wide range of other cell types, has been reported, and the firs I studies showing proof of principle of the. application of hESC- derivcd cells in preclinical animal models of disease have recently been published.

While this record is impressive, very significant challenges remain ahead if hESC are actually going to fulfill their potential. The reality is that even our basic understanding of the phenυtype of human pluripotent stem cells is limited. hESC are characterized by their immunological profile, by transcriptional analysis, and by biological assay of their capability for self-renewal and multilineage differentiation. Most work carried out on hESC has made the tacit assumption that the canonical hESC phenotype-a cell positive for specific surface antigens (SSEA-3, SSEA-4, TRA-1-60, CD9), expressing genes specific to pluripotent cells (Oct-4, nanog) and capable of indefinite renewal and differentiation into derivatives of all three embryonic germ layers-essctitially describes a single discrete cellular entity. However, the canonical description of the phenotype of the hESC in fact describes the properties of a. heterogeneous population of cells, some of which have embarked on the pathway to differentiation. Because of this, and because the early stages of hESC commitment and differentiation are largely uncharted, present studies at the cellular, molecular and biochemical level, which treat hESC cultures as a homogeneous population of cells, are capable of providing only limited insight into the control of stem cell renewal and differentiation. In particular, the numerous studies of the hESC transcriptome and proteome, (Wei, CL. , Mitira, T., Robson, P., Urn, S.K., Xu, X.Q., Lee, M.Y., Gupla, S., Stanton, U, Luo, Y., Schmitt, J-, Thics, S., Wang, W., Khrebtutcova, I., Zhou, D., Liu, ET. Ruan, YJ., Rao, M., and TJm, B. (2005). Transcriptome profiling of human and murine ESCs identifies divergent paths required to maintain UlC stem cell state. Stem Cells 23, 166-185; Xu, R.H., Chen, X., Li, D.S., Li, R., Addicks, G.C., Glennon, C₇ Zwaka, T.P., and Thomson, J.A. (2002). 1ΪMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat Biotechnol 20, 1261-1264; Rosier, -FJS-, Fisk, GJ., Ares, X., Irving, L, Miura, T., Rao, M.S., and Carpenter, M.K. (2004). Long- term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn 229, 259-274; Robson, P. (2004). The maturing of the human embryonic stem cell transcriptome profile. Trends Biotechnol 22, 609-612; Rao, R.R., Calhoun, J.D., Qin, X., Rekaya, R.. Clark, J.K., and Slice, S.L. (2(K)4). Comparative transcriptional profiling of two human embryonic stem cell lines. Biotechnol Bioeng 88, 273-286; .Gtuis, L₇ Luo, Y., Miura, T., Thies, S., Brandenberger, R., Gerecht-Nir, S., Amit, M., Hokβ, A., Carpenter, M.K., Itskovitz-Eldor, J., and Rao, M.S. (2004). Differences between human and mouse embryonic stem cells. Dev Biol 269, 360-380; Carpenter, M.K., Rosier, E.S., Fisk, GJ,, Brandenberger, R., Ares. X., Miura, T., Lucero, M., and Rao, M.S. (2004), Properties of four human embryonic stem cell lines maintained in a feeder-free culture system, Dev Dyn 229, 243-258; Brandenberger, R., Khrebtukova, 1., Thics, R.S., Miura, T., Jingli, C, Puri, R., Vasicek, T., Lebkowski, J ., and Rao, M. (2004). MPSS profiling of human embryonic stem cells. BMC Dev Biol 4, 10; Bhattacharya, B,, Qu, J., Luo, Y., Miura, T,, Mejido, J., Brimble, S.N., Zeng, X., Schulz, T.C., Rao, M.S., and Puri, R-K. (2005). Comparison of the gene expression profile of Undifferentiated human embryonic stem cell lines and differentiating embryoid bodies. BMC Dev Biol 5, 22; Bhallauharya, B., Miura, T., Brandenberg, R., Mcjidu, J... Luo, Y., Yang, A.X., Joshi, B.H,, Irene, G., Thies, R.S., Amit, M., Lyons, I., Condie, B.G., Iskovirv.-Eldor, J., Rao, M.S., and Puri, R.K. (2003), Gene Expression in Human Embryonic Stem Cell Lines: Unique Molecular Signature. Blood; 133Spergcr, J.M., Chen, X., Draper, J.S., Antosiewicz, J.E ., Chon, CH., Jones, S.B., Brooks, J.D., Andrews, F.W., Brown, P.O., and Thomson, J.A. (2003). Gene expression patterns in human embryonic stem cells and human pluripotent. germ cell tumors. Proc Natl Acad Sci U S A 100, 13350-13355; Richards, M., Tan, S.P., Tan, J.H., Chan, W.K., and Bongso, A. (2004). The transcriptome profile of human embryonic stem cells as defined by SAGE. Stem Cells 22, 5l-64)which generally have compared hESC populations grown under conditions that support renewal to cultures undergoing overt differentiation, have produced a molecular blueprint of the pluripotent state, but this blueprint is limited in its resolution due to the inherent complexity of the cell populations under comparison.

The structure of stem cell differentiation hierarchies in general, and that of hBSC in particular, is often depicted as a scries of binary choices between alternate and discrete cell states, driven by a serial cascade of expression of specific transcription factors. However, other data indicate that for pluripotent stem cells at least, the catly progression through a differentiation hierarchy is in fac( a continuum that may be reversibly traversed (Rathjen, J., Lake, J.A., Bcttcss, M.D., Washington, J.M., Chapman, G., and Rathjen, P.D. ( 1999), Formation of a primitive ectoderm like cell population, EPL cells, from ES cells in response to biologically derived factors. J Cell Sci 1 12 (Pt.5), 601-612). In fact, emerging concepts regarding cell fate choice in the preimplantation mouse embryo support a less rigid interpretation of the process of lineage commitment. A newer model (Rossant, J. (2004). Lineage development and polar asymmetries in the peri-implantation mouse blastocyst. Semin CeIl Dev Biol 15, 573-581) depicts the formation of three specific lineages of tlic mammalian peri-implantation embryo, inner cell mass, trophectoderm, and extraembryonic endoderm, not as a sequence of binary decisions, but as the result of a dynamic interplay of expression of a network of particular regulatory genes. Specifically, networks of key transcriptional regulators, including Oct-4, nanog, cdx-2 and GATA -4 and -6, interact in a spatially restricted fashion in the preimplantation embryo to determine fate, rather than acting in a sequential and binary mode. This model implies that the process of lineage choice begins early, before overt loss of all stem cell maintenance factors. These concepts arc reminiscent of the model of lineage priming, derived from studies of hematopoicsis, in which expression of genes characteristic of multiple differentiation lineages is observed in stem or progenitor cells that have not yet undertaken overt commitment (Hu, M., Krause, D., Greaves, M., Sharkis, LS., Dexter, M., Heyworth, C, and Enver, T. (1997). Multilineage gene expression precedes commitment in the hemopoietic system. Genes Dev 1 1, 774-785; Greaves, M.F., Chan, L.C., Furley, AJ., Watt, S,M., and Molgaard, H.V. ( 1986). Lineage promiscuity in hemopoietic differentiation and leukemia. Blood 07, 1-11; Orkin, S.H. (2003), Priming the hematopoietic pump. Immunity 19, 633-634). A recent study (Boyer, L.A., Lee, T.T., Cole, ME, Johnstone, S.E.. Lcvinc, S.S., Zncker, J.P , Guenther, M.G., Kumar, R.M., Murray, H.L., Jenner, R.G., Gifford, E>.K., Melton, D.A., Jacniseh, R., and Young, R.A. (2005). Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947-956) of the transcriptional regulatory circuits in hESC predicts that cell fate is the result of a dynamic interplay between key regulatory factors, and that alterations in stotchiomctry between these factors lead to global changes in gene expression and ultimately cell commitment to specific lineages. However, this study highlighted a key role for pluripotency genes in the suppression of the expression of many lineage specific transcription factors.

The present invention is directed toward a method of determining if a particular type of stem cell has maintained its ability to differentiate into virtually any type of cell. This includes methods for isolating and identifying specific pluripotent coll populations based on the presence of immunological markers. Using this method the present invention establishes a classification system for the pluripotency of a particular type of stem cell. SUMMARY OF THE INVENTION

The invention carries out an analysis of stages in hESC differentiation through immunotranscriptional profiling. The invention uses flow cytometry to fractionate hESC populations grown under conditions that support sLem cell renewal on thϋ basis of their levels of expression of at least two surface markers. Following fractionation, each population is subjected to transcriptome analysis via microarray. Critical findings are confirmed by low density array quantitative RT-PCR. The invention provides a new method of soiling a stem cell population, determined by the expression of a plurality of markers of the pluripotent state and potential paracrine regulators of cell fate. The method resulting in a more refined stem cell population characiemcd by the continuum of expression of pluripotcncy genes and. lineage specific transcription factors across the population.

Stem cell populations, and in particular selected human pluripotent stem cell populations, are identified, isolated, and classified using binding agents such as antibodies which bind to markers are an indication of the cell's pluripotency. The present invention provides distinct subsets of human pluripotent stem cells characterized by particular molecular markers and/or cell surface antigens characteristic of pluripotent stem cells, or alternatively associated with early differentiated derivatives that have a certain level of commitment to a certain lineage. The human embryonic stem cells (hESC) ace grown under substantially the same conditions and are derived from the same or a similar source. Accordingly, the cells share many characteristics including cell surface markers. These similarities increase the difficulty in separating the cells based on characteristics which are an indication of the pluripotency of the cells. The invention uses a plurality of antibodies which bind to a plurality of different cell surface markers. By determining which markers are present on the cell surfaces and the amounts and relative amounts of such markers determinations can be made as to the pluripotency of the cells.

In the present invention, distinct cell populations are grown and the potential of such cells confirmed prior to use or manipulation. The propagation of human pluripotent stem cell subpopulations as cell cultures with confirmed attributes permits investigation of fundamental questions regarding the biochemical and cellular properties of these cells and the dynamics of interaction in their cellular and chemical environment. In addition, useful tissues and substances can be produced using the human pluripotent stem cells of the invention, and lhe methods for identification, isolation and classification as described herein will allow better control over the starting material for regulatory purposes.

Th e .present invention also provides a purified preparation of human pluripotent slem cells that. has the ability to differentiate into cells derived from each of the three germ layers.

Alternatively, using the classification scheme of the present invention, celts can be provided that are primed to differentiate down a specific lineage.

In one aspect the invention provides a method of sorting cells, the method comprising providing a sample of cells; and sorting cells in the sample based on quantitative levels of expression of at least 2 markers selected from the group consisting of GCTM-2, GD9, TG343, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, HSPA8 and SSEA-I^".

In a preferred embodiment the two Markers are GCTM-2 (or Tra 1-60 or Tra 1-81 or TG343) and CD9, GCTM-2 (or Tra 1-60 or Tra 1-81 or TG343) and SSEA-3, GCTM-2 (or Tra 1-60 or Tra 1-81 or TG343) and SSEA-4, GCTM-2 (or Tra 1-60 or Tra 1-81 or TG343) and HSPA8, CP9 and SSEA-3, CD9 and SSEA-4, CD9 and HSPA8, SSEA-3 and SSEA-4, SSEA-3 and HSP AS, or SSEA-4 and HSPAK. It is particularly preferred that, the sorting is done based on the quantitative levels of GCTM-2 and CD9 expression.

As will be understood by those skilled in this field TG343, Tra 1-60 and Tra 1-81 detect related molecules to GCTM-2 (Martin F. Pera, Adam Filipezyk, Susan M. Hawes, and Andrew L. Laslett, (2003) Isolation, characterization, and differentiation of human embryonic stem cells. Methods in Enzymology 365: 429-46.).

Preferably the sorting is carried out using a formulation of a plurality of different antibodies. It is also preferred that the method further comprises assigning the sorted cells to subsets related to a stage of will differentiation. The sorting is preferably cytometric sorting and the cells aϊc preferably stem cells, more preferably embryonic stem cells, preferably human embryonic stem cells.

In a preferred form the method further comprises sorting the cells based on quantitative levels of at least one additional markers selected from the group consisting of CD9, GCTM-2, Oct-4, TG343, SSEA-3, SSBA-4, Tra 1-60, Tra 1-81, HSPA8, SSEA- 1- and combinations thereof. Preferably the cell sorting is based on quantitative levels of each of CD9⁺, GCTM-2⁺, 0ct-4⁺ and SSEA-1 . It is preferred that the sample consists essentially of hES cells, or cells derived therefrom, grown under substantially similar conditions.

In another aspect the invention provides a method of classifying human embryonic stem (hES) cells, comprising; contacting a population of hES cells grown under substantially similar conditions with a formulation comprised of a plurality of different antibodies which bind to markers on hES cells which comprise at least 2 markers selected f roxn the group consisting of GCTM-2, CD9. TG343, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, HSPA8 a nd SSEA-1 ; allowing the antibodies to bind to markers on the population of hES cells; and determining relative levels of binding of the different antibodies to different antigens. ln a preferred embodiment the two markers are GCTM-2 (or Tra 1-60 or Tra ! -81 or TG343) and CD9, GCTM-2 (or Tra 1-60 or Tra 1-K 1 or TG343) and SSEA-3, GCTM-2 (or IYa 1 -60 or Tra 1-81 or TG343) and SSEA-4, GCTM-2 (or Tra 1-60 or Tra I -Si orTG343) and TTSPAδ, CD9 and SSEA-3, CD9 and SSEA-4, CD9 and HSPA8, SSEA-3 and SSEA-4, SSEA-3 and HSPΑ8, or SSEA-4 and HSPA8. It is particularly preferred that the sorting is done based on the quantitative levels of GCTM-2 and CD9 expression.

It is preferred that the method further comprises associating the determined relative levels of binding with a quantitative amount of the different antigens on the hES cells; and classifying the pluripotency of cells in the population of hES cells based on a determined quantitative level of GCTM-2 and CD-9 on each cell.

In another embodiment the method further comprises contacting the hES cells with at least one additional antibody, wherein the antibody binds an antigen selected from the group consisting of Oct-4, CD9, GCTM-2, TG343. SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, IISPA8 and SSEA-1^'. Preferably the hES cells are contacted with a plurality different antibodies which together bind to each of CD9⁺, GCTM-2⁺, Oct-4 ⁺ and SSEA- I-,

It is also preferred that the antibodies are monoclonal antibodies.

In another aspect the invention provides a method of treatment, comprising the steps of; growing a plurality of human pluripolent cells, (hP) cells, under substantially similar conditions; contacting the hP cells with a plurality of different antibodies which bind to a plurality of different antigens associated with hP cell pluripotency; determining hP cells destined to differentiate into a known cellular lineage based on binding to the antibodies and isolating those hP cells; formulating the isolated hP cells; and administering the formulated cells.

Preferably the antibodies bind to epitopes of markers chosen from CD9⁺, G CTM-2⁺, 0ct-4⁺ and SSEA-1 ,

It is preferred that the isolated hP cells are destined to differentiate into an ectodermal lineage, or a mesodermal lineage, or an endodermal lineage, or an extraembryonic lineage.

In still another aspect the invention provides a method of identifying pluripotent human embryonic stem (hES), the method comprising: contacting a population of hES cells grown under substantially similar conditions with a formulation comprised of a plurality of different antibodies which bind to at least two markers on hES cells selected from the group consisting of GCTM-2, CD9, TG343, SSRA-3, SSEA-4, Tra 1-60, Tra 1-81, HSPA8 and SSEA-1-; allowing the antibodies to bind to markers on the population of hES cells; and determining relative levels of binding of the different antibodies to different antigens.

In a preferred embodiment the two markers are GCTM-2 (or Tra 1-60 or Tra 1 -81 or TG343) and CD9, GCTM-2 (or Tra 1 -60 or Tra 1 -81 or TG 343) and SSEA-3, GCTM-2 (or Tra 1 -60 or Tra 1 -81 or TG343) and SSEA-4, GCTM-2 (or Tra 1-60 or Tra 1 -81 or TG343) and HSPA8, CD9 and SSEA-3, CD9 and SSEA-4, CD9 and HSPA8, SSEA-3 and SSEA-4, SSRA-3 and IISPA 8, or SSEA-4 and HSPA8. It is particularly preferred that the sorting is done based on the quantitative levels of GCTM-2 and CD9 expression.

It is preferred that the method further comprises: IO

associating the determined relative levels of binding with a quantitative amount of the different antigens on the ( hES) cells; and classifying the pluripotency of cells in the population of hES cells based on a determined, quantitative level of the at least two markers. Preferably the method further comprises contacting the (hES) cells with at least one additional antibody, wherein the antibody binds an antigen selected from the group consisting of CD9, GCTM-2, Oct-4, TG343, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, HSPA8 and SSEA- 1-. It is preferred that the antibodies comprises a formulation of different antibodies which together bind to each of CD9⁺, GCTM-2⁺, Oct-4¹ and SSEA-I-. It is further preferred that the antibodies are monoclonal antibodies.

The invention also provides a method of sorting cells comprising providing a sample of celts; and sorting cells in the sample based on quantitative levels of expression of at least one gene, wherein the gene is selected from the genes set out in Table 1 and combinations thereof.

It is preferred that the sorting is carried out using a formulation of a plural ity of different antibodies. Preferably the method further comprises assigning the sorted cells to subsets related to a stage of cell differentiation.

It is preferred that the sorting is cytometric sorting. It is also preferred thai lhe cells are stem cells, preferably embryonic stern cells, preferably human embryonic stem cells.

It is also preferred that the method further comprises sorting the cells based on quantitative levels of at least three genes wherein in two of the genes are GCTM-2 and CD9.

Preferably the method further comprises sorting the cells based on quantitative levels an additional markers selected from the group consisting of Oct-4, TG343, SSEA-3, SSEA-4, Tra 1-60, Tra 1-8 L, HSPA8, SSFA-I- and combinations thereof. Preferably the cell sorting is based on quantitative levels of at least five genes wherein four of the genes are CD9, GCTM- 2, Oct-4 and SSEA-1.

It is also preferred that at least one of the genes is selected from the group consisting of six transmembrane epithelial antigen of the prostate, supervillin, the chondroitin sulphate proteoglycans barnacan, versican, and opticin, SIDF, Intm2a and combinations thereof. In another aspect the invention provides a population of cells sorted, classified or identified by the method of the present invention.

In one aspect of the invention a defined pluripotent stem cell subpopulation of the invention or cells derived therefrom is used, to stably incorporate genetic sequences encoding various proteins to ensure maintenance of pluripotency (e.g., telomerase), receptors, ligands, etc.

In another aspect of the invention a defined pluripυlent stem cell subpopulation of the invention or cells derived thcfcfrottt can be used to deliver gene silencing elements such as siRNAs to specific tissues.

In another aspect of the invention cells identified by specific markers am used to treat patients with various disorders and for identifying compounds and small molecules that interact with the modified cells.

The present invention discloses and describes novel potential growth regulators of human pluripotent stem cell populations. The present invention is thus also a method of isolating a cell line derived from human pluripotent cells with a particular, defined potential. In one aspect, the invention provides a method for screening to identify compounds that cause cells with defined pluripotcncy to differentiate along a specific pathway. Starting with a defined cell population rather than a more heterogenous cell population will allow improved identification of factors that function. Alternatively, the defined cell populations can be used to better identify factors and molecules to retain the defined pluripotcncy of a specific cell population.

In a particular embodiment, the factors identified in the cell classification system, which are involved in the maintenance of pluripotency, are chromatin remodeling factors which arc modulated at very early stages of differentiation.

In one specific embodiment, the invention sets forth a classification system providing a more refined definition of stem cell phenotype, based on the presence of and/or relative amounts of surface markers used to characterize the most primitive cells in the human pluripotent stem cell differentiation hierarchy.

Using the methods of the present invention, a cell classification system is provided whereby cells are placed into one of a plurality of classes based on the cell's pluripotency. The classification system allows researchers, regulatory authorities and others involved in the stem cell industry to convey large amounts of information regarding a pluripotent stem cell and in particular informalion on the potential of that stem cell to further differentiate into other types of cells.

In another embodiment, the invention provides an assay method comprising incubating a candidate molecule with a defined cell population of the invention under conditions sufficient to allow the molecule and cells in the defined population to interact; and determining the effect of the molecule on the defined pluripotentiality of the cell population. A cell function that may be modulated (e.g., inhibited or stimulated) by the molecule includes, but is not limited to, differentiation, gene expression, production of growth factors, response to growth factors, chromatin remodeling, and modulation of cell membrane permeability.

In another aspect, the present invention provides useful pharmaceutical products produced using the defined cell populations or derived from specific classified cell populations. This "includes classified cells and cell lines comprising one or more genetic modifications or including gene silencing technologies (RNAi molecules). It is one object of the present invention to provide a primate embryonic stem cell line characterized by the following markers: CD9⁺ and GCTM-2⁺; these cells are optionally also confirmed to be Oct-4⁺ and SSEA-I-.

In another aspect, the invention provides a method of using the isolation and classification methods of the present invention to produce cell populations and cell lines with a restricted developmental lineage. These cells can be identified by the presence of specific cell markers that indicate maintenance of a certain level of potential, but also early differentiation towards a specific lineage. For example, cells of the ectodermal lineage can be identified by their expression of anteriorising factors such as Wnt and TGF beta antagonists.

In another aspect, a selectable marker is expressed in a restricted developmental lineage cell, The restricted developmental lineage cell contains a recombinant polynucleotide that encodes the selectable marker such that the marker is expressed from a restricted developmental lineage cell specific promoter. The restricted developmental lineage cells may be, for example, destined to differentiate into a neural lineage (e.g., neurons, glia, or epithelial cells) or a mesodermal lineage (e.g., cardiomyocytes or skeletal muscle cells). In another embodiment, the invention provides a method for identifying a compound that can de-diffcrentiate a restricted developmental lineage cell to provide a defined pluripotent cell population according to the classification system, e.g., cells that are CD9⁺ and GCTM-2⁺ In one embodiment of the method, components, including the compound and at least one human restricted developmental lineage cell, arc incubated under conditions sufficient to allow the components to interact. The effect of the compound on the human restricted developmental lineage cell is deteimined before and after incubating in the presence of the compound. The appearance in culture of a celI having the characteristics of a defined pluripotent cell population indicates the compound is effective to induce de-differentiation of the restricted developmental lineage cell.

An aspect of the invention is a method whereby stem cell markers are used to identify stem cells with respect, to their pluripotency.

Another aspect of the invention is such a method wherein the markers comprise a cell surface protein chosen from any of CD9', GCTM-2⁺, Oct-4⁺ and SSRA-1^' which may be recognized by a monoclonal antibody or other molecule-specific binding moiety.

Yet another aspect of the invention is a classification system whereby cells are classified into a class based on markers which are associated with the ability of the cell to differentiate in other types of cells.

These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the method and system as more fully described below. BRIEF DESCRIPTION OF TH E DRAWINGS

The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings, It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures:

Figure 1. Combined flow cytometric analysis of hESC for GCTM-2, TG 30 (CD9) and Oct-4 Gates are set relative to isotype controls.

A. Yellow represents cells with high staining intensity for both GCTM-2 andTG30 (R14)

B. Percentages of cells staining for Oct-4 from regions R2, R3, R4 and R14 of Figure 1A. Figure 2. FACS of hESC for array analysis Gates are set relative to isotype controls.

A. I IESC were separated by FACS according to staining intensity for GCTM-2 and TG30 (CD9) into 4 populations; P 4 (GCTM-2-CD9-), P5 (GCTM--2LOWCD9LOW), P 6 (GC.TM- 2MIDCD9MID) and P7 (GCTM-2HΪGHCD9HIGH). B . Heat map depicting normalized intensity of gene expression for genes_, from P7 V P4 experiment with a B stat greater than zero.

Figure 3. Relative gene expression levels of combined array and qPCR analyses of P4, P5, P6 and F7.

Stem cell markers are presented relative to P7 (set at 100) A: Stem ecll markers: Cell surface or secreted factors B: Stem cell markers: Transcription factors

Figure 4. Relative gene expression levels of combined array and qPCR analyses of P4, P5, P6 and P7.

Stem cell markers are presented relative to P7 (set at 100) A: Stem cell markers: Genes involved in chromatin structure B: Stem cell markers: miscellaneous

Figure 5. Relative gene expression levels of combined array and qPCR analyses of P4, P5, P6 and P7.

Differentiation markers are presented relative to P4 (set at 100 ) A: Differentiation markers: extraembryonic B: Differentiation markers: neural

Figure 6. Relative gene expression levels of combined array and qPCR analyses of P4, P5, P6 and P7.

Differentiation markers are presented relative to P4 (set at 100). Differentiation markers: miscellaneous

DETAILED DESCRIPTION OF THE INVENTION

Before the present method and classification system are described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, idl technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications arc cited. Il is understood thai the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

It must be noted thai as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the step" includes reference Io one Or more steps and equivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently continued.

Definitions The "transcriptome" of a cell is defined as the complete collection of transcribed elements of the genome. In addition to mRNAs, it also represents non-coding RNAs which are used for structural and regulatory purposes. " Pluripotent" refers to cells that retain the developmental potential to differentiate into a wide range of cull lineages including the germ line. The term "human pluripotent stem cell" as used here includes hES cells, human embryonic germ cells, or any cell that is modified, de- differentiated or in any other way modified such that it has the potential to produce any cell in the human body.

The term "cell" as used herein also refers to individual cells, cell lines, or cultures derived from such cells. The term "cell line" as used herein refers to human pluripotent stem cells or cells derived therefrom such as are maintained in in vilxo culture.

The term "antibody" as used in this invention includes intact molecules as well as fragments thereof, such as Fab, Fab', F(ab')2. and Fv that can bind the desired epitope. If needed, polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a substrate to which the polypeptide or a peptide to which the antibodies were raised is bound. Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (Sec, e.g., Coligan, et al., Current Protocols in Immunology, Wiley Interscience, current edition, incorporated by reference). "Purified antibody" means an antibody that is at least 60%, by weight, free from proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody, e.g., an anti-CD9 specific antibody. A purified antibody may be obtained, for example, by affinity chromatography using recombinantly-produced protein or conserved motif peptides and standard techniques. The invention can employ not only intact monoclonal or polyclonal antibodies, but also an immunologically-active antibody fragment, such as a Fab, Fab' or (Fab')2 fragments, or a genetically engineered Fv fragment (Ladner et al., U.S. Pat. No. 4,946,788).

The term "Epitope" means any antigenic determinant on an antigen to which an antibody or the active fragment of an antibody binds. Epitopes usually comprise a chemically active surface of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Throughout this specification the word "comprise", or variations such as "comprises" or

"comprising", will be understood to imply the inclusion of a slated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other clement, integer or step, or group of elements, integers or steps. All publications mentioned in this specification arc herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form 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.

The Invention in General

The invention combines immunological and transcriptional analysis (immunυtranscriptional profiling) to compare gene expression in hESC populations at very early stages of difrerentiation lmmunotranscriptional profiling identifies marker for stem cells and their differentiated progeny, and identifies potential regulators of hESC commitment and differentiation. The data show clearly that genes associated with the pluripυleπl state are downregutated in a coordinated fashion, and that they are co-expressed with lineage specific transcription factors in a continuum across the early stages of stem cell differentiation. These findings have important practical implications for propagation and directed differentiation of hESC, and for the interpretation of mechanistic studies of hESC renewal and commitment-

The phcnotype of pluripotent cell lines derived from the human embryo have been defined at the immunological, molecular, and biological levels, and a number of studies of the Human embryonic stem ( hES) cell transcriptome have now been reported. However, the work to date has described the properties of a population of cells grown in vitro known to be heterogenous, at least in terms of expression of various immunologically defined surface markers. Using appropriate cell surface markers, such as CD9. the GCTM-2 antigen, and others, the inventors have shown that human pluripotenl cells grown under standard conditions of propagation in vitro consist of heterogenous and immunologically distinct subpυpulations of cells. These cell populations vary in their levels of expression of xnolccularly defined markers of the pluripotent stale, such as the transcription factor Oct-4,

Research on the hESC transcriptome has primarily focused on differences between cell populations grown under different conditions, and typically one condition supporting stem cell renewal versus one favoring differentiation. In developing the methods and classification system of the present invention, the inventors instead compared closely related cell populations grown under identical conditions. By using fractionation technologies such as FACS analysis to isolate subsets of cells, the inventors have isolated immunologically defined subpopulations which represent distinct cell populations in molecular terms. Analysis of these subpopulations has allowed the inventors to define the isolated cell populations wilh respect to different stages in an early differentiation hierarchy. These populations differ quantitatively in their levels of expression of stem cell markers, making it possible to classify cells based on the cell's level of pluripυlency- Human JTS cell cultures are characterized by their expression of surface markers, including the pericellular matrix proteoglycan recognized by the antibodies TKA -1-60 and GCTM-2, and the tetraspanin membrane protein CD9, Our previous indirect immunofluorescence and flow cytometric analyses have indicated thitt human ES cultures are heterogeneous with respect to expression of these surface markers. In our investigations we used three monoclonal antibodies recognising canonical markers of hESC. The first was the monoclonal antibody GCTM-2, which recognises an epitope on the protein core of a high molecular weight pericellular matrix keratan sulphate/chondroitin sulphate proteoglycan [Cooper, S., Pcra, M.F., Bennett, W., and Finch, J.T. (1992). A novel keratan sulphate proteoglycan from a human embryonal carcinoma cell line. Biochem J 286 ( It 3), 959-966.J. A recent study identified the GCTM-2 antigen as the CP34 related sialomucin podocalyxin [Schopperle, W.M., Kershaw, D.B., and DeWoIf, W.C. (2003). Human embryonal carcinoma tumor antigen, Gρ2(X)/GCTM-2, is podocalyxin. Biochem Biophys Res Commun 300, 285-290.]. To assess this conclusion, wliich was based primarily on co-purification of the GCTM-2 antigen and podocalyxin on lectin affinity columns, we transfected a mouse kidney cell line with human podocalyxin cDNA. Anti-podocalyxin antibodies detected a band of the appropriate molecular weight in imtnunobtots of extracts, but there was no reaction with either GCTM-2 or the antibody TRA-1-60, which reacts with a carbohydrate epitope on the proteoglycan. While both GCTM-2 and anti-podocalyxin antibodies stained cells in hESC cultures, the populations stained were distinct, and in the human kidney, GCTM-2 stained tubular epithelium weakly whilst anti-podocalyxin antibodies stained podocytes as expected. Thus it is unlikely the GCTM-2 antigen and podocalyxin represent the same molecular entity.

We also used monoclonal antibody TG30, produced by immunisation of mice with a partially purified preparation of the GCTM-2 antigen. TG30 reacts with a ceil surface epitope on a 25 kDa protein. This epitope was identified as the tetraspanin protein CD9 following transfection of mouse STO cells with a human CD9 cDNA clone and demonstration of reactivity of the transfected cells with TG30. Others have reported expression of CD9 in hESC [Carpenter, M.K., Rosler, E.S., Fisk, G J- Brandenberger, R-, Ares, X., Miura, T., Lucero, M., and Rao, M.S. (2004). Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev Dyn 229, 243-258.1. Finally we used a monoclonal antibody against the transcription factor Oct3/4, a molecule with long established function in the maintenance of pluripotcncy in moutse BS cells[Nichυl$, J., Zevnik, β., Anaslassiadis, K,, Niwa, 11., Klewe- Nebenius, D,, Chambers, T., Scholer, H., and Smith, A, (1998). Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. C eIl 95, 379- 391.1. Recent data strongly supports a similar role for Ih is transcription factor in hESC [Zaehres, Tl., Lensch, M. W ., Daneron. L,. Stewart, S.A., Ifskovitz-Eldor, J,, and Daley, G.Q. (200.5). Higli-effϊciency RNA interference in human embryonic stem cells. Stem Cells 23, 299-305; Matin, M.M., Walsh, J .R., Gokhak, PJ ., Draper, J.S., Bahrami, AJR., Morion, I., Moore, ILD., and Andrews, P.W. (2004). Specific knockdown of Oct4 and bela2- microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells. Stem Cells 22, 659-6681.

HESC grown in scrum-containing medium in the presence of mouse embryonic -fibroblast feeder cell support were examined by double and triple label indirect immunofluorescence. The cultures were heterogeneous in their expression of these surface markers. In healthy growing colonies prior to overt cellular differentiation, only those cells at the edge of the colony expressed all three markers. Further in towards the colony centre, GCTM-2 staining was reduced, but CD9 and Oct-4 staining remained high. As differentiation proceeded, the interior of the colony became negative for all of these markers bul the outer rim remained positive.

This result was confirmed by analyzing human ES cells by flow cytometry using the same three antibodies (Figure 1). Results showed that GCTM-2- TG30- human ES cells were less than 5% positive for Oct-4. In contrast, GCTM-2⁺ TG30⁺ human ES cells were 80-90% positive for Oct-4. GCTM-2 ^f TG30- cells were -30% positive for Oct-4 and very few cells were GCTM-2- TG30⁺. Within the TG30⁺ population there is a subset of cells that show only weak surface reactivity with GCTM-2. Gating on the brightest TG-30⁺ GCTM-2⁺ cells outlined a population of cells >95% positive for Oct-4. This combination of cell surface markers was used for FACS selection of cells negative for both markers (P4), GCTM- 2hiTG30hi (P7), GCTM-2midTG-30mid ( 1*6) and GCTM-2loTG30lo (P5) subpopulations of human ES cells (Figure 2A).

Total RNA was extracted and amplified from the sorted subsets and each population compared by microarray analysis. Most genes showed consistent increases or decreases across the overall population, though a few fluctuate (Figure 2B), CD9, which was used to select the cell populations, shows a modest drop in transcript levels as expected. The comparison υf the two most closely related subpopulations of cells, P7 and P6, both of which express relatively high levels of CD9 and G CTM-2 antigen, is highly informative and shows that the L'7 population expresses higher levels of α modest number of genes (271 in all at twofold or higher level) on the array, and that an even smaller number of genes are activated in P6 compared co P7 (47 at twofold higher levels -TaWe 1), While the HBI of genes differing between P7 and P6 contains seme lhaf have been associated with stem cell phenotype in previous studies, such as TDGF-I, DNMT3B. and TERF-I, many novel molecules potentially critical to the earliest stages of stem cell differentiation are also identified in this comparison including a number of cell surface markers not previously associated wilh hESC phenotype. As CD9/GCTM-2 antigen immunoreactivily decreases, the cell population begins to express Wnt and TGF beta antagonists, including dickopf, gremlin and follistatin. These genes are known as anteriorising factors from animal embryology, and they and others related to them are involved in induction of ihc formation of the nervous system. The early activation of these neuralising factors accounts for the tendency of hESC cells to undergo "spontaneous" neural differentiation under these conditions of culture, and indeed, amongst the most, strongly induced genes in the early stages of differentiation arc transcription factors characteristic of early neural lineages such as Pax-6. This finding suggests strongly that to obtain other differentiation outcomes, it will be necessary to override these endogenous inhibitory signals, for example through combination of Wnt and BMP signaling. Moreover, several surface markers of these early differentiating populations are identified, including sπiootheπed and cadherin 6 type 2. These surface markers may be used to isolate the early differentiating cells from the most primitive stem cell population. Identification of Defined Cell Populations

Identification of pluripotent cell populations based on specific cell surface markers provides a novel and potent means of improving various in vitro manipulations, including but not limited to genetic manipulation υf stem cells. Enriched pluripotent sub- populations of cells may be created via selection using markers indicative of a greater level of pluripotency, e.g., CD9 or GCTM-2. The level of pluripotency is optionally confirmed following isolation by examining expression of other markers, such as Oct-4. By use of various techniques described more fully below, a highly enriched pluripotent stem cell population may be obtained. Various techniques may be employed to separate the cells, including direct separation using an appropriate binding moiety (e.g., an antibody to CD9 attached to a magnetic bead) or an initial separation of the cells from cells of dedicated lineage ("lineage-committed" cells) followed by direct separation with the binding moiety. Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation. The antibodies may be attached to a solid support to allow for crude separation. The separation techniques employed should maximize the viability of the fraction to be collected.

The use of separation techniques include those based on differences in physical (density gradient centrifugation and counter-flow centrifugal elutriation), cell surface (lectin and antibody affinity), and vital staining properties (mitochondria-binding dye rhυdamine 123 and DNA-binding dye Hoechst 33342). Procedures for separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, including complement and cytotoxics, and "panning" with antibody attached to a solid matrix or any other convenient technique. Techniques providing accurate separation include flow cytometry which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc.

Concomitantly or subsequent to a gross separation providing for positive selection, a negative selection may be carried out, where antibodies to markers indicating early differentiation and/or lineage-specific markers present on dedicated cells are employed. A large proportion of the differentiated cells may be removed by initially using a relatively heterogenous population, where major cell population lineages identified as being along the differentiation pathway

(e.g., cells positive For SSEA- 1 ). The purified cells should display low side scatter and low to medium forward scatter profiles by FACS analysis. Cells may be selected based on light- scatter properties as well as their expression of various cell surface antigens.

Cells can be initially separated by a coarse separation, followed by a fine separation, with positive selection of a marker associated with human pluripotent stem cells and negative selection for markers associated with early differentiation and/or lineage committed cells. Compositions highly enriched in human pluripotent stem cells may be achieved in this manner. The desired stem cells are exemplified by a population with the CD9 ^' GCTM-2 ⁺ SSEA-1 phcnotype, and are characterized by having a higher level of pluripotency. Cell Surface Antigens and Cell Fractionation

Cell-surface antigens provide invaluable tools for the identification of cells and for the analysis of cell differentiation. In particular, antigens that are developmentally regulated during early differentiation into the three lineages - mesoderm, ectoderm and endoderm - can be used to identify human embryonic stem cell subpopulalions that have taken the first steps towards differentiation of the particular lineage. It is known that human ES cells are characterized by the expression of The cell-surface antigens (for example. GCTM -2, CD9, SSEA-3, SSEA-4, TRA- 1-60, and TR A- 1-81), and by the lack of expression of other cell surface markers (e.g., SSEA-1), The present invention sets forth other cell surface markers which can be used to identify cells with higher levels of pluripotency, and to establish a classification system using both known markers and newly defined markers to establish a differentiation hierarchy in the earliest stages of human pluripotent stem cell differentiation. In accordance with one embodi menl of the invention, a heterogenous pluripolcnt cell population is fractionated into one or more subsets of interest using flow cytometry. This can be performed by isolation of immunologically defined subpopulations representing distinct cell populations which correspond to particular stages in a differentiation hierarchy, as judged by their patterns of gene expression and cell surface antigen expression. hESC grown under standard conditions supportive of stem cell maintenance were harvested and sorted by flow cytometry into populations expressing various levels of the both stem cell markers CD9 and the pericellular keralan/chondroitin sulphate proteoglycan recognized by the monoclonal antibody CKJTM-2 (Figure IA). It was shown that cells expressing the highest levels of both surface markers also contained the highest proportion υf cells expressing lhe transcription factor Oct-4 (Figure 1 B), thus linking stiτfaee antigen expression with that of a known molecular determinant of pluripotentiality. Declining levels of expression of both surface markers were correlated with declining levels of Oct-4 expression. Cells were sorted into four separate populations, according to their expression of both surface markers, IiNA was isolated, subjected to linear amplification, cDNA prepared, then analysed by Cornpugeit microarray.

A number of genes were found to have significantly different levels of expression in P7 (the population expressing the highest levels of lhc stem cell surface antigens) versus P4, P5 and P6. Most genes show consistent increases or decreases across the overall population, though a few fluctuate. CDP, which was used to select the cell populations, shows a modest drop in transcript levels as expected. The comparison of the two most closely related subpopulations of cells, P7 and P6, both of which express relatively high levels of CD9 and GCTM-2 antigen, is highly informative (Table 1). The comparison shows thai the P7 population expresses higher levels of a modest number of genes (271 in all at twofold or higher level) on the microarray, and that an even smaller number of genes are activated in P6 compared to P7 (47 at twofold higher levels). While the list of genes differing between P7 and P6 contains some that have been associated with stem cell phenotype in previous studies, such as TDGF-I, ZFP42, DNMT3B, and TERF-I , many novel molecules potentially critical to the earliest stages of stem cell differentiation are identified in this comparison.

For example, a number of cell surface markers not previously associated with the pluripoient phenotype have been identified by the inventors, including six transmembrane epithelial antigen of the prostate, supervillin, the chondroitin sulphate proteoglycans bamacan, versican, and opticin, SIDP, and IntnV2a. In addition, a number of polypeptide regulatory factors and receptors are also identified as being associated with the pluripotent state, including neurotensin, adrenomedullin, endothelin, and the endothelin receptor. The expression of these molecules can be used in conjunction with the cell surface molecules such as CD9 to confirm cells with a higher level of pluripotency. As CD9 and C1CTM-2 antigen expression decreases, the cell population begins lυ express WiH and TGF beta antagonists, including dikkopf, gremlin and follistatin. These genes are known as anteriυrising factors from animal embryology, and they and others related to them are involved in induction of the formation of the nervous system.

The early activation of these neuralising factors accounts fur the tendency of I)ESC cells to undergo "spontaneous" neural differentiation under these conditions of culture, and indeed, amongst tlicmost strongly induced genes in the early stages of differentiation are transcription factors characteristic of early neural lineages such as Pax-6. This finding suggests strongly that to obtain other differentiation outcomes, it will be necessary to override these endogenous inhibitory signals, for example through a combination of Wnt and BMP signaling. Moreover, several surface markers of these early differentiating populations are identified, including betaglycan, srnoothened, cadherin 6 type 2 and FGFR3, These surface markers may be used to isolate the early differentiating ceils from the most primitive stem cell population.

In addition to these surface molecules and receptors and ligands, a number of genes with known or suspected function in chromatin remodeling arc downregulated during the early phases of stem cell differentiation. Although chromatin remodeling is clearly important during differentiation, and chromatin remodeling proteins arc thought to be critical components of oocyte cytoplasm for the reprogramming process that occurs in the donor nucleus during cloning by somatic cell nuclear transfer, it was an unexpected result to show in the present methods that these molecules display an overall downregulation during stem cell differentiation. Thus, chromatin plasticity is an essential feature of the pluripotenl state and that expression of remodeling factors is important to stem cell maintenance. One particularly interesting factor found TO be associated with pluripotency is the E 1 A like inhibitor of differentiation ElD-I or CRI-I. This factor is responsible for the E1A like activity in stem cells lhat interacts with Oct-4 to drive expression of target genes. E1D-1 is a general suppressor of differentiation, including differentiation induced by BMPs, and down regulates histone acetyltransferase activity. This protein and other remodeling factors arc essential for the maintenance of the pluripotent state have an important role in rcprogramimπg of adult stem cells. Specifically, introduction of this protein into tissue cells along with other chromatin remodeling factors leads to reprogramming of gene expression and expression of pluripotentiality in these cell types. These proteins thus play an important role in the classification system of the present invention, and their expression can be used to confirm or repudiate the pluripotency of selected pluripotent cell populations.

Uses of Pluripotent Stem Cell Populations

As described, the present invention provides human pluripotent stem cell populations, methods of isolating and confirming the pluripotent status of such cells in culture, and a classification system whereby specific markers are used to group pluripotent cell populations according into their commitment, to a specific germ line differentiation pathway. An advantage of the invention is that human pluripotent cells with defined pluripotency can be efficiently grown and the potential of such cells confirmed prior to use or manipulation. The propagation of selected human pi uri potent stem cell populations as cell cultures with confirmed attributes permits investigation of fundamental questions regarding the biochemical and cellular properties of these cells and the dynamics of interaction in their cellular and chemical environment In addition, useful tissues and substances can he produced using the human plurjpotcnt stem cells of the invention, and the classification system will allow better control over the starling material for regulatory purposes.

As one example, a defined pluripotent stem cell subpυpulation of the invention or cells derived therefrom can be used to siably incorporate genetic sequences encoding various proteins to ensure maintenance of pluripotency (e.g., telomerase), receptors, ligands, etc. In another example, a defined pluripotent stem cell subpopulation of the invention or cells derived therefrom can be used to deliver gene silencing elements such as siRNAs to specific tissues. Both of these techniques could be used, for example, to treat patients with various disorders and for identifying compounds and small molecules that interact with the modified cells.

The hESC phenotype has been defined at the immunological, transcriptional, and biological levels. The present invention shows that flow cytometric sorting based on quantitative levels of expression of two surface markers, the GCTM-2 antigen and CD9, allows fractionation of the hHSC population into subsets expressing varying levels of pluripotcncy genes. The GCTM-2 antibody reacts with a protein epitope on lhe core of a large pericellular matrix proteoglycan found on the surface of monkey ES cells, human EC] cells and human ES cells. The marker is not exclusive to primate pluripotent stem cells, but its expression is informative of stem cell status in a restricted context. The function of this proteoglycan is unknown. Contrary to u previous report (Schopperle, W.M., Kershaw, D.B., and DcWoIf, W.C. (2003). ' Human embryonal carcinoma tumor antigen, Gp200/GCTM-2, is podocalyxin. Biochem Biυphys Res Commun 300, 285-290), the GCTM-2 antigen is unlikely to be podocalyxin, since the antibody fails to recognize human recombinant podocalyxin expressed in mouse cell derived from metanephric mesenchyme; the molecular weight of the podocalyxin expressed in these cells was consistent with that of the mature glycosylated protein. It is of interest that our array studies found that several known chondroitin sulphate proteoglycan core proteins were highly expressed in stem cells and rapidly downregulated during the early stages of differentiation. It is now appreciated that, like the better-studied heparan sulphate proteoglycans, chondroitin sulphate proteoglcyans can function to present growth factors to cells (Sugahara, K., Mikami, T., Uyama, T., Mizuguchi, S., Nomura, K., and Kitagawa, H. (2003). Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate. Curr Opin Struct Biol 13, 6.12-620). These chondroitin sulphate proteoglycans appear to represent an important component of the hESC inicroenvironment. CD9 is a tetraspanin protein thought to function in organizing integrins and other receptors at the cell surface. There is some evidence to implicate the molecule in ES cell maintenance in the mouse (Oka, M., Tagoku, K., Russell, T.L., Nakano, Y., Hamazaki, T., Meyer, E.M., Yokota, T., and Terada, N. (2002). CD9 is associated with leukemia inhibitory factor-mediated maintenance of embryonic stem ceils, MoI Biol Cell 13, 1274-1281 ). CD9 transcript levels, measured by microarray or Q-RTPCR, correlated well with levels of the protein as determined by flow cytometry.

The use of these markers enabled separation of the hESC population into subsets of cells at various stages of differentiation, as indicated by their expression of stem cell markers and lineage specific transcription factors. Perhaps because the cell populations we examined are so closely related, relatively few genes showed significant changes in transcript levels across the cell populations studied, and some novel candidate surface markers and stem cell regulators were identified. Novel surface markers will facilitate identification of stem cell sυbpopulations and early differentiated cells. Ligands such as endothelin and their cognate receptors may have roles in stem cell regulation.

In addition to these surface molecules and receptors and ligands, it is notable that a number of genes with known or suspected function in chromatin remodeling were downregulaied during the early phases of stem cell differentiation. Although chromatin remodeling is clearly important during differentiation, and chromatin remodeling proteins are thought to be critical components of oocyte cytoplasm for the reprogramming process that occurs in the donor nucleus during cloning by somatic cell nuclear transfer, previous studies have not reported an overall downregulation of these molecules during stem cell differentiation. The study of Boyer el al. (Royer, L.A., Lee, T.I., Cole, M. F., Johnstone, S.E., Lcvinc, S.S., Zucker, J.P., Guenther, M.G., Kumar, R.M., Murray, H.L., Jcnncr, R-CL, Clifford, D.K., Mellon, D.A., Jacnisch, R., and Young, R, A- (2005). Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947-956) identified chromatin remodeling complexes as targets of Oct-4. It is possible that chromatin plasticity is an essential feature of the pluripotent state and that expression of remodeling factors is therefore important to stem cell maintenance. In Arabadopsis, maintenance of the pluripotent stem cell population of the apical meristem depends on expression of both specific transcription factors and a set of chromatin remodeling factors (Guyomarc'h, S., Bertrand, C, Dclarue, M., and Zhou, D.X. (2005). Regulation of rαeristem activity by chromatin remodeling. Trends Plant Sci 10, 332- 338). The present invention shows that stem cell maintenance factors are co-expressed at early stages of the differentiation process along with a number of transcription factors characteristic of commitment to extraembryonic endoderm and neural lineages. The co-expression of stem cell and lineage specific transcription factors was first described in stem cells of the hematopoietic lineage (Hu, M., Krause, D., Greaves, M., Shurkis, S., Dexter, M., Heyworth, C, and Enver, T. (1997). Multilincage gene expression precedes commitment in the hemopoietic system. Genes Dev 11, 774-785; Greaves, M.K, Chan, L.C., Furley, AJ-, Watt, S.M., and Motgaard, H.V, (1986). Lineage promiscuity in hemopoietic differentiation and leukemia. Blood 67, 1-11 ), where the phenomenon has come to be known as lineage priming. In essence the concept states that stem cells, rather than completely repressing lineage specific commitment genes, express these genes at low levels pending internal or external signals that activate specific differentiation programs fully. Although the lineage priming model has been questioned, on the grounds thai the cells expressing lineage specific markers might represent cells already committed to differentiation, specific marking of hematopoietic stem cells using are recombinase driven by the myeloid lineage specific promoter for lysozyme, showed that marked cells which activated the gene were capable of long term renewal and multipoint differentiation (Ye, M., Iwasaki, H., Laiosa, C.V., Stadtfeld, M., Xie, H., Heck, S., Clausen, B., Akashi, K., and Graf. T. (2003). Hematopoietic stem cells expressing the myeloid lysozyme gene retain long-term, multilineage repopulation potential. Immunity 19, 689-699). EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that, the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

EXAMPLK 1 : Im m unocharacterization of subsets of human embryonic stem cells Cell culture hESC lines HES-2, -3, and -4 were grown as previously described (Reubtnoff, B.E., Fera, M-F-, Fong, C.Y., Trounson, A., and Bongso, A. (2000). Embryonic stem cell lines from human blastocysts: somalie differentiation in vitro. Nat Biotech no! IS, 399-404), using serum containing medium, mouse embryonic fibroblast feeder cell support, and mechanical dissection of colonics for subculture. This methodology was used for hESC culture because in our hands this approach provides for long-term maintenance of pluripotent stem cells with a normal diploid karyotype.

Indirect immunofluorescence

The primary antibodies used were GCT M-2, TG30 (this laboratory) and Oct-4 (C- 10, Santa Cruz, CA, USA), fixation with 100% ethanoi was used unless otherwise specified. Triple indirect imm unofluorescent staining for GCTM-2, Oct-4 and TG30 was carried out by utilizing isotype specific secondary antibodies. Oct-4 was detected with anti-mouse lgG2bAF568 , TG30 with anti-mouse lgG2aAF488 and GCTM-2 with biυlitiylated anti- mouse IgM (Dako, CA, USA) followed by streplavidin AF350 (all Alcxa Fluor (AP) anliliodies from Molecular Probes, OR, USA). Triple stained slides were mounted in ProT-ong Antifade (Molecular Probes,OR, USA). Controls Tor primary and secondary antibodies revealed neither nonspecific staining nor antibody cross-reactivity.

Flow cytometric analyses Human ES cell colonics were harvested and fixed with 100% methanol. Cells were then stained with a mixture of mouse IgM GCTM-2, mouse TG30 lgG2a and mouse Oct-4 IgG2b or a mixture of class matched negative control anti bodies (Dako, CA, USA) and detected by incubation with biotinylated rabbit anti-mouse IgM, followed by a mixture of goat anti-mouse IgG2u-AF488, goat anti-mouse IgG2b AF647 and streptavidin-PE (BD Pharmingen). Samples were assayed on a flow cytometer (MoFIo, Dako Cylomation, Ft Collins, CO, USA). Cells were gated initially using forward and right angle light scatter and AF488, AF647 and PE fluorescence signals were collected (Figure 1). Human ES cells analyzed via the above method were compared to single color controls tor TG30, GCTM-2 and Oct-4 and parallel analyses examined live, non-fixed, human HS cells for the presence of the cell surface markers GCTM-

2 and TG30. Co-incubation of the primary antibodies with each other and with fixed or non- fixed human ES cells did not affect the percentages of cells displaying immunofluorescence for each antibody

FACS analysis

Unfixed human ES cells were harvested and stained in solution for GCTM-2, TG30 and Thyl.2-PE as above except that secondary antibodies used were goat anti-mouse igG2a- AF48S (Molecular probes, Oregon, USA) and goat anti-nwuse IgM AF647 (Molecular probes, Oregon, USA). Cells were sorted four ways into eppendorf tubes (P4, P5, F6 and P7 see

Figure 2A) using the FACS Vantage-DIVA (BDBiosdences). Sorted cells were initially gated using forward and side scatter, followed by the removal of clumps and doublets by gating on single cells (FSC-A vs FSCH, and the removal of MEF feeder cells using negative selection for Thyl.2-PE. Unfixed human ES cells were harvested and stained in solution for GCTM-2, TG30 and

Th.yl.2-PE and the secondary antibodies used were goat anti-mouse Ig(J2a-AF488 (Molecular probes, Oregon, USA) for TG30 and goat anti-mouse IgM AF647 (Molecular probes, Oregon, USA) for GCTM-2. Cells were sorted four ways into oppendorf tubes (P4, P5, P6 and P7 see Figure 2A) using the FACSVantage-DlVA (BDBiosciences).

Sorted cells were initially gated using forward and side scatter, followed by the removal of clumps and doublets by gating on single cells ( FSC-A vs FSC-H), and the removal of MBF feeder cells using negative selection for Thy 1.2-PE. Collected cells were washed once in PBS, pelleted, lysed in RNA lysis buffer (Qiagen) and stored at -80"C prior to total RNA purification using the RNAesay microRNA kit (Qiagen).

EXAMPLE 2: Transcriptome Analysis of Subsets of Human Embryonic Stem Cells RNA amplification and target labeling Total RNA from the sorted fractions described in Example I was linearly amplified using the message AMP aRNA kit (Aπϊbion) yielding a minimum of 10 micrograms of amino-allyl labeled anti-sense aRNA. The quantity and integrity of these aRNAs were compared via running each sample on a bio-analyser RNA micro-fluidic chip (Agilent) prior to labeling. 5 micrograms of each aRNA sample was then labeled by covalent linking Cy5- or Cy3-labcllcd UTP (Amersham). Finally the labeled material was hydrolysed and used for hybridization.

Array fabrication and generation The arrays used in this study were all obtained from the SRC Microarray Facility, University of Queensland (ARC Centre for Functional and Applied Genomics) and comprised of 17260 human gene-specific oligonucleotides (Compugen) spotted onto epoxy-silane coated slides (Fullmoon). Arrays were hybridised for a minimum of 16 hours at 45oC using previously described conditions (Challen, GA., Martinez, G., Davis, M.J., Taylor, D.F., Crowe, M., Teasdalc, R.D., Grimmond, S.M., and Little, M.H, (2(KH). Identifying the molecular phenotype of renal progenitor cells. J Am Soc Nephrol 15, 2344-2357,)- Image analysis, normalisation and analysis

Hybridised slides were washed, dried and scanned in a 600B array scanner (Agilent). The images were analyzed with Imagene 5.5 (BioDiscovery Inc) to determine mean foreground and background for both channels. Print tip intensity independent Lowess normalization and scaling between arrays was performed, using the R statistical software from the LIMMA package (.Smyth, G. K. (2004). Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology 3, No. 1, Article 3; Smyth, G, K., Michaud, L, and Scott, H. (2005). The use of within-array replicate spots for assessing differential expression in microarray experiments, Bioinformatics 21 (9), 2067-2075.)

Experimental design: The four isolated subpopulations were compared to one another in a boxed experimental design whece each sample was compared to the other in tit least triplicate and included a dyeswap to account for dye bias..

Ranking of differential Iy expressed genes:

Differential expression was defined using a robust statistical method rather than simple fold change, All genes were ranked using the B statistic (Lonnstedt, I. and Speed, T. P. (2002). Replicated microarray data. Statistica Sinica 12, 31-46.) Method where both fold change and variance of signals in replicates is used to determine the likelihood that genes are truly differentially expressed. A threshold in the β statistic of 0.0 was adopted as genes with a B score>0 have a >50% probability of being truly expressed (Smyth G K, Yang YH and Speed f (2003) Statistical issues in cDNA microarray data analysis. Methods MoI Biol. 2003; 224:1 1 I- 36.). This analysis was executed using the Bio-conductor package that has been implemented as a plug in tool in BASE. EXAMPLE 3: Immunohistochemistry

Normal human kidney tissue was obtained froin the uninvolved pole of renal carcinoma nephrectomy specimens (approved by the Monash Medical Centre Human Research Ethics Committee). Sections (4μm) of formalin-fixed, paraffin embedded tissue were dewaxed, hydrated in PBS and then blocked in 10% normal sheep scrum and 10% foetal calf serum in PBS for 30 min. Sections then were incubated overnight with GCTM-2 or PHM-5 (Hancock, W. W., and Atkins, R-C- (1983). Monoclonal antibodies to human glomerular cells: a marker for glomerular epithelial cells. Nephron 33, 83-90) antibody at 4ºC, washed (x3) in PBS; endogenous peroxidase was inactivated in 0.6% H₂O₂ in methanol for 20.nin, and the sections were washed in P BS. Sections were then incubated with either horseradish peroxidase (HRP)- conjugated goat anti-mouse IgG (Dako, Glostrup, Denmark) (for detection of PHM-5) or goat anti-mouse IgM (Serotec, Oxford, UK) (for detection of CϊCTM-2) in 5% normal sheep serum and 3% bovine scrum albumin in PBS for 40 min, washed (x3) in PBS, incubated with complexes of HRP-conjugated mouse anti-HRP IgG complexes (Dako), washed (x3)in PBS and developed with the diaminobenzidine substrate (Sigma-Aldrich, Castle Hill, NSW, Australia) to produce a brown colour.

EXAMPLE 4:

FACS analysis and cell sorting

For analysis of compression of Oct-4 and surface markers, harvested hESC were dissociated into single cell suspension by trituration and fixed with 100% methanol. Cells were then stained with a mixture of mouse TgM GCTM-2, mouse TG30 (CD9) TgCτ2a and mouse Oct-4 IgG2b or a mixture of class matched negative, control antibodies (Figure J). Binding of primary antibodies was detected by incubation with biolinylated rabbit anti-mouse IgM, followed by a mixture of goat anti-mouse IgG2a-AF4H8, goat anti-mouse IgG2b AF647 and streptavidin-PE. Samples were assayed on a flow cytometer (FACS Aria Vantage, Becton Dickinson). Cells were gated initially using forward and right angle light scatter and AF488, AF647 and PE fluorescence signals were collected. hESC cells analysed via the above method were compared to single color controls for TG30, CϊCTM-2 and Oct-4 and parallel analyses examined live, non-fixed, human ES cells for lhe presence of the cell surface markers GCTM- 2 and TG30. Co-incubation of the primary antibodies with each other unci with fixed or nort- fixed human HS cells did not affect the percentages of cells displaying immunofluorescence for each antibody.

For preparative isolation of discrete cell populations for RNA analysis, unfixed human ES cells were harvested and stained in solution for GCTM-2, TG30 (CD9) and Thy 1.2-PH (Io gate out any remaining mouse embryo fibroblasts) as above, except, that the secondary antibodies used were goal anti -mouse IgG2a-AF48K (Molecular Probes, Oregon, USA) and goat anti- mouse IgM AF647 (Molecular probes, Oregon, USA), Cells were sorted four ways inlo microfuge tubes (P4, P5, P6 and P7, see Figure 2A above) using the PACSVantage-DIVA (BDBiosciences). Sorted cells were initially gated using forward and side scatter, followed by the removal of clumps and doublets by gating on single cells (FSC-A vs. FSC-H), and the removal of MEF feeder cells using negative selection for Thy 1.2-PE.

Microarray analysis

KNA amplification and target labelling:

Total RNA from the sorted fractions described above was isolated using Trizol and linearly amplified using the messagcAMP aRNA kit (Ainbton) yielding a minimum of 10 micrograms of ainino-allyl labeled anti-sense aRNA. The quantity and integrity of lhesc aRNAs was compared via running each sample on a bio-analyser RNA micro-fluidic chip (Agilent) prior to labeling. 5 micrograms of each aRNA sample was then labeled by covalent linking Cy5- or Cy3-labelled UTP (Amersham). Finally the labeled material was hydrolysed and used for hybridisation.

Array fabrication and generation: The arrays used were obtained from (he SRC Microarray Facility, University of Queensland (ARC Centre for Functional and Applied Genomics) and comprised 17260 human gene- specific oligonucleotides (Compugen) spotted onto epoxy-silane coated slides (FuI I moon). Arrays were hybridised for a minimum of 16 hours at 4SoC using previously described conditions (Challen, G .A., Marlines, G., Davis, M.J., Taylor, D.F., Owe, M., Teasdale, R.D., Grimmond, S.M., and Little, M.H. (2004). Identifying the molecular phenolype of renal progenitor cells. J Am Soc Nephrol 15, 2344-2357). linage analysis, normalization analysis:

Hybridised slides were washed, dried and scanned in a 600B array scanner (Agilent). The images were analyzed with lmagene 5.5 (BioDiscovcry Inc) Io determine mean foreground and background for both channels. Print tip intensity independent Lowcss normalization and scaling between arrays was performed, using the R statistical software from the LlMMA package | Smyth, G. K. (2004). Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology 3, No. 1, Article 3. Smyth, G. K., Mϊchaud, J., and Scott, H. (2005). The use of within-array replicate spots for assessing differential expression in microarray experiments. Bioϊnformatics 21(9), 2067-2075.]

Experimental design:

The four FACS sorted populations were compared to one another in a boxed experimental design where each sample is compared to the other in al least triplicate; a dyeswap was included to account for dye bias. These results are then compared between experiments on cells grown under different conditions. Differential expression was defined using a robust statistical method rather than simple fold change. All genes were ranked using the B statistic method where both fold change and variance of signals iα replicates is used to determine the likelihood that genes are truly differentially expressed. A threshold in the B statistic of 0,0 was adopted as genes with a B score>0 have a >50% probability of being truly expressed (Smyth, G.K., and Speed, T. (2003). Normalization of cDNA inicroarray data. Methods 31 , 265-273). This analysis was executed using the Bio-conductor package that has been implemented as a plug in tool in BASE.

Q-RT-PCR confirmation of gene expression patterns:

We used the ABI Microfluidic Card system for quantitative RT-rCR validation of patterns of gene expression. We designed a 384 well format card enabling assay ot^~96 target genes on four samples. The card incorporates proprietary validated RT-PCR primm for key genes identified in our original studies as strongly up- or down-reguiated during early phases of hESC differentiation, plus a number of classical hESC genes and a number of genes characteristic of early differentiation pathways. To carry out analyses, total RNA (1 ng) was isolated, reverse transcribed, and 100ng introduced into the gene card in PCR masiermix. Amplification was carried out in the ABI Prism 7900 HT system at the Australian Genome Research Facility Melbourne Node, and anatysed using the comparative CT method using the proprietary sequence detection software.

The comparison of expression levels was carried out with reference calibration to the population of cells with the highest level of stem cell marker expression.

These results were confirmed and extended by QRT-PCR using AEI Microfluidic card analysis of 96 genes selected in part from those identified in the array analysts, and some key stem cell maintenance factors and differentiation markers that were not represented on the array or failed to read out. Two separate JTESC lines were immunologically fractionated as described above, RNA was prepared, cDNA was synthesised, and analysed for expression of the genes shown. Downregulation of transcript levels of a number of known and novel markers of the pluripotent state was confirmed (Figures 3 & 4). Transcripts for CD9, which was used for flow cytometry sorting of the population, showed the expected reduction, as did many other markers of the pluripotent state. The QRT-PCR analysis highlighted that genes that are activated early on after loss of stem cell markers include genes expressed in extraembryonic endoderm and neural genes (Figure 5). Thus, the transcription factors GATA- 4, GATA-6, and F OXA1 are all activated, as is the Wnt antagonist dickopf; these transcription factors are critical Io the differentiation of primitive endoderm in the mammalian embryo, and dickopf is secreted by this tissue. The analysis also confirmed the induction of BMP and TGF-beta antagonists including chordin, noggin, gremlin, and follistatin. These genes arc transcribed in tissues involved in patterning in the mouse postimplantation embryo, the node and the anterior mesendoderm , and they function to drive commitment of pluripotent cei! populations to neural fates. Possibly as a consequence of the production of these anteriorising factors, many transcription factors characteristic of neureetoderm are activated early on during differentiation (Figure 5B). The picture is consistent with the early commitment of ES cells to the extraembyonic lineage and the subsequent elaboration of factors from these cells and other cell types that drive neural commitment. It is important to note that the expression of most of these lineage specific transcription factors begins to rise well before extinction of expression of markers of pi uri potency.

Expression of certain surface markers for ES and differentiated cells was also confirmed (Figures 3 A & 6), as was the downreguiution of a number of genes involved in chromatin Structure (Figure 4A). Example 5: Antibodies to Epitopes expressed on the Surface of Primate Pluripotent Stem Cells Reveal Heterogeneity in hESC Cultures

Three monoclonal antibodies recognising canonical markers of hESC were used. Thy first was the monoclonal antibody GCTM-2, which recognises an epitope on the protein core of a high molecular weight pericellular matrix keratan sulphate/chondroitin sulphate proteoglycan (Cooper, S., Peru, M. K, Bennett, W., and Finch, J.T. (1992). A novel keratan sulphate proteoglycan from a human embryonal carcinoma cell line. Biochem J 286 (It 3), 959-966). A recent study identified the GCTM-2 antigen as the CD34 related sialomucin podocalyxin (Schopperle, W.M., Kershaw, D.B., and DeWoIf, W.C. (2003). Human embryonal carcinoma tumor antigen, Gp200/GCTM-2, is podocalyxin. Biochem Biophys Res Commun 300, 285- 290). To assess this conclusion, which was based primarily on co-purification of the GCTM-2 antigen and podocalyxin on lectin affinity columns, we transfected the mouse kidney cell line with human podocalyxin cDNA. Anti podocalyxin antibodies detected a band of the appropriate molecular weight in immunoblots of extracts, but there was no reaction with either GCTM-2 or the antibody TRA- 1-60, which reacts with a carbohydrate epitope on the proteoglycan. White both GCTM-2 and antipodoculyxin. antibodies stained cells in hESC cultures, the populations stained were distinct, and in the human kidney, GCTM-2 stained tubular epithelium weakly whilst anti-podocalyxϊn antibodies stained podocytes as expected. Thus it is unlikefy the GCTM-2 antigen and podocidyxin represent the same molecular entity.

Monoclonal antibody TG30, produced by immunisation of mice with a partially purified preparation of the GCTM-2 antigen was used, TG30 reacts with a cell surface epitope on a 25 kDa protein. This epitope was identified as the tetraspannin protein CD9 following transection of mouse STO cells with a human CD9 cDNA clone and demonstration of reactivity of the transfected cells with TG30. Others have reported expression of CD9 in hESC (Carpenter, M.K., Rosier, H.S.. Fisk, G.J., Brandenberger, R., Arcs, X., Miura, T., Lucero, M., and Rao, M.S. (20Ω4). Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev Dyn 229, 243-25«)- Finally we used a monoclonal antibody against the transcription factor Ocl3/4, a molecule with long established 5 function in the maintenance of pluripotency in mouse ES cells (Nichols, J., Zevnik, B., Amstassiadis, K., Niwa. H., Klewe-Nebenius, I)., Chambers, L, Scholcr, π., and Smith, A. (1998). Formation of pluripotent stem cells in the mammalian embryo depends on lhc POU transcription factor Ocl4, Cell 95, 379-391); recent data strongly supports a similar role for this transcription factor in hESC (Zaehres, H., Lensch, M.W., Daheron, L., Stewart, S. A., 0 Itskovitz- Eldor, J., and Daley, G.Q. (2005). High-efficiency RNA interference in human embryonic stem cells. Stem Cells 23, 299-305; Matin, M.M., Walsh, JR., Gukhale, PJ., Draper, J.S., Bahrami, A.R., Morton, L , Moore, U.D., and Andrews, F.W. (2004). Specific knockdown of Oct4 and beta2- microglobulin expression by RNA interference in human embryonic stem cells and embryonic carcinoma cells. Stem Cells 22, 659-668). S Mammalian expression of recombinant podocalyxin and iimminoblot analysis

Preliminary attempts to express recombinant human podocalyxin in mouse STO cells resulted in the production of a protein immunoreactive with monoclonal antibodies specific for human podocalyxin, but this protein was much smaller than the canonical form, suggesting either premature termination of transcription or translation, partial degradation, or incomplete 0 glycosylation. We reasoned that a mouse cell line that normally expresses the protein might produce mature full-length human podocalyxin when transfected with recombinant cDNA. Mouse M15 cells, derived from embryonic mesonephros {Larsson, 1995 #333}, were grown in Dulbccco's Modified Eagle's Medium (high glucose formulation) supplemented with 20% foetal calf serum, penicillin and streptomycin and L-glutamine. For Ixansfectioπ, M15 cells 5 were plated on T-75 flasks the day before to allow 50-80% confluency at the time of transfection. An hour before Iransfeclioπ, media was replaced witb scrum-free, supplemented media. For each transfection, 30μL Fugene 6 was diluted in 470μL of serum-free media. DNA at a ratio of (v/w) 3:1 (Fugene 6: DNA) was then added, and incubated for. 15 mins at room temperature. The mixture was added dropwise to the cells which were incubated at 37°C with 0 5% CO2. 5 hours after transfection, the media was replaced with serum-containing, supplemented media. Cells were lysed 48 hours post-transfection using lysis buffer containing 10mM Tris-HCl buffer (pll 7.0), 1% Triton X-100 detergent and ImM EDTA in water. 1 mM phenylmethanesulfonyl flouride diluted in isopropanol was added to the lysis buffer prior to use. Protein lysate samples were separated in a 6% reducing SDS-PAGE gel and transferred for 1 hour at 75 mAmps onto a PVDF membrane. Blots were blocked in 5% sk im milk powder in PBS-Tween overnight and incubated with the monoclonal antibodies GCTM2, podocalyxin (PHM-5) and TG343 tor 1 hour. Washed blots were then incubated with the secondary antibody anti-mouselg-HRP for an hour and the blots were developed with ECL. HESC grown in serum-containing medium in the presence of mouse embryonic fibroblast feeder cell support were examined by double and triple label indirect immunofluorescence. The cultures were heterogeneous in their expression of these surface markers. In healthy growing colonies prior to overt cellular differentiation, only those cells at the edge of the colony expressed all three markers. Further in towards the colony centre, GCTM-2 staining was reduced, but CD9 and Oct-4 staining remained high. As differentiation proceeded, the interior of the colony became negative for all of these markers but the outer rim remained positive.

Example 6: Levels of expression of C-CTM-2 and CD30 correlate with levels ot^'Oct-4 expression The results above were suggestive of a gradient of antigen expression ill growing colonies of riESC, but these indirect immunofluorescence data were qualitative only. The heterogeneity of antibody staining apparent on indirect immunofluorescence prompted us to carry out quantitative analysis of antigen expression by flow cytometry. We sought to establish the relationship between staining levels for the two surface antigens and intracellular protein levels of Oct-4. Flow cytometric; analysis of healthy growing colonies of hESC prior to overt differentiation showed that there was a gradient in staining levels for the surface antigens; most cells expressed both antigens, but some expressed only one or the other (Figure IA). There was a strong relationship between the levels of expression of Oct-4 and the intensity of double surface staining for GCTM-2 and CD9 (Figure 1B). Thus, the cell population expressing the highest level of both surface markers also expressed the highest level of Oct-4 positivity.

EXAMPLE 7: Microarray analysis of gene expression in immunologically defined subpopulations of hESC shows coordinated regulation of gene expression

The relationship between levels of expression of two stem cell markers and the transcription factor Oct-4 suggested the possibility that there might be an overall gradient of expression of stern cell markers in the hESC populations. To assess this we used microarray analysis to examine global gene expression patterns in immunologically defined subpopulalions of hESC. Cells were sorted into four separate populations (Figure 2A), according to their expression of both surface markers, RNA was isolated, cDNA prepared, subjected to linear amplification, and then analysed by Compugen microarray.

We carried out global analysis of those genes showing significantly different levels of expression in P7 (GCTM-2111GTTCD9HIGH) versus P4 (GCTM-2-CD9 -), P5 (GCTM- 21LOWCD9LOW) and P6 (GCTM-2M1DCD9MID). A two-fold difference in expression levels and a B-statistic greater than zero were the criteria used to identify significant changes.

Most genes whose expression levels changed significantly showed consistent increases or decreases across the overall population, and only a small minority fluctuated up and down (Figure 2B). The comparison of the two most closely related subpopulations of cells, P7 and P6, both υf which expressed relatively high levels of CD9 and GCTM-2 antigen, was highly informative. This comparison showed that the P7 population expressed higher levels of a modest number of genes (271 in all at twofold or higher level) on the microarray, and that an even smaller number of genes were activated in P6 compared to P7 (47 at twofold higher levels) (Table 1). While the list of genes expressed more strongly in P7 versus P6 contained many that have been associated with stem cell phenotype in previous studies, such as CD9, TDG F- 1 , ZEP42, DNMT3B, and TERF-1 (Robson, P. (2004). The maturing of the human embryonic stem cell transcriptome profile. Trends Biotechnol 22, 609-612), many novel molecules potentially critical to the earliest stages of stem cell differentiation were identified through this comparison. A number of cell surface markers not previously associated with hESC were identified, including six transmembrane epithelial antigen of the prostate, supervillin, the chondroitin sulphate proteoglycans, bamacan, versican, and opticin, SIDF, and Intm2a. In addition, a number of polypeptide regulatory factors and receptors are also identified, including neurotensin, adrenomedullin, and the endothelin receptor. It is also of interest that a number of genes involved in chromatin remodeling were expressed at highest levels in the P7 population and were downregulated as stem cell surface marker decreases.

Analysis of genes upregulated as cell surface marker expression decreases pointed to neural differentiation as a key pathway undergoing activation. Amongst the most strongly induced genes in the early stages of differentiation are transcription factors characteristic of early neural lineages such as Pax-6. The early activation of these neuralising factors may account for the tendency of hESC cells to undergo "spontaneous" neural differentiation under these conditions of culture (Reubinoff, B.E., [tsykson, P., Turetsky, T., Pera M. F., Reinhartz, E., Itzik, A., and Ben-Hur, T. (2001 ). Neural progenitors from human embryonic stem cells. Nat Biotechnol 19, 1134-1140) At the same time, as CD9/GCTM-2 antigen expression decreased from P7 to P6, the cell population levels of transcripts for Wnt and TGF beta antagonists, including dickopf, gremlin and fσllistatin rose, along with the BMPs themselves. These antagonists are known as anleriorising factors from animal embryology, and they and other molecules related to them are involved in induction of the formation of the nervous system. Moreover, several surface markers of these early differentiating populations were identified, including betaglycan. smoothened, cadherin 6 type 2 and FGFR3. These cell surface molecules may serve to mark the early differentiating cells from the most primitive stem cell population. EXAMPLE 8:- QRT-PCR confirms coordinated downregulation of pluripotency genes and concomitant activation of transcription factors associated with induction of extraembryonic endoderm and neurectoderm

These results were confirmed and extended by .QRT-PC R using ABI Microfluidic card analysis of 96 genes selected in part from tho.se identified in the array analysis, and some key stem cell maintenance factors and differentiation markers that were not represented on the array or failed to read out. Two separate hESC lines were immunologically fractionated as described above, RNA was prepared, cDNA was synthesised, and analysed for expression ol the genes shown. Downregulation of transcript levels of a number of known and novel markers of the pϊuripotcnt state was confirmed (Figures 3 & 4). Transcripts for CR9, which was used for flow cytometry sorting of the population, showed the expected reduction, as did many other markers of the pluripolent state. The QRT-PCR analysis highlighted that genes that arc activated early on after toss of stem cell markers include genes expressed in extraembryonic endodertn and neural genes (Figure 5). Thus, the transcription factors GATA- 4, GATA-6, and FOXA1 are all activated, as is the Wnt antagonist dickopf; these transcription factors are critical to the differentiation of primitive endoderm in the mammalian embryo, and dickopf is secreted by this tissue. The analysis also confirmed the induction of BMP and TGF-beta antagonists including ehordin, noggin, gremlin, and follistatin. These genes are transcribed in tissues involved in patterning in the mouse postimplantation embryo, the node and the anterior mesendoderm, and they function to drive commitment of pluripotent cell populations to neural fates. Possibly as a consecμience of the production of these anteriorising factors, many transcription factors characteristic of neurectoderm arc activated early on during differentiation (Figure 5B). The picture is consistent with the early commitment of HS ceils to the extraembyonic lineage and the subsequent elaboration of factors from these cells and other cell types that drive neural commitment. It. is important to note that the expression of most of these lineage specific transcription factors begins to rise well before extinction of expression of markers of plurtpotency. Expression of certain surface markers for ES and differentiated cells was also confirmed (Figures 3A & 6), as was the downregulation of a. number of genes involved in chromatin structure (Figure 4A). The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described Or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples add conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Table 1

Fold chuiige B Accession number . UGl number

2.1 4.2 NM 016505 Hs.524094

2.1 3.7 NM_019079 Hs.135693

2.1 2.8 AK021466 Hs.51093B

2.1 1.3 NM_OOB407 Hs.518060

2.1 1.3 AL110180 Hs.29464

2.1 3.6 AL050124 Hs.498661

2.1 0.9 AK026659 Hs.79881

2.1 0.2 NM.019845 Hs, 100890

2 1.2 NM_00485Q Hs.58617

2 1.4 AH6143S Hs.104347

2 4 AU 17661 Hs.527688

2 3.1 NWL017489 Hs,442707

2 2.1 U56251 Hs.542315

2 3.8 AL137730 Hs-512635

2 1.7 NM 006709 Hs.520038

2 4.9 AK025047 Hs.272416

2 2.7 NIVL016333 Hs.433343

2 1.2 NM_018227 H3.212774

2 4.6 NM 000416 Hs.520414

.2 3.4 NM_002045 Hs.134974

2 3 NM..004774 Hs.462956

2 0.2 NM.005903 Hs.167700

2 0.8 AF055018 Hs.130195

2 2,2 NM_006479 Hs.50455O

2 1 NM_ 003074 Hs.476179

2 1.6 NM_003026 Hs.75149

2 3.4 NM_018489 Hs.491060

2 2.9 AL3S5518 Hs.517293

2 2.4 NM.006325 Hs.10842

2 4.8 NM ...005802 Hs.533976

2 1.1 NM_00θ892 Hs.251673

2 3.8 NM_018155 Hs.144130

0.5 1.8 AY007135 Hs.350927

0.5 4.1 AK001115 Hs.4862β5

0-5 3.6 NM..018660 Hs.435535

O.S 3.5 NM_003633 Hs.104925

0.5 1.2 AL359620 Hs.490512

0.5 2,7 NM_006312 Hβ.137510

0.5 1.2 NM_014341 Hs.485262

0.5 1.2 NM_019B54 Hs.504530

0.5 2.8 NM_005777 Hs.188879

0.5 2.7 ABO 19566 Hs.528374

0.5 1.8 NM_003243 Hs.482390

0-5 0.3 NM_002046 Hs.479728

0.5 2.9 NM_014522 Hs,496456

0.5 2.7 AF086037 Hs.15069

0.5 1.8 NM 001104 Hs.445037

0.5 4.9 AK023283 Hs.116750

Claims

1. A method of sorting cells, the method comprising providing a sample of cells; and sorting cells in the sample based on quantitative levels of expression of at least 2 markers selected from the group consisting of GCTM-2, CD9 , TG343, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, HSPΑ8 and SSEA- 1.

2. The method of claim 1 wherein the two markers are GCTM-2 (or Tra 1-60 or Tra 1- 81 orTG343) and .D9, GCTM-2 (or Tra 1-60 or Tra 1-81 or TG343) and SSEA-3, GCTM-2 (or Tra 1 -60 or Tra 1 -81 or TG343) and SSEA-4, GCTM-2 (or Tra 1 -60 or Tra 1 -81 or TG343) and HSPA8, CD9 and SSEA-3, CD9 and SSEA -4, CD9 and HSPA8, SSEA-3 and SSEA-4, SSEA -3 and HSPAS, or SSEA-4 and HSPA8. ,

3. The method of claim I or 2 wherein the sorting is done based on the quantitative levels of GCTM-2 and CD9 expression.

4. The method of any one of claims 1 to 3, wherein the sorting is carried out using a formulation of a plurality of different antibodies.

5. The method of any one of claims 1 to 4, further comprising assigning the sorted cells to subsets related to a stage of cell differentiation.

6. The method of any one of claims 1 to 5, wherein the sorting is cytometric sorting.

7. The method of any one of claims 1 to 6, wherein the cells are stem cells.

8. The method of claim 7, wherein the stem cells are embryonic stem cells.

9. The method of claim 8, wherein the stem cells are human embryonic stem cells.

10. The method of any one of claims 1 to 9, further comprising sorting the cells based on quantitative levels of at least one additional markers. selected from the group consisting of CD9, GCTM-2, Oct-4, TG343, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, HSPA8. SSEA -1- and combinations thereof,

11. The method of any one of claims 1 to 10, wherein the cell sorting is based on quantitative levels of each of CD9, GCTM-2¹, Oct-4* and SSEA-1 ^'.

12. The method of any one of claims I to 11, wherein the sample consists essentially of hES cells, or cells derived therefrom, grown under substantially similar conditions.

13. A method of classifying human embryonic stem (hES) cells, comprising: contacting a population of hES cells grown under substantially similar conditions with a formulation comprised υf a plurality of different antibodies which bind to markers on hES cells which comprise at least 2 markers selected from the group consisting of GCTM-2, CD9, TG343, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, HSPA8 and SSEA-1-; allowing the antibodies to bind to markers on the population of hES cells; and determining relative levels of binding of the different antibodies to different antigens.

14. The method of claim 13 wherein the two markers are GCTM-2 (or Tra 1-60 or Tra I- 81 or TG343) and CD9, GCTM-2 (or Tra 1-60 or Tra 1-81 or TG343) and SSEA-3, GCTM -2 (or Tra 1-60 or Tra 1-81 orTG343) and SSEA-4, GCTM-2 (or Tra 1-60 or Tra 1-81 or

• TG343) and HSPA8, CD9 and SSEA-3, CD9 and SSRA-4, CD9 and HSPA8, SSEA-3 and SSEA-4, SSEA-3 and HSPA8, or SSEA-4 and HSPA8.

15. The method of claim 13 or 14 wherein the sorting is done based on the quantitative levels of GCTM-2 and CD9 expression.

16. The method of any one of claims 13 to 15, further comprising associating the determined relative levels of binding with a quantitative amount of the different antigens on the hES cells; and classifying the pluripotency of cells in the population of hES cells based on a determined quantitative: level of GCTM-2 and CD-9 on each cell.

17. The metJiod of any one of claims 13 to 16, further comprising contacting the hES cells with at least one additional antibody, wherein the antibody binds an antigen selected from the group consisting of CD9, GCTM-2, Oct-4, TG343, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81 , HSPA8 and SSEA- 1 -

18. The method of any one of claims 13 to 17, wherein the hES cells are contacted with a plurality different antibodies which together bind to each of CD9*, GCTM-2⁺, Oct-4⁺ and SSEA- 1-.

19. The method of any one of claims 13 to 18, wherein the antibodies are monoclonal antibodies.

20. A method of treatment, comprising the sreps of: growing a plurality of human pluripotent cells, (hP) cells, under substantially similar conditions; contacting the hP cells with a plurality of different antibodies which bind to a plurality of different antigens associated with hP cell pluripotency; determining hP cells destined to differentiate into a known cellular lineage based on binding to the antibodies and isolating those hP cells; fom ulating the isolated hP cells; and administering the formulated cells.

■ 21. The method of claim 20, wherein the antibodies bind to epitopes of markers chosen from CD9\ GCTM-2⁺, Oct-4⁺ and SSEA-1\

22, The method, of claim 20 or 21, whtsrdu the isolated hP cells are destined to differentiate into an ectodermal 1 ineage.

23, The method of claim 20 or 21, wherein the isolated hP cells are destined to differentiate into a mesodermal lineage.

24, The method of claim 20 or 21, wherein the isolated hP cells are destined to differentiate into un endodeπnal lineage.

25. The method of claim 20 or 21, wherein the isolated hP cells are destined to differentiate into an extraembryonic lineage.

26. A method of identifying pluripotent human embryonic stem (hES), the method comprising: contacting a population of hES cells grown under substantially similar conditions with a formulation comprised of a plurality of different antibodies which bind to at least two markers on hES cells selected from) the group consisting of GCTM-2, CD9, TG343, SSEA-3, SSR A-4, Tra 1-60, Tra 1-81. HSPA8 and SSEA-1^"; allowing the antibodies to bind to markers on the population of hES cells; and determining relative levels of binding of the different antibodies to different, antigens.

27. The method of claim 26 wherein the two markers arc GCTM-2 (or Tra 1-60 or Tra 1- 81 or TG343) and CD9, GCTM-2 (or Tra 1-60 or Tra 1-81 or TG343) and SSEA-3, GCTM-2 (or Tra I -60 or Tra I -81 or TG343) and SSEA-4, GCTM-2 (or Tra I -60 or Tra 1 -81 or . TG343) and H SP AS, CD9 and SSEA-3, CD9 and SSEA-4, CD9 and HSPA8, SSEA-3 and SSEA-4, SSEA-3 and HSPA8, or SSEA-4 and HSPAH.

28. The method of claim 26 or 27 wherein the identification is done based on the quantitative levels of GCTM-2 and CD9 expression.

29. The method of any one of claims 26 to 28, further comprising: associating the determined relative levels of binding with a quantitative amount of the different antigens on the (hES) cells; and classifying the pluripotency of cells in the population of hES cells based on a determined quantitative level of GCTM-2 and CD-9 on each cell.

30. The method of any one of claims 26 to 29, further comprising contacting the (hES) cells with at least one additional antibody, wherein the antibody binds an antigen selected from the group consisting of CD9, GCTM-2. Oct-4, TG343, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, HSPAS and SSEA- 1-.

31. The method of claim 30, wherein the antibodies comprises a formulation of different antibodies which together bind to each of CD9⁺, GCTM-2' , Oct-4⁺ and SSEA- I -.

32. The method of any one of claims 26 to 31 , wherein the antibodies are monoclonal antibodies.

33. A cell population identified by the method according to any one of claims 26 to 32.

34. A method of sorting cells, the method comprising providing a sample of cells; and sorting cells in the sample based on quantitative levels of expression of at least one gene, wherein the gene is selected from the genes set out in Table 1 and combinations thereof.

35. The method of claim 34, wherein the sorting is carried out using a formulation of a plurality of different antibodies.

36. The method of claim 34 or 35, further comprising assigning the sorted cells Io subsets related to a stage of cell differentiation.

37. The method of any one of claims 34 to 36, wherein the sorting is cytometric sorting.

38. The method of any one of claims 34 to 37, wherein the cells, are stow cells.

39. The method of claim 38, wherein the stem cells arc embryonic stem cells.

40. The method of claim 38, wherein the stem cells are human embryonic stem cells.

41. The method of any one of claims 34 to 40, further comprising sorting the cells based on quantitative levels of at least three genes wherein in two of the genes are GCTM-2 and

CD9.

42. The method of claim 41, further comprising sorting the cells based on quantitative levels an additional markers selected from the group consisting of Oct-4, TG343., SSEA-3, SSEA-4, Tra 1 -60, Tra 1-81, HSPA8, SSEA-1- and combinations thereof.

43, The method of claim 42, wherein the cell sorting is based on quantitative levels of at leant five genes wherein four of the genes are CD9, GCTM-2, Oct-4 and SSEA- 1.

44. The method of any one of. claims 34 to 43, at least one of the genes is selected from the group consisting of six transmembrane epithelial antigen of the prostate, supervillin, the chondroitin sulphate proteoglycans bamacan, versican, and opticin, SIDF, Intm 2a and combinations thereof-

45. The method of any one of claims 34 to 44, wherein the sample consists essentially of hES cells, or cells derived therefrom, grown under substantially similar conditions.