WO2011058300A2

WO2011058300A2 - Induced pluripotent stem cell

Info

Publication number: WO2011058300A2
Application number: PCT/GB2010/002051
Authority: WO
Inventors: Tyson Valentine Sharp; Tristan Rowntree Mckay
Original assignee: The University Of Nottingham; The University Of Manchester
Priority date: 2009-11-12
Filing date: 2010-11-08
Publication date: 2011-05-19
Also published as: GB0919773D0; WO2011058300A3

Abstract

The disclosure relates to modified mammalian cells which show an enhanced induced pluripotent stem cell phenotype, expression cassettes and vectors that include nucleic acids useful in deriving induced pluripotent cells; methods to derive said cells and induced pluripotent cells obtained by said methods.

Description

Induced Pluripotent Stem Cell

The term "stem cell" represents a generic group of undifferentiated cells that possess the capacity for self-renewal while retaining varying potentials to form differentiated cells and tissues. Stem cells can be pluripotent or multipotent. A pluripotent stem cell is a cell that has the ability to form all tissues found in an intact organism although the pluripotent stem cell cannot form an intact organism. A multipotent cell has a restricted ability to form differentiated cells and tissues. Typically, adult stem cells are multipotent stem cells and are the precursor stem cells or lineage restricted stem cells that have the ability to form some cells or tissues and replenish senescing or damaged cells/tissues. Generally they cannot form all tissues found in an organism, although some reports have claimed a greater potential for such 'adult' stem cells than originally thought. A totipotent cell is a cell that has the ability to form all the cells and tissues that are found in an intact organism, including the extra-embryonic tissues (i.e. the placenta). Totipotent cells comprise the very early embryo (8 cells) and have the ability to form an intact organism and are not as such considered stem cells.

Embryonic stem cells 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). A problem associated with the use of human embryonic stem cells is that it is necessary to sacrifice a human embryo to obtain embryonic stem cell-lines with pluripotent potential. This raises moral and ethical issues with respect to isolation of human embryonic stem cells. It is known that human somatic cells can be re-programmed to an undifferentiated state similar to an embryonic stem cell. For example, WO2007/069666 describes re- programming of differentiated cells. Nuclear re-programming is achieved by transfection of somatic cells that encode nuclear re-programming factors, for example Oct family, Sox family, Klf family and Myc family of transcription factors. The somatic cells dedifferentiate and express markers of human embryonic stem cells to produce an "induced pluripotent cell". In Takahashi er a/ [Cell vol 131 , p861-872, 2007] adult human dermal fibroblasts with the four transcription factors: Oct3/4, Sox2, Klf4, and c-Myc dedifferentiate to human ES cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes and telomerase activity. Furthermore, these iPS cells could differentiate into cell types of the three germ layers in vitro. In a later published manuscript Nakagawa er a/ [Published online: 30 November 2007 |doi: 10.1038/nbt1374] show that c-Myc can be omitted from the re-programming cocktail and still achieve de-differentiation to a pluripotential state although efficiency is substantially reduced. This disclosure relates to Lim domain containing proteinl (LIMD1 ) family member proteins, and their use in somatic cell re-programming. LIMD1 is a nucleo-cytoplasmic shuttling protein, co-ordinating cues from the cell surface with nuclear responses [ ]. In the nucleus LIMD1 acts as a co-repressor of cell cycle progression with the retinoblastoma protein (pRB) [ ]. It also acts to down-regulate E-cadherin expression by repressing the Snail/Slug family of transcriptional regulators which are required to promote Epithelial-Mesenchymal Transition (EMT) and neural crest development [ ]. The function of the cytoplasmic pool of LIMD1 in regulating growth control has remained more elusive. We have discovered that down regulation of LIMD1 and family member proteins in combination with the expression of two or more re-programming factors leads to enhanced somatic cell re-programming.

According to an aspect of the invention there is provided an isolated mammalian somatic cell wherein said cell is modified which modification reduces the expression or activity of a polypeptide that comprises a Lim domain amino acid motif.

In a preferred embodiment of the invention the modification in expression of said Lim domain polypeptide is regulatable, for example inducible or repressible. In a preferred embodiment of the invention said polypeptide comprises the amino acid motif: C(X)2C{X) ie-23(H/C)(X)2/4(C/H/E)(X)2C{X)2C(X) i4-2i(C/H)(X)2/i/3{C/H/D/E)X, wherein X is any amino acid residue.

In a preferred embodiment of the invention said polypeptide is selected from the group consisting of: LIMD1 , Ajuba, WTIP, Zyxin, LPP, TRIP6 and Migfilin

In a preferred embodiment of the invention said polypeptide is LIMD1 and is represented by the nucleotide sequence presented in Figure 4d.

In a preferred embodiment of the invention said polypeptide is LIMD1 and is represented by the amino acid sequence presented in Figure 4c. In an alternative preferred embodiment of the invention said polypeptide is Ajuba and is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 10.

In an alternative preferred embodiment of the invention said polypeptide is WTIP and is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 11.

In an alternative preferred embodiment of the invention said polypeptide is Zyxin and is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 12.

In an alternative preferred embodiment of the invention said polypeptide is LPP and is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 13.

In an alternative preferred embodiment of the invention said polypeptide is TRIP6 and is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 14. In an alternative preferred embodiment of the invention said polypeptide is Migfilin and is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 15. In a preferred embodiment of the invention the expression of the Lim domain polypeptide is reduced or inhibited by transfection of a siRNA or shRNA.

A technique to specifically ablate gene function is through the introduction of double stranded RNA, also referred to as small inhibitory or interfering RNA (siRNA), into a cell which results in the destruction of mRNA complementary to the sequence included in the siRNA molecule. The siRNA 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 siRNA molecule is typically derived from exons of the gene which is to be ablated. The mechanism of RNA interference is being elucidated. Many organisms respond to the presence of double stranded RNA by activating a cascade that leads to the formation of dsRNA. The presence of double stranded RNA activates a protein complex comprising RNase III which processes the double stranded RNA into smaller fragments (siRNAs, approximately 21-29 nucleotides in length) which become part of a ribonucleoprotein complex. The siRNA acts as a guide for the RNase complex to cleave mRNA complementary to the antisense strand of the siRNA thereby resulting in destruction of the mRNA.

Preferably said siRNA molecule is between 18bp and 29bp in length. More preferably still said siRNA molecule is between 21 bp and 27bp in length. Preferably said siRNA molecule is about 21 bp in length.

In a preferred embodiment of the invention said siRNA includes modified nucleotides.

The term "modified" as used herein describes a nucleic acid molecule in which;

i) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide). Alternatively or preferably said linkage may be the 5' end of one nucleotide linked to the 5' end of another nucleotide or the 3' end of one nucleotide with the 3' end of another nucleotide; and/or ii) a chemical group, such as cholesterol, not normally associated with nucleic acids has been covalently attached to the double stranded nucleic acid. iii) Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, phosphate triesters, acetamidates, peptides, and carboxymethyl esters.

The term "modified" also encompasses nucleotides with a covalently modified base and/or sugar. For example, modified nucleotides include nucleotides having sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position.

Thus modified nucleotides may also include 2' substituted sugars such as 2'-0-methyl-;

2-O-alkyl; 2-O-allyl; 2'-S-alkyl; 2'-S-allyl; 2'- fluoro-; 2'-halo or 2;azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose.

Modified nucleotides are known in the art and include, by example and not by way of limitation, alkylated purines and/or pyrimidines; acylated purines and/or pyrimidines; or other heterocycles. These classes of pyrimidines and purines are known in the art and include, pseudoisocytosine; N4, N4-ethanocytosine; 8-hydroxy-N6-methyladenine; 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil; 5-fluorouracil; 5-bromouracil;5- carboxymethylaminomethyl-2-thiouracil; 5 carboxymethylaminomethyl uracil; dihydrouracil; inosine; N6-isopentyl-adenine; l-methyladenine; 1-methylpseudouracil; 1- methylguanine; 2,2-dimethylguanine; 2-methyladenine; 2-methylguanine; 3- methylcytosine; 5-methylcytosine; N6-methyladenine; 7-methylguanine; 5- methylaminomethyl uracil; 5-methoxy amino methyl-2-thiouracil; β-D-mannosylqueosine; 5-methoxycarbonylmethyluracil; 5-methoxyuracil; 2 methylthio-N6-isopentenyladenine; uracil-5-oxyacetic acid methyl ester; psueouracil; 2-thiocytosine; 5-methyl-2 thiouracil, 2- thiouracil; 4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic acid methylester; uracil 5— oxyacetic acid; queosine; 2-thiocytosine; 5-propyluracil; 5-propylcytosine; 5-ethyluracil; 5-ethylcytosine; 5-butyluracil; 5-pentyluracil; 5-pentylcytosine; and 2,6,-diaminopurine; methylpsuedouracil; 1 -methylguanine; 1-methylcytosine. Modified double stranded nucleic acids also can include base analogs such as C-5 propyne modified bases (see Wagner et al., Nature Biotechnology 14:840-844, 1996). In a preferred embodiment of the invention said somatic cell is transiently transfected to reduce Lim domain expression.

In an alternative preferred embodiment of the invention said somatic cell is stably transfected to reduce Lim domain expression.

In a preferred embodiment of the invention said somatic cell is further modified to express at least one re-programming factor. In a preferred embodiment of the invention said somatic cell expresses at least Oct 4 and Sox 2.

In a preferred embodiment of the invention Oct 4 is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 5.

In a preferred embodiment of the invention Sox 2 is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 6.

In a preferred embodiment of the invention said cells express at least Oct 4, Sox 2, c- MYC, Klf4 and/or LIN28.

In a preferred embodiment of the invention c-MYC is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 7. In a preferred embodiment of the invention Klf4 is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 8.

In a preferred embodiment of the invention Lin28 is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 9.

In an embodiment of the invention said somatic cell is transfected with a vector that includes said re-programming factor.

In a preferred embodiment of the invention said somatic cell is a mammalian cell; preferably a human somatic cell. In a preferred embodiment of the invention said somatic cell is selected from the group consisting of: a hematopoietic cell, e.g. lymphocyte, myeloid cell; a buccal mucosa cell, an epidermal cell, a mesenchymal cell, a keratinocyte. In a preferred embodiment of the invention said somatic cell is a fibroblast; preferably an adult or embryonic fibroblast.

In a preferred embodiment of the invention said somatic cell is selected from the group consisting of: a dermal fibroblast, a foetal fibroblast, a corneal fibroblast, an intestinal mucosa fibroblast, an oral mucosa fibroblast and urethral fibroblast.

According to an aspect of the invention there is provided a transcription cassette wherein said cassette includes a nucleotide sequence designed with reference to Figure 4d and is adapted for expression by provision of at least one promoter operably linked to said nucleic acid sequence such that both sense and antisense molecules are transcribed from said cassette.

In a preferred embodiment of the invention said cassette is adapted such that both sense and antisense nucleic acid molecules are transcribed from said cassette wherein said sense and antisense nucleic acid molecules are adapted to anneal over at least part or all of their length to form a siRNA or shRNA.

In a further preferred embodiment of the invention said cassette is provided with at least two promoters adapted to transcribe both sense and antisense strands of said nucleic acid molecule.

In a preferred embodiment of the invention said cassette comprises a nucleic acid molecule wherein said molecule comprises a first part linked to a second part wherein said first and second parts are complementary over at least part of their sequence and further wherein transcription of said nucleic acid molecule produces an RNA molecule which forms a double stranded region by complementary base pairing of said first and second parts thereby forming an shRNA.

Preferably said cassette includes one or more of the following nucleotide sequences selected from the group consisting of: GGTTAGTGCTCGAGTGAAA

GGGAAGAGCTGCCCGCATA TGTTAGGACTTAAGAGAAT GAGCAGAGATCCAGGCCAT CATCATAGCACCAAAGGAA GCAGAGTGATCGTAACTAA GGACAAGGGTGCAGGCAAC CTTTGGAGCCTGTGTGAAA GAAAATACCTATACCCAAA GCACAAGACCACAAATTAT TCACAGAGCTTCACCAAAT

CAGATAGGTTTGCGAATAT

In a further preferred embodiment of the invention said cassette comprises nucleic acid molecules that encode at least the re-programming factors Oct 4 and Sox 2.

Preferably, Oct 4 is encoded by a nucleic acid molecule comprising the nucleotide sequence in Figure 5.

Preferably, Sox 2 is encoded by a nucleic acid molecule comprising the nucleotide sequence in Figure 6.

According to a further aspect of the invention said cassette is part of an expression vector wherein said cassette is operably linked to a promoter sequence. In a preferred embodiment of the invention said promoter is a regulatable promoter; preferably an inducible promoter and/or a tissue/cell specific promoter.

"Promoter" is an art recognised term and, for the sake of clarity, includes the following features which are provided by example only. Enhancer elements are cis acting nucleic acid sequences often found 5' to the transcription initiation site of a gene (enhancers can also be found 3' to a gene sequence or even located in intronic sequences). Enhancers function to increase the rate of transcription of the gene to which the enhancer is linked. Enhancer activity is responsive to trans acting transcription factors which have been shown to bind specifically to enhancer elements. The binding/activity of transcription factors (please see Eukaryotic Transcription Factors, by David S Latchman, Academic Press Ltd, San Diego) is responsive to a number of physiological/environmental cues. Promoter elements 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.

In a preferred embodiment of the invention said vector is a viral based vector.

A number of viruses are commonly used as vectors for the delivery of exogenous nucleic acid molecules. Commonly employed vectors include recombinantly modified enveloped or non-enveloped DNA and RNA viruses, preferably selected from retroviridae parvoviridiae, picornoviridiae, herpesveridiae, poxviridae, adenoviridiae, or picornnaviridiae.

Chimeric vectors may also be employed which exploit advantageous elements of each of the parent vector properties (See e.g., Feng, et al.(1997) Nature Biotechnology 15:866- 870). Such viral vectors may be wild-type or may be modified by recombinant DNA techniques to be replication deficient, conditionally replicating or replication competent.

Conditionally replicating viral vectors are used to achieve selective expression in particular cell types. Examples of conditionally replicating vectors are described in Pennisi, E. (1996) Science 274:342-343; Russell, and S.J. (1994) Eur. J. of Cancer 30A(8):1 65-1 171. Additional examples of selectively replicating vectors include those vectors wherein a gene essential for replication of the virus is under control of a promoter which is active only in a particular cell type or cell state such that in the absence of expression of such gene, the virus will not replicate. Examples of such vectors are described in Henderson, et al., United States Patent No. 5,698,443 issued December 16, 1997 and Henderson, et al., United States Patent No. 5,871 ,726 issued February 16, 1999 the entire teachings of which are herein incorporated by reference.

Additionally, the viral genome may be modified to include inducible promoters which achieve replication or expression only under certain conditions. Examples of inducible promoters are known in the scientific literature (See, e.g. Yoshida and Hamada (1997) Biochem. Biophys. Res. Comm. 230:426-430; lida, et al. (1996) J. Virol. 70(9):6054- 6059; Hwang, et al.(1997) J. Virol 71 (9):7128-7131 ; Lee, et al. (1997) Mol. Cell. Biol. 17(9):5097-5105; and Dreher, et al.(1997) J. Biol. Chem 272(46); 29364-29371. In a preferred embodiment of the invention said viral based vector is a lentiviral vector.

According to an aspect of the invention there is provided a cell culture comprising a somatic cell according to the invention.

According to an aspect of the invention there is provided a method for the reprogramming of a mammalian somatic cell comprising contacting said cell with an agent that inhibits the expression or activity of a Lim domain protein in combination with the expresssion of at least two re-programming factors.

According to a further aspect of the invention there is provided a method for the re- programming of a somatic cell comprising:

i) providing a cell preparation comprising a mammalian somatic cell wherein said cell expresses one or more re-programming factors;

i) contacting said cell with an agent that inhibits the expression or activity of a Lim domain protein; and

ii) culturing said cell preparation and optionally monitoring the differentiation state of said somatic cell. In a preferred method of the invention said re-programming factors is Oct 4 and Sox 2.

In a preferred method of the invention Oct 4 is encoded by a nucleotide sequence as represented in Figure 5. In a preferred method of the invention Sox 2 is encoded by a nucleotide sequence as represented in Figure 6.

Acording to a further aspect of the invention there is provided an induced pluripotent cell obtained or obtainable by the method according to the invention.

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

According to a further aspect of the invention there is provided a method for the preparation of a library comprising induced stem cell specific gene expression products comprising the steps: i) providing a preparation comprising an induced stem cell according to the invention;

ii) extracting nucleic acid from said cell preparation;

iii) preparing cDNA from ribonucleic acid contained in said extracted nucleic acid; and

iv) ligating cDNA formed in (iii) into a vector.

In a preferred method of the invention said vector is a phage based vector. According to a further aspect of the invention there is provided a method for the identification of genes associated with induced stem cells comprising:

i) providing a preparation comprising an induced stem cell according to the invention;

ii) extracting nucleic acid from said cell preparation;

iii) contacting said extracted nucleic acid with a nucleic acid array; and iv) detecting a signal which indicates the binding of said nucleic acid to a binding partner on said nucleic acid array.

Preferably said method includes the additional steps of:

i) collating the signal(s) generated by the binding of said nucleic acid to said binding partner;

ii) converting the collated signal(s) into a data analysable form; and optionally;

iii) providing an output for the analysed data.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

An embodiment of the invention will now be described by example only and with reference to the following figures:

Figure 1 illustrates the co-association of LIMD1 with the RISC complex and P- bodies in US20S cells;

Figure 2 illustrates that LIMD1 is necessary for Let-7 miRNA mediated repression of a luciferase reporter construct in U20S cells. In the left upper panel Limdl knockdown (red) or a dominant negative mutant (blue) increases expression of a luciferase construct with a 3' UTR containing Let-7a binding sites in an miRNA dependent manner. Again, in the right panel siRNA knockdown of LIMD1 de-represses the activity of the RLuc-miLet7 but not the RLuc-siLet7 construct;

Figure 3 illustrates lentiviral shRNA knockdown in hDFs reduces LIMD1 protein to levels similar to that seen in hES cells when compared to actin controls. Differentiation of HUES7 hES cells leads to upregulation of LIMD1 (left panel). HDF shLIMDI cells co- transduced with lentiviral vectors expressing OCT4 and SOX2 generate iPS colonies that activate the OCT4 promoter (OCT4-GFP) with great efficiency (middle panel). Endogenous mRNA of pluripotency markers is upregulated in Ips containing OS shLIMDI hDF cultures (RNA collected from total cultures containing ~23% iPS colonies (right panel);

Figure 4a illustrates the amino acid domains in LIMD1 including the consensus motif founf in related proteins; Figure 4b is a comparison of conserved domains LIM domain containing proteins; Figure 4c is the amino acid sequence of human LIMD1 ; Figure 4d is the nucleotide sequence of human LIMD1 ;

Figure 5 is the nucleotide sequence of human Oct 4;

Figure 6 is the nucleotide sequence of human Sox2; Figure 7 is the nucleotide sequence of human cMYC; Figure 8 is the nucleotide sequence of human Klf4; Figure 9 is the nucleotide sequence of human Ln28;

Figure 10 is the nucleotide sequence of human Ajuba; Figure 1 1 is the nucleotide sequence of human WTIP; Figure 12 is the nucleotide sequence of human Zyxin; Figure 13 is the nucleotide sequence of human LPP; Figure 14 is the nucleotide sequence of human TRIP 6; and

Figure 15 is the nucleotide sequence of human Migfilin.

Figure 16 illustrates lllumina miRNA array data obtained from U20S cells depleted for LIMD1 indicated specific down-regulation of the Let-7 family of miRNAs associated with a pro- differentiated cellular phenotype and conversely up-regulation of miRNAs associated with stemness/pluripotency (miRNAs 200c, 302b, 302d).

Materials and Methods

Human iPS Reprogramming

Primary human Dermal Fibroblasts (hDF) were obtained under a non-restrictive Material Transfer Agreement (MTA) from Intercytex, UK. These cells were cultured in hDF growth medium (DMEM containing 1 x penicillin/streptomycin, L-glutamine, non-essential amino acids (NEAA) and 10% Fetal Bovine Serum; all Invitrogen) on tissue culture plasticware pre-coated with 0.1 % porcine gelatine. hDFs were transduced with a lentiviral vector expressing shRNA directed against the human LIMD1 transcript and a YFP reporter gene (a kind gift from Dr. Greg Longmore, Washington University, St Louis, MO, USA). Cells were transduced at a Multiplicity of Infection (MOI) of 10 and then cells expressing high levels of the YFP reporter purified by Fluorescence Activated Cell Sorting (FACS). This population of hDFs (hDF-shl_IMD1) was initially analysed by western blot for LIMD1 protein before and after knockdown and LIMD1 protein was found to be substantially reduced in hDF-shLIMD1 cells. The cells were then subjected to transduction with combinations of lentiviral vectors expressing the human transgenes OCT4, SOX2, KLF4 and C-MYC (obtained from Addgene (Cambridge, MA, USA) under an academic MTA. Transduced hDF-shLIMD1 cells were subsequently cultured in human Embryonic Stem (hES) cell growth medium (Knockout DMEM supplemented with 205 Knockout Serum Replacement, L-Glutamine, Penicillin/Streptomycin, NEAA, 100μΜ β-mercaptoethanol and 8ng/ml FGF-2) replenishing growth medium every 48 hours. Cells were split 1 :10 when confluent using trypsin and replated on gelatine coated plates. This occurred once every 7 days. After 14 days colonies began to form. Lentiviral production

Lentivectors were prepared as follows: Producer 293T cells were seeded at 2x10⁷ cells per T-150 flask. Plasmid DNA was mixed in the following amounts per T-150 flask; vector construct 40Mg, pMDG ^*\ 0vg, p8.91 30 g to a final volume of 5 ml in OptiMEM (Invitrogen, Paisley, UK). Polyethylenimine (PEI) (Sigma, Poole, UK) was added to 5 ml of OptiMEM to a final concentration of 2 nM and filtered through a 0.22 μιη filter. The DNA was added dropwise to the PEI solution and incubated at room temperature for 20 minutes. The DNA PEI solution was added to the 293T cells and incubated for 4 hours at 37°C, 5% C0₂ before being replaced by complete DMEM (Invitrogen). Growth medium was changed after 24 h and supernatant harvested after a further 24 h and replaced with growth medium for a second collection if necessary. Viral supernatant was initially centrifuged at 2500 rpm using a desktop centrifuge (MSE, Germany) for 10 minutes and then filtered through a 0.22pm filter prior to ultracentrifugation (Sorvall, UK) at 23,000 rpm (~50,000 xg), 4°C, for 2 h. Medium was carefully decanted and viral pellets resuspended in 50μΙ of CellGro medium (CellGro, VDG, USA). Finally, viral suspensions were centrifuged at 4,000 rpm for 10 minutes using a desktop microfuge. All viral preparations were used fresh and titered on 293T cells for biological titre by limiting dilution and FACS analysis for GFP or physical titre by p24 ELISA assay as previously described (Demaison et al.). Western blot

Cell lysates were prepared from cells in monolayer using standard methodology. Cell lysate was then resuspended 1 :1 in RIPA solution containing protease inhibitors (Invitrogen) and incubated on ice for 30 minutes. Cells were centrifuged at 7300 rpm at 4°C for 10 minutes in a desktop microfuge, supernatant collected and stored at -80°C. Samples were subjected to elecrophoresis on 5-15% SDS-polyacrylamide gels (Invitrogen) at 10V/cm² for 30-50 minutes. Following electrophoresis the protein was transferred to a PVDF membrane (Invitrogen) by semi-dry blotting as per manufacturer's instructions. Membrane transfer was carried out at 25V for 1 hour. The membrane was blocked at 4°C overnight in 5 % Milk / TBS block solution washed in 1 x TBS at room temperature whilst shaking before being incubated at room temperature for 2 hours with primary antibody. Following three washes with 1 x TBS-0.1 % Tween and one wash with 1 xTBS the membrane was then incubated with the appropriate secondary antibody for 1 hour at room temperature. Following three washes with 1 x TBS-Tween and one wash with 1 x TBS protein could then be detected using the ECL kit (Amersham, Bucks, UK) and subsequent exposure to Biomax MR Scientific imaging film (Kodak, Herts, UK).

Example 1 We have a comprehensive unpublished dataset showing that the tumour suppressor gene LIMD1 is a novel component of miRNA-mediated transcriptional repression complex. Mature double stranded miRNAs are dissociated within the cytoplasmic RISC complex and bind with incomplete homology to a target mRNA, usually in its 3' UTR. The nature of this incomplete complementarity (resulting in a small bleb) differentiates miRNA repression, where the mRNA is prevented from entering the translational machinery, from siRNA where complete homology targets the mRNA for degradation (25). The miRNA-RISC complexes have recently been shown to be the main and common component of cytoplasmic P-bodies ( 17). Our data show that LIMD1 associates with protein components of the P-body associated RNAi pathway; the elF4E ribosome docking protein, the mRNA decapping protein DCP2, the cap-dependent translational inhibitor RCK and the RNAi-mediated translational repressor AG02 (Fig. 1 ). In order to show that LIMD1 is directly involved in miRNA-mediated transcriptional repression we carried out luciferase derepression experiments by transducing a U20S cell line stably expressing the luciferase gene under the post-transcriptional control of serial Let-7a binding domains in a synthetic 3' UTR. These cells were transduced with lentiviral vectors expressing a LIMD1 specific shRNA, a control scrambled shRNA, full length LIMD1 and a dominant-negative LIMD1.

Example 2

The shLIMDI construct effectively "de-repressed" luciferase activity by greater than 3- fold, as did the dominant-negative LIMD1 construct. The other constructs had no effect (Fig. 2 Left Panel). To delineate between miRNA and siRNA mechanisms we transfected U20S cells containing a Renilla luciferase vector with either miRNA or siRNA Let-7a binding domains controlled against constitutive firefly luciferase expression. These cells were co-transfected with siRNAs against LIMD1 , AG02 or the P-body specific GW182. Both LIMD1 and GW182 showed miRNA-specific de- repression of RLuc whereas AG02 showed a siRNA-specifc de-repression (Fig. 2 Right Panel).

Example 3

We transduced shLIMDI knock down human Dermal Fibroblasts (hDF) with single or combinations of the iPS factors; OCT4/SOX2/KLF4/LIN28 and C-MYC. We found that OCT4/SOX2 transduced hDF-shl_IMD1 (OS shLIMDI ) cells formed iPS-like cells with high efficiency. Colonies show activation of an OCT4-GFP reporter (Fig. 3, Middle Panel) and express increased levels of endogenous OCT4, SOX2, Nanog and C-MYC by qPCR (Fig. 3, Right Panel). Their characterisation is ongoing. Interestingly, these colonies show proliferation kinetics similar to those of hES cells and vastly different to the parental hDF or hDF-shLIMD1 knock down cells alone. Finally, and importantly, the efficiency of generation of these cells is beyond the realms of current iPS efficiency. I have generated and characterised 4 other iPS cell lines from the same hDFs using lentiviral vectors expressing the Yamanaka cocktail of factors (0CT4/S0X2/KLF4/C- MYC). These iPS colonies arose with an efficiency of roughly 1 in 400,000. Using OS shLIMDI transduction I have calculated by serial counts that 23%±8 of all cells (>10 colonies per well) have iPS morphology 21 days after transduction. This startling increase in efficiency, if fully validated, could result in a paradigm shift in the way that iPS cells are generated.

Example 4

In order to show that specific depletion of LIMD1 creates a microRNA profile that is anti- differentiation and pro-pluripotency, we compared illumina miRNA array analysis data obtained from the U20S cells depleted for LIMD1 to scramble control shRNA (Fig. 15). The data indicated specific down-regulation of the Let-7 family of miRNAs is associated with a pro-differentiated cellular phenotype and conversely up-regulation of miRNAs is associated with stemness/pluripotency. Total RNA were extracted by Trizol (Invitrogen, Carlsbad, CA, USA). The lllumina's MicroRNA Expression Profiling Panels (Illumina, San Diego, CA, USA) containing probes for 470 human miRNAs from the Sanger database v10.1 were used. Overall miRNA hybridization signal distribution was analyzed GraphPad Prism 4 statistics software (GraphPad Software Inc., La Jolla, CA, USA).

References

Demaison, C, Parsley, K., Brouns, G., Scherr, M., Battmer, K., Kinnon, C, Grez, M., and Thrasher, A.J. 2002. High-level transduction and gene expression in hematopoietic repopulating cells using a human immunodeficiency virus type 1 -based lentiviral vector containing an internal spleen focus forming virus promoter. Hum Gene Ther 13:803-813.

Claims

1 An isolated mammalian somatic cell wherein said cell is modified which modification reduces the expression or activity of a polypeptide that comprises a Lim domain amino acid motif.

2. A cell according to claim 1 wherein the modification in expression of said Lim domain polypeptide is regulatable.

3. A cell according to claim 1 or 2 wherein In a preferred embodiment of the invention said polypeptide comprises: the amino acid motif:

C(X)2C(X) i 6-23(H/C)(X)2/4(C/H/E)(X)₂C(X)2C(X) i4-2i(C/H)(X)2/i/3(C/H/D/E)X, wherein X is any amino acid residue.

4. A cell according to claim 3 wherein said polypeptide is selected from the group consisting of: LIMD1 , Ajuba, WTIP, Zyxin, LPP, TRIP6 and Migfilin

5. A cell according to any of claims 1-4, wherein said polypeptide is LimD1 and is represented by the amino acid sequence presented in Figure 4c.

6. A cell according to any of claims 1-4, wherein said polypeptide is LimD1 and is represented by the nucleotide sequence presented in Figure 4d.

7. A cell according to any of claims 1-4 wherein said polypeptide is Ajuba and is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 10.

8. A cell according to any of claims 1-4 wherein said polypeptide is WTIP and is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 11.

9. A cell according to any of claims 1-4 wherein said polypeptide is Zyxin and is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 12.

10. A cell according to any of claims 1-4 wherein said polypeptide is LPP and is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 13.

11. A cell according to any of claims 1-4 wherein said polypeptide is TRIP6 and is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 14.

12. A cell according to any of claims 1-4 wherein said polypeptide is Migfilin and is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 15.

13. A cell according to any of claims 1-12 wherein the expression of the Lim domain polypeptide is reduced or inhibited by transfection of a siRNA or shRNA.

14. A cell according to claim 13 wherein said siRNA molecule is between 18bp and 29bp in length.

15. A cell according to claim 13 or 14 wherein said siRNA includes modified nucleotides.

16. A cell according any of claims 13-15 wherein said somatic cell is transiently transfected to reduce Lim domain expression.

17. A cell according to any of claims 13-15 wherein said somatic cell is stably transfected to reduce Lim domain expression.

18. A cell according to any of claims 1-17 wherein said somatic cell is further modified to express at least one re-programming factor.

19. A cell according to claim 18 wherein said somatic cell expresses at least Oct 4 and Sox 2.

20. A cell according to claim 19 wherein Oct 4 is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 5.

21. A cell according to claim 19 wherein Sox 2 is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 6.

22. A cell according to any of claims 18-21 wherein said cells express at least Oct 4, Sox 2, c-MYC, Klf4 and/or LIN28.

23. A cell according to claim 22 wherein c-MYC is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 7.

24. A cell according to claim 22 wherein Klf4 is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 8.

25. A cell according to claim 22 wherein Lin28 is encoded by a nucleic acid molecule comprising a nucleotide sequence as represented in Figure 9.

26. A cell according to any of claims 18-25 wherein said somatic cell is transfected with a vector that includes said re-programming factor.

27. A cell according to any of claims 1-26 wherein said mammalian cell is a human somatic cell.

28. A cell according to claim 27 wherein said somatic cell is selected from the group consisting of: a hematopoietic cell, a lymphocyte, myeloid cell; a buccal mucosa cell, an epidermal cell, a mesenchymal cell, a keratinocyte.

29. A cell according to claim 28 wherein said somatic cell is a fibroblast; preferably an adult or embryonic fibroblast.

30. A cell according to claim 29 wherein said fibroblast cell is selected from the group consisting of: a dermal fibroblast, a foetal fibroblast, a corneal fibroblast, an intestinal mucosa fibroblast, an oral mucosa fibroblast and urethral fibroblast.

31. A transcription cassette wherein said cassette includes a nucleotide sequence designed with reference to Figure 4d and is adapted for expression by provision of at least one promoter operably linked to said nucleic acid sequence such that both sense and antisense molecules are transcribed from said cassette.

32. A cassette according to claim 31 wherein said cassette is adapted such that both sense and antisense nucleic acid molecules are transcribed from said cassette wherein said sense and antisense nucleic acid molecules are adapted to anneal over at least part or all of their length to form a shRNA.

33. A cassette according to claim 31 or 32 wherein said cassette is provided with at least two promoters adapted to transcribe both sense and antisense strands of said nucleic acid molecule.

34. A cassette according to any of claims 31-33 wherein said cassette comprises a nucleic acid molecule wherein said molecule comprises a first part linked to a second part wherein said first and second parts are complementary over at least part of their sequence and further wherein transcription of said nucleic acid molecule produces an RNA molecule which forms a double stranded region by complementary base pairing of said first and second parts thereby forming an shRNA.

35. A cassette according to any of claims 31-34 wherein said cassette includes one or more of the following nucleotide sequences:

GGTTAGTGCTCGAGTGAAA GGGAAGAGCTGCCCGCATA

!TGTTAGGACTTAAGAGAAT

iGAGCAGAGATCCAGGCCAT

jCATCATAGCACCAAAGGAA

iGCAGAGTGATCGTAACTAAj

jGGACAAGGGTGCAGGCAAC

!CTTTGGAGCCTGTGTGAAA!

:GAAAATACCTATACCCAAA:

GCACAAGACCACAAATTAT TCACAGAGCTTCACCAAAT jCAGATAGGTTTGCGAATATj

36. A cassette according to any of claims 31-35 wherein said cassette further comprises nucleic acid molecules that encode at least the re-programming factors Oct 4 and Sox 2.

37. A cassette according to claim 36 wherein Oct 4 is encoded by a nucleic acid molecule comprising the nucleotide sequence in Figure 5.

38. A cassette according to claim 36 wherein Sox 2 is encoded by a nucleic acid molecule comprising the nucleotide sequence in Figure 6.

39. An expression vector comprising a cassette according to any of claims 31-38 wherein said cassette is operably linked to a promoter sequence.

40. A vector according to claim 39 wherein said promoter is a regulatable promoter; preferably an inducible promoter.

41. A vector according to claim 40 wherein said vector is a viral based vector.

42. A vector according to claim 41 wherein said viral based vector is a lentiviral vector.

43. A cell culture comprising a cell according to any of claims 1-42.

44. A method for the reprogramming of a mammalian somatic cell comprising contacting said cell with an agent that inhibits the expression or activity of a Lim domain protein in combination with the expresssion of at least two re-programming factors.

45. The method according to claim 44 wherein said re-programming factors is Oct 4 and Sox 2.

46. The method according to claim 45 wherein Oct 4 is encoded by a nucleotide sequence as represented in Figure 5.

47. The method according to claim 45 wherein Sox 2 is encoded by a nucleotide sequence as represented in Figure 6.

48. An induced pluripotent cell obtained or obtainable by the method according any of claims 44-47.

49. A cell culture obtained or obtainable by the method according to any of claims 44-47.

50. A method for the preparation of a library comprising induced stem cell specific gene expression products comprising the steps: i) providing a preparation comprising an induced stem cell according to the invention;

ii) extracting nucleic acid from said cell preparation;

iii) preparing a cDNA from ribonucleic acid contained in said extracted nucleic acid; and

iv) ligating cDNA formed in (iii) into a vector.

51. A method for the identification of genes associated with induced stem cells comprising:

ii) extracting nucleic acid from said cell preparation;

i) contacting said extracted nucleic acid with a nucleic acid array; and ii) detecting a signal which indicates the binding of said nucleic acid to a binding partner on said nucleic acid array.

52. A method according to claim 51 wherein said method includes the additional steps of:

iii) providing an output for the analysed data.