Patent Publication Number: US-2004053869-A1

Title: Stem cell differentiation

Description:
[0001] The invention relates to a method to modulate stem cell differentiation comprising introducing inhibitory RNA (RNAi) into a stem cell to ablate mRNA&#39;s which encode polypeptides which are involved in stem cell differentiation. Typically these mRNA&#39;s encode negative regulators of differentiation the removal of which promotes differentiation into a particular cell type(s).  
       [0002] A number of techniques have been developed in recent years which purport to specifically ablate genes and/or gene products. For example, the use of anti-sense nucleic acid molecules to bind to and thereby block or inactivate target mRNA molecules is an effective means to inhibit the production of gene products. This is typically very effective in plants where anti-sense technology produces a number of striking phenotypic characteristics. However, antisense is variable leading to the need to screen many, sometimes hundreds of, transgenic organisms carrying one or more copies of an antisense transgene to ensure that the phenotype is indeed truly linked to the antisense transgene expression. Antisense techniques, not necessarily involving the production of stable transfectants, have been applied to cells in culture, with variable results.  
       [0003] In addition, the ability to be able to disrupt genes via homologous recombination has provided biologists with a crucial tool in defining developmental pathways in higher organisms. The use of mouse gene “knock out” strains has allowed the dissection of gene function and the probable function of human homologues to the deleted mouse genes, (Jordan and Zant, 1998).  
       [0004] A much more recent technique to specifically ablate gene function is through the introduction of double stranded RNA, also referred to as inhibitory RNA (RNAi), into a cell which results in the destruction of mRNA complementary to the sequence included in the RNAi molecule. The RNAi molecule comprises two complementary strands of RNA (a sense strand and an antisense strand) annealed to each other to form a double stranded RNA molecule. The RNAi molecule is typically derived from exonic or coding sequence of the gene which is to be ablated.  
       [0005] Recent studies suggest that RNAi molecules ranging from 100-1000 bp derived from coding sequence are effective inhibitors of gene expression. Suprisingly, only a few molecules of RNAi are required to block gene expression which implies the mechanism is catalytic. The site of action appears to be nuclear as little if any RNAi is detectable in the cytoplasm of cells indicating that RNAi exerts its effect during mRNA synthesis or processing.  
       [0006] The exact mechanism of RNAi action is unknown although there are theories to explain this phenomenon. For example, all organisms have evolved protective mechanisms to limit the effects of exogenous gene expression. For example, a virus often causes deleterious effects on the organism it infects. Viral gene expression and/or replication therefore needs to be repressed. In addition, the rapid development of genetic transformation and the provision of transgenic plants and animals has led to the realisation that transgenes are also recognised as foreign nucleic acid and subjected to phenomena variously called quelling (Singer and Selker, 1995), gene silencing (Matzke and Matzke, 1998), and co-suppression (Stam et. al., 2000).  
       [0007] Initial studies using RNAi used the nematode  Caenorhabditis elegans.  RNAi injected into the worm resulted in the disappearance of polypeptides corresponding to the gene sequences comprising the RNAi molecule(Montgomery et. al., 1998; Fire et. al., 1998). More recently the phenomenon of RNAi inhibition has been shown in a number of eukaryotes including, by example and not by way of limitation, plants, trypanosomes (Shi et. al., 2000) Drosophila spp. (Kennerdell and Carthew, 2000). Recent experiments have shown that RNAi may also function in higher eukaryotes. For example, it has been shown that RNAi can ablate c-mos in a mouse ooctye and also E-cadherin in a mouse preimplanation embryo (Wianny and Zernicka-Goetz, 2000). This suggests that it may be possible to influence the developmental fate of early embryonic cells.  
       [0008] During mammalian development those cells that form part of the embryo up until the formation of the blastocyst are said to be totipotent (e.g. each cell has the developmental potential to form a complete embryo and all the cells required to support the growth and development of said embryo). During the formation of the blastocyst, the cells that comprise the inner cell mass are said to be pluripotential (e.g. each cell has the developmental potential to form a variety of tissues).  
       [0009] Embryonic stem cells (ES cells, those with pluripotentiality) may be principally derived from two embryonic sources. Cells isolated from the inner cell mass are termed embryonic stem (ES) cells. In the laboratory mouse, similar cells can be derived from the culture of primordial germ cells isolated from the mesenteries or genital ridges of days 8.5-12.5 post coitum embryos. These would ultimately differentiate into germ cells and are referred to as embryonic germ cells (EG cells). Each of these types of pluripotential cell has a similar developmental potential with respect to differentiation into alternate cell types, but possible differences in behaviour (eg with respect to imprinting) have led to these cells to be distinguished from one another.  
       [0010] Typically ES/EG cell cultures have well defined characteristics. These include, but are not limited to;  
       [0011] i) maintenance in culture for at least 20 passages when maintained on fibroblast feeder layers;  
       [0012] ii) produce clusters of cells in culture referred to as embryoid bodies;  
       [0013] iii) ability to differentiate into multiple cell types in monolayer culture;  
       [0014] iv) can form embryo chimeras when mixed with an embryo host;  
       [0015] v) express ES/EG cell specific markers.  
       [0016] Until very recently, in vitro culture of human ES/EG cells was not possible. The first indication that conditions may be determined which could allow the establishment of human ES/EG cells in culture is described in WO96/22362. The application describes cell lines and growth conditions which allow the continuous proliferation of primate ES cells which exhibit a range of characteristics or markers which are associated with stem cells having pluripotent characteristics.  
       [0017] More recently Thomson et al (1998) have published conditions in which human ES cells can be established in culture. The above characteristics shown by primate ES cells are also shown by the human ES cell lines. In addition the human cell lines show high levels of telomerase activity, a characteristic of cells which have the ability to divide continuously in culture in an undifferentiated state. Another group (Reubinoff et. al., 2000) have also reported the derivation of human ES cells from human blastocyts. A third group (Shamblott et. al., 1998) have described EG cell derivation.  
       [0018] A feature of ES/EG cells is that, in the presence of fibroblast feeder layers, they retain the ability to divide in an undifferentiated state for several generations. If the feeder layers are removed then the cells differentiate. The differentiation is often to neurones or muscle cells but the exact mechanism by which this occurs and its control remain unsolved.  
       [0019] In addition to ES/EG cells a number of adult tissues contain cells with stem cell characteristics. Typically these cells, although retaining the ability to differentiate into different cell types, do not have the pluripotential characteristics of ES/EG cells. For example haemopoietic stem cells have the potential to form all the cells of the haemopoietic system (red blood cells, macrophages, basophils, eosinophils etc). All of nerve tissue, skin and muscle retain pools of cells with stem cell potential. Therefore, in addition to the use of embryonic stem cells in developmental biology, there are also adult stem cells which may also have utility with respect to determining the factors which govern cell differentiation. Further recent studies have suggested that some stem cells previously thought to be committed to a single fate, (e.g neurons) may indeed possess considerable pluripotentcy in certain situations. Neural stem cells have recently been shown to chimerise a mouse embryo and form a wide range of non-neural tissue (Clark et. al., 2000).  
       [0020] A further group of cells which have relevance to developmental biology are teratocarcinoma cells (EC cells). These cells form tumours referred to as teratomas and have many features in common with ES/EG cells. The most important of these features is the characteristic of pluripotentiality.  
       [0021] Teratomas contain a wide range of differentiated tissues, and have been known in humans for many hundreds of years. They typically occur as gonadal tumours of both men and women. The gonadal forms of these tumours are generally believed to originate from germ cells, and the extra gonadal forms, which typically have the same range of tissues, are thought to arise from germ cells that have migrated incorrectly during embryogenesis. Teratomas are therefore generally classed as germ cell tumours which encompasses a number of different types of cancer. These include seminoma, embryonal carcinoma, yolk sac carcinoma and choriocarcinoma.  
       [0022] The similar biology of EC cells with ES/EG cells has been exploited to study the developmental fates of cells and to identify cell markers commonly expressed in EC cells and ES/EG cells. For example, and not by way of limitation, the expression of specific cell surface markers SSEA-3 (+), SSEA-4 (+), TRA-1-60 (+), TRA-1-81 (+) (Shevinsky et al 1982; Kannagi et al 1983; Andrews et al 1984a; Thomson et al 1995); alkaline phosphatase (+) (Andrews et. al., 1996); and Oct. 4 (Scholer et. al., 1989; Kraft et. al., 1996; Reubinoff et. al., 2000; Yeom et. al., 1996).  
       [0023] We have accumulated expression studies which identify a number of genes thought to be involved in determining the developmental fate of stem cells, particularly embryonic stem cells. By Northern blotting we have identified the expression of human homologs of two signalling pathways believed to be critical in cell fate determination. Expression of ligands, receptors and downstream components of the Notch and Wingless signalling cascades have been elucidated. Using the model system NTERA2/D1 embryonal carcinoma cells we have recorded changes in the expression of some of these components as the cells differentiate. Baring in mind the role these cascades play in embryonic development throughout the animal kingdom, these changes suggest a significant role for both the wingless and Notch signalling pathways in differentiation of stem cells. Furthermore the activity of some genes are required for differentiation to occur along specific pathways e.g. the myogenic gene MyoD1. Other genes have activity which inhibits cellular differentiation along particular pathways. We envisage regulation of stem cell differentiation to yield a specific cell type could be achieved by:  
       [0024] (i) inhibition of certain genes that normally promote differentiation along particular pathways; therefore promoting differentiation to alternate cell phenotypes;  
       [0025] (ii) inhibition of gene activity that prevents differentiation into particular cell types; and  
       [0026] (iii) a combination of (i) and (ii), see FIG. 1  
       [0027] The differentiation of stem cells during embryogenesis, during tissue renewal in the adult and wound repair is under very stringent regulation: aberrations in this regulation underlie the formation of birth defects during development and are thought to underlie cancer formation in adults. Generally, it is envisaged that such stem cells are under both positive and negative regulation which allows a fine degree of control over the process of cell proliferation and cell differentiation: excess proliferation at the expense of cell differentiation can lead to the formation of an expanding mass of tissue—a cancer—whereas express differentiation at the expense of proliferation can lead to the loss of stem cells and production of too little differentiated tissue in the long term, and especially the loss of regenerative potential. Certain genes have already been identified to have a negative role in preventing stem cell differentiation. Such genes, like those of the Notch family, when mutated to acquire activity can inhibit differentiation; such mutant genes act as oncogenes. On the contrary, loss of function of such genes on their inhibition results in stem cell differentiation. We propose to use EC cells has our model cell system to follow the effects of RNAi on cell fate.  
       [0028] According to a first aspect of the invention there is provided a method to modulate the differentiation state of a stem cell comprising:  
       [0029] (i) contacting a stem cell with at least one inhibitory RNA (RNAi) molecule comprising a sequence of a gene, or the effective part thereof, which mediates at least one step in the differentiation of said cell;  
       [0030] (ii) providing conditions conducive to the growth and differentiation of the cell treated in (i) above; and optionally  
       [0031] (iii) maintaining and/or storing the cell in a differentiated state.  
       [0032] The stem cell in (i) above may be a teratocarcinoma cell.  
       [0033] In a preferred method of the invention said conditions are in vitro cell culture conditions.  
       [0034] In a preferred method of the invention said stem cell is selected from: pluripotent stem cells such as an embryonic stem cell or embryonic germ cell; and lineage restricted stem cells such as, but not restricted to; haemopoietic stem cell; muscle stem cell; nerve stem cell; skin dermal sheath stem cell.  
       [0035] It will be apparent that the method can provide stem cells of intermediate commitment. For example, embryonic stem cells could be programmed to differentiate into haemopoietic stems cells with a restricted commitment. Alternatively, differentiated cells or stem cells of intermediate commitment could be reprogrammed to a more pluripotential state from which other differentiated cell lineages can be derived.  
       [0036] In a further preferred method of the invention said stem cell is an embryonic stem cell or embryonic germ cell.  
       [0037] In a yet further preferred method of the invention said gene encodes a cell surface receptor expressed by the stem cell.  
       [0038] In a further preferred method of the invention said cell surface receptor is selected from: human Notch 1(hNotch 1); hNotch 2; hNotch 3; hNotch 4; TLE-1; TLE-2; TLE-3; TLE-4; TCF7; TCF7L1; TCFFL2; TCF3; TCF19; TCF1; mFringe; lFringe; rFringe; sel 1; Numb; Numblike; LNX; FZD1; FZD2; FZD3; FZD4; FZD5; FZD6; FZD7; FZD8; FZD9; FZD10; FRZB.  
       [0039] In an alternative preferred method of the invention said gene encodes a ligand.  
       [0040] Typically, a ligand is a polypeptide which binds to a cognate receptor to induce or inhibit an intracellular or intercellular response. Ligands may be soluble or membrane bound.  
       [0041] In a further alternative preferred method of the invention said ligand is selected from: D11-1; D113; D114; Dlk-1; Jagged 1; Jagged 2; Wnt 1; Wnt 2; Wnt 2b; Wnt 3; Wnt 3a; Wnt5a; Wnt6; Wnt7a; Wnt7b; Wnt8a; Wnt8b; Wnt10b; Wnt11; Wnt14; Wnt15.  
       [0042] Alternatively, said gene is selected from: SFRP1; SFRP2; SFRP4; SFRP5; SK; DKK3; CER1; WIF-1; DVL1; DVL2; DVL3; DVL1L1; mFringe; lFringe; rFringe; sel11; Numb; LNX Oct4; NeuroD1; NeuroD2; NeuroD3; Brachyury; MDFI.  
       [0043] In a further preferred method of the invention of the invention said sequence comprises at least one of the sequences identified in Table 4 which are incorporated by reference.  
       [0044] In a yet further preferred method according to the invention said gene is selected from the group consisting of: DLK1; Oct 4; hNotch 1; hNotch 2; RBPJk; and CIR.  
       [0045] In a further preferred method according to the invention said gene is DLK1. Preferably the DLK1 RNAi molecule is derived from the nucleic acid sequence comprising the sequence presented in FIG. 2 a.    
       [0046] In a further preferred method according to the invention said gene is Oct 4. Preferably the Oct 4 RNAi molecule is derived from the nucleic acid sequence comprising the sequence presented in FIG. 2 b.    
       [0047] In a further preferred method according to the invention said gene is hNotch 1. Preferably said hNotch 1 RNAi molecule is derived from the nucleic acid sequence comprising the sequence presented in FIG. 2 c.    
       [0048] In a further preferred method according to the invention said gene is hNotch 2. Preferably said hNotch 2 RNAi molecule is derived from the nucleic acid sequence comprising the sequence presented in FIG. 2 d.    
       [0049] In a further preferred method according to the invention said gene is RBPJk. Preferably said RBPJk RNAi molecule is derived from the nucleic acid sequence comprising the sequence presented in FIG. 2 e . RBPJk is also referred to as CBF-1.  
       [0050] In a further preferred method according to the invention said gene is CIR. Preferably said CIR RNAi molecule is derived from the nucleic acid sequence comprising the sequence presented in FIG. 2 f.    
       [0051] Many methods have been developed over the last 30 years to facilitate the introduction of nucleic acid into cells which are well known in the art and are applicable to RNAi&#39;s.  
       [0052] Methods to introduce nucleic acid into cells typically involve the use of chemical reagents, cationic lipids or physical methods. Chemical methods which facilitate the uptake of DNA by cells include the use of DEAE-Dextran (Vaheri and Pagano Science 175: p434). DEAE-dextran is a negatively charged cation which associates and introduces the nucleic acid into cells. Calcium phosphate is also a commonly used chemical agent which when co-precipitated with nucleic acid introduces the nucleic acid into cells (Graham et al Virology (1973) 52: p456).  
       [0053] The use of cationic lipids (eg liposomes (Felgner (1987) Proc. Natl. Acad. Sci USA, 84:p7413) has become a common method. The cationic head of the lipid associates with the negatively charged nucleic acid backbone to be introduced. The lipid/nucleic acid complex associates with the cell membrane and fuses with the cell to introduce the associated nucleic acid into the cell. Liposome mediated nucleic acid transfer has several advantages over existing methods. For example, cells which are recalcitrant to traditional chemical methods are more easily transfected using liposome mediated transfer.  
       [0054] More recently still, physical methods to introduce nucleic acid have become effective means to reproducibly transfect cells. Direct microinjection is one such method which can deliver nucleic acid directly to the nucleus of a cell (Capecchi (1980) Cell, 22:p479). This allows the analysis of single cell transfectants. So called “biolistic” methods physically shoot nucleic acid into cells and/or organelles using a particle gun (Neumann (1982) EMBO J, 1: p841). Electroporation is arguably the most popular method to transfect nucleic acid. The method involves the use of a high voltage electrical charge to momentarily permeabilise cell membranes making them permeable to macromolecular complexes.  
       [0055] More recently still a method termed immunoporation has become a recognised techinque for the introduction of nucleic acid into cells, see Bildirici et al Nature (2000) 405, p298. The technique involves the use of beads coated with an antibody to a specific receptor. The transfection mixture includes nucleic acid, antibody coated beads and cells expressing a specific cell surface receptor. The coated beads bind the cell surface receptor and when a shear force is applied to the cells the beads are stripped from the cell surface. During bead removal a transient hole is created through which nucleic acid and/or other biological molecules can enter. Transfection efficiency of between 40-50% is achievable depending on the nucleic acid used. In addition the specificity of cell delivery of RNAi&#39;s can be enhanced by association or linkage of the RNAi to specific antibodies, ligands or receptors.  
       [0056] According to a further aspect of the invention there is provided an RNAi molecule characterised in that it comprises the coding sequence of at least one gene which mediates at least one step in stem cell differentiation.  
       [0057] In a preferred embodiment said coding sequence is an exon.  
       [0058] Alternatively said RNAi molecule is derived from intronic sequences or the 5′ and/or 3′ non-coding sequences which flank coding/exon sequences of genes which mediate stem cell differentiation.  
       [0059] In a further preferred embodiment of the invention the length of the RNAi molecule is between 100 bp-1000 bp. More preferably still the length of RNAi is selected from 100 bp; 200 bp; 300 bp; 400 bp; 500 bp; 600 bp; 700 bp; 800 bp; 900 bp; or 1000 bp. More preferably still said RNAi is at least 1000 bp.  
       [0060] In an alternative preferred embodiment of the invention the RNAi molecule is between 15 bp and 25 bp, preferably said molecule is 21 bp.  
       [0061] In a further preferred embodiment of the invention said RNAi molecule comprises sequences identified in Table 4 which are incorporated by reference.  
       [0062] In a preferred embodiment of the invention said RNAi molecule is derived from a gene selected from the group consisting of: DLK1; Oct 4; hNotch 1; hNotch 2; RBPJk; and CIR. Preferably said RNAi molecule comprise a nucleic acid sequence selected from the group consisting of the nucleic acid sequences presented in FIGS. 2 a - 2   f.    
       [0063] In yet a further preferred embodiment of the invention said RNAi molecules comprise modified ribonucleotide bases.  
       [0064] It will be apparent to one skilled in the art that the inclusion of modified bases, as well as the naturally occuring bases cytosine, uracil, adenosine and guanosine, may confer advantageous properties on RNAi molecules containing said modified bases. For example, modified bases may increase the stability of the RNAi molecule thereby reducing the amount required to produce a desired effect.  
       [0065] According to a further aspect of the invention there is provided an isolated DNA molecule comprising a sequence of a gene which mediates at least one step in stem cell differentiation as represented by the DNA accession numbers identified in Table 4 characterised in that said DNA is operably linked to at least one further DNA molecule capable of promoting transcription (“a promoter”) of said DNA linked thereto.  
       [0066] In a preferred embodiment of the invention said gene is selected from the group consisting of: DLK1; Oct 4; hNotch 1; hNotch 2; RBPJk; and CIR. Preferably said DNA comprises a sequence selected from the group consisting of the sequences as represented in FIGS. 2 a - 2   f.    
       [0067] In a further preferred embodiment of the invention said gene is provided with at least two promoters characterised in that said promoters are oriented such that both DNA strands comprising said DNA molecule are transcribed into RNA.  
       [0068] It will be apparent to one skilled in the art that the synthesis of RNA molecules which form RNAi can be achieved by providing vectors which include target genes, or fragments of target genes, operably linked to promoter sequences. Typically, promoter sequences are phage RNA polymerase promoters (eg T7, T3, SP6). Advantageously vectors are provided with with multiple cloning sites into which genes or gene fragments can be subcloned. Typically, vectors are engineered so that phage promoters flank multiple cloning sites containing the gene of interest. Phage promoters are oriented such that one promoter synthesises sense RNA and another phage promoter, antisense RNA. Thus, the synthesis of RNAi is facilitated.  
       [0069] Alternatively target genes or fragments of target genes can be fused directly to phage promoters by creating chimeric promoter/gene fusions via oligo-synthesising technology. Constructs thus created can be easily amplified by polymerase chain reaction to provide templates for the manufacture of RNA molecules comprising RNAi.  
       [0070] According to a further aspect of the invention there is provided a vector including a DNA molecule according to the invention.  
       [0071] According to a farther aspect of the invention there is provided a method to manufacture RNAi molecules comprising:  
       [0072] (i) providing DNA molecule or vector according to the invention;  
       [0073] (ii) providing reagents and conditions which allow the synthesis of each RNA strand comprising said RNAi molecule; and  
       [0074] (iii) providing conditions which allow each RNA strand to associate over at least part of their length, or at least that part corresponding to the nucleic acid sequence encoding said stem cell gene which mediates stem cell differentiation.  
       [0075] Preferably said gene, or gene fragment is selected from those genes represented in table 4.  
       [0076] In vitro transcription of RNA is an established methodology. Kits are commercially available which provide vectors, ribonucleoside triphosphates, buffers, Rnase inhibitors, RNA polymersases (eg phage T7, T3, SP6) which facilitate the production of RNA.  
       [0077] According to a further aspect of the invention there is provided an in vivo method to promote the differentiation of stem cells comprising administering to an animal an effective amount of RNAi according to the invention sufficient to effect differentiation of a target stem cell. Preferably said method promotes differentiation in vivo of endogenous stem cells to repair tissue damage in situ.  
       [0078] It will be apparent to one skilled in the art that RNAi relies on homology between the target gene RNA and the RNAi molecule. This confers a significant degree of specificity to the RNAi molecule in targeting stem cells. For example, haemopoietic stem cells are found in bone marrow and RNAi molecules may be administered to an animal by direct injection into bone marrow tissue.  
       [0079] RNAi molecules may be encapsulated in liposomes to provide protection from an animals immune system and/or nucleases present in an animals serum.  
       [0080] Liposomes are lipid based vesicles which encapsulate a selected therapeutic agent which is then introduced into a patient. Typically, the liposome is manufactured either from pure phospholipid or a mixture of phospholipid and phosphoglyceride. Typically liposomes can be manufactured with diameters of less than 200 nm, this enables them to be intravenously injected and able to pass through the pulmonary capillary bed. Furthermore the biochemical nature of liposomes confers permeability across blood vessel membranes to gain access to selected tissues. Liposomes do have a relatively short half-life. So called STEALTH R  liposomes have been developed which comprise liposomes coated in polyethylene glycol (PEG). The PEG treated liposomes have a significantly increased half-life when administered intravenously to a patient. In addition STEALTH R  liposomes show reduced uptake in the reticuloendothelial system and enhanced accumulation selected tissues. In addition, so called immuno-liposomes have been develop which combine lipid based vesicles with an antibody or antibodies, to increase the specificity of the delivery of the RNAi molecule to a selected cell/tissue.  
       [0081] The use of liposomes as delivery means is described in U.S. Pat. No. 5,580,575 and U.S. Pat. No. 5,542,935.  
       [0082] It will be apparent to one skilled in the art that the RNAi molecules can be provided in the form of an oral or nasal spray, an aerosol, suspension, emulsion, and/or eye drop fluid. Alternatively the RNAi molecules may be provided in tablet form. Alternative delivery means include inhalers or nebulisers.  
       [0083] According to a yet further aspect of the invention there is provided a therapeutic composition comprising at least one RNAi molecule according to the invention.  
       [0084] Preferably said RNAi molecule is for use in the manufacture of a medicament for use in promoting the differentiation of stem cells to provide differentiated cells/tissues to treat diseases where cell/tissues are destroyed by said disease. Typically this includes pernicious anemia; stroke, neurodegenerative diseases such as Parkinson&#39;s disease, Alzhiemer&#39;s disease; coronary heart disease; cirrhosis; diabetes. It will also be apparent that differentiated stem cells may be used to replace nerves damaged as a consequence of (eg replacement of spinal cord tissue).  
       [0085] In a further preferred embodiment of the invention said therapeutic composition further comprises a diluent, carrier or excipient.  
       [0086] According to a further aspect of the invention there is provided a therapeutic cell composition comprising a differentiated cell produced by introduction of a RNAi molecule or composition according to the invention.  
       [0087] According to a further aspect of the invention there is provided a cell obtainable by the method according to the invention.  
       [0088] In a preferred embodiment of the invention said cell is selected from the group consisting of: a nerve cell; a mesenchymal cell; a muscle cell (cardiomyocyte); a liver cell; a kidney cell; a blood cell (eg erythrocyte, CD4+ lymphocyte, CD8+ lymphocyte; panceatic β cell; epithelial cell (eg lung, gastric,); and a endothelial cell.  
       [0089] According to a further aspect of the invention there is provided a cell culture obtainable by the method according to the invention.  
       [0090] According to a yet further aspect of the invention there is provided at least one organ comprising at least one cell according to the invention.  
       [0091] An embodiment of the invention will now be described by example only and with reference to the following figures and tables wherein:  
       [0092] Table 1 represents a selection of antibodies used to monitor stem cell differentiation;  
       [0093] Table 2 represents nucleic acid probes used to assess mRNA markers of stem differentiation;  
       [0094] Table 3 represents protein markers of stem cell differentiation;  
       [0095] Table 4 represents specific primers used to generate RNAi for gene specific inhibition and gene sequences with DNA database accession numbers;  
       [0096] Table 5 represents a summary of FACS data presented in FIG. 3; 
     
    
    
     [0097]FIG. 1 illustrates stem cell differentiation is controlled by positive and negative regulators (A). The specific cell phenotypes that are derived are a direct result of positive and negative regulators which activate or suppress particular differentiation events. RNAi can be used to control both the initial differentiation of stem cells (A) and the ultimate fate of the differentiated cells D1 and D2 by repression of positive activators which would normally promote a particular cell fate;  
     [0098]FIG. 2 a  represents the forward and reverse primers used to amplify delta-like 1 (DLK1) and the amplified sequence; FIG. 2 b  represents the forward and reverse primers used to amplify Oct 4 and the amplified sequence; FIG. 2 c  represents the forward and reverse primers used to amplify Notch 1 and the amplified sequence; FIG. 2 d  represents the forward and reverse primers used to amplify Notch 2 and the amplified sequence; FIG. 2 e  represents the forward and reverse primers used to amplify RBPJK and the amplified sequence; and FIG. 2 f  represents the forward and reverse primers used to amplify CIR and the amplified sequence;  
     [0099]FIG. 3 represents a FACS scan of monitoring the expression of SSEA3 by NTERA2cl D1 human EC cells following RNAi to Notch (A), RBPJk(B), Oct 4 (C) and control RNAi (D). Flow cytofluorimetric analysis of SSEA3 expression by NTERA2 cl.D1 human EC cells, 4 days following transfection with RNAi directed to a) Notch1 and Notch2; b) RBPJk; c) Oct4; d) control RNAi. Each panel shows two histograms of cell number against log fluorescence intensity (arbitrary units), after staining cells with monoclonal antibody MC631 (anti SSEA3) followed by FITC labelled goat anti-mouse IgM. In each panel, one histogram was derived from ‘mock’ transfected cells that had been treated with all relevant reagents except RNAi; the second histogram in each panel was derived from cells treated with RNAi directed to the set of genes as described above. Note that the cells exhibit a bimodal histogram in all cases representing SSEA3+ and SSEA3− populations (regions marked M1 and M2 respectively). Note that following treatment with RNAi to Notch1 and Notch2 (Panel A) and Oct4 (Panel c), there was a marked downward shift in the fluorescence intensity of the SSEA3+ population, denoting evidence of stem cell differentiation. A smaller shift, also downwards, was evident in cells treated with RBPJk (Panel B). Such results would be anticipated if these gene products play a role in maintenance of an undifferentiated EC cell phenotype, and if treatment with RNAi directed to the corresponding mRNA results in down regulation of these key regulatory proteins. By contrast, treatment with control RNAi (Panel D) did not result in any down regulation of SSEA3. Expression of SSEA3 appears to be a very sensitive marker of an undifferentiated EC stem cell phenotype and is one of the most rapid markers to disappear upon differentiation (Fenderson et al 1987; Andrews et al 1996). Likewise SSEA3 is expressed by human ES cells (Thomson et al 1998) and also disappears rapidly upon their differentiation (P W Andrews and J S Draper, unpublished results);  
     [0100]FIG. 4 represents (A) a schematic diagram illustrating the Notch and Wnt signalling pathways. The Notch and Wnt signaling pathways are shown. Ligands of the Delta/Serrate/Lag (DSL) family bind Notch receptors, leading to activation of Suppressor of Hairless (Su-H)/CBF1/RBPJk and enhanced transcription of target genes. (B) a northern blot analysis of the expression of the DLS ligand Dlk and the Notch target gene TLE1 in NTERA2 EC cells. TLE1 was identified as a target gene of the Notch pathway in NTERA2 EC cells. TLE1 shows a pattern of expression highly similar to that of the DSL ligand, Dlk1, during retinoic acid-induced differentiation. At 3 days following RA treatment (RA3), both genes are substantially downregulated. At subsequent time points, a progressive recovery in expression is seen, through to 21 days after RA treatment (RA21). The downregulation of TLE1 indicates that the cells have entered a differentiation pathway. (C) RT PCR analysis of TLE1 and HASH1 in RNAi treated ES cells. RT-PCR was performed for TLE1 and HASH1 3 days after dsRNA treatment. Lane 1: water; lane 2: untreated ES cells; lane 3: mock transfection; lane 4: Notch 1&amp; 2 dsRNA; lane 5: DlkI dsRNA; lane 6: RBPJk dsRNA; lane 7: CIR dsRNA; lane 8: Oct4 dsRNA; lane 9: control dsRNA. Note the specific reduction of TLE1 expression in lanes 5 and 6, corresponding to samples in which components of the Notch signaling pathway have been targeted by dsRNA. Also note the appearance of HASH1 in lane 5. These data indicate that the cells are embarking on a program of neural differentiation (de la Pompa et al, Conservation of the Notch signalling pathway in mammalian neurogenesis. Development 124, 1139-1148 (1997). The failure of Notch1&amp;2 dsRNA to induce a similar effect is due to functional redundancy of the receptor system, or a high abundance of receptor in relation to other pathway components.  
     [0101]FIG. 5 represents RNAi of human ES cells using RNAi molecules derived from different genes involved in stem cell differentiation using RT PCR to monitor steady-state levels of mRNA. RT-PCR analysis of targeted transcript abundance in human embryonic stem cells 3 days after dsRNA treatment. Lane 1: water; lane 2: untreated ES cells; lane 3: mock transfection; lane 4: Notch 1&amp;2 dsRNA; lane 5: Dlk1 dsRNA; lane 6: RBPJk (CBF]) dsRNA; lane 7: CIR dsRNA; lane 8: Oct4 dsRNA; lane 9: control dsRNA. Note that specific reduction in targeted transcript abundance persists for at least 3 days after dsRNA treatment. The effect is especially prominent in cells treated with the Notch 1&amp;2, RBPJk (CBF1) and Oct4 dsRNAs. Beta Actin PCR was used as a template loading control for PCR.  
     [0102]FIG. 6 represents RNAi of NTERA2/D1 using RNAi molecules derived from different genes involved in stem cell differentiation using RT PCR to monitor steady-state levels of mRNA. RT-PCR analysis of targeted transcript abundance in the human embryonal carcinoma cell line, NTERA2, 17 hours after dsRNA treatment. Lane 1: water; lane 2: untreated EC cells; lane 3: Oct4 dsRNA; lane 4:control dsRNA; lane 5: RBPJk dsRNA; lane 6: Notch 1&amp;2 dsRNA; lane 7: mock transfection. Note the specific and substantial reduction of targeted transcript abundance. Beta Actin PCR was used as a template loading control.  
    
    
     MATERIALS AND METHODS  
     [0103] Cell Culture  
     [0104] NTERA2 and 2102Ep human EC cell lines were maintained at high cell density as previously described (Andrews et al 1982, 1984b), in DMEM (high glucose formulation) (DMEM)(GIBCO BRL), supplemented with 10% v/v bovine foetal calf serum (GIBCO BRL), under a humidified atmosphere with 10% CO 2  in air.  
     [0105] Double Stranded RNA Synthesis  
     [0106] PCR primers were designed against the mRNA sequence of interest to give a product size of around 500 bp. At the 5′ end of each primer was added a T7 RNA polymerase promoter, comprising one or other of the following sequences: TAATACGACTCACTATAGGG; AATTATAATACGACTCACTATA. PCR was performed using these primers on an appropriate cDNA source (e.g. derived from the cell type to be targeted) and the product cloned and sequenced to confirm its identity. Using the sequenced clone as a template, further PCRs were performed as required to generate template DNA for RNA synthesis. In each case, a quantity of the PCR was electrophoresed through agarose to verify product size and abundance, whilst the remainder was purified by alkaline phenol/chloroform extraction. RNA was synthesized using the Megascript kit (Ambion Inc.) according to the manufacturer&#39;s protocol and acid phenol/chloroform extracted. The simultaneous synthesis of complementary strands of RNA in a single reaction circumvents the requirement for an annealing step. However, the quality and duplexing of the synthesized RNA was confirmed by agarose gel electrophoresis, with the desired products migrating as expected for double stranded DNA of the same length.  
     [0107] Treatment of Human Cells with dsRNA to Produce RNAi  
     [0108] The following method describes RNAi of cells cultured in 6 well plates. Volumes and cell numbers should be scaled appropriately for larger or smaller culture vessels. Cells were seeded at 500,000 per well on the day prior to treatment and grown in their normal medium. For each well to be treated, 9.5 μg of the double stranded RNA of interest was diluted in 300 μl of 150 mM NaCl. 21 μl of ExGen 500 (MBI Fermentas) was added to the diluted RNA solution and mixed by vortexing. The dsRNA/ExGen 500 mixture was incubated at room temperature for 10 minutes. 3ml of fresh cell growth medium was then added, producing the RNAi treatment medium. Growth medium was aspirated from the culture vessel and replaced with 3ml of RNAi treatment medium per well. Culture vessels were then centrifuged at 280 g for 5 minutes and returned to the incubator. After 12-18 hrs, RNAi treatment medium was replaced with normal growth medium and the cells maintained as required.  
     [0109] Oct 4 RNAi Production  
     [0110] PCR primers were designed against the Oct 4 mRNA sequence of interest to give a product size of around 500 bp. At the 5′ end of each primer was added a T7 RNA polymerase promoter, comprising the following sequence: taatacgactcactataggg. PCR was performed using these primers on an appropriate cDNA source (e.g. derived from the cell type to be targeted) and the product cloned and sequenced to confirm its identity. Using the sequenced clone as a template, further PCRs were performed as required to generate template Oct 4 DNA for RNA synthesis. In each case, a quantity of the PCR was electrophoresed through agarose to verify product size and abundance, whilst the remainder was purified by alkaline phenol/chloroform extraction. RNA was synthesized using the Megascript kit (Ambion Inc.) according to the manufacturer&#39;s protocol and acid phenol/chloroform extracted. The simultaneous synthesis of complementary strands of RNA in a single reaction circumvents the requirement for an annealing step. However, the quality and duplexing of the synthesized RNA was confirmed by agarose gel electrophoresis, with the desired products migrating as expected for double stranded DNA of the same length.  
     [0111] Treatment of Human EC Cells with Oct 4 dsRNA to Produce RNAi  
     [0112] The following method describes Oct 4 RNAi of cells cultured in 6 well plates. Volumes and cell numbers should be scaled appropriately for larger or smaller culture vessels.  
     [0113] Cells were seeded at 500,000 per well on the day prior to treatment and grown in their normal medium. On the day of treatment, a 15 ul aliquot of Lipofectin (Gibco BRL) was added to 100 μl of Optimem (Gibco BRL) for each well to be treated. Concurrently, 6 ug of Oct 4 dsRNA was added to 300 ul of Optimem for each well to be treated. The Lipofectin-Optimem and dsRNA-Optimem solutions were incubated at room temperature for 40 minutes, then mixed to produce RNAi treatment medium with a total volume of around 415 ul for each well. The treatment medium was incubated at room temperature for 10 minutes prior to use. During this time, growth medium was aspirated from the cells and each well washed with 3 ml of PBS. The PBS wash was then replaced with RNAi treatment medium, supplemented with a further 0.5 ml of Optimem per well. Culture vessels were returned to the incubator for 6.5 hours, after which the treatment medium was aspirated and replaced with normal growth medium. Target mRNA inhibition was assayed 3 days after treatment by PCR.  
     [0114] RNAi Introduction to Cell Lines  
     [0115] Human EC stem cells were seeded at 2×10 5  cells/well of a 6 well plate in 3 cm 3  of Dulbecco&#39;s modified Eagles medium and allowed to settle for 3 hrs. 6 μg RNAi was added to the medium and the cells were agitated for 30 mins at room temperature.  
     [0116] Foetal calf serum (GIBO BRL) was added to the medium to a concentration of 10% and the cells were grown on.  
     [0117] Total RNA Production  
     [0118] Growing cultures of cells were aspirated to remove the DME and foetal calf serum. Trace amounts of foetal calf serum was removed by washing in Phosphate-buffered saline. Fresh PBS was added to the cells and the cells were dislodged from the culture vessel using acid washed glass beads. The resulting cell suspension was centrifuged at 300×g. The pellets had the PBS aspirated from them. Tri reagent (Sigma, USA) was added at 1 ml per 10 7  cells and allowed to stand for 10 mins at room temperature. The lysate from this reaction was centrifuged at 12000×g for 15 minutes at 4° C. The resulting aqueous phase was transferred to a fresh vessel and 0.5 ml of isopropanol/ml of trizol was added to precipitate the RNA. The RNA was pelleted by centrifugation at 12000×g for 10 mins at 4° C. The supernatant was removed and the pellet washed in 70% ethanol. The washed RNA was dissolved in DEPC treated double-distilled water.  
     [0119] Analysis of the Differentiation of EC Stem Cells Induced by Exposure to RNAi  
     [0120] Following exposure to RNAi corresponding to specific key regulatory genes, the subsequent differentiation of the EC cells was monitored in a variety of ways. One approach was to monitor the disappearance of typical markers of the stem cell phenotype; the other was to monitor the appearance of markers pertinent to the specific lineages induced. The relevant markers included surface antigens, mRNA species and specific proteins.  
     [0121] Analysis of Transfectants by Antibody Staining and FACS  
     [0122] Cells were treated with trypsin (0.25% v/v) for 5 mins to disaggregate the cells; they were washed and re-suspended to 2×10 5  cells/ml. This cell suspension was incubated with 50 μl of primary antibody in a 96 well plate on a rotary shaker for 1 hour at 4° C. Supernatant from a myeloma cell line P3X63Ag8, was used as a negative control. The 96 well plate was centrifuged at 100 rpm for 3 minutes. The plate was washed 3 times with PBS containing 5% foetal calf serum to remove unbound antibody. Cell were then incubated with 50 μl of an appropriate FITC-conjugated secondary antibody at 4° C. for 1 hour. Cells were washed 3 times in PBS +5% foetal calf serum and analysed using an EPICS elite ESP flow cytometer (Coulter eletronics, U.K).(Andrews et. al., 1982)  
     [0123] Northern Blot Analysis of RNA  
     [0124] RNA separation relies on the generally the same principles as standard DNA but with some concessions to the tendancy of RNA to hybridise with itself or other RNA molecules. Formaldehyde is used in the gel matrix to react with the amine groups of the RNA and form Schiff bases. Purified RNA is run out using standard agarose gel electrophresis. For most RNA a 1% agarose gel is sufficiant. The agarose is made in 1×MOPS buffer and supplemeted with 0.66M formaldehyde. Dryed down RNA samples are reconstituted and denatured in RNA loading buffer and loaded into the gel. Gels are run out for apprx. 3 hrs (until the dye front is ¾ of the way down the gel).  
     [0125] The major problem with obtaining clean blotting using RNA is the presence of formaldehyde. The run out gel was soaked in distilled water for 20 mins with 4 changes, to remove the formaldehyde from the matrix. The transfer assembly was assembled in exactly the same fashion as for DNA (Southern) blotting. The transfer buffer used however was 10×SSPE. Gels were transfered overnight. The membrane was soaked in 2×SSPE to remove any agarose from the transfer assembly and the RNA was fixed to the memebrane. Fixation was acheived using short-wave (254 nM) UV light. The fixed membrane was baked for 1-2 hrs to drive off any residual formaldehyde.  
     [0126] Hybridisation was acheived in aqueous phase with formamide to lower the hybridisation temperatures for a given probe. RNA blots were prehybridised for 2-4 hrs in northern prehybridisation soloution. Labelled DNA probes were denatured at 95° C. for 5 mins and added to the blots. All hybridisation steps were carried out in rolling bottles in incubation ovens. Probes were hybridised overnight for at least 16 hrs in the prehybridisation soloution. A standard set of wash soloutions were used. Stringency of washing was acheived by the use of lower salt containing wash buffers. The following wash procedure is outlined as follows  
                                                            2X SSPE   15 mins   room temp             2X SSPE   15 mins   room temp             2X SSPE/0.1% SDS   45 mins   65° C.             2X SSPE/0.1% SDS   45 mins   65° C.           0.1X SSPE   15 mins   room temp                      
 
     [0127] Preparation of Radiolabelled DNA Probes  
     [0128] The method of Feinberg and Vogelstein (Feinberg and Vogelstein, 1983) was used to radioactively label DNA. Briefly, the protocol uses random sequence hexanucleotides to prime DNA synthesis at numerous sites on a denatured DNA template using the Klenow DNA polymerase I fragment. Pre-formed kits were used to aid consistency. 5-100 ng DNA fragment (obtained from gel purifcation of PCR or restriction digests) was made up in water,denatured for 5 mins at 95° C. with the random hexamers. The mixture was quench cooled on ice and the following were added,  
     [0129] 5 μl[α-32P] DATP 3000 Ci/mmol  
     [0130] 1 μl of Klenow DNA polymerase (4U)  
     [0131] The reaction was then incubated at 37° C. for 1 hr. Unincorporated nucleotide were removed with spin columns (Nucleon Biosciences).  
     [0132] Production of cDNA  
     [0133] The enzymatic conversion of RNA into single stranded cDNA was achieved using the 3′ to 5′ polymerase activity of recombinant Moloney-Murine Leukemia Virus (M-MLV) reverse transcriptase primed with oligo (dT) and (dN) primers. For Reverse Transcription-Polymerase Chain Reaction, single stranded cDNA was used. cDNA was synthesised from 1 μg poly (A)+ RNA or total RNA was incubated with the following  
                                      1.0 μM   oligo(dT) primer for total RNA or random hexcamers for mRNA       0.5 mM   10 mM dNTP mix         1 U/μl   RNAse inhibitor (Promega)       1.0 U/μl   M-MLV reverse transcriptase in manufacturers supplied buffer           (Promega)                          
 
     [0134] Fluorescent Automated Sequencing  
     [0135] To check the specificity of the PCR primers used to generate the template used in RNAi production automatic sequencing was carried out using the prism fluorescently labelled chain terminator sequencing kit (Perkin-Elmer) (Prober et al 1987). A suitable amount of template (200 ng plasmid, 100 ng PCR product), 10 μM sequencing primer (typically a 20 mer with 50% G-C content) were added to 8 μl of prism pre-mix and the total reaction volume made up to 20 μl. 24 cycles of PCR (94° C. for 10 seconds, 50° C. for 10 seconds, 60° C. for 4 minutes). Following thermal cycling, products were precipitated by the addition of 2 μl of 3M sodium acetate and 50 μl of 100 % ethanol. DNA was pelleted in an Eppendorf microcentrifuge at 13000 rpm, washed once in 70% ethanol and vacuum dried. Samples were analysed by the in-house sequencing Service (Krebs Institute). Dried down samples were resuspended in 4 μl of formamide loading buffer, denatured and loaded onto a ABI 373 automatic sequencer. Raw sequence was collected and analysed using the ABI prism software and the results were supplied in the form of analysed histogram traces.  
     [0136] Detection of Specific Protein Targets by SDS-PAGE and Western Blotting  
     [0137] To obtain cell lysates monolayers of cells were rinsed 3 times with ice-cold PBS supplemented with 2 mM CaCl 2 . Cells were incubated with 1 ml/75 cm 2  flask lysis buffer (1% v/v NP40, 1% v/v DOC, 0.1 mM PMSF in PBS) for 15 min at 4° C. Cell lysates were transferred to eppendorf tubes and passed through a 21 gauge needle to shear the DNA. This was followed by freeze thawing and subsequent centrifugation (30 min, 4° C., 15000 g) to remove insoluble material. Protein concentrations of the supernatants were determined using a commercial protein assay (Biorad) and were adjusted to 1.3 mg/ml. Samples were prepared for SDS-PAGE by adding 4 times Laemmli electrophoresis sample buffer and boiling for 5 min. After electrophoresis with 16 μg of protein on a 10% polyacrylamide gel (Laemmli, 1970) the proteins were transferred to nitro-cellulose membrane with a pore size of 0.45 μm. The blots were washed with PBS and 0.05% Tween (PBS-T). Blocking of the blots occurred in 5% milk powder in PBS-T (60 min, at RT). Blots were incubated with the appropriate primary antibody. Horseradish peroxidase labelled secondary antibody was used to visualise antibody binding by ECL (Amersham, Bucks., UK). Materials used for SDS-PAGE and western blotting were obtained from Biorad (California, USA) unless stated otherwise.  
               TABLE 1                          Antibodies used to detect stem cell differentiation                                                 Cell                           phenotype   Changes on       Antibody   Class   Species   detected   Differentiation   Reference               TRA-1-   IgM   Mouse   Human EC,   ↓   Andrews et.       60           ES cells.   differentiation   al., 1984a       TRA-l-   IgM   Mouse   Human EC,   ↓   Andrews et.       81           ES cells.   differentiation   al., 1984a       SSEA3   IgM   Rat   Human EC,   ↓   Shevinskyet                   ES cells.   differentiation   et al 1982,                           Fenderson                           et al 1987       SSEA4   IgG   Mouse   Human EC,   ↓   Kannagi et                   ES cells.   differentiation   al 1983                           Fenderson                           et al 1987       A2B5   IgM   Mouse       ↑   Fenderson                       differentiation   et al 1987       ME311   IgG   Mouse       ↑   Fenderson                       differentiation   et al 1987       VIN-IS-   IgM   Mouse       ↑   Andrews       56               differentiation   et al 1990       VIN-IS-   IgG   Mouse       ↑   Andrews       53               differentiation   et al 1990                  
 
     [0138]               TABLE 2                          Probes used to assess mRNA markers of differentiation                             Gene   Cell Type                       Synaptophysin   Neuron           NeuroD1   Neuron           MyoD1   Muscle           Collagens   Cartlidge           Alpha-actin   Skeletal muscle           Smooth-muscle actin   Smooth muscle                        
     [0139]               TABLE 3                          Protein markers of differentiation, detected by       Western Blot and/or immununofluorescence.       The following antibodies were detected by the       appropriate commercially available antibodies                             Cell Type   Antigen                       Neurons   Neurofilaments           Glial cells   GFAP           Epithelial cells   Cytokeratins           Mesenchymal cells   Vimentin           Muscle   Desmin           Muscle   Tissue specific actins           Connective tissue cells   Collagens                        
     [0140]               TABLE 4                          Specific Primers used to generate dsRNA for gene specific inhibition       All sequences written 5′ to 3′                             Gene   Accession               Name   number   PCR primer Sequences   Position               Notch                   Pathway       Ligands:       Dll-1   AF003522       Dll3   NM_016941       Dll4   NM_019454       Dlk-1   NM_003836   taatacgactcactatagggcctcttgctcct               gctggcttt               taatacgactcactatagggatgggt               tgggggtgcagctgtt       Receptors:       Notch1   M73980   gcggccgcctttgtggttctgttc   5224-5726               gccggcgcgtcctcctcttcc       Notch2   Inhouse   gccagaatgatgctacctgt           sequence   tagagcagcaccaatggaac       Notch3   U97669   aagttacccccaagaggcaagtgtt   7013-7348               aaggaaatgagaggccagaagga               ga       Notch4   U95299   ggctgcccctcccactctcg   3727-4132               cagcccgggccccaggatag       Down-       stream:       TLE-1   NM_005077       TLE-2   M99436       TLE-3   M99438       TLE-4   M99439       TCF7   NM_003202       TCFFL2   Y11306       TCF3   M31523       TCF19   NM_007109       TCF1   NM_000545       mfringe   NM_002405       lfringe   U94354       rFringe   AF108139       Se11   AF157516       Numb   NM_003744       LNX   NM_010727       Wingless       Pathway       Ligands       Wnt1   NM_005430       Wnt2   NM_003391       Wnt2B   NM_004185   tgagtggttcctgtactctg   1159-1503               actcacactgggtaacacgg       Wnt5A   L20861       Wnt6   AF079522       Wnt7A   NM_004625       Wnt8B   NM_003393       Wnt10B   NM_003394       Wnt11   NM_004626       Wnt14   AF028702       Wnt15   AF028703       Wnt16   AF169963       Receptors       FZD1   NM_003505       FZD2   NM_001466   tacccagagcggcctatcattttt   955-1439               acgaagccggccaggaggaaggac       FZD3   NM_017412       FZD4   NM_012193       FZD5   NM_003468       FZD6   NM_003506   tggcctgaggagcttgaatgtgac   607-1026               atcgcccagcaaaaatccaatgaa       FZD7   NM_003507       FZD8   AA481448       FZD9   NM_003508       FZD10   NM_007197       FRZB   NM_001463       Extra-       cellular       Effectors       SFRP1   NM_003012       SFRP2   AFO 17986       SFRP4   AF026692   agaggagtggctgcaatgaggtc   877-1178               gcgcccggctgttttctt       SFRP5   NM_003015       SK   AB020315       CER1   NM_005454       WIF-1   NM_007191       DVL1   U46461       DVL2   NM_004422       DVL3   NM_004423       Tran-       scription       Factors       Oct4   Z11899   taatacgactcactatagggagcag               cttgggctcgagaag               taatacgactcactatagggccctttg               tgttcccaattcc       Brachyury   NM_003181       NeuroD1   NM_002500       NeuroD2   NM_006160       NeuroD3   U63842       MyoD   NM_002478       MDFL   NM_005586       REST   NM_005612                    
     [0141]               TABLE 5                          Mean Fluorescence Intensity of SSEA-3 (+) and SSEA-3 (−)       (M1 and M2) subpopulations of NTERA2 cells treated with dsRNA,       as described in the legend to FIG. 3                         Mean Fluorescence Intensity           (Log scale, Arbitary Units)                         Treatment   M1 = SSEA3 (+)   M2 = SSEA3 (−)                                 Mock (control)   319   2.0       RNAi (Notch 1 + Notch 2)   195   1.7       RNAi (RBPJk)   267   1.8       RNAi (Oct4)   181   1.6       RNAi control   354   1.7                    
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     [0144] Andrews P. W., Banting G. S., Damjanov I., Arnaud D. and Avner P. 1984a. Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells.  Hybridoma.  3: 347-361.  
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     [0147] Fenderson B. A., Andrews P. W., Nudelman E., Clausen H. and Hakomori S.-i. 1987. Glycolipid core structure switching from globo- to lacto- and ganglio-series during retinoic acid-induced differentiation of TERA-2-derived human embryonal carcinoma cells.  Dev. Biol.  122: 21-34.  
     [0148] Kannagi, R., Levery, S. B., Ishigami, F., Hakomori, S., Shevinsky, L. H., Knowles, B. B. and Solter, D. (1983) New globoseries glycosphingolipids in human teratocarcinoma reactive with the monoclonal antibody directed to a developmentally regulated antigen, stage-specific embryonic antigen 3.  J. Biol. Chem.  258, 8934-8942.  
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taatacgact cactataggg cctcttgctc ctgctggctt t                         41 

 
           
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taatacgact cactataggg atgggttggg ggtgcagctg tt                        42 

 
           
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cctcttgctc ctgctggctt tcggccacag cacctatggg gctgaatgct tcccggcctg     60 

caacccccaa aatggattct gcgaggatga caatgtttgc aggtgccagc ctggctggca    120 

gggtcccctt tgtgaccagt gcgtgacctc tcccggctgc cttcacggac tctgtggaga    180 

acccgggcag tgcatttgca ccgacggctg ggacggggag ctctgtgata gagatgttcg    240 

ggcctgctcc tcggccccct gtgccaacaa caggacctgc gtgagcctgg acgatggcct    300 

ctatgaatgc tcctgtgccc ccgggtactc gggaaaggac tgccagaaaa aggacgggcc    360 

ctgtgtgatc aacggctccc cctgccagca cggaggcacc tgcgtggatg atgagggccg    420 

ggcctcccat gcctcctgcc tgtgcccccc tggcttctca ggcaatttct gcgagatcgt    480 

ggccaacagc tgcaccccca acccat                                         506 

 
           
             4  
             40  
             DNA  
             Homo sapiens  
           
            4 

taatacgact cactataggg agcagcttgg gctcgagaag                           40 

 
           
             5  
             40  
             DNA  
             Homo sapiens  
           
            5 

taatacgact cactataggg ccctttgtgt tcccaattcc                           40 

 
           
             6  
             423  
             DNA  
             Homo sapiens  
           
            6 

agcagcttgg gctcgagaag gatgtggtcc gagtgtggtt ctgtaaccgg cgccagaagg     60 

gcaagcgatc aagcagcgac tatgcacaac gagaggattt tgaggctgct gggtctcctt    120 

tctcaggggg accagtgtcc tttcctctgg ccccagggcc ccattttggt gccccaggct    180 

atgggagccc tcacttcact gcactgtact cctcggtccc tttccctgag ggggaagcct    240 

ttccccctgt ctctgtcacc actctgggct ctcccttgca ttcaaactga ggtgcctgcc    300 

tgcccttcta ggaatggggg acagggggag gggaggagct agggaaagaa aacctggagt    360 

ttgtgccagg gtttttggat taagttcttc attcactaag gaaggaattg ggaacacaaa    420 

ggg                                                                  423 

 
           
             7  
             43  
             DNA  
             Homo sapiens  
           
            7 

aattataata cgactcacta tacgtgggct gcggggtgct gct                       43 

 
           
             8  
             42  
             DNA  
             Homo sapiens  
           
            8 

aattataata cgactcacta tatgcaggag gcgatcatga gc                        42 

 
           
             9  
             425  
             DNA  
             Homo sapiens  
           
            9 

cgtgggctgc ggggtgctgc tgtcccgcaa gcgccggcgg cagcatggcc agctctggtt     60 

ccctgagggc ttcaaagtgt ctgaggccag caagaagaag cggcgggagc ccctcggcga    120 

ggactccgtg ggcctcaagc ccctgaagaa cgcttcagac ggtgccctca tggacgacaa    180 

ccagaatgag tggggggacg aggacctgga gaccaagaag ttccggttcg aggagcccgt    240 

ggttctgcct gacctggacg accagacaga ccaccggcag tggactcagc agcacctgga    300 

tgccgctgac ctgcgcatgt ctgccatggc ccccacaccg ccccagggtg aggttgacgc    360 

cgactgcatg gacgtcaatg tccgcgggcc tgatggcttc accccgctca tgatcgcctc    420 

ctgca                                                                425 

 
           
             10  
             41  
             DNA  
             Homo sapiens  
           
            10 

taatacgact cactataggg tcgtgcaaga gccagttacc c                         41 

 
           
             11  
             41  
             DNA  
             Homo sapiens  
           
            11 

taatacgact cactataggg aatgtcatgg ccgcttcaga g                         41 

 
           
             12  
             537  
             DNA  
             Homo sapiens  
           
            12 

tcgtgcaaga gccagttacc cacccacagg tccccctact tcctgccaag cattccattg     60 

actgcctgta tggaacacat ttgtcccaga tctgagcatt ctaggcctgt ttcactcact    120 

cacccagcat atgaaactag tcttaactgt tgagcctttc ctttcatatc cacagaagac    180 

actgtctcaa atgttgtacc cttgccattt aggactgaac tttccttagc ccaagggacc    240 

cagtgacagt tgtcttccgt ttgtcagatg atcagtctct actgattatc ttgctgctta    300 

aaggcctgct caccaatctt tctttcacac cgtgtggtcc gtgttactgg tatacccagt    360 

atgttctcac tgaagacatg gactttatat gttcaagtgc aggaattgga aagttggact    420 

tgttttctat gatccaaaac agccctataa gaaggttgga aaaggaggaa ctatatagca    480 

gcctttgcta ttttctgcta ccatttcttt tcctctgaag cggccatgac attccct       537 

 
           
             13  
             40  
             DNA  
             Homo sapiens  
           
            13 

taatacgact cactataggg tcctgtgcct gtggtagaga                           40 

 
           
             14  
             42  
             DNA  
             Homo sapiens  
           
            14 

taatacgact cactataggg actgtggctg tagatgatgt ga                        42 

 
           
             15  
             438  
             DNA  
             Homo sapiens  
           
            15 

tcctgtgcct gtggtagaga gccttcagtt gaatggcggt ggggacgtag caatgcttga     60 

acttacagga cagaatttca ctccaaattt acgagtgtgg tttggggatg tagaagctga    120 

aactatgtac aggtgtggag agagtatgct ctgtgtcgtc ccagacattt ctgcattccg    180 

agaaggttgg agatgggtcc ggcaaccagt ccaggttcca gtaactttgg tccgaaatga    240 

tggaatcatt tattccacca gccttacctt tacctacaca ccagaaccag ggccacggcc    300 

acattgcagt gtagcaggag caatccttcc agccaattca agccaggtgc cccctaacga    360 

atcaaacaca aacagcgagg gaagttacac aaacgccagc acaaattcaa ccagtgtcac    420 

atcatctaca gccacagt                                                  438 

 
           
             16  
             42  
             DNA  
             Homo sapiens  
           
            16 

taatacgact cactataggg agtagtgaga gtgagagtaa ca                        42 

 
           
             17  
             43  
             DNA  
             Homo sapiens  
           
            17 

taatacgact cactataggg ctctatacaa gtctgtgcca tgg                       43 

 
           
             18  
             463  
             DNA  
             Homo sapiens  
           
            18 

agtagtgaga gtgagagtaa caataaagaa aaaaaaatac aaaggaagaa aagaaagaaa     60 

aacaagtgtt cagggcataa caacagtgat tctgaagaga aggacaagtc taagaagaga    120 

aagcttcatg aagaactttc tagcagtcac cataaccggg aaaaagccaa ggaaaagccc    180 

aggttcttaa aacacgagag ttctagggag gacagcaaat ggagccattc tgattctgac    240 

aaaaagtcca gaacccataa acatagccca gagaagagag gctctgaaag aaaggagggg    300 

agcagcagaa gccacggcag ggaggaaagg agccggagaa gccggagcag aagtcctggt    360 

agttacaagc aaagggagac aaggaaacgg gcacagcgaa atcctggtga agagcaaagc    420 

agaagaaatg acagcagaag ccatggcaca gacttgtata gag                      463 

 
           
             19  
             24  
             DNA  
             Homo sapiens  
           
            19 

gcggccgcct ttgtggttct gttc                                            24 

 
           
             20  
             21  
             DNA  
             Homo sapiens  
           
            20 

gccggcgcgt cctcctcttc c                                               21 

 
           
             21  
             20  
             DNA  
             Homo sapiens  
           
            21 

gccagaatga tgctacctgt                                                 20 

 
           
             22  
             20  
             DNA  
             Homo sapiens  
           
            22 

tagagcagca ccaatggaac                                                 20 

 
           
             23  
             25  
             DNA  
             Homo sapiens  
           
            23 

aagttacccc caagaggcaa gtgtt                                           25 

 
           
             24  
             25  
             DNA  
             Homo sapiens  
           
            24 

aaggaaatga gaggccagaa ggaga                                           25 

 
           
             25  
             20  
             DNA  
             Homo sapiens  
           
            25 

ggctgcccct cccactctcg                                                 20 

 
           
             26  
             20  
             DNA  
             Homo sapiens  
           
            26 

cagcccgggc cccaggatag                                                 20 

 
           
             27  
             20  
             DNA  
             Homo sapiens  
           
            27 

tgagtggttc ctgtactctg                                                 20 

 
           
             28  
             20  
             DNA  
             Homo sapiens  
           
            28 

actcacactg ggtaacacgg                                                 20 

 
           
             29  
             24  
             DNA  
             Homo sapiens  
           
            29 

tacccagagc ggcctatcat tttt                                            24 

 
           
             30  
             24  
             DNA  
             Homo sapiens  
           
            30 

acgaagccgg ccaggaggaa ggac                                            24 

 
           
             31  
             24  
             DNA  
             Homo sapiens  
           
            31 

tggcctgagg agcttgaatg tgac                                            24 

 
           
             32  
             24  
             DNA  
             Homo sapiens  
           
            32 

atcgcccagc aaaaatccaa tgaa                                            24 

 
           
             33  
             23  
             DNA  
             Homo sapiens  
           
            33 

agaggagtgg ctgcaatgag gtc                                             23 

 
           
             34  
             18  
             DNA  
             Homo sapiens  
           
            34 

gcgcccggct gttttctt                                                   18