WO2003012082A2

WO2003012082A2 - Method for modulating stem cell differentiation using stem loop rna

Info

Publication number: WO2003012082A2
Application number: PCT/GB2002/003409
Authority: WO
Inventors: Peter Andrews; James Walsh; Paul Gokhale
Original assignee: Axordia Limited
Priority date: 2001-07-26
Filing date: 2002-07-25
Publication date: 2003-02-13
Also published as: WO2003012082A3; GB0118223D0

Abstract

This invention relates to a method to promote the differentiation of stem cells, typically embryonic stem cells, through the use of RNA interference, by the introduction of stem loop RNA into a cell.

Description

Method for Modulating Stem Cell Differentiation Using Stem Loop RNA

The invention relates to a method to modulate stem cell differentiation comprising introducing stem loop containing RNA into a stem cell to ablate mRNA's which encode polypeptides which are involved in stem cell differentiation; stem loop RNA's ; and nucleic acid molecules and vectors encoding stem loop RNA's.

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.

hi 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).

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.

Surprisingly, 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.

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, hi addition, the rapid development of genetic transfoπnation 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).

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. (Kermerdell 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).

The use of RNAi to ablate stem cell RNA is disclosed in our co-pending application, WO 02/16620, which is incorporated by reference. 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).

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, i 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 .

Typically ES/EG cell cultures have well defined characteristics. These include, but are not limited to;

i) maintenance in culture for at least 20 passages when maintained on fibroblast feeder layers; ii) produce clusters of cells in culture referred to as embryoid bodies; iii) ability to differentiate into multiple cell types in monolayer culture; iv) can form embryo chimeras when mixed with an embryo host; v) express ES/EG cell specific markers.

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.

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, hi 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. Shamblott et. al., 1998 have also described EG cell derivation, hi Lake et al J Cell Science 2000, 113:555-66 and Rathjen et al J Cell Science 1999, 112: 601-12, ectodermal stem cells are disclosed. The above references are each both incorporated by reference in their entirety.

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.

hi 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 determimng 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).

A further group of cells which have relevance to developmental biology are pluripotent embryonal carcinoma cells (EC cells) which are stem cells of teratocarcinomas, also referred to as teratomas, which are able to differentiate into all cell types found in these tumours. A teratocarcinoma also includes teratocarcinoma cells which do not have the full pluripotential characteristics of an EC cell but nevertheless can differentiate into a restricted number of differentiated tissues. These cells have many features in common with ES/EG cells. The most important of these features is the characteristic of pluripotentiality.

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.

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). 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. Bearing 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 myo genie gene MyoDl. 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:

(i) inhibition of certain genes that normally promote differentiation along particular pathways; therefore promoting differentiation to alternate cell phenotypes; (ii) inhibition of gene activity that prevents differentiation into particular cell types; and

(iii) a combination of (i) and (ii), see figure 1

hi our co-pending application, WO02/16620, we introduce RNAi molecules homologous to genes encoding factors involved in stem cell differentiation. 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 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 a model cell system to follow the effects of perturbations in stem cell differentiation. We further propose an alternative approach to introduce double stranded RNA molecules into stem cells to ablate mRNA's.

The invention relates to the provision of stem-loop RNA structures which can either be synthesised in vitro followed by transfection into a stem cell, or alternatively, synthesised in vivo by the stem cell from vectors which are provided with expression cassettes which include a DNA molecule which includes the coding sequence for the stem-loop RNA.

The DNA molecule encoding the stem-loop RNA is constructed in two parts, a first part which is derived from a gene the regulation of which is desired. The second part is provided with a DNA sequence which is complementary to the sequence of the first part. The cassette is typically under the control of a promoter which transcribes the DNA into RNA. The complementary nature of the first and second parts of the RNA molecule results in base pairing over at least part of the length of the RNA molecule to form a double stranded hairpin RNA structure or stem-loop. The first and second parts can be provided with a linker sequence. According to a first aspect of the invention there is provided a method to modulate the differentiation state of a stem cell comprising:

(i) contacting a stem cell with at least one nucleic acid molecule comprising a sequence of a gene which mediates at least one step in the differentiation of said cell which nucleic acid molecule consists of a first part linked to a second part wherein said first and second parts are complementary over at least part of their length and further wherein said first and second parts form a double stranded region by complementary base pairing over at least part of their length; (ii) providing conditions conducive to the growth and differentiation of the cell treated in (i) above; and optionally (iii) maintaining and/or storing the cell in a differentiated state.

In a preferred method of the invention said first and second parts are linked by at least one nucleotide base.

The provision of first and second sequences which are complementary to one another and which comprise at least part of the coding sequence of a gene involved in stem cell differentiation means that when the sequence is transcribed into RNA the complementarity between first and second sequences allows base pairing between first and second sequences to form a double stranded RNA structure, see Figure 1. The optional provision of a linking region bewteen first and second parts results in the formation of a so called "hair-pin" loop structure. The transcription of the nucleic acid provides many copies of the hair-pin loop RNA which effectively functions as a RNAi molecule.

In a preferred method of the invention said nucleic acid molecule is a stem loop RNA molecule. Alternatively, said nucleic acid molecule is a DNA molecule which encodes said stem loop RNA. Ideally said DNA molecule is a vector adapted for expression of said stem loop RNA. The stem cell in (i) above may be a teratocarcinoma cell.

In a preferred method of the invention said conditions are in vitro cell culture conditions.

hi a further preferred method of the invention said stem cell is selected from: pluripotent stem cells such as embryonic stem cell; embryonic germ cell and embryonal carcinoma cells; 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; liver stem cell; and teratocarcinoma cells.

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.

In a further preferred method of the invention said stem cell is an embryonic stem cell or embryonic germ cell.

In a yet further preferred method of the invention said stem loop RNA molecule is derived from a gene which encodes a cell surface receptor expressed by a stem cell.

In a further preferred method of the invention said cell surface receptor is selected from: human Notch l(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. hi an alternative preferred method of the invention said stem loop RNA molecule is derived from a gene which encodes a ligand.

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.

In a further alternative preferred method of the invention said ligand is selected from: DI M; D113; D114; Dlk-1; Jagged 1; Jagged 2; Wnt 1; Wnt 2; Wnt 2b; Wnt 3; Wnt 3a; Wnt5a; Wnt6; Wnt7a; Wnt7b; Wntδa; Wnt8b; WntlOb; W tll; Wntl4; Wntl5.

Alternatively, said gene is selected from: SFRP1; SFRP2; SFRP4; SFRP5; SK; DKK3; CER1; WIF-1; DVL1; DVL2; DVL3; DVLlLl;mFringe; lFringe; rFringe; selll; Numb; LNX Oct4;NeuroDl; NeuroD2; NeuroD3; Brachyury; MDFI.

In a further preferred method of the invention said stem loop RNA molecule is derived from at least one of the sequences identified in Table 4 or Figures 4-54.

In a yet futher preferred embodiment of the invention said sequence is derived from Oct 4. Preferably the Oct 4 sequence corresponds to nucleotide sequence about 610 to about 1032 of the Oct 4 sequence found in GenBank accession number NM_ 002701.

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 the stem loop RNA structures disclosed herein or the vectors which encode said stem loop structures.

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).

The use of cationic lipids (eg liposomes ( Feigner (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.

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.

More recently still a method termed immunoporation has become a recognised technique 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's can be enhanced by association or linkage of the RNAi to specific antibodies, ligands or receptors.

There are also a number of commercially available transfection kits which purport to provide high efficiency transfection of cells. A kit which is particularly preferred is sold under the tradename ExGen SOO⁴"¹ by MBI Fermentas, Lithuania. ExGen is a polyethylenimine, non-liposomal transfection reagent.

According to a further aspect of the invention there is provided a stem loop RNA molecule derived from a coding sequence of at least one gene involved in stem cell differentiation comprising a first part linked to a second part wherein said first and second parts are complementary over at least part of their length and further wherein, said first and second parts form a double stranded region by complementary base pairing over at least part of their length.

hi a preferred embodiment of the invention said first and second parts are linked by at least one nucleotide base. In a further preferred embodiment of the invention said first and second parts are linked by 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide bases. In a yet further preferred embodiment of the invention said linker is at least 10 nucleotide bases.

In a preferred embodiment said coding sequence is an exon.

Alternatively said RNA 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.

In a further preferred embodiment of the invention the length of the RNA molecule is between 10 nucleotide bases (nb) -lOOOnb. More preferably still the length of the RNA molecule is selected from lOnb; 20nb; 30nb; 40nb; 50nb; 60nb; 70nb; 80nb; 90nb. More preferably still said RNA molecule is 21nb in length.

In a further preferred embodiment of the invention said RNA molecule is lOOnb; 200nb; 300nb; 400nb; 500nb; 600nb; 700nb; 800nb; 900nb; or lOOOnb. More preferably still said RNA molecule is at least lOOOnb.

In a further preferred embodiment of the invention said RNA molecule comprises sequences identified in Table 4 or Figures 4-54.

In yet a further preferred embodiment of the invention said RNA molecules comprise modified nucleotide bases.

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 guanosme, may confer advantageous properties on RNA molecules containing said modified bases. For example, modified bases may increase the stability of the RNA molecule thereby reducing the amount required to produce a desired effect. The provision of modified bases may also provide stem-loop structures which are more or less stable.

According to a further aspect of the invention there is provided a nucleic acid molecule encoding at least part of a gene which mediates at least one step in stem cell differentiation comprising a first part linked to a second part which first and second parts are complementary over at least part of their length, wherein said nucleic acid molecule is operably linked to at least one further nucleic acid molecule capable of promoting transcription of said nucleic acid linked thereto and further wherein said first and second parts form a double stranded region by complementary base pairing over at least part of their length as or when said nucleic acid molecule is transcribed.

In a preferred embodiment of the invention said first and second parts are linked by linking nucleotides as hereinbefore described. It will be apparent to one skilled in the art that the synthesis of RNA molecules which form RNA stem loops 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 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.

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 stem loop RNA's.

According to a further aspect of the invention there is provided a vector including an expression cassette comprising a first sequence linked to a second sequence wherein said first and second sequences are complementary over at least part of their lengths and further wherein the expression cassette is transciptionally linked to a promoter sequence.

In a preferred embodiment of the invention said first and second parts are linked by linking nucleotides as hereinbefore described.

Vectors including expression cassettes encoding stem-loop RNA's are adapted for eukaryotic gene expression. Typically said adaptation includes, by example and not by way of limitation, the provision of transcription control sequences (promoter sequences) which mediate cell/tissue specific expression. These promoter sequences may be cell/tissue specific, inducible or constitutive. Promoter elements typically also include so called TATA box and RNA polymerase initiation selection sequences which function to select a site of transcription initiation. These sequences also bind polypeptides which function, inter alia, to facilitate transcription initiation selection by RNA polymerase.

Adaptations also include the provision of selectable markers and autonomous replication sequences which both facilitate the maintenance of said vector in either the eukaryotic cell or prokaryotic host. Vectors which are maintained autonomously are referred to as episomal vectors. Further adaptations which facilitate the expression of vector encoded genes include the provision of transcription termination sequences.

These adaptations are well known in the art. There is a significant amount of published literature with respect to expression vector construction and recombinant DNA techniques in general. Please see, Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, NY and references therein; Marston, F (1987) DNA Cloning Techniques: A Practical Approach Vol HI IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, hιc.(1994).

According to a further aspect of the invention there is provided a cell transfected with the nucleic acid or vector according to the invention. Preferably said cell is an embryonic stem cell or embryonic germ cell. Alternatively said cell is an embryonal carcinoma cell.

According to a further aspect of the invention there is provided a method to manufacture stem loop RNA molecules comprising:

(i) providing a vector or promoter/gene fusion according to the invention; (ii) providing reagents and conditions which allow the synthesis of the RNA molecule comprising a stem loop RNA molecule according to the invention; and

(iii) providing conditions which allow the RNA molecule to base pair over at least part of its length, or at least that part corresponding to the nucleic acid sequence encoding said stem cell gene which mediates stem cell differentiation.

Preferably said gene, or gene fragment is selected from those genes represented in table 4 or Figures 4-54.

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.

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 stem loop RNA molecule, or vector encoding a stem loop RNA molecule 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.

It will be apparent to one skilled in the art that stem loop RNA relies on homology between the target gene RNA and double stranded region of the stem loop in a similar way to conventional RNAi. This confers a significant degree of specificity to the stem loop RNA molecule in targeting stem cells. For example, haemopoietic stem cells are found in bone marrow and stem loop RNA molecules may be administered to an animal by direct injection into bone marrow tissue. Stem loop RNA molecules may be encapsulated in liposomes to provide protection from an animals immune system and/or nucleases present in an animals serum.

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 200nm, 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.

The use of liposomes as delivery means is described in US5580575 and US 5542935.

It will be apparent to one skilled in the art that the stem loop RNA molecules can be provided in the form of an oral or nasal spray, an aerosol, suspension, emulsion, and/or eye drop fluid. Alternatively the stem loop RNA molecules may be provided in tablet form. Alternative delivery means include inhalers or nebulisers.

According to a yet further aspect of the invention there is provided a therapeutic composition comprising a stem loop RNA molecule according to the invention or a vector encoding a stem loop RNA according to the invention.

Preferably said stem loop RNA molecule or vector 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's disease, Alzhiemer'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).

In a further preferred embodiment of the invention said therapeutic composition further comprises a diluent, carrier or excipient.

According to a further aspect of the invention there is provided a cell obtainable by the method according to the invention.

It will be apparent that a cell obtainable by the method according to the invention has useful applications . For example, a stably transfected cell under the control of a regulatable promoter (ie inducible, repressible, developmentally regulated, cell lineage regulated, cell-cycle regulated) offers the opportunity to modulate the expression of the stem-loop RNA in said cell thereby modulating the differentiation state, or not as the case maybe, in culture or in viyo.

According to a yet further aspect of the invention there is provided at least one organ comprising at least one cell obtainable by the method according to the invention.

According to a yet further aspect of the invention there is provided a non-human transgenic animal comprising a RNA molecule according to the invention, or a nucleic acid molecule according to the invention, or a vector according to the invention.

An embodiment of the invention will now be described by example only and with reference to the following figures and tables wherein: Table 1 represents a selection of antibodies used to monitor stem cell differentiation;

Table 2 represents nucleic acid probes used to assess mRNA markers of stem differentiation;

Table 3 represents protein markers of stem cell differentiation;

Table 4 represents specific primers used to generate stem loop RNA for gene specific inhibition;

Table 5 represents vectors used for the expression of stem loop RNA in cells including the promoters used to drive transcription of stem loop RNA's.

Figure 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. Stem loop RNA can be used to control both the initial differentiation of stem cells (A) and the ultimate fate of the differentiated cells Dl and D2 by repression of positive activators which would normally promote a particular cell fate;

Figure 2 represents the Oct 4 nucleic acid sequence from position 610-1032 of the sequence found in GenBank accession number NM_002701.

Fig 3A illustrates a transcription cassette comprising a promoter sequence operable linked to a nucleic acid encoding a stem loop RNA; Fig 3B illustrates a stem loop RNA synthesised from the cassette illustrated in Fig 1 A;

Figure 4 is the nucleic acid sequence of murine notch ligand delta-like 1 ;

Figure 5 is the nucleic acid sequence of murine notch ligand jagged 1; Figure 6 is the nucleic acid sequence of human notch ligand jagged 1 (alagiUe syndrome) (JAG1);

Figure 7 is the nucleic acid sequence of human notch ligand jagged 2 (JAG2)

Figure 8 is the nucleic acid sequence of murine notch ligand jagged 2;

Figure 9 is the nucleic acid sequence of human notch ligand delta-like 3 (DLL3);

Figure 10 is the nucleic acid sequence of human notch ligand delta-1 (DLLl);

Figure 11 is the nucleic acid sequence of human notch ligand delta-like 4 (DLL4);

Figure 12 is the nucleic acid sequence of murine notch ligand delta-like 4(DLL4);

Figure 13 represents the nucleic acid sequence of human Wnt 13;

Figure 14 represents the nucleic acid sequence of human dickkopfl;

Figure 15 represents the nucleic acid sequence of human dickkopf2;

Figure 16 represents the nucleic acid sequence of human dickkopβ; and

Figure 17 represents the nucleic acid sequence of human dickkopfl;

Figure 18 represents the nucleic acid sequence of WNT-1;

Figure 19 represents the nucleic acid sequence of WNT-2;

Figure 20 represents the nucleic acid sequence of WNT 2B; Figure 21 represents the nucleic acid sequence of WNT 3;

Figure 22 represents the nucleic acid sequence of WNT 4;

Figure 23 represents the nucleic acid sequence of WNT 5 A;

Figure 24 represents the nucleic acid sequence of WNT 6;

Figure 25 represents the nucleic acid sequence of WNT 7A;

Figure 26 represents the nucleic acid sequence of WNT 8B;

Figure 27 represents the nucleic acid sequence of WNT 10B;

Figure 28 represents the nucleic acid sequence of WNT 11;

Figure 29 represents the .nucleic acid sequence of WNT 14

Figure 30 represents the nucleic acid sequence of WNT 16;

Figure 31 represents the nucleic acid sequence of FZD 1;

Figure 32 represents the nucleic acid sequence of FZD 2;

Figure 33 represents the nucleic acid sequence of FZE 3;

Figure 34 represents the nucleic acid sequence of FZD 4;

Figure 35 represents the nucleic acid sequence of FZD 5;

Figure 36 represents the nucleic acid sequence of FZD 6; Figure 37 represents the nucleic acid sequence of FZD 7;

Figure 38 represents the nucleic acid sequence of FZD 8;

Figure 39 represents the nucleic acid sequence of FZD 9;

Figure 40 represents the nucleic acid sequence of FZD 10;

Figure 41 represents the nucleic acid sequence of FRP;

Figure 42 represents the nucleic acid sequence of SARP 1;

Figure 43 represents the nucleic acid sequence of SARP 2;

Figure 44 represents the nucleic acid sequence of FRZB;

Figure 45 represents the nucleic acid sequence of FRPHE;

Figure 46 represents the nucleic acid sequence of SARP 3;

Figure 47 represents the nucleic acid sequence of CER 1;

Figure 48 represents the nucleic acid sequence of DKK1;

Figure 49 represents the nucleic acid sequence of DKK 2;

Figure 50 represents the nucleic acid sequence of DKK 3;

Figure 51 represents the nucleic acid sequence of DKK 4; Figure 52represents the nucleic acid sequence of WIF-1;

Figure 53 represents the nucleic acid sequence of SRFP 1;

Figure 54 represents the nucleic acid sequence of SRFP 4;

Materials and Methods

Cell Culture

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)(GTBCO BRL), supplemented with 10% v/v bovine foetal calf serum (GTBCO BRL), under a humidified atmosphere with 10% CO₂ in air.

Stem Loop RNA Production

Primers were designed against specific target genes with T7 bacteriophage promoters at their 5' ends . The primers consist of typically 18- 25 bp against the target gene, a linker sequence of variable length (indicated by N in primer sequence) followed by the reverse complement of the gene specific sequence. The primers were used in a standard RNA in vitro, transcription reaction using a MEGASCRIPT kit following manufacturers protocols (Ambion, USA). Longer siRNA templates were produced buy cloning head-to -tail the sense and anti-sense gene specific sequences to generate a palindromic template from which RNA could be synthesized.

The following primers were used

Capital letters indicate the T7 polymerase promoter sequence.

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'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.

Stem Loop RNA introduction to Cell Lines

Human EC stem cells were seeded at 2 XI 0⁵ cells/well of a 6 well plate in 3 cm³ of Dulbecco's modified Eagles medium and allowed to settle for 3 hrs. Appx. 9.5μg of DNA was incubated with an optimised amount of ExGEN 500 for each well of a 6-well plate. Previously cells were seeded 1 day before. This gives apprx. a 70% confluent culture. The DNA/ExGen mixture was added to the cells and the culture vessel spun at 280g for 5 mins.

Total RNA production 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 300xg. The pellets had the PBS aspirated from them. Tri reagent (Sigma, USA) was added at 1ml per 10⁷ cells and allowed to stand for 10 mins at room temperature. The lysate from this reaction was centrifuged at 12000 x 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 x 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.

Analysis of the differentiation of EC stem cells induced by exposure to Stem Loop RNA

Following exposure to stem loop RNA 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 disapearance 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.

Analysis of Transfectants by Antibody Staining and FACS

Cells were treated with trypsin (0.25% v/v) for 5 mins to disaggregate the cells; they were washed and re-suspended to 2x10⁵ cells/ml. This cell suspension was incubated with 50μl 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 lOOrpm 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)

Northern blot Analysis of RNA

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 ran out using standard agarose gel electrophresis. For most RNA a 1% agarose gel is sufficiant. The agarose is made in IX 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 3/4 of the way down the gel).

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 10X SSPE. Gels were transfered overnight. The membrane was soaked in 2X 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.

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/ 0.1% SDS 45 mins 65°C

0. IX SSPE 15 mins room temp

Preparation of radiolabelled DNA probes

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 . 5-100ng 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, 5 μl [α-32P] dATP 3000 Ci/mmol 1 μl of Klenow DNA polymerase (4U)

The reaction was then incubated at 37°C for 1 hr. Unincorporated nucleotide were removed with spin columns ( Nucleon Biosciences).

Production of cDNA

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 lμg poly (A)+ RNA or total RNA was incubated with the following 1.OμM oligo(dT) primer for total RNA or random hexcamers for mRNA 0.5mM lOmM dNTP mix lU/μl RNAse inhibitor (Promega) l.OU/μl M-MLV reverse transcriptase in manufacturers supplied buffer

(Promega) The reaction was incubated for 2-3 hours at 42°C Fluorescent Automated Sequencing

To check the specificity of the PCR primers used to generate the template used in stem loop RNA 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 (200ng plasmid, lOOng PCR product), 10 μM sequencing primer (typically a 20mer 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 2μl 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.

Detection of specific protein targets by SDS-PAGE and Western Blotting

To obtain cell lysates monolayers of cells were rinsed 3 times with ice-cold PBS supplemented with 2 mM CaCl₂. Cells were incubated with 1 ml/75 cm² 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 ) to remove insoluble material. Protein concentrations of the supernatants were determined using a commercial protein assay (Biorad). Samples were prepared for SDS-PAGE by adding 6 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 PVDF membrane. 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

Table 2: Probes used to assess mRNA markers of differentiation

Table 3: Protein markers of differentiation, detected by Western Blot and/or immunofluorescence.

The following antibodies were detected by the appropriate commercially available antibodies

Table 4: Specific Primers used to generate Stem Loop RNA for gene specific inhibition

All sequences written 5' to 3'

Table 5

Listed are examples of vector systems that are to be used in cells to direct the production of stem loop RNA.

References

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Claims

1. A method to modulate the differentiation state of .a stem cell comprising: i) contacting a stem cell with at least one nucleic acid molecule comprising a sequence of a gene which mediates at least one step in the differentiation of said cell which nucleic acid molecule consists of a first part linked to a second part wherein said first and second parts are complementary over at least part of their length and further wherein said first and second parts form a double stranded region by complementary base pairing over at least part of their length; (ii) providing conditions conducive to the growth and differentiation of the cell treated in (i) above; and optionally (iii) maintaining and/or storing the cell in a differentiated state.

2. A method according to Claim 1 wherein said first and second parts are linked by at least one nucleotide base.

3 A method according to Claim 1 or 2 wherein said nucleic acid molecule is a stem loop RNA molecule or a nucleic acid molecule or a vector encoding said stem loop RNA.

4. A method according to any of Claims 1-3 wherein said conditions are in vitro cell culture conditions.

5. A method according to any of Claims 1-4 wherein said stem cell is selected from the group consisting of: an embryonic stem cell; an embryonic germ cell; an embryonal carcinoma cell; a haemopoietic stem cell; a muscle stem cell; a nerve stem cell; a skin dermal sheath stem cell; a liver stem cell; a teratocarcinoma cell.

6. A method according to any of Claims 1-5 wherein said stem cell is an embryonic stem cell or embryonic germ cell.

7. A method according to any of Claims 1-6 wherein said nucleic acid molecule is derived from at least one nucleic acid sequence as represented by Figures 4- 54.

8. A RNA molecule derived from a coding sequence of at least one gene involved in stem cell differentiation comprising a first part linked to a second part wherein said first and second parts are complementary over at least part of their length and further wherein said first and second parts form a double stranded region by complementary base pairing over at least part of their length.

9. A RNA molecule according to Claim 8 wherein said first and second parts are linked by at least one nucleotide base (nb).

10. A RNA molecule according to Claim 9 wherein said first and second parts are linked by 2, 3, 4, 5, 6, 7, 8, 9, or lOnb in length.

11. A RNA molecule according to Claim 9 wherein said linker is at least lOnb in length.

12. A RNA molecule according to any of Claims 8-11 wherein the length of the RNA molecule is between 1 Onb -1 OOOnb in length.

13. A RNA molecule according to Claim 12 wherein the length of the RNA molecule is selected from lOnb; 20nb; 3 Onb; 40nb; 50nb; 60nb; 70nb; 80nb; 90nb in length.

14. A RNA molecule according to Claim 12 wherein said RNA molecule is lOOnb; 200nb; 300nb; 400nb; 500nb; 600nb; 700nb; 800nb; 900nb; or lOOOnb in length.

15. A RNA molecule according to Claim 8 wherein said RNA molecule is at least lOOOnb in length.

16. A RNA molecule according to Claim 8 wherein said RNA molecule is

21nb in length.

17. A RNA molecule according to any of Claims 8 -16 wherein said RNA molecule comprises sequences identified in Figures 4-54.

18. A RNA molecule according to any of Claims 8-17 wherein said RNA molecules comprise modified nucleotide bases.

19. A nucleic acid molecule which encodes an RNA molecule according to any of Claims 8-18 wherein said nucleic acid molecule is operably linked to at least one further nucleic acid molecule capable of promoting transcription of said nucleic acid linked thereto.

20. A nucleic acid molecule according to Claim 19 wherein said further nucleic acid molecule is a promoter capable of inducible transcription.

21. A vector including a nucleic acid molecule according to Claim 19 or 20.

22. A cell transfected with an RNA molecule according to any of Claims 8-18, nucleic acid molecule according to Claim 19 or 20 or a vector according to Claim 21.

23. A cell according to Claim 22 wherein said cell is an embryonic stem cell or embryonic germ cell.

24. A cell according to Claim 22 wherein said cell is an embryonal carcinoma cell.

25. A method to manufacture stem loop RNA molecules comprising: (i) providing a nucleic acid molecule according to Claim 19 or 20 or a vector according to Claim 21;

(ii) providing reagents and conditions which allow the synthesis of the RNA molecule comprising a RNA molecule according to any of Claims 8-18; and

26. An in vivo method to promote the differentiation of stem cells comprising administering to an animal an effective amount of an RNA molecule according to any of Claims 8-18, a nucleic acid molecule according to Claim 19 or 20 or a vector according to Claim 21, sufficient to effect differentiation of a target stem cell.

27. A RNA molecule according to any of Claims 8-18, a nucleic acid molecule according to Claim 19 or 20 or a vector according to Claim 21 for use as a pharmaceutical.

28. A pharmaceutical composition comprising a RNA molecule according to any of Claims 8-18, a nucleic acid molecule according to Claim 19 or 20 or a vector according to Claim 21.

29. Use of a RNA molecule according to any of Claims 8-18, a nucleic acid molecule according to Claim 19 or 20 or a vector according to Claim 21 for 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.

30 Use according to Claim 29 wherein said disease is selected from the group consisting of: pernicious anemia; stroke, neurodegenerative diseases such as Parkinson's disease, Alzhiemer's disease; coronary heart disease; cirrhosis; diabetes; nerves damaged as a consequence of trauma (e.g. replacement of spinal cord tissue).

31. A cell obtainable by the method according to any of Claims 1 -7.

32. An organ comprising at least one cell according to Claim 31.

33. A non-human transgenic animal comprising a RNA molecule according to any of Claims 8-18, or a nucleic acid molecule according to Claim 19 or 20, or a vector according to Claim 21.