WO2012007725A2

WO2012007725A2 - Method of reprogramming a cell

Info

Publication number: WO2012007725A2
Application number: PCT/GB2011/001063
Authority: WO
Inventors: Yen Choo; Mylvaganam Jeyakumar
Original assignee: Plasticell Ltd
Priority date: 2010-07-16
Filing date: 2011-07-14
Publication date: 2012-01-19
Also published as: WO2012007725A3

Abstract

The present invention relates to a method of reprogramming a cell via a chemically induced multi-lineage potential (CIMP) cell comprising the steps of: (i) contacting the cell to be reprogrammed with a medium comprising one or more, preferably, two or more chemical reprogramming agents (CRAs); (ii) obtaining a CIMP cell in which the expression of one or more pluripotency genes is induced therein and one or more pluripotency proteins are expressed by the one or more induced pluripotency genes following contact with the one more CRA(s) such that the cell has gained potency and the capacity to differentiate into an increased number of cell types as compared to the cell of step (i); and (iii) differentiating the CIMP cell into a more lineage restricted cell, preferably, wherein the cell to be reprogrammed has not been genetically manipulated to induce pluripotency and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

Description

METHOD OF REPROGRAMMING A CELL

SUMMARY

The present invention relates to methods and compositions for reprogramming a cell. In particular, the present invention relates to methods, compositions and kits for chemically reprogramming a cell into a chemically induced multi-lineage potential (CIMP) cell. From this reprogrammed state, the CIMP cell may be differentiated into different cell types.

BACKGROUND

Regenerative medicine is a growing field of research involving the repair, replacement or regeneration of damaged or diseased cells, tissues and organs. Healthy cells or tissue generated in the laboratory from the stem cells of a compatible donor or the patient's own stem cells are transplanted into the patient. Stem cells are undifferentiated cells which can either renew their own cell population or differentiate and give rise to specialized, differentiated cells. Sources of stem cells which can be used in such therapies include embryonic stem cells (ESCs), adult stem cells (ASCs) or umbilical cord blood stem cells. Due to the controversial nature of ESC derivation from human embryos, the resulting opposition on ethical and religious grounds as well as restricted funding for research, scientists have sought to identify new avenues for generating stem cells from tissues that are not of fetal origin. One approach involves the manipulation of autologous adult stem cells. The advantage of using autologous adult stem cells for regenerative medicine lies in the fact that they are derived from and returned to the same patient, and are therefore not subject to immune-mediated rejection. The major drawback is that these cells typically lack the plasticity and pluripotency of ES cells and thus their potential is uncertain. Another approach is aimed at reprogramming somatic cells from adult tissues to create pluripotent ES-like cells. However, this approach has been difficult as each cell type within a multi-cellular organism has a unique epigenetic signature that is thought to become fixed once cells differentiate or exit from the cell cycle.

A lot of progress has been made in the field of ESC research, particularly in deciphering the genetic machinery and signaling mechanisms that enable these cells to either maintain their pluripotent state or are responsible for driving them down the differentiation pathway for a particular cell type. Key genes which regulate pluripotency have been identified. This has lead to the generation of a new type of pluripotent cell, the induced pluripotent stem (iPS) cell. iPS cells are adult somatic cells whose genome has been reprogrammed to an embryonic-like pluripotent state in vitro by the introduction and forced expression of certain genes and factors deemed crucial for the maintenance of ES cell pluripotency.

Although patient-specific pluripotent cell lines have great potential for medical use some of the existing technologies to derive such cells have a number of severe limitations. By way of example, the technologies require the use of retroviral vectors in order to introduce and express reprogramming factors that can randomly integrate into the genome in multiple copies. They may integrate into the vicinity of or even into active endogenous genes and may cause the activation or inactivation of mutations in cancer or tumour suppressor genes. Such genetic modifications and the continuous presence of retroviral vectors may therefore lead to the development of cancerous cells and is therefore, a highly undesirable route. By way of further example, the continuous presence of sequences of expression vectors for reprogramming factors, for example, the coding sequences for the reprogramming factors and their regulatory sequences, could interfere with the proper differentiation and/or function of the derived cells which may limit their utility for regenerative medicine. To try and address the safety issues that arise from harbouring integrated exogenous sequences into the target cell genome, a number of modified genetic methods have been developed w ith potentially reduced risks - such as the use of nonintegrating adenoviruses to deliver reprogramming genes; transient transfection of reprogramming plasmids; a piggyBac transposition system; Cre-excisable viruses; and or/P/EBNA1 -based episomal expression systems. However, all of these methods still involve the use of genetic materials and thus the potential for unexpected genetic modifications by the exogenous sequences in the target cells.

A recent development which aims to avoid introducing exogenous genetic modifications is to deliver the reprogramming proteins directly into cells, rather than relying on transcription from delivered genes. Zhou ef al. (2009) Cell Stem Cell 4, 1 -4) describe the generation of protein induced pluripotent stem cells (PiPSCs) from murine embryonic fibroblasts using recombinant cell-penetrating reprogramming proteins. However, like current methods for generating iPS cells, this methodology has many drawbacks. By way of example, the reprogramming proteins are expensive to make since they are of recombinant origin and the proteins need to be transduced into cells which means that the methodology is complex.

The use of chemical biology to advance regenerative medicine has been reported . For example, efforts have been made to try and identify chemicals that can replace the introduced reprogramming factors to make iPS cells. In one study, an inhibitor of the TGF-beta receptor has been shown to be capable of replacing Sox2 in the reprogramming process (lchida JK ef al. (2009) Cell Stem Cell 5, 491 -503). Other researchers have tried to use chemicals to substitute each of the reprogramming genes in order to create cells with increased differentiation potential. However, when the chemicals are used together to try to reprogram the cell they have not resulted in cells that have gained poten cy.

US2009/0253203 describes methods for inducing the expression of at least one gene which contributes to a cell being pluripotent or multipotent using small molecule modulators without genetically manipulating the cell to induce pluripotency. The methods require that the cells are grown in a culture medium in the presence of a small molecule modulator. Certain cells cultured in the presence of RG108 showed upregulated Nanog gene expression. Certain cells cultured in the presence of one of hydralazine HCL, ALA, biotin, nicotinamide, procaine HCL, sodium phenylbutyrate, tranylcypromine, SIRT1 activator 3, CAY 10433 , depudicin, EX-527, splitomycin, ITSA, valproxam, resveratrol or 2-PCPA showed upregulated or increased Oct4 gene expression. Certain cells cultured in the presence of valproic acid (VPA) or RG108 showed upregulated or increased Nanog and/or Oct4 gene expression. Certain cells cultured in the presence of VPA showed a change in expression of Oct-4, Nanog and Sox-2. Cells treated with VPA were further studied to determ ine if the increase in expression of pluripotency genes correlated with morphological changes consistent with embryonic cells. In this method, human dermal fibroblasts were cultured in the presence of VPA. After 5 days, cells were then transferred to a different culture medium in the presence of the same amount of the VPA for a period of 16 days. Cells resembling embryoid-like bodies were observed. However, no positive pluripotent protein staining was detected. Whilst it was concluded that individual small molecule modulators can be used to transform a differentiated cell into a cell with embryonic-like morphology, it was not shown that the cells had gained potency since no positive pluripotent protein staining could be detected in these cells.

Aside from reprogramming, lost or damaged cells can be replaced via the processes of dedifferentiation or transdifferentiation. Dedifferentiation involves a terminally differentiated cell reverting back to a less-differentiated stage from within its own lineage. This process allows the cell to proliferate again before redifferentiating , leading to the replacement of those cells that have been lost. Transdifferentiation is another naturally occurring mechanism that was first observed in the regenerating lens of the newt. This process takes dedifferentiation a step further in that cells regress to a point where they can switch lineages thereby allowing them to differentiate into another cell type. In contrast, reprogramming aims to induce differentiated cells into reverting to a more potent state - such as a pluripotent state. From the potent state, the cells can then differentiate into a different cell types. Reprogramming can sidestep the requirement to use embryos for regenerative therapies by using differentiated cells taken from a patient. This can also prevent immunological problems associated with engraftment.

There is a need in the art for methods that can reprogra m a cell in order to change the differentiation potential thereof without the need to genetically manipulate the cell or contact the cell with exogenous proteins to induce pluripotency. From this reprogrammed state, it would be desirable if the cells could be differentiated into different cell types.

SUMMARY ASPECTS AND EMBODIMENTS

The present invention is based, at least in part, on the finding that chemicals alone - such as small molecules - can be used to reprogram a cell without the need to either genetically manipulate the cell or to contact the cell with one or more exogenous reprogramming proteins to induce pluripotency. The chemicals used to reprogram the cells are referred to herein as chemical reprogramming agents (CRAs). The cell that is obtained following contact with the CRA(s) is more primitive than the cell from which it was derived and has gained potency. The more primitive cell is referred to herein as a chemically induced multi-lineage potential (CIMP) cell. The methods provide a powerful way to de-differentiate a cell whose developmental fate has been previously determined. Advantageously, the chemical derivation of CIMP cells solves many of the problems currently faced by cell researchers. By way of example, the generation of CIMP cells is convenient and flexible since the genetic manipulation of the cell to induce pluripotency (for example, via the exogenous expression of one or more gene(s) - such as one or more genes encoding one or more pluripotency genes or one or more transcription factors) is not required; the effects of small molecules are typically reliable and reproducible and their effects can be precisely tuned by varying the concentration. This may therefore be an important step in the development of stem cell based therapies. In addition, the derivation of CIMP cells does not require the use of early embryos or germ cells, thereby avoiding ethical issues associated with ES cells. Advantageously, the CIMP cell can be differentiated. The CIMP cell can be differentiated into a cell that is different to the CIMP cell and/or different to the cell from which the CIMP cell was derived. For example, the CIMP cell can be differentiated into a cell that is of a different germ cell layer to the CIMP cell and/or the cell from which the CIMP cell was derived.

Aspects and embodiments of the present invention are set forth in the accompanying claims. In one aspect, there is provided a method of reprogramming a cell via a chemically induced multi-lineage potential (CIMP) cell comprising the steps of: (i) contacting the cell to be reprogrammed with a medium comprising one or more, preferably two or more, chemical reprogramming agents (CRAs); (ii) obtaining a CIMP cell in which the expression of one or more pluripotency genes is induced therein and one or more pluripotency proteins are expressed by the one or more induced pluripotency genes following contact with the one more CRAs such that the cell has gained potency and the capacity to differentiate into an increased number of cell types as compared to the cell of step (i); and (iii) differentiating the CIMP cell into a more lineage restricted cell, preferably, wherein the cell to be reprogrammed has not been genetically manipulated to induce pluripotency and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

In one embodiment, the one or more pluripotency proteins that are expressed in step (ii) comprise, consist ot consist essentially of pluripotency proteins selected from the group consisting of alkaline phosphatase (ALP), SSEA1 , Oct4 or Nanog or a combination of two or more thereof, preferably, wherein the one or more pluripotency proteins that are expressed in step (ii) comprise, consist or consist essentially of alkaline phosphatase (ALP), SSEA1 , Oct4 and Nanog . In one embodiment, the CRA(s) are small molecules.

In one embodiment, the CIMP cell is phenotypically at an earlier developmental stage than the cell from which it was derived and is morphologically different to the cell from which it was derived.

In one embodiment, the CRA(s) is selected from the group consisting of: a DNA methyltransferase inhibitor(s); a histone deacetylase inhibitor(s); a KLF4 inducer(s), a retinoic acid receptor agonist(s); an HDAC1 inhibitor(s); a protein tyrosine phosphatase PTP inhibitor(s), a MEK/ERK inhibitor(s), a protein tyrosine phosphatase PTP inhibitor(s); a H2 histamine receptor antagonist(s), pregnant mare serum Gonadotropin(s) (PMSG), an NADPH oxidase inhibitor(s), a histone methyltransferase inhibitor(s), a calcium channel agonist(s), a demethylating agent(s), an inhibitor of Rho-associated protein kinase(s), a voltage gated sodium channel stabiliser(s), a GPR modulator(s), a P53 inhibitor(s), a Peroxisome Proliferator- Activated Receptor (PPAR) gamma agonist(s), a caspase-3 Inhibitor(s) and a xanthine oxidase inhibitor(s) or a combination of two or more thereof.

In one embodiment, the CRA(s) is selected from the group consisting of: a PPAR gamma agonist(s), a histone deacetylase inhibitor(s), a DNA methyltransferase inhibitor(s), and a P13K inducer(s); preferably, wherein the CRA(s) are selected from a combination of: a PPAR gamma agonist(s), a histone deacetylase inhibitor(s), a DNA methyltransferase inhibitor(s), a P13K inducer(s), an NADPH oxidase inhibitor(s), a GPR modulator(s) and a p53 inhibitor(s) or a combination of two or more thereof; preferably, wherein the CRA(s) are selected from: a PPAR gamma agonist(s), a histone deacetylase inhibitor(s), a DNA methyltransferase inhibitor(s), a P13K inducer(s), an NADPH oxidase inhibitor(s), a GPR modulator(s), a p53 inhibitor(s) and an anti-apoptosis agent(s) or a combination of two or more thereof; preferably wherein the CRA(s) are selected from: a PPAR gamma agonist(s), a histone deacetylase inhibitor(s), a DNA methyltransferase inhibitor(s), a P1 3K inducer(s), an NADPH oxidase inhibitor(s), a GPR modulator(s), a p53 inhibitor(s), an anti-apoptosis agent(s) and a histone methyltransferase inhibitor(s) or a combination of two or more thereof.

In one embodiment, said cell to be reprogrammed is contacted with the CRA(s) simultaneously, separately or sequentially.

In one embodiment, said cell to be reprogrammed is incubated in the absence of one or more of the CRA(s) for a period of time.

In one embodiment, said cell to be reprogrammed is contacted with the following combinations of CRA(s): (i) an HDAC inhibitor, a PPAR gamma agonist, a DNA methyltransferase inhibitor, a NADPH oxidase inhibitor and a P13K inducer; (ii) an HDAC inhibitor, a PPAR gamma agonist, a DNA methyltransferase inhibitor, a histone methyltransferase inhibitor and an anti-apoptosis agent; and (iii) an HDAC inhibitor, a PPAR gamma agonist, a DNA methyltransferase inhibitor, a NADPH oxidase inhibitor, a GPR modulator and a p53 inhibitor. In one embodiment, said cell to be reprogrammed is contacted with these combinations of CRA(s) step wise.

In one embodiment, said CIMP cell is differentiated in step (iii) into a cell that is different to the cell of step (i) an/or step (ii), preferably, wherein the CIMP cell is differentiated in step (iii) into a cell of a different germ layer to the cell of step (i) and/or step (ii).

In one embodiment, after the reprogramming, said CIMP cell is expanded , preferably, wherein said expanded CIMP cell is a population of homogenous cells.

In a further aspect, there is provided a cell obtained or obtainable by the method of the present invention.

In a further aspect, there is provided a method of reprogramming a cell into a chemically induced multi-lineage potential (CIMP) cell comprising the steps of: (i) contacting the cell to be reprogrammed with a medium comprising one or more, preferably, two or more, CRA(s); and (ii) obtaining a CIMP cell in which the expression of one or more pluripotency genes is induced therein and one or more pluripotency proteins are expressed by the one or more induced pluripotency genes following contact with the CRA(s) such that the cell has gained potency and the capacity to differentiate into an increased number of cell types as compared to the cell of step (i), preferably, wherein the cell to be reprogrammed has not been genetically manipulated and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

In a further aspect, there is provided a CIMP cell obtained or obtainable by the method of the present invention .

In a further aspect, there is provided a CIMP cell, wherein said cell is more potent than the cell from which it was derived following contact with one or more, preferably two or more, CRA(s) and wherein the expression of one or more pluripotency genes is induced in the CIMP cells and one or more proteins are expressed by the one or more induced pluripotency genes such that the cell has gained potency and the capacity to differentiate into an increased number of cell types as compared to the cell from which it was derived, wherein said CIMP cell has the capacity to differentiate into a more lineage restricted cell and preferably, wherein the cell to be reprogrammed has not been genetically manipulated to induce pluripotency and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

In a further aspect, there is provided a method for identifying one or more, preferably two or more, CRA(s) for reprogramming a cell into a CIMP cell comprising the steps of: (i) contacting the cell to be reprogrammed with a medium comprising one or more, preferably two or more, chemicals, preferably small molecules; (ii) determining if the expression of one or more pluripotency genes is induced therein and one or more proteins are expressed by the one or more induced pluripotency genes following contact with the chemicals such that the cell has gained potency and the capacity to differentiate into an increased number of cell types as compared to the cell of step (i); and (iii) determining if said cell of step (i) has been reprogrammed in to a CIMP cell, wherein the presence of a CIMP cell is indicative that the chemicals are CRA(s) able to reprogram the cell into a CIMP cell, preferably, wherein the cell to has not been genetically manipulated and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

In a further aspect, there is provided a combination comprising, consisting or consisting essentially of: (i) a DNA methyltransferase inhibitor; a histone deacetylase inhibitor; a KLF4 inducer, a retinoic acid receptor agonist; an HDAC1 inhibitor; and a protein tyrosine phosphatase PTP inhibitor; (ii) a histone deacetylase inhibitor; a KLF4 inducer, a MEK/ERK inhibitor; a protein tyrosine phosphatase PTP inhibitor; a H2 histamine receptor antagonist and Pregnant mare serum Gonadotropin (PMSG); (iii) an NADPH oxidase inhibitor; a MEK/ERK inhibitor; a histone methyltransferase inhibitor; a protein tyrosine phosphatase PTP inhibitor; and a calcium channel agonist.; or (iv) a demethylating agent; a histone deacetylase inhibitor; a KLF4 inducer, an inhibitor of Rho-associated protein kinase; a histone methyltransferase inhibitor and a voltage gated sodium channel stabiliser; (v) a KLF4 inducer, an NADPH oxidase inhibitor, an HDAC1 inhibitor; a GPR modulator, a P53 inhibitor and a demethylating agent; (vi) a DNA methyltransferase inhibitor, a deacetylase inhibitor, a PPAR gamma agonist, an NADPH oxidase inhibitor, a PKC activator and an a1 -adrenoceptor antagonist; (vii) a histone deacetylate inhibitor, a PPAR gamma agonist, a retinoic acid receptor agonist, a caspase-3 Inhibitor, a xanthine oxidase inhibitor; and a H2 histamine receptor antagonist; (viii) a demethylating agent, a KLF4 inducer, an NADPH oxidase inhibitor and a HDAC1 inhibitor; (ix) a PPAR gamma agonist, a histone deacetylase inhibitor, a DNA methyltransferase inhibitor, or a P13K inducer; (x) a PPAR gamma agonist, a histone deacetylase inhibitor, a DNA methyltransferase inhibitor, a P13K inducer, an NADPH oxidase inhibitor, a GPR modulator and a p53 inhibitor; (xi) a PPAR gamma agonist, a histone deacetylase inhibitor, a DNA methyltransferase inhibitor, or a P13K inducer, an NADPH oxidase inhibitor, a GPR modulator, a p53 inhibitor and an anti-apoptosis agent; (xii) a PPAR gamma agonist, a histone deacetylase inhibitor, a DNA methyltransferase inhibitor, a P13K inducer, an NADPH oxidase inhibitor, a GPR modulator, a p53 inhibitor, an anti-apoptosis agent and a histone methyltransferase inhibitor; (xiii) an HDAC inhibitor, a PPAR gamma agonist, a DNA methyltransferase inhibitor, a NADPH oxidase inhibitor and a P13K inducer; (xiv) an HDAC inhibitor, a PPAR gamma agonist, a DNA methyltransferase inhibitor, a histone methyltransferase inhibitor and an anti-apoptosis agent; or (xv) an HDAC inhibitor, a PPAR gamma agonist, a DNA methyltransferase inhibitor, a NADPH oxidase inhibitor, a GPR modulator and a p53 inhibitor. In a further aspect, there is provided a pharmaceutical composition comprising one or more of the combinations admixed with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

In a further aspect, there is provided a method for generating a transgenic non-human animal comprising the steps of: (i) providing a CIMP cell or a composition according to the invention ; (ii) introducing the cell into a non-human blastocyst; (iii) transferring the blastocyst into the uterus of a female non-human animal; and (iv) allowing the blastocyst to develop into an embryo.

In a further aspect, there is provided a transgenic non-human animal obtained or obtainable by the method of the present invention

In a further aspect, there is provided the CIMP cell or a cell derived or derivable from said cell or said CIMP cell or the composition for use in the treatment of disease; or for the treatment of disease.

In a further aspect, there is provided the use of the cell, the CIMP cell or a cell derived or derivable from said cell or said CIMP cell or the composition of the present invention in the manufacture of a composition for the treatment of a disease that results from malfunctioning, damaged or failing tissue.

In a further aspect, there is provided a method of treating a disease in a subject comprising administering the pharmaceutical composition to said subject.

In a further aspect, there is provided one or more combinations or the pharmaceutical composition for treating a disease.

In a further aspect, there is provided the use of one or more combinations or the pharmaceutical composition in the manufacture of a composition for the treatment of a disease that results from malfunctioning, damaged or failing tissue.

In a further aspect, there is provided a method for reprogramming a cell into a CIMP cell comprising the use of one or more of the combinations, preferably, wherein the cell to be reprogrammed is or is derived from the ectoderm, endoderm or mesoderm lineage.

In a further aspect, there is provided the use of one or more of the combinations for reprogramming a cell.

In a further aspect, there is provided a kit for reprogramming a cell into a CIMP cell comprising one or more of the combinations and optionally a set of instructions for performing same.

In a further aspect, there is provided a method, cell, combination, composition, transgenic non- human animal, use or kit substantially as described herein and with reference to the accompanying drawings.

FIGURES

Figure 1 Generation of CIMP cells from primary mouse neural stem cell (NSC) line via CombiCult and small molecular compounds. (A) CIMP bearing beads identified through ALP screen; antibody staining of SSEA1 , Oct4 and Nanog in CIMP bearing beads.

Figure 2

NSC derived CIMP cells in monolayer cultures. Representative light m icrograph images are shown. (A) bright field images showing morphology of CIMP cells; (B) Alkaline phosphatase (ALP) staining of CIMP cells; (C) immunocytochemical staining of CI MP colonies for SSEA1 , Oct4 and Nanog.

Figure 3

Haematopoietic differentiation of NSC CIMP cells. (A) Various CFU generated from NSC-CIMP in MethoCult cultures; (B) CD34 immunostaining of CFU in MethoCult cultures; (C) Wrights- Giesma stain of Cytospin preparations of MethoCult cultures, showing various blood forming cells, including megakaryocytes, pre-erythroblast, granulocytes, monoblast, myeloblast, lymphoblast and erythrocytes.

Figure 4

CombiCult derived NSC CIMP cells differentiate into phagocytes, as determined by a phagocytosis functional assay. CIMP bearing beads were cultured in differentiation media for 14 days (see materials and methods), incubated with E. coli BioParticles® tetramethylrhodamine conjugates (Invitrogen, E2862) and the phagocytic cells signals identified under a fl uorescence microscope.

Figure 5

Adipo and osteogenic differentiation of NSC CIMP cells. Adipogenesis is determined using oil red staining; Osteogenesis is determined using Alizarin Red staining.

Figure 6

Generation of CIMP cells from primary mouse dermal fibroblast (MDF) via CombiCult and small molecular compounds. (A) CIMP bearing beads identified through ALP screen. (B) bright field images showing morphology of MDF derived CIMP cells; (B) ALP staining of MDF derived CIMP cells.

Figure 7

MDF CIMP colonies showing positive staining for Oct4 and Nanog.

Figure 8

Adipo and osteogenic differentiation of MDF CIMP cells. Adipogenesis is determined using oil red staining; Osteogenesis is determined using Alizarin Red staining.

Figure 9

Neuroectoderm differentiation into neurons and astroglia of MDF-derived CIMP cells.

DETAILED DESCRI PTION Definitions

Reprogram ming. The term "reprogramming" as used herein relates to the process of changing the differentiation potential of a cell. A cell may be reprogrammed such that it is at an earlier developmental stage than the cell from which it was derived and will be a more primitive cell compared to the cell from which it was derived. Thus, the cell that has been reprogrammed may be a de-differentiated cell. An example of this process is reprogramming a cell from a differentiated or semi-differentiated cell into a CIMP cell. In one embodiment, the cell may be reprogrammed such that it is phenotypically and/or epigenetically at an earlier developmental stage than the cell from which it was derived. An example of a phenotypic change is the morphology of the cell whereby the cell that it is phenotypically at an earlier developmental stage than the cell from which it was derived is morphologically different. Examples of epigenetic changes are the methylation of DNA, including but not limited to the methylation of promoter regions of genes, and/or changes in higher order chromatin structure known as chromatin remodelling. Epigenetic changes may lead to a difference (eg. an increase) in expression of one or more pluripotency genes/markers (a pluripotency gene including a gene that contributes to a cell being pluripotent); or a difference (eg. an increase) in expression of one or more proteins expressed from the pluripotency genes/markers; or a difference (eg. an increase) in the developmental potential of the cell. Epigenetic changes may be passed on from a cell to its progeny, hence for instance resulting in patterns of gene expression that persist from one generation to the next. The CIMP cell can be differentiated into a cell - such as a more lineage restricted cell - when incubated under appropriate conditions and with appropriate agents that are known in the art.

Chem ical reprogramm ing agent. The term "chemical reprogramming agent (CRA)" refers to a chemical that is contacted with a medium or a cell that is contained or about to be contained in the medium. Thus, for example, the CRA(s) may be added (eg. exogenously added or supplemented) to the medium in which the cell to be reprogrammed or the cell that is being reprogrammed is contained. The CRA(s) may not normally be present in the cell or the medium in which the cell is contained. The CRA(s) may be a single chemical, preferably a single small molecule or a combination of chemicals, preferably, a combination of small molecules - such as a combination of two or more different chemicals/small molecules. In one embodiment, the CRA(s) is not a protein (eg. a growth factor or a pluripotency protein), a nucleic acid (eg. a nucleic encoding a growth factor or a pluripotency protein), or a polysaccharide. In particular, the CRA(s) is not a reprogramming protein that has been modified to penetrate a cell or a nucleic acid encoding same. The CRA(s) may be a chemical, preferably a small molecule, with a distinct composition. Preferably, the CRA(s) is or is derived from a small molecule. In one embodiment, when a single CRA is used to reprogram a cell the CRA is not a DNA methyltransferase inhibitor (eg. RG108 or epigallocatechin-3-gallate or hydralazine HCI or procaine HCI); or a histone deacetylase inhibitor (eg. ALA or biotin); or a sirtuin inhibitor (eg. nicotinamide or EX-527); or a histone deacetylase inhibitor (eg. sodium phenylbutyrate or CAY 10433 or depudicin or splitomycin or valproxam or valproic acid); or a lysine demethylase inhibitor (eg. tranylcypromine); or a sirtuin activator (eg. SIRT1 activator 3 or resveratrol); or a Trichostatin A inhibitor (eg. ITSA); or a histone/lysine 1 demethylase inhibitor (eg. 2-PCPA). Smal l molecu le. The term "small molecule" refers to a low molecular weight organic compound . The molecular weight limit for the small molecule is approximately 1500 Daltons or less or 1000 Daltons or less or 900 Daltons or less or 800 Daltons or less or 700 Daltons or less or 600 Daltons or less or 500 Daltons or less, which allows for the possibility of diffusion across cell membranes and into cells. The small molecules may be natural or artificial. The constituent monomers of proteins, nucleic acids and polysaccharides - such as ribo- or deoxyribonucleotides, amino acids, and monosaccharides - are not small molecules in the context of this definition.

Cell. The methods described herein can be used to reprogram cells. The cell may be, for example, a somatic cell, a progenitor cell, an adult stem cell, an organ specific stem cell, a stem like cell, a cell line or a primary cell line. The cell may be a differentiated cell (eg. a non- pluripotent cell) or a terminally differentiated cell. The cell may be a semi-differentiated cell or an incompletely differentiated cell. The cell may be a non-diseased cell or a diseased cell. The cell may be obtained from any organ or tissue. The cell may be from humans, or animals - such as a mouse, a guinea pig, a rat, cattle, horses, pig, sheep or non-human primates and the like. The cell may be or may be derived from a wild type cell. Typically, the cell will not be a pluripotent cell. Suitably, the cell to be reprogrammed is free of one or more genetic modifications that induce pluripotency. Suitably, the cell to be reprogrammed is free of one or more exogenous pluripotency proteins or nucleic acids encoding same to induce pluripotency. Suitably, the cell to be reprogrammed will comprise, consist or consist essentially of a genetically unmodified set of endogenous pluripotency genes. Suitably, the cell to be reprogrammed will have no or substantially no detectable artificially induced pluripotency gene and/or protein expression, obtained, for example, through genetic manipulation and/or contact with one or more exogenous reprogramming proteins or nucleic acids that induce pluripotency. Stem cell . This term refers to a cell that can self-renew indefinitely and differentiate to form specialised cells of tissues and organs and includes a pluripotent or multipotent cell. A stem cell can divide to produce two daughter stem cells, or one daughter stem cell and one progenitor cell, which then differentiates into the tissue's mature, fully formed cells.

Somatic cell. The term "somatic cell" has the same meaning as is understood in the art ie. any cell forming the body of an organism other than a germ line cell. In mammals, germ line cells (also known as gametes) are the spermatozoa and ova which fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Every other cell type in the mammalian body - apart from the sperm and ova, the cells from which they are made (gametocytes) is a somatic cell. The term "somatic cell" also includes adult stem cells - such as hematopoietic stem cells, neural stem cells, and mesenchymal stem cells.

Progenitor cell. The term "progenitor cell" has the same meaning as is understood in the art ie. any cell that has the capacity to differentiate into a specific type of cell and that is more restricted than a stem cell, ie. they are further differentiated towards a terminally differentiated cell. An important difference between stem cells and progenitor cells is that stem cells can replicate indefinitely, whereas progenitor cells can typically only divide a limited number of times. Most progenitor cells are described as unipotent or multipotent and as such they may be compared to adult stem cells, but progenitors are in a farther stage of cell differentiation. Their differentiation state lies in between a stem cell and a fully or terminally differentiated cell. Progenitor cells are found in adult organisms and form part of the maintenance and repair system of the body. They are most abundant in tissues capable of turning over, e.g. the skin, gut, blood etc.

CIMP cel l. The term " chemically induced multi-lineage potential (CIMP) cell" refers to cells that are obtained by the methods described herein through the addition of one or more CRA(s) to a medium. Suitably, the formation of CIMP cells does not require the need for the cell to be genetically manipulated to induce the expression of one or more pluripotency genes therein. Suitably, the formation of CIMP cells does not require contact with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency. A cell is considered to be a CIMP cell when at least: (i) it has been obtained by the addition of one or more CRA(s) to the cell or the medium in which the cell is contained without the need for the cell to be genetically manipulated to induce the expression one or more pluripotency genes therein and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency; (ii) the expression of one or more, two or more, three or more or four or more or five or more pluripotency genes has been induced therein by the addition of one or more CRA(s); (iii) the expression of one or more, two or more, three or more, four or more or five or more proteins expressed by the one or more induced pluripotency genes is detectable (for example, detectable by an antibody); and (iv) the cell has gained potency such that it has the capacity to differentiate into an increased number of cell types. As used herein, the term "gained potency" means that a cell has gained the capacity to differentiate into a cell that it could not previously differentiate into under normal circumstances. Suitably, the cell that the CIMP cell is differentiated into is a cell of a different germ layer to the cell from which it was derived. In one embodiment, the one or more pluripotency genes that have been induced by the addition of one or more CRA(s) is selected from the group consisting of alkaline phosphatase (ALP), SSEA1 , Oct4 or Nanog or a combination of two or more thereof - such as ALP and SSEA1 ; ALP and Oct4; ALP and Nanog; ALP, SSEA1 and Oct4; ALP, SSEA1 and Nanog; SSEA1 , Oct4 and Nanog; Oct4, ALP and Nanog. Other examples of pluripotency genes that may be induced in CIMP cells may include, but are not limited to, Oct3, ABCG2, stage specific embryonic antigen-1 (SSEA-1 ), SSEA-3, SSEA-4, Tra-1 -60, Tra-1 -81 , Tra-2- 49/6E, ERas/ECAT5, E-cadherin fibroblast growth factor 4 (Fgf4), Cripto, Dax1 , zinc finger protein 296 (Zfp296), N-acetyltransferase-1 (Natl ), ES cell associated transcript 1 (ECAT1 ), ESG1/DPPA5/ECAT2, ECAT3, ECAT6, ECAT7, ECAT8, ECAT9, ECAT10, ECAT15-1 , ECAT15-2, Fthl17, Sail 4, undifferentiated embryonic cell transcription factor (Utf1 ), Rex1 , p53, G3PDH. telomerase, including TERT, silent X chromosome genes, Dnmt3a, Dnmt3b, TRIM28, F-box containing protein 15 (Fbx15), Sox2, Klf4, Esrrb, TDGF1 , GABRB3, Zfp42, FoxD3, GDF3, CYP25A1 , developmental pluripotency-associated 2 (DPPA2), and T-cell lymphoma breakpoint 1 (Tcl1 ), DPPA3/Stella, DPPA4, Dnmt3L, Sox15, Stat3, Grb2, beta-catenin and Bmi1 . In one embodiment, pluripotency will not have been induced in the cell to be reprogrammed prior to contact with the one or more CRA(s). A CIMP cell is not considered to be a PiPSC since one or more exogenously added reprogramming proteins are not contacted with a cell to induce pluripotency.

Geneitc manipulation. This term, as used in the context of a cell that has been genetically manipulated to induce the expression of one or more pluripotency genes/markers (eg. transcription factors) therein, refers in its broadest sense to any kind of genetic manipulation that results in one or more of the genes/markers being induced (eg. artificially induced) to express proteins encoded thereby in the cell. Examples of genetic manipulation include, but are not limited to, the use of expression elements (eg. exogenous genes - such as exogenous genes encoding one or more reprogramming proteins) that are transfected or transduced into cells through the use of, for example, vectors (eg. viral vectors), plasmids, non-integrating episomal vectors, non-integrating viral vectors, vesicles and the like. Another example of genetic manipulation is the artificial induction of endogenous pluripotency genes/markers into the cell to express proteins encoded thereby. Thus, for example, a promoter may be introduced into the genome of the cell to induce the expression of one or more endogenous genes. Further non-limiting examples include the transient transfection of one or more reprogramming plasmids, the use of the piggyBac transposition system, Cre-excisable viruses; and or/P/EBNA1 -based episomal expression system. The nucleic acid that is introduced can be, for example, a homologous or heterologous encoding nucleic acid. The term "heterologous" refers to a nucleic acid derived from a source other than the referenced species whereas "homologous" refers to a nucleic acid derived from the host cell. As described in detail herein, a CIMP cell is not genetically manipulated to induce pluripotency therein since CRA(s) alone can be used to reprogram a cell into a CIMP cell. Exogenous. As used herein, the term "exogenous" in the context of one or more reprogramming proteins that are contacted with a cell means that the protein is introduced into the cell to be reprogrammed. The protein can be introduced, for example, by transducing the protein into the cell. Protein transduction refers to the internalisation of a protein into a cell, from the external environment. This process typically relies on the inherent property of a small number of proteins and peptides of being able to penetrate the cell membrane. The transducing property of these molecules can be conferred upon proteins which are expressed as fusions with them and thus offers an alternative to gene therapy for the delivery of therapeutic proteins into target cells. The protein may also be introduced, for example, by introduction of an encoding nucleic acid and will therefore involve the 'genetic manipulation' of the cell as discussed above. The protein can be, for example, a homologous or heterologous protein. Transgenic non-human anim al. This term refers to a non-human animal in which there has been a deliberate modification of its genome. Obtaining a CIMP cell

In one aspect, there is provided a method of reprogramming a cell into a CIMP cell comprising the steps of: (i) contacting the cell to be reprogrammed with a medium comprising one or more CRA(s); and (ii) obtaining a CIMP cell, preferably, wherein the cell to be reprogrammed has not been genetically manipulated and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

The cell to be reprogrammed may be a somatic cell - such as a primary cell or an immortalised cell. Such cells may be primary cells (non-immortalised cells) isolated or derived from a mammal - such as animal (eg. mouse) or a human. The somatic cell may be a primary cell or a progeny of a primary or secondary cell. The somatic cell may be or may derived by well-known methods, from different organs, such as, but not limited to skin, lung, pancreas, liver, stomach, intestine, blood, heart, reproductive organs, bladder, kidney, urethra and other urinary organs, or generally from any organ or tissue containing living somatic cells. Mammalian somatic cells include Sertoli cells, endothelial cells, granulosa epithelial cells, neurons, pancreatic islet cells, epidermal cells, epithelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), erythrocytes, macrophages, monocytes, mononuclear cells, fibroblasts, cardiac muscle cells, other known muscle cells, and generally any live somatic cells. The somatic cell may be obtained from a sample - such as a tissue sample, a biopsy sample, a blood sample, a swab sample or the like. Methods for obtaining samples from various tissues and methods to establish primary cell lines are well-known in the art (see for example, Jones GE, Wise CJ., "Establishment, maintenance, and cloning of human dermal fibroblasts." Methods Mol Biol. 1997;75: 13-21 ). Somatic cell lines may be purchased from a number of suppliers such as, for example, the American tissue culture collection (ATCC), the German Collection of Microorganisms and Cell Cultures (DSMZ) or PromoCell GmbH, Sickingenstr. 63/65, D-69126 Heidelberg , for example.

In one embodiment, the cell to be reprogrammed is or is derived from the mesoderm. In another embodiment, the cell to be reprogrammed is or is derived from endoderm or the ectoderm.

The cell to be reprogrammed may be a stem cell or a progenitor cell - such as an adult stem cell or a multipotent progenitor cell - that can give rise to terminally differentiated cells.

In one embodiment, one or more pluripotency genes have not been artificially induced in the cell

(eg. by genetically altering the cell) to be reprogrammed prior to contact with the one or more

CRA(s).

Several genes have been found to be associated with pluripotency and include, but are not limited to, Oct3/4, Sox-2, HDAC1 1 , Klf-4, Myc, Lin28 and Nanog.

Oct-3/4 is one of the family of octamer transcription factors, and plays a role in maintaining pluripotency. The knockdown of Oct-3/4 expression in Oct-3/4⁺ cells, such as blastomeres and embryonic stem cells, leads to spontaneous differentiation, and presence of Oct-3/4 thus gives rise to the pluripotency and differentiation potential of embryonic stem cells.

The Sox family of genes is associated with maintaining pluripotency similar to Oct-3/4, although it is associated with multipotent and unipotent stem cells in contrast with Oct-3/4, which is exclusively expressed in pluripotent stem cells. Genes in the Sox family have been found to work as well in the induction process including Sox1 , Sox2, Sox3, Sox15, and Sox18.

In addition to Klf4, other members of the Klf family of genes include Klf1 ,Klf2, Klf4 and Klf5. The Myc family of genes are proto-oncogenes implicated in cancer. C-myc is a factor implicated with pluripotency. N-myc and L-myc have been identified to induce pluripotency instead of c-myc with similar efficiency.

In embryonic stem cells, Nanog, along with Oct-3/4 and Sox2, are believed to be necessary in promoting pluripotency.

LIN28 is an mRNA binding protein expressed in embryonic stem cells and embryonic carcinoma cells and is associated with differentiation and proliferation.

Thus, examples of pluripotency genes include SOX family genes - such as SOX1 , SOX2, SOX3, SOX15, SOX18, KLF family genes - such as KLF1 , KLF2, KLF4, KLF5, SALL4, OCT4, NANOG, HDAC1 1 , LIN28, STELLA, NOBOX Esrrb or a STAT family gene. STAT family members may include, for example STAT1 , STAT2, STAT3, STAT4, STAT5 (STAT5A and STAT5B), and STAT6, FoxD3, UTF1 , Rex1 , ZNF206, Myb12, DPPA2, ESG1 , Otx2 and combinations thereof. Whilst one gene may be associated with pluripotency, the expression of more than one gene is typically associated with pluripotency . For example, two or more genes may be associated with pluripotency - such as OCT4 and SOX2 or OCT4 and Nanog . By way of further example, three or more genes may be associated with pluripotency - such as OCT4, SOX2 and KLF4 or OCT4, SOX2 and Nanog .

Thus, in one embodiment, one or more genes associated with pluripotency are not artificially upregulated in the cells to be reprog rammed (eg. by genetically altering the cell) prior to contact with the one or more CRA(s), wherein said gene(s) is selected from the group consisting of the SOX family genes, the KLF family genes, SALL4, OCT3/4, NANOG, LIN28, HDAC1 1 , STELLA, NOBOX, Esrrb, a STAT family gene, FoxD3, UTF1 , Rex1 , ZNF206, Myb12, DPPA2, ESG1 , Otx2 or a combination thereof.

In one embodiment, one or more genes associated with pluripotency are not artificially upregulated in the cells to be reprog rammed (eg. by genetically altering the cell) prior to contact with the one or more CRA(s), wherein said gene(s) is selected from the group consisting of SOX1 , SOX2, SOX3, SOX15, SOX18, KLF1 , KLF2, KLF4, KLF5, SALL4, OCT4, NANOG, LIN28, HDAC1 1 , STELLA, NOBOX, Esrrb, STAT1 , STAT2, STAT3, STAT4, STAT5 {eg. STAT5A and STAT5B), and STAT6, FoxD3, UTF1 , Rex1 , ZNF206, Myb12, DPPA2, ESG1 , Otx2 or a combination thereof.

More than one gene may be associated with pluripotency. For example, two or more genes may be associated with pluripotency - such as OCT4 and SOX2 or OCT4 and Nanog. By way of further example, three or more genes may be associated with pluripotency - such as OCT4, SOX2 and KLF4 or OCT4, Nanog and SOX2. By way of further example, three or more genes may be associated with pluripotency - such as OCT3/4, SOX2 and KLF4; OCT4, Nanog , SOX2 and KLF4; or OCT4, Nanog SOX2 and HDAC1 1 .

Thus, in a further embodiment, one or more genes associated with pluripotency are not artificially upregulated in the cells to be reprog rammed (eg. by genetically altering the cell) prior to contact with the one or more CRA(s), wherein said gene is selected from the group consisting of OCT4, SOX2 and KLF4 or a combination of two or more thereof - such as OCT4 and SOX2; OCT4 and KLF4; SOX2 and KLF; or OCT4, KLF4 and SOX2.

Thus, in a further embodiment, one or more genes associated with pluripotency are not artificially upregulated in the cells to be reprog rammed (eg. by genetically altering the cell) prior to contact with the one or more CRA(s), wherein said gene is selected from the group consisting of OCT3/4, SOX2 and KLF4. or a combination of two or more thereof - such as OCT3/4 and SOX2; OCT3/4 and KLF4; SOX2 and OCT3/4; and OCT3/4, SOX2 and KLF4.

Thus, in a further embodiment, one or more genes associated with pluripotency are not artificially upregulated in the cells to be reprog rammed (eg. by genetically altering the cell) prior to contact with the one or more CRA(s), wherein said gene is selected from the group consisting of OCT4, Nanog, SOX2 and HDAC1 1 or a combination of two or more thereof - such as OCT4 and Nanog; OCT4 and SOX2; OCT4 and HDAC1 1 ; Nanog and SOX2; Nanog and HDAC 1 1 ; SOX2 and HDAC1 1 ; OCT4, Nanog and SOX2; OCT4, Nanog and HDAC1 1 ; Nanog, SOX2 and HDAC1 1 ; or OCT4, Nanog, SOX2 and HDAC1 1 .

The nucleic acid and amino sequences of these genes are widely available in databases.

For some embodiments, it may be desirable to use a cell that is to be reprogrammed which has been genetically manipu lated to artificially induce the expression of one or more pluripotency genes therein. This is because one or more {eg. two or more) of the CRA(s) described herein may improve the efficiency of reprogramming into a desired cell type. One or more of the CRA(s) described herein may improve the speed of reprogramming into a desired cell type. One or more of the CRA(s) described herein may improve the efficiency and speed of reprogramming into a desired cell type. Accordingly, a further aspect relates to a method for improving the efficiency and/or speed of reprogramming a cell into a desired cell type comprising the steps of: (i) providing a cell which has been genetically manipulated to induce the expression of one or more pluripotency genes therein; (ii) contacting the cell to be reprogrammed with a medium (eg. a culture medium) comprising one or more CRA(s); and (ii) obtaining a reprogrammed cell. A further aspect relates to the use of one or more CRA(s) and/or combinations of CRA(s) as described herein for increasing the efficiency and/or speed of reprogramming a cell.

Contacti ng a cell with one or more CRA(s)

In one embodiment, the method described herein comprises contacting the cell to be reprogrammed with a medium comprising one or more CRA(s). In one embodiment, the one or more CRA(s) are added to a culture medium.

The medium - such as the culture medium - comprising the one or more CRA(s) may be replaced with the same or another culture medium or a maintenance medium lacking the one or more CRA(s). Suitably, the medium comprising the one or more CRA(s) is replaced once the reprogramming has been completed. This may be after 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45 or 50 more days.

The cell may be placed in contact with the one or more CRA(s) for a period of time. The cell may be placed in contact with the one or more CRA(s) for at least about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 16 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours or about 120 hours. In one embodiment, the cell is placed in contact with the one or more CRA(s) for at least about 24 to 72 hours, suitably, at least about 48 to 72 hours, or more suitably, at least about 72 hours.

The cell may be placed in contact with the one or more CRA(s) for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days or 30 days (eg. about 1 to 7 days, about 1 to 6 days, about 1 to 5 days, about 1 to 4 days or about 1 to 3 days).

The cell may be placed in contact with the one or more CRA(s) until such time as the one or more CRA(s) have had their desired effect on the cell. Thus, for example, the CRA(s) may be placed in contact with the cell for a period of time that is long enough for the CRA(s) to remodel chromatin and prime CIMP cell formation. The CRA(s) may be placed in contact with the cell for a period of time that is long enough for the CRA(s) to induce CIMP cell formation. The CRA(s) may be placed in contact with the cell for a period of time that is long enough for the CRA(s) to stabilise and to optionally expand said CIMP cell.

The CRA(s) may be placed in contact with the cell for at least about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 16 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours or about 120 hours, suitably, at least about 24 to 72 hours, more suitably, at least about 48 to 72 hours, or more suitably, at least about 72 hours to remodel chromatin and prime CIMP cell formation. The CRA(s) may be placed in contact with the cell for at least about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 16 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours or about 120 hours, suitably, at least about 24 to 72 hours, more suitably, at least about 48 to 72 hours, or more suitably, at least about 72 hours to induce CIMP cell formation.

The CRA(s) may be placed in contact with the cell for at least about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 16 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours or about 120 hours, suitably, at least about 24 to 72 hours, more suitably, at least about 48 to 72 hours, or more suitably, at least about 72 hours to stabilise and to optionally expand the CIMP cell.

If the CRA is a single chemical then it may be contacted with the cell only once. If the CRA is a single chemical then it is may be contacted with the cell more than once using the same or a different (eg. a higher or lower) amount or concentration of chemical for each of the times that the cell is contacted with the chemical. Thus the contact with the cell may be repeated one or more times - such as repeated sequentially using the same or a different (eg. a higher or lower) amount or concentration of chemical for each of the repeats.

The cell may be contacted with two or more CRA(s) - such as 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or 100 or more CRA(s). The CRA(s) may be used individually and/or together as a combination of CRA(s) - such as a combination of 2, 3, 4, 5, 6, 7, 8, 9 , 10 15, 20, 25, 30, 40, 50 or 100 or more CRA(s). One or more single CRA(s) may be used together with a combination of CRA(s) or a combination of different CRA(s). One or more different single CRA(s) may be used together with a combination of CRA(s) or a combination of different CRA(s). Mixtures of combinations may also be used. If the CRA is a combination of chemicals then it is may be contacted with the cell once or more than once using the same or a different (eg. a higher or lower) amount or concentration of one or more of the combinations of chemicals for each of the times that the cell is contacted with the chemical. Thus the contact with the cell may be repeated one or more times - such as repeated separately or sequentially using the same or a different (eg. a higher or lower) amount or concentration of combinations of chemical.

If the CRA is a combination of chemicals then it is may be contacted with the cell once or more than once using different combinations of chemicals for each of the times that the cell is contacted with the chemical. Thus the contact with the cell may be repeated one or more times - such as repeated simultaneously, separately or sequentially using different combinations of chemicals.

Each of the CRA(s) (eg. single CRA(s) and/or combinations of CRA(s)) may be used separately. Each of the CRA(s) (eg. single CRA(s) and/or combinations of CRA(s)) may be used simultaneously or simultaneously separately. For example, a first CRA may be contacted with a cell, together with a second CRA and so on. By way of further example, a first combination of CRA(s) and a second combination of CRA(s) may be added - each of the combination of CRA(s) may be the same or different. Combinations of CRA(s) may be used together with one or more single CRA(s). Thus, for example, a first CRA and a combination of two or more CRA(s) may be added- each of the CRA(s) or combinations of CRA(s) may be the same or different.

Each of the CRA(s) (eg. single CRA(s) and/or combinations of CRA(s)) may be used sequentially. For example, a first CRA may be contacted with a cell, followed by a second CRA, followed by a third CRA and so on - each of the CRA(s) may be the same or different. By way of further example, a first combination of CRA(s) may be contacted with a cell, followed by a second combination of CRA(s), followed by a third combination of CRA(s) and so on - each of the combination of CRA(s) may be the same or different. Combinations of CRA(s) may be used together with the single CRA. Thus, for example, a first CRA may be contacted with a cell, followed by a combination of two or more CRA(s), followed by a third CRA and so on - each of the CRA(s) or combinations of CRA(s) may be the same or different.

If one or more CRA(s) are to be contacted with a cell more than once, then for some embodiments, it is desirable to have an interval or a gap in time between one or more of the times that the CRA(s) are contacted with the cell. The interval or the gap may be reproduced each time the CRA(s) are contacted with the cell or only some of the times. The interval or the gap in time may be the same as the amount of time that the CRA(s) are contacted with the cell. ef The interval or the gap in time may be a different amount of time compared to the time that the CRA(s) are contacted with the cell. Suitably, the medium used during the interval or gap is the same medium as used for the reprogramming only lacking all of the CRA(s) used the previous step. In one embodiment, the interval or the gap in time may be in the absence of all CRA(s) used to reprogram the cells. Thus, according to this embodiment, the cells are contacted with a culture medium that does not comprise CRA(s) during this interval or gap in time.

Suitably, the interval or gap is at least about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 16 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours or about 120 hours, suitably, at least about 24 to 72 hours, more suitably, at least about 48 to 72 hours, or more suitably, at least about 72 hours.

Suitably, the interval or gap is at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days or 30 days (eg. about 1 to 7 days, about 1 to 6 days, about 1 to 5 days, about 1 to 4 days or about 1 to 3 days). In one embodiment, the interval or gap is the same amount of time that the cells are contacted with the one or more CRA(s) used to reprogram the cells, a smaller amount of time that the cells are contacted with the one or more CRA(s) used to reprogram the cells, a larger amount of time that the cells are contacted with the one or more CRA(s) used to reprogram the cells, or a combination thereof for one or more of the repeats.

This iterative procedure of incubating the cells in the presence and absence of one or more CRA(s) used to reprogram the cells may be repeated as desired using various combinations thereof.

In one embodiment, one or more CRA(s) are contacted with a cell for about 36 hours, followed by an interval time of about 36 hours in the absence of CRA(s) used to reprogram the cells and so on.

In one embodiment, one or more CRA(s) are contacted with a cell for about 36 hours, followed by an interval time of about 36 hours in the absence of CRA(s) used to reprogram the cells, followed by incubation with one or more (of the same or different) CRA(s) for about 36 hours, followed by an interval of about 36 hours without CRA(s) used to reprogram the cells, followed by incubation with one or more (of the same or different) CRA(s) for about 36 hours, followed by an interval time of about 36 hours in the absence of CRA(s) used to reprogram the cells.

In one embodiment, one or more CRA(s) are contacted with a cell for about 24 hours, followed by an interval time of about 48 hours in the absence of CRA(s) used to reprogram the cells and so on.

In one embodiment, one or more CRA(s) are contacted with a cell for about 24 hours, followed by an interval time of about 48 hours in the absence of CRA(s) used to reprogram the cells, followed by incubation with one or more (of the same or different) CRA(s) for about 24 hours, followed by an interval of about 48 hours without CRA(s) used to reprogram the cells, followed by incubation with one or more (of the same or different) CRA(s) for about 24 hours, followed by an interval time of about 48 hours in the absence of CRA(s) used to reprogram the cells.

Absence of CRA(s)

There is also provided a method of reprogramming a cell into a CIMP cell comprising the steps of: (i) contacting the cell to be reprogrammed with a medium comprising one or more CRA(s); and (ii) obtaining a CIMP cell, wherein said cell is incubated in the substantial absence or absence of the one or more (eg. all) CRA(s) for a period of time.

As used herein, the term "substantial absence" means that the cell is incubated in the presence of such a low concentration of CRA(s) that it does not have a reprogramming effect on the cell. Suitably, said method comprises: (i) incubating said cell in the presence of one or more CRA(s); (ii) incubating said cell in the absence of one or more (eg. all) of the CRA(s); (iii) optionally repeating either step (i) or step (ii) one or more times; and (iii) obtaining a CIMP cell

There is also provided a method of reprogramming a cell into a CIMP cell comprising the steps of: (i) providing a cell; (ii) contacting said cell with one or more CRA(s) that remodel chromatin and initiate CIMP cell formation; (ii) contacting said cell with one or more CRA(s) that induce CIMP cell formation; (iii) contacting said cell with one or more CRA(s) that stabilise and expand said CIMP cell; and (iv) obtaining a CIMP cell, wherein said cell is incubated in the substantial absence or absence of the one or more CRA(s) after step (i), step (ii) and/or step (iii).

There is also provided a method for identifying one or CRA(s) for reprogramming a cell into a CIMP cell comprising the steps of: (i) providing a cell; (ii) incubating said cell in the presence of one or more chemicals; and (iii) determ ining if said cell has been reprogrammed, wherein said cell is incubated in the substantial absence or absence of the one or more chemicals, and wherein the presence of a CIMP cell is indicative that the one or more chemicals are CRA(s) able to reprogram the cell.

Types of CRA

In one embodiment, the CRA may be or may be derived from a small molecule. Small molecules may target one or more (eg. multiple) epigenetic pathways and may modulate (eg. override) epigenetic silencing .

If the small molecule is an organic compound then it may comprise two or more hydrocarbyl groups. Here, the term "hydrocarbyl group" means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. The CRA may comprise at least one cyclic group. The cyclic group may be a polycyclic group, such as a non-fused polycyclic group. The CRA may comprise at least one of said cyclic groups linked to another hydrocarbyl group. The CRA may contain halo groups. Here, "halo" includes halogen compounds eg. halides and includes fluoro, chloro, bromo or iodo groups. The CRA may contain one or more of alkyl, alkoxy, alkenyl, alkylene and alkenylene groups- which may be unbranched-or branched-chain.

The small molecule may be any compound from or derived from a small molecule library. Several small molecule libraries are available in the art including those from Enzo Life Science - such as the Protease Inhibitor Library, Endocannabinoid Library, ICCB Known Bioactives Library, Ion Channel Ligand Library, Fatty acid library, Kinase Inhibitor Library, Neurotransm itter Library, Natural Products Library, Kinase/Phosphatase Inhibitor Library, Nuclear Receptor Library, Orphan Ligand Library, Phosphatase Inhibitor Library, and the Rare Natural Products Library.

The small molecule may be prepared by chemical synthesis techniques and may be separated and purified by conventional methods.

The small molecule may be a salt - such as a pharmaceutically acceptable salt. Pharmaceutically-acceptable salts are well known to those skilled in the art, and for example include those mentioned by Berge et al, in J. Pharm. Sci., 66, 1 -19 (1977). Suitable acid addition salts are formed from acids which form non-toxic salts and include the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, hydrogenphosphate, acetate, trifluoroacetate, gluconate, lactate, salicylate, citrate, tartrate, ascorbate, succinate, maleate, fumarate, gluconate, formate, benzoate, methanesulphonate, ethanesulphonate , benzenesulphonate and p-toluenesulphonate salts. When one or more acidic moieties are present, suitable pharmaceutically acceptable base addition salts can be formed from bases which form non-toxic salts and include the aluminium, calcium, lithium, magnesium, potassium, sodium, zinc, and pharmaceutically-active amines such as diethanolamine, salts. A pharmaceutically acceptable salt of an agent may be readily prepared by mixing together solutions of an agent and the desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. The small molecule may be an isotopic variation thereof ie. one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that may be incorporated include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as ²H, ³H, ¹³C, ¹ C, ¹⁵N, ¹⁷0, ¹⁶0, ³¹ P, ³²P, ³⁵S, ¹⁸F and ³⁶CI, respectively. Zwitterionic forms are also contemplated. Any number, combination or concentration of small molecules may be used. The small molecule may be directed towards one or more specifics genes, nucleic acids and/or proteins, one or more specific classes of genes, nucleic acids and/or proteins, or one or more specific families of genes, nucleic acids and/or proteins.

In one embodiment, the CRA is selected from the group consisting of: a PPAR gamma agonist, a histone deacetyiase inhibitor, a DNA methyltransferase inhibitor, a P13K inducer, an NADPH oxidase inhibitor, a GPR modulator, a p53 inhibitor, an anti-apoptosis agent and a histone methyltransferase inhibitor or a combination of two or more thereof. In another embodiment, a combination of CRA(s) is used comprising, consisting or consisting essentially of a PPAR gamma agonist, a histone deacetyiase inhibitor, a DNA methyltransferase inhibitor, a P13K inducer, an NADPH oxidase inhibitor, a GPR modulator, a p53 inhibitor, an anti-apoptosis agent and a histone methyltransferase inhibitor.

In another embodiment, the CRA is selected from the group consisting of: a DNA methyltransferase inhibitor; a histone deacetyiase inhibitor; a KLF4 inducer, a retinoic acid receptor agonist; an HDAC1 inhibitor; and a protein tyrosine phosphatase PTP inhibitor, a MEK/ERK inhibitor, a protein tyrosine phosphatase PTP inhibitor; a H2 histamine receptor antagonist, pregnant mare serum Gonadotropin (PMSG), an NADPH oxidase inhibitor, a histone methyltransferase inhibitor, a calcium channel agonist, a demethylating agent, an inhibitor of Rho-associated protein kinase, a voltage gated sodium channel stabiliser, a GPR modulator, a P53 inhibitor, a PPAR gamma agonist, a caspase-3 Inhibitor, a xanthine oxidase inhibitor or a combination of two or more thereof.

In one embodiment, the combination of CRA(s) is a combination of: a PPAR gamma agonist(s) (eg. 15d-PGJ2 and/or ciglitazone and/or compound P), a histone deacetyiase inhibitor(s) (eg. valproic acid and/or Scriptaid and/or sodium butyrate and/or Trichostatin A and/or HDAC inhibitor III), a DNA methyltransferase inhibitor(s) (eg. 5-aza-2'deoxycytidine and/or RG108), and a P13K inducer(s) (eg. sodium orthovandate and/or PMA).

In another embodiment, the combination of CRA(s) is a combination of: a PPAR gamma agonist(s) (eg. 1 5d-PGJ2 and/or ciglitazone and/or compound P), a histone deacetyiase inhibitor(s) (eg. valproic acid and/or Scriptaid and/or sodium butyrate and/or Trichostatin A and/or HDAC inhibitor III), a DNA methyltransferase inhibitor(s) (eg. 5-aza-2'deoxycytidine and/or RG108), a P13K inducer(s) (eg. sodium orthovandate and/or PMA), an NADPH oxidase inhibitor(s) (eg. sinomenine), a GPR modulator(s) (eg. GPR30 agonist) and a p53 inhibitor(s) (eg. pifithrin-a cyclic).

In another embodiment, the combination of CRA(s) is a combination of: a PPAR gamma agonist(s) (eg. 15d-PGJ2 and/or ciglitazone and/or compound P), a histone deacetyiase inhibitor(s) (eg. valproic acid and/or Scriptaid and/or sodium butyrate and/or Trichostatin A and/or HDAC inhibitor III), a DNA methyltransferase inhibitor(s) (eg. 5-aza-2'deoxycytidine and/or RG108) , a P13K inducer(s) (eg. sodium orthovandate and/or PMA), an NADPH oxidase inhibitor(s) (eg. sinomenine), a GPR modulator(s) (eg. GPR30 agonist), a p53 inhibitor(s) (eg. pifithrin-a cyclic) and an anti-apoptosis agent(s) (eg. Y-27632 and/or casp-3 inhibitor).

In another embodiment, the combination of CRA(s) is a combination of: a PPAR gamma agonist(s) (eg. 15d-PGJ2 and/or ciglitazone and/or compound P), a histone deacetylase inhibitor(s) (eg. valproic acid and/or Scriptaid and/or sodium butyrate and/or Trichostatin A and/or HDAC inhibitor III), a DNA methyltransferase inhibitor(s) (eg. 5-aza-2'deoxycytidine and/or RG108), a P13K inducer(s) (eg. sodium orthovandate and/or PMA), an NADPH oxidase inhibitor(s) (eg. sinomenine), a GPR modulator(s) (eg. GPR30 agonist), a p53 inhibitor(s) (eg. pifithrin-a cyclic), an anti-apoptosis agent(s) (eg. Y-27632 and/or casp-3 inhibitor) and a histone methyltransferase inhibitor(s) (eg. BIX 01294).

In another embodiment, the combination of CRA(s) is a combination of: a PPAR gamma agonist(s) (eg. 15d-PGJ2 and/or ciglitazone and/or compound P), a histone deacetylase inhibitor(s) (eg. valproic acid and/or Scriptaid and/or sodium butyrate and/or Trichostatin A and/or HDAC inhibitor III), a DNA methyltransferase inhibitor(s) (eg. 5-aza-2'deoxycytidine and/or RG108) , a P13K inducer(s) (eg. sodium orthovandate and/or PMA) and one or more of an NADPH oxidase inhibitor(s) (eg. sinomenine), a GPR modulator(s) (eg. GPR30 agonist) and a p53 inhibitor(s) (eg. pifithrin-a cyclic).

In another embodiment, the combination of CRA(s) is a combination of: a PPAR gamma agonist(s) (eg. 15d-PGJ2 and/or ciglitazone and/or compound P), a histone deacetylase inhibitor(s) (eg. valproic acid and/or Scriptaid and/or sodium butyrate and/or Trichostatin A and/or HDAC inhibitor III), a DNA methyltransferase inhibitor(s) (eg. 5-aza-2'deoxycytidine and/or RG108), a P13K inducer(s) (eg. sodium orthovandate and/or PMA), an NADPH oxidase inhibitor(s) (eg. sinomenine) and one or more of a GPR modulator(s) (eg. GPR30 agonist), a p53 inhibitor(s) (eg. pifithrin-a cyclic), an anti-apoptosis agent(s) (eg. Y-27632 and/or casp-3 inhibitor) and a histone methyltransferase inhibitor(s) (eg. BIX 01294).

In another embodiment, the combination of CRA(s) is a combination of: a histone deacetylase inhibitor(s) (eg. valproic acid and/or Scriptaid and/or sodium butyrate and/or Trichostatin A and/or HDAC inhibitor III), a PPAR gamma agonist (eg. 15d-PGJ2 and/or ciglitazone and/or compound P), a DNA methyltransferase inhibitor (eg. 5-aza-2'deoxycytidine and/or RG1 08), a NADPH oxidase inhibitor (eg. sinomenine), a GPR modulator(s) (eg. GPR30 agonist) and a P13K inducer (eg. sodium orthovandate and/or PMA).

In another embodiment, the combination of CRA(s) is a combination of: a histone deacetylase inhibitor(s) (eg. valproic acid and/or Scriptaid and/or sodium butyrate and/or Trichostatin A and/or HDAC inhibitor III), a PPAR gamma agonist (eg. 1 5d-PGJ2 and/or ciglitazone and/or compound P), a DNA methyltransferase inhibitor (eg. 5-aza-2'deoxycytidine and/or RG1 08), a histone methyltransferase inhibitor (eg. BIX 01294) and an anti-apoptosis agent (eg. Y-27632 and/or casp-3 inhibitor).

In another embodiment, the combination of CRA(s) is a combination of: a histone deacetylase inhibitor(s) (eg. valproic acid and/or Scriptaid and/or sodium butyrate and/or Trichostatin A and/or HDAC inhibitor III), a PPAR gamma agonist (eg. 15d-PGJ2 and/or ciglitazone and/or compound P), a DNA methyltransferase inhibitor (eg. 5-aza-2'deoxycytidine and/or RG108), a NADPH oxidase inhibitor (eg. sinomenine), a GPR modulator (eg. a GPR30 agonist) and a p53 inhibitor (eg. pifithrin-a cyclic).

In another embodiment, one or more CRA(s) may be or may be derived from a chromatin modifying chemical. The structure of chromatin is subject to modification by processes known as chromatin remodelling. Chromatin remodelling may involve a number of processes - such as a change in the spacing between nucleosomes; the removal of nucleosomes from a region of DNA; the movement of nucleosomes from one region of DNA to another; and/or the addition of nucleosomes to a region of DNA in the chromosome. Chromatin remodelling may result in changes in higher order structure, thereby changing the balance between transcriptionally active chromatin (eg. open chromatin or euchromatin) and transcriptionally inactive chromatin {eg. closed chromatin or heterochromatin).

The chromatin modifying chemical may be or may be derived from a DNA methyltransferase inhibitor, a histone deacetylase inhibitor, a histone methyltransferase inhibitor or an ATP dependent chromatin remodelling agent or a combination of one or more thereof.

DNA-methyltransferase inhibitors may be or may be derived from nucleoside analogues and non-nucleoside analogues. Examples of nucleoside analogs include 5-Azacytidine, 5-Aza-2'- deoxycytidine, 5-Fluoro-2'-deoxycytidine, 5,6-Dihydro-5-azacytidine and Zebularine and derivatives thereof. Examples of non-nucleoside analogues include Hydralazine, Procainamide, EGCG, Psammaplin A, MG98 and RG108 (N-Phthalyl-L-tryptophan) and derivatives thereof. A combination of one or more DNA-methyltransferase inhibitors may be used.

Histone-deacetylase inhibitors may be or may be derived from cyclic tetrapeptides, short chain fatty acids, hydroxamic acids, and/or a derivative of benzoic acid (eg. benzamide) . Examples of cyclic tetrapeptides and benzamides include Apicidin, Depsipeptide, TPX-HA analogue (CHAP) and Trapoxin. Exemplary Benzamides include CI-994 (N-acetyldinaline) and MS-275 and derivatives thereof. Examples of short chain fatty acids include Butyrate and Valproic acid (2- Propylpentanoic acid - (CH3CH2CH2)2CHC02H)) and derivatives thereof. Examples of hydroxamic acids include m-Carboxy cinnamic acid bishydroxamic acid (CBHA), Oxamflatin, PDX 101 , Pyroxamide, Scriptaid (N-Hydroxy-1 ,3-dioxo-1 H-benz[de]isoquinoline-2(3H)-hexan amide), suberoylanilide hydroxamic acid (SAHA), Trichostatin A ([R-(E,E)]-7-[4- (Dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxo-2,4-heptadienamide (TSA)), LBH589, NVP-LAQ824, MS-275, AN-9, apicidin derivatives, Baceca, CBHA, CHAPs, chlamydocin, CS- 00028, CS-055, EHT-0205, FK-228, FR-135313, G2M-777, HDAC-42, LBH-589, MGCD-0103, NSC-3852, PXD-101 , pyroxamide, SAHA derivatives, suberanilohydroxamic acid, tacedinaline, VX-563, zebularine and/or organosulfur compounds - such as allyl isothiocyanate and derivatives thereof. Further examples are described WO 97/1 1366. A combination of two or more histone-deacetylase inhibitors may be used.

Histone-methyltransferase inhibitors may be or may be derived from 2-(Hexahydro-4-methyl-1 H- 1 ,4-diazepin-1 -yl)-6,7-dimethoxy-N-[1 -(phenylmethyl)-4-piperidinyl] 4-quinazolinamine (BIX01294), Parnate (Tranylcypromine hydrochloride), and/or sc-202651 (diazepinyl- quinazolinamine). A combination of two or more histone-methyltransferase inhibitors may be used.

ATP dependent chromatin remodelling agents may be or may be derived from adenosine 3', 5'- cyclic monophosphate (cAMP) and/or 8-Bromo-cAMP. A combination of two or more ATP dependent chromatin remodelling agents may be used.

The CRA may be or may be derived from a Nanog inducer, a Klf4 inducer, a P13K inducer; a MEK/ERK inhibitor, a Telomerase activator, a GPR modulator, a Wnt modulator, a RAR signalling agent, a calcium signalling agent, a p53 inhibitor, or an anti-apoptotic/anti-oxidant compound.

Examples of Nanog inducers include, but are not limited to cyclic Pifithrin-a (PFT; C16H16N2S HB r), Dexamethasone, (+)-4-hydroxy- 3,7-dimethoxy- 17-methylmorphin- 7-en- 6- one (Sinomenine), Flurbiprofen, Theanine, prostaglandins A1/A2 (PGA1/PGA2) and/or Caspase-3 Inhibitor VII and derivatives thereof. A combination of two or more nanog inducers may be used.

Examples of Klf inducers include, but are not limited to 15-Deoxy-Delta-12,14-prostaglandin J2 (15d-PGJ2), Compound P, 5-{4-[(1 -methylcyclohexyl)methoxy]benzyl}-1 ,3-thiazolidine-2,4-dione (Ciglitazone, PPAR gamma agonist), Troglitazone, Acrolein and/or Zebularine and derivatives thereof. A combination of two or more Klf inducers may be used.

Examples of PI3K inducers include, but are not limited to,: Inosine, PMA, GPR30 Agonist G1 (1 - (4-(6-Bromobenzo[1 ,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopentatc]quinolin-8-yl)- ethanone), GPR40 agonist (3-(4-(((3-(Phenoxy)phenyl)methyl)amino)phenyl)propanoic acid), sodium orthovanadate (Na3V04), HDAC inhibitors and derivatives thereof. A combination of two or more PI3K inducers may be used.

Examples of MEK/ERK inhibitors include, but are not limited to 2 -amino-3 -methoxyflavone (PD98059), U0126 and/or PD0325901 and derivatives thereof. A combination of two or more MEK/ERK inhibitors may be used.

Examples of telomerase activators include, but are not limited to trans-2-Phenyl- cyclopropylamine (CPL) (Inhibits LSD1 , a histone H3 demethylase), astragaloside IV, cycloastragenol and/or astragenol and derivatives thereof. A combination of two or more telomerase activators may be used.

Examples of GPR modulators include, but are not limited to GPR30 Agonist and/or GPR40 agonist and derivatives thereof. A combination of two or more GPR modulators may be used. Examples of Wnt modulators include, but are not limited to 6-bromoindirubin-3'-oxime (BIO), GSK-3beta Inhibitor XII (TWS1 19), Wnt agonist, lndirubin-3 -monoxime (I3M), Kenpaullone (KP), butyric acid, sodium butyrate (NaBu) , CHIR99021 and/or LiCI and derivatives thereof. A combination of two or more Wnt modulators may be used .

Examples of RAR signalling agents include, but are not limited to,: retinoic acid, arotinoid acid (4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1 -propenyl]benzoic acid, TTNPB), and/or L-3,3',5-Triiodothyronine (T3) and derivatives thereof. A combination of two or more RAR signalling agents may be used.

Examples of calcium signalling agents include, but are not limited to 3-Pyridinecarboxylic acid, 1 ,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, methyl ester (Bay K8644), (RS)- 2-(3,4-dimethoxyphenyl)-5-[[2-(3,4-dimethoxyphenyl)ethyl]-(methyl)amino]-2

isopropylpentanenitrile (Verapamil) and/or Phenytoin and derivatives thereof. A combination of two or more calcium signalling agents may be used.

Examples of anti-apoptotic agents include, but are not limited to (R)-(+)-trans-4-(1 -Aminoethyl)- N-(4-Pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, a ROCK inhibitor) , and/or Caspase-3 Inhibitor VII and derivatives thereof. A combination of two or more anti-apoptotic agents may be used.

Examples of inhibitors of p53 include, but are not limited to cyclic Pifithrin-a (PFT), prostaglandin A2 (PGA2) and/or A1 (PGA1 ) and derivatives thereof. A combination of two or more inhibitors of p53 may be used.

Examples of Peroxisome Proliferator-Activated Receptor (PPAR) gamma agonists include, but are not limited to Ciglitizone, GW1929, Troglitazone, Pioglitazone, Rosiglitazone (BRL49653), Fmoc-Leu, Azelaoyl PAF, Bezafibrate, BVT.13 hydrate, Pioglitazone hydrochloride, Indomethacin and/or Carnosic acid and derivatives thereof. A combination of two or more PPAR gamma agonist of p53 may be used.

Other examples of CRA(s) may include, but are not limited to, L-ascorbic acid, Progesterone Synthetic (P8783), Metformin (stimulate AMPK), T3 (thyroid hormone analoque) , 5-aza-2'- deoxycytidine, RG108, Folic Acid, Scriptaid, 4-(Diethylamino)-N-[7-(hydroxyamino)-7- oxoheptyl]benzamide (HDAC Inhibitor III (M344)), Trichostatin A, butyric acid, sodium butyrate, cAMP and/or 8-Bromo-cAMP, Dexamethasone, Sinomenine, Flurbprofen, Theanine, 15d-PGJ2, Compound P, Ciglitazone, trans-2-Phenyl-cyclopropylamine (CPL), cyclic Pifithrin-a (PFT), 5- aza-2'-deoxycytidine, valproic acid, TTNPB, Y-27632, BIX01294, Phenytoin, Folic acid, Trichostatin A, Phenytoin, PD 98059, 1 ,4-diamino-2,3-dicyano-1 ,4-bis(2- aminophenylthio)butadiene (U0126), BIX01294, Inosine, PMA, GPR30 Agonist, GPR40 agonist, Sodium orthovanadate in combination with HDAC inhibitors, Y-27632, Caspase-3 Inhibitor VII, CHIR99021 , 6-bromoindirubin-3'-oxime (BIO), PD173074 and/or PD0325901 and derivatives thereof or a combination of two or more thereof.

Examples of chemicals for chromatin remodelling and CIMP priming include but are not limited to 5-aza-2'-deoxycytidine, RG108, Folic Acid, Scriptaid, HDAC Inhibitor III (M344), Trichostatin A, Sodium Butyrate, cAMP and/or 8-Bromo-cAMP and derivatives thereof or a combination of two or more thereof.

Examples of chemicals for CIMP induction include but are not limited to Dexamethasone, Sinomenine, Flurbprofen, Theanine, 1 5d-PGJ2, Compound P, Ciglitazone, trans-2-Phenyl- cyclopropylamine (CPL), cyclic Pifithrin-a (PFT), 5-aza-2'-deoxycytidine, valproic acid, TTNPB, Y-27632, BIX01294, Phenytoin, Folic acid, Trichostatin A and/or Phenytoin and derivatives thereof or a combination of two or more thereof.

Examples of chemicals for CIMP stabilization and/or expansion include but are not limited to PD 98059, U0126, BIX01294, Inosine, PMA, GPR30 Agonist, GPR40 agonist, Sodium orthovanadate in combination with HDAC inhibitors, Y-27632, Caspase-3 Inhibitor VII, CHIR99021 , 6-bromoindirubin-3'-oxime (BIO), PD173074 and/or PD0325901 and derivatives thereof or a combination of two or more thereof. For some embodiments, the small molecule is not a small molecule that blocks signalling of Wnt and/or Nodal - such as CKI-7 and/or SB- 431542. Obtaining a CIMP cell

The methods described herein can be used to identify agents that can reprogram a cell into a CIMP cell or can be used to reprogram CIMP cells.

Cells may be screened to determine if they are CIMP cells. This can be achieved by a variety of techniques, for instance by visual inspection of the cells under a microscope, or by determining a product characteristic of a CIMP cell. For example, this may be an endogenous marker such as a particular nucleic acid sequence, or a cell protein which can be detected by a ligand or antibody. Live cells may be visualised using a variety of stains, or conversely dead cells can be labelled using a variety of methods, for instance using propidium iodide. Depending on the application it may be possible to use standard laboratory equipment, or it may be advantageous to use specialised instrumentation. For instance, certain analysis and sorting instruments (eg. see Union Biometrica Inc., Somerville MA, USA) have flow cell diameters of up to one millimeter, which allows flow sorting of beads with diameters up to 500 microns.

Genotype determination may be carried out using well known techniques such as the polymerase chain reaction (PCR) , fluorescence in situ hybridisation (FISH), DNA sequencing, and others. Phenotype determination may be carried out by a variety of techniques, for instance by visual inspection of the cells under a microscope, or by detecting a marker product characteristic of the cell. This may be an endogenous marker such as a particular DNA modification or RNA sequence, or a cell protein which can be detected by a ligand, conversion of an enzyme substrate, or antibody that recognises a particular phenotypic marker (for instance see Appendix E of Stem Cells : Scientific Progress and Future Research Directions. Department of Health and Human Services. June 2001 ).

More particularly, a CIMP cell may be identified using at least some of the methods used for identifying an iPS cell, using, for example, methods that include the use of various nucleic acids (eg. RNA, mRNA and/or DNA and the like) and/or protein markers specific for undifferentiated cells. By way of example, markers include, but are not limited to Oct3/4, Nanog, alkaline phosphatase (ALP), ABCG2, stage specific embryonic antigen-1 (SSEA-1 ), SSEA-3, SSEA-4, Tra-1 -60, Tra-1 -81 , Tra-2-49/6E, ERas/ECAT5, E-cadherin, fibroblast growth factor 4 (Fgf4), Cripto, Dax1 , zinc finger protein 296 (Zfp296) , N-acetyltransferase-1 (Natl ), ES cell associated transcript 1 (ECAT1 ), ESG1/DPPA5/ECAT2, ECAT3, ECAT6, ECAT7, ECAT8, ECAT9, ECAT10, ECAT15-1 , ECAT15-2, Fthl17, Sal14, undifferentiated embryonic cell transcription factor (Utf1 ), Rex1 , p53, G3PDH. telomerase, including TERT, silent X chromosome genes, Dnmt3a, Dnmt3b, TRIM28, F-box containing protein 15 (Fbx15) , Nanog/ECAT4, Oct3/4, Sox2, Klf4, c-Myc, Esrrb, TDGF1 , GABRB3, Zfp42, FoxD3, GDF3, CYP25A1 , developmental pluripotency-associated 2 (DPPA2), and T-cell lymphoma breakpoint 1 (Tell ), DPPA3/Stella, DPPA4. Other markers can include Dnmt3L, Sox15, Stat3, Grb2, beta-catenin and Bmi1 .

In one embodiment, the marker is alkaline phosphatase and/or SSEA1 and/or Oct4 and/or Nanog. In another embodiment, the markers SSEA1 , Oct4 and Nanog are used to identify a CIMP cell. In another embodiment, the markers alkaline phosphatise, SSEA1 , Oct4 and Nanog are used to identify a CIMP cell. Suitably, these markers are upregulated in the CIMP cell. Immunocytochemistry methods that are well known in the art may be used to identify a CIMP cell.

CIMP cells may be further characterized by the down-regulation of markers characteristic of the cell from which it was derived. Additional markers include, but are not limited to, cell surface markers, antigens, and other gene products including RNA (including microRNAs and antisense RNA) and DNA (including genes and cDNAs).

CIMP cells may be further identified by exponential cell proliferation, pluripotency, or cell morphology (see Takahashi, K. et al. , Cell 131 :861 -872 (2007)). Alternatively, high telomerase activity may be detected by the telomeric repeat amplification protocol (TRAP) since CIMP cells may have high telomerase activity. Pluripotency may be determined by forming teratoma and identifying tissues or cells of three embryonic germ layers (ie. ectoderm, mesoderm, and endoderm). In one example, cells are injected intradermaly into a nude mouse (where the cells are induced from murine somatic cells) or in the spermary of a SCID mouse (where the cells are induced from human somatic cells), followed by monitoring the formation of tumours and then confirming that the tumour tissues are composed of tissues including neural rosettes (ectoderm), cartilage (mesoderm), cardiac myocyte (mesoderm), gut-like epithelium (endoderm), adipose (mesoderm), and the like. Germline transmission can be used to test for pluripotency of mouse cells.

Culture medium/maintenance medium

In one embodiment, cells are reprog rammed in a culture medium comprising one or more CRA(s). In another embodiment, after the reprogramming in the presence of the CRA(s), the culture medium is replaced with a maintenance medium that does not comprise any of the CRA(s). Without wishing to be bound by theory, it is believed that the use of a maintenance medium after the reprogramming stabilises the CIMP cells in their reprogrammed state and may help in the selective expansion of CIMP cells. The use of a maintenance medium is also advantageous since it avoids the use of medium with undefined (and undesirable) serum components therein.

Many different types of culture medium are widely available in the art and the choice of culture medium will typically be determined by the choice of cell that is to be reprogrammed. One example of culture medium is DMEM culture medium as described herein in Table 6.

Maintenance media are also widely available in the art.

A preferred maintenance medium is iSTEM^® (Stem Cell Sciences) which is a serum-free, medium for feeder-free culture that is designed to capture cells - such as mouse ES cells - in their basal state by blocking the inductive pathways of differentiation with selective small molecule inhibitors . The medium contains three selective small molecule inhibitors that act to eliminate differentiation-inducing signals and promote cell survival, enabling the maintenance of the pluripotent, ground state without the requirement for stimulatory cytokines. The three inhibitors are CHIR99021 (3 μM), PD0325901 (0.4 μM) and PD173074 (100 μM) (Ying er a/., (2008) Nature 453: 519-523). CHIR99021 is a potent and highly selective inhibitor of glycogen synthase kinase-3beta (GSK-3beta). In combination with the MEK/MAPK inhibitor, PD184352, and fibroblast growth factor receptor (FGFR) inhibitor PD173074 , the medium has been shown to allow for long-term expansion of murine embryonic stem cells (Ying et al., (2008 ) Nature 453: 519-523). Without wishing to be bound by theory, these inhibitors may eliminate differentiation signals by inhibiting cell signalling, thereby sustaining mouse ES cell self-renewal and their ground state pluripotency.

Another preferred maintenance mediu m is ESGRO Complete PLUS Clonal Grade Medium, also known as Chemicon (Millipore). This medium is a defined serum-free medium comprising BMP4 and LIF and is designed to enhance viability of cells- such as mouse ES cells - and increases maintenance of pluripotency in the absence of serum and feeder cells (Ying ef a/., (2003) Cell 115:281 -292).

Another example of a maintenance medium is mTeSR™1 maintenance medium (05850, Stem Cell Technologies) . This is a complete, defined and serum-free maintenance medium designed to maintain and expand hES cells in an undifferentiated state.

Accordingly, in one aspect, there is provided a method of reprogramming a cell into a CIMP cell comprising the steps of: (i) contacting the cell to be reprogrammed with a culture medium comprising one or more CRA(s); (ii) obtaining a CIMP cell; and (iii) replacing the culture medium with a maintenance medium that is free or substantially free of the CRA(s) used to reprogram the cell, preferably, wherein the cell to be reprogrammed has not been genetically manipulated and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

CIMP cells obtained or obtainable by the methods descri bed herein

In addition to the various markers and the like that can be used to identify CIMP cells as discussed above, a further characteristics of the CIMP cells is that they have not been (knowingly) manipulated (eg. genetically manipulated or genetically engineered) and/or contacted with one or more exogenous reprogramming proteins or nucleic acids to induce pluripotency since CRA(s) alone can be used to reprogram cells into CIMP cells as described herein. The CIMP cell may be a stem cell.

As described herein, several genes (eg. genes expressing transcription factors and/or other factors known in the art) have been found to be associated with pluripotency and include, but are not limited to, the SOX family genes - such as SOX1 , SOX2, SOX3, SOX 15, SOX18, KLF family genes - such as KLF1 , KLF2, KLF4, KLF5, MYC family genes - such as C-MYC, L-MYC, N-MYC, SALL4, OCT4, NANOG, LIN28, STELLA, NOBOX Esrrb or a STAT family gene. STAT family members may include, for example STAT1 , STAT2, STAT3, STAT4, STAT5 (STAT5A and STAT5B), and STAT6, FoxD3, UTF1 , Rex1 , ZNF206, Myb12, DPPA2, ESG1 , Otx2 and combinations thereof. Whilst only one gene may be required to induce pluripotency, the expression of more than one gene is typically required. For example, two or more genes may need to be expressed in a cell to induce pluripotency - such as OCT4 and SOX2. By way of further example, three or more genes may need to be expressed in a cell to induce pluripotency - such as OCT4, SOX2 andKLF4. It has been shown previously that as few as two factors may be sufficient to reprogram somatic cells, e.g., using OCT4 and SOX2, however, as few as one factor may be sufficient to reprogram the cells.

Thus, in one embodiment, the CIMP cells do not have one or more genes genetically manipulated to induce pluripotency wherein said gene is selected from the group consisting of the SOX family genes, the KLF family genes, SALL4, OCT3/4, NANOG, LIN28, HDAC1 1 , STELLA, NOBOX, Esrrb, a STAT family gene, FoxD3, UTF1 , Rex1 , ZNF206, Myb12, DPPA2, ESG1 , Otx2 or a combination thereof and/or have not been contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency . In one embodiment, the CIMP cells do not have one or more genes genetically manipulated to induce pluripotency wherein said gene is selected from the group consisting of SOX1 , SOX2, SOX3, SOX15, SOX18, KLF1 , KLF2, KLF4, KLF5, SALL4, OCT4, NANOG, LIN28, HDAC1 1 , STELLA, NOBOX, Esrrb, STAT1 , STAT2, STAT3, STAT4, STAT5 (eg. STAT5A and STAT5B), and STAT6, FoxD3, UTF1 , Rex1 , ZNF206, Myb12, DPPA2, ESG1 , Otx2 or a combination thereof and/or have not been contacted with one or more exogenou s reprogramming proteins or nucleic acids encoding same to induce pluripotency.

More than one gene may be associated with pluripotency. For example, two or more genes may be associated with pluripotency - such as OCT4 and SOX2 or OCT4 and Nanog . By way of further example, three or more genes may be associated with pluripotency - such as OCT4, SOX2 and KLF4 or OCT4, Nanog and SOX2. By way of further example, three or more genes may be associated with pluripotency - such as OCT3/4, SOX2 and KLF4; OCT4, Nanog, SOX2 and KLF4; or OCT4, Nanog SOX2 and HDAC1 1 and/or have not been contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

Thus, in a further embodiment, the CIMP cells do not have one or more genes genetically manipulated to induce pluripotency wherein said gene is selected from the group consisting of OCT4, SOX2 and KLF4 or a combination of two or more thereof - such as OCT4 and SOX2; OCT4 and KLF4; SOX2 and KLF; or OCT4, KLF4 and SOX2 and/or have not been contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

In a further embodiment, the CIMP cells do not have one or more genes genetically manipulated to induce pluripotency wherein said gene is selected from the group consisting of OCT3/4, SOX2 and KLF4. or a combination of two or more thereof - such as OCT3/4 and SOX2; OCT3/4 and KLF4; SOX2 and OCT3/4; and OCT3/4, SOX2 and KLF4 and/or have not been contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

In a further embodiment, the CIMP cells do not have one or more genes genetically manipulated to induce pluripotency wherein said gene is selected from the group consisting of OCT4, Nanog, SOX2 and HDAC1 1 or a combination of two or more thereof - such as OCT4 and Nanog; OCT4 and SOX2; OCT4 and HDAC1 1 ; Nanog and SOX2; Nanog and HDAC1 1 ; SOX2 and HDAC1 1 ; OCT4, Nanog and SOX2; OCT4, Nanog and HDAC1 1 ; Nanog, SOX2 and HDAC1 1 ; or OCT4, Nanog, SOX2 and HDAC1 1 and/or have not been contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

For some embodiments, it may be desirable to use a cell that is to be reprogrammed which has been genetically manipulated to induce the expression of one or more pluripotency genes therein. This is because one or more {eg. two or more) of the CRA(s) described herein may improve the efficiency of reprogramming into a desired cell type. One or more of the CRA(s) described herein may improve the speed of reprogramming into a desired cell type. One or more of the CRA(s) described herein may improve the efficiency and speed of reprogramming into a desired cell type. Accordingly, a further aspect relates to a method for improving the efficiency and/or speed of reprogramming a cell into a desired cell type comprising the steps of:

(i) providing a cell which has been genetically manipulated to induce the expression of one or more pluripotency genes therein; (ii) contacting the cell to be reprogrammed with a medium (eg. a culture medium) comprising one or more CRA(s); and (ii) obtaining a reprogrammed cell. Likewise, for some embodiments, it may be desirable to use a cell that has been contacted with one or more exogenou s reprogramming proteins or nucleic acids encoding same to induce pluripotency which may improve the efficiency and/or speed of reprogramming into a desired cell type. Accordingly, a further aspect relates to a method for improving the efficiency and/or speed of reprogramming a cell into a desired cell type comprising the steps of: (i) providing a cell which has been contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency; (ii) contacting the cell to be reprogrammed with a medium (eg. a culture medium) comprising one or more CRA(s); and (ii) obtaining a reprogrammed cell.

A further aspect relates to a method for improving the efficiency and/or speed of reprogramming a cell into a desired cell type comprising the steps of: (i) providing a cell which has been genetically manipulated to induce the expression of one or more pluripotency genes therein and has been contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency; (ii) contacting the cell to be reprogrammed with a medium (eg. a culture medium) comprising one or more CRA(s); and (ii) obtaining a reprogrammed cell.

A further aspect relates to the use of one or more CRA(s) and/or combinations of CRA(s) as described herein for increasing the efficiency and/or speed of reprogramming a cell.

The CI MP cells described herein may, in certain embodiments, have properties that are similar to or substantially the same as iPS cells. An iPS cell is a cell that exhibits characteristics similar to embryonic stem (ES) cells and is produced by de-differentiating adult cells. Said characteristics of iPS cells may include but are not limited to: (i) unlimited self renewal in vitro;

(ii) a normal karyotype; (iii) a characteristic gene expression pattern including stem cell marker genes like Oct3/4, Sox2, Nanog, alkaline phosphatase (ALP) and stem cell-specific antigen 3 and 4 (SSEA3/4); and (iv) the capacity to differentiate into specialized cell types (eg. see Hanna, J., et al. (2007). Science 318 (5858): 1920-3; and Takahashi, K., et al. (2007) Cell 131 (5): 861 -72). One of the prevailing methods to establish iPS cells has been to infect somatic cells with retroviruses expressing exogenous genes encoding transcription factors. The infected cells are then cultured in standard ES medium and drug selection is applied for the activation of pluripotency-associated promoters - such as Oct4 and Nanog pro moters. Once morphologically ES-like colonies are obtained, pluripotency of iPS cells is evaluated. The pluripotency of murine iPS cells may tested by in vitro differentiation into differentiated cells of all three germ layers and/or the production of germline chimeric mice through blastocyst injection. Human iPS cells may be analyzed through in vitro differentiation into differentiated cells of all three germ layers and their in vivo differentiation capacity can be tested by injection into immunodeficient SCID mice and the characterisation of resulting tumours as teratomas. Reprogramming

In a further aspect there is provided a method for reprogramming a cell - such as cell of the ectoderm, endoderm or mesoderm lineage - into a CIMP cell.

Without wishing to be bound by theory, cells may be reprogrammed by: (i) contacting said cell with one or more CRA(s) that remodel chromatin and prime CIMP cell formation; (ii) contacting said cell with one or more CRA(s) that induce CIMP cell formation; and (iii) contacting said cell with one or more CRA(s) that stabilise and optionally expand said CIMP cell.

The step of CIMP priming may be characterised by overriding certain epigenetic silencing with synergistic effects between HDAC inhibitors and DNA-demethylating agents, and de-repression of endogenous promoters controlling the ES cell specific genes. The step of CIMP induction may be characterised by one or more of ES marker genes beginning to be expressed, a transition state to pluripotency. The gene expression may be transient in nature. The step of CIMP stabilisation may be characterised by a reprogrammed cell having achieved a ground state of pluripotency and/or stable expression of, for example, at least 3 or more pluripotency marker genes, and/or a clonally expandable population of cells.

In a further aspect there is provided a method for reprogramming a cell into a CIMP cell comprising the steps of: (i) contacting the cell to be reprogrammed with one or more of the CRA(s) (eg. combinations of CRA(s)) that have been identified and described herein; and (ii) obtaining a CIMP cell. As discussed herein, the CIMP cell may subsequently be differentiated . (Protocol 1 ) In one embodiment, the method comprises contacting the cell with a CRA selected from the group consisting of a DNA methyltransferase inhibitor (eg. RG108); a histone deacetylase inhibitor (eg. Scriptaid and/or Trichostatin A (TCA)); a KLF4 inducer (eg. 15d- PGJ2), a retinoic acid receptor agonist (eg. TTNPB); and a HDAC1 inhibitor (eg. valproic acid); and a protein tyrosine phosphatase PTP inhibitor (eg. Sodium Orthovanadate (SOV)); a MEK/ERK inhibitor (eg. PD98059); a H2 histamine receptor antagonist (eg. Ranitidine) and PMSG or a combination thereof.

(Protocol 1 ) Suitably, the method comprises contacting the cell with a combination of CRA(s) comprising, consisting or consisting essentially of a DNA methyltransferase inhibitor (eg. RG108); a histone deacetylase inhibitor (eg. Scriptaid); a KLF4 inducer (eg. 15d-PGJ2), a retinoic acid receptor agonist (eg. TTNPB); an HDAC1 inhibitor (eg. valproic acid); and a protein tyrosine phosphatase PTP inhibitor (eg. Sodium Orthovanadate (SOV); and (eg. followed by) a combination of CRA(s) comprising, consisting or consisting essentially of a histone deacetylase inhibitor (eg. Trichostatin A (TCA)); a KLF4 inducer (eg. 15d-PGJ2), a MEK/ERK inhibitor (eg. PD98059); a protein tyrosine phosphatase PTP inhibitor (eg. Sodium Orthovanadate (SOV)); a H2 histamine receptor antagonist (eg. Ranitidine) and PMSG.

(Protocol 1 ) More suitably, the method comprises contacting the cell with a combination of CRA(s) comprising, consisting or consisting essentially of a DNA methyltransferase inhibitor (eg. RG108); a histone deacetylase inhibitor (eg. Scriptaid); a KLF4 inducer (eg. 15d-PGJ2), a retinoic acid receptor agonist (eg. TTNPB); an HDAC1 inhibitor (eg. valproic acid); and a protein tyrosine phosphatase PTP inhibitor (eg. Sodium Orthovanadate (SOV)); incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days; incubating the cell in the presence of a combination of CRA(s) comprising, consisting or consisting essentially of a DNA methyltransferase inhibitor (eg. RG108); a histone deacetylase inhibitor (eg. Scriptaid); a KLF4 inducer (eg. 15d-PGJ2), a retinoic acid receptor agonist (eg. TTNPB); an HDAC1 inhibitor (eg. valproic acid); and a protein tyrosine phosphatase PTP inhibitor (eg. Sodium Orthovanadate (SOV)) ; incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days; and incubating the cell in the presence of a combination of CRA(s) comprising, consisting or consisting essentially of a histone deacetylase inhibitor (eg. Trichostatin A (TCA)); a KLF4 inducer (eg. 15d-PGJ2), a MEK/ERK inhibitor (eg. PD98059); a protein tyrosine phosphatase PTP inhibitor (eg. Sodium Orthovanadate (SOV)); a H2 histamine receptor antagonist (eg. Ranitidine) and PMSG; and optionally incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days;. The exposure in the presence of the chemicals may be for about 1 , 2 or 3 or more days at a time. The exposure in the absence of the chemicals may be for about 1 , 2 or 3 or more days at a time.

(Protocol 2) In one embodiment, the method comprises contacting the cell with a CRA selected from the group consisting of a NADPH oxidase inhibitor (eg. Sinomenine); a MEK/ERK inhibitor (eg. PD98059 and U0126); a histone methyltransferase inhibitor (eg. BIX 01294)); a protein tyrosine phosphatase PTP inhibitor (eg. Sodium Orthovanadate (SOV)); and a calcium channel agonist (eg. a dihydropyridine derivative - such as Bay K8644); a demethylating agent (eg. 5- aza-2'-deoxycytidine (5-AZA)); a histone deacetylase inhibitor (eg. Scriptaid); a KLF4 inducer (eg. 15d-PGJ2), an inhibitor of Rho-associated protein kinase (eg. Y-27632); a voltage gated sodium channel stabiliser (eg. 5,5-Diphenylhydantoin sodium salt (Phenytoin); an HDAC1 inhibitor (eg. valproic acid); a GPR modulators (eg. GPR30 Agonist) and a P53 inhibitor (eg. Pifithrin-a, Cyclic (PFT)) or a combination of two or more thereof.

(Protocol 2) Suitably, the method comprises contacting the cell with a combination of CRA(s) comprising, consisting or consisting essentially of an NADPH oxidase inhibitor (eg. Sinomenine) ; a MEK/ERK inhibitor (eg. PD98059 and U0126); a histone methyltransferase inhibitor (eg. BIX 01294)); a protein tyrosine phosphatase PTP inhibitor (eg. Sodium Orthovanadate (SOV)); and a calcium channel agonist (eg. a dihydropyridine derivative - such as Bay K8644); and a combination of CRA(s) comprising, consisting or consisting essentially of a demethylating agent (eg. 5-aza-2'-deoxycytidine (5-AZA)); a histone deacetylase inhibitor (eg. Scriptaid); a KLF4 inducer (eg. 15d-PGJ2), an inhibitor of Rho-associated protein kinase (eg. Y- 27632); a histone methyltransferase inhibitor (eg. BIX 01294)) and a voltage gated sodium channel stabiliser (eg. 5,5-Diphenylhydantoin sodium salt (Phenytoin); and a combination of CRA(s) comprising, consisting or consisting essentially of a KLF4 inducer (eg. 15d-PGJ2), an NADPH oxidase inhibitor (eg. Sinomenine); an HDAC1 inhibitor (eg. valproic acid); a GPR modulator (eg. GPR30 Agonist), a P53 inhibitor (eg. Pifithrin-a, Cyclic (PFT)) and a demethylating agent (eg. 5-aza-2'-deoxycytidine (5-AZA)) or a combination of two or more thereof.

(Protocol 2) More suitably, the method comprises contacting cell with a combination of CRA(s) comprising, consisting or consisting essentially of an NADPH oxidase inhibitor (eg. Sinomenine); a MEK/ERK inhibitor (eg. PD98059 and U0126); a histone methyltransferase inhibitor (eg. BIX 01294)); a protein tyrosine phosphatase PTP inhibitor (eg. Sodium Orthovanadate (SOV)); and a calcium channel agonist (eg. a dihydropyridine derivative - such as Bay K8644); incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days; incubating the cell in the presence of a combination of CRA(s) comprising, consisting or consisting essentially of a demethylating agent (eg. 5-aza-2'-deoxycytidine (5- AZA)); a histone deacetylase inhibitor (eg. Scriptaid); a KLF4 inducer (eg. 15d-PGJ2), an inhibitor of Rho-associated protein kinase (eg. Y-27632); a histone methyltransferase inhibitor (eg. BIX 01294) and a voltage gated sodium channel stabiliser (eg. 5,5-Diphenylhydantoin sodium salt (Phenytoin); incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days; incubating the cell in the presence of a combination of CRA(s) comprising, consisting or consisting essentially of a KLF4 inducer (eg. 15d-PGJ2), an NADPH oxidase inhibitor (eg. Sinomenine); an HDAC1 inhibitor (eg. valproic acid); a GPR modulator (eg. GPR30 Agonist), a P53 inhibitor (eg. Pifithrin-a, Cyclic (PFT)) and a demethylating agent (eg. 5-aza-2'-deoxycytidine (5-AZA)); and optionally incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days. The exposure in the presence of the chemicals may be for about 1 , 2 or 3 or more days at a time. The exposure in the absence of the chemicals may be for about 1 , 2 or 3 or more days at a time.

(Protocol 3) In one embodiment, the method comprises contacting the cell with a CRA selected from the group consisting of a DNA methyltransferase inhibitor (eg. RG108); deacetylase inhibitor (eg. butyric acid - such as Sodium butyrate (NaBu)); a PPAR gamma agonist (eg. Ciglitazone); an NADPH oxidase inhibitor (eg. Sinomenine) ; a PKC activator (eg. Phorbol-12- myristate-13-acetate (PMA)); an σ1 -adrenoceptor antagonist (eg. verapamil hydrochloride); a KLF4 inducer (eg. 15d-PGJ2), an HDAC1 inhibitor (eg. valproic acid); a GPR modulator (eg. GPR30 Agonist), a P53 inhibitor (eg. Pifithrin-a, Cyclic (PFT)) and a demethylating agent (eg. 5- aza-2'-deoxycytidine (5-AZA)) or a combination of two or more thereof.

(Protocol 3) Suitably, the method comprises contacting the cell with a combination of CRA(s) comprising, consisting or consisting essentially of a DNA methyltransferase inhibitor (eg. RG108); deacetylase inhibitor (eg. butyric acid - such as Sodium butyrate (NaBu)); a PPAR gamma agonist (eg. Ciglitazone); an NADPH oxidase inhibitor (eg. Sinomenine); a PKC activator (eg. Phorbol-l 2-myristate-13-acetate (PMA)); and an a1 -adrenoceptor antagonist (eg. verapamil hydrochloride); and a combination of CRA(s) comprising, consisting or consisting essentially of a KLF4 inducer (eg. 15d-PGJ2); an NADPH oxidase inhibitor (eg. Sinomenine); an HDAC1 inhibitor (eg. valproic acid); a GPR modulator (eg. GPR30 Agonist), a P53 inhibitor (eg. Pifithrin-a, Cyclic (PFT)) and a demethylating agent (eg. 5-aza-2'-deoxycytidine (5-AZA)).

(Protocol 3) More suitably, the method comprises contacting cell with a combination of CRA(s) comprising, consisting or consisting essentially of a DNA methyltransferase inhibitor (eg. RG108); deacetylase inhibitor (eg. butyric acid - such as Sodium butyrate (NaBu)); a PPAR gamma agonist (eg. Ciglitazone); an NADPH oxidase inhibitor (eg. Sinomenine); a PKC activator (eg. Phorbol-12-myristate-13-acetate (PMA)); and an a1 -adrenoceptor antagonist (eg. verapamil hydrochloride); incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days; incubating the cell in the presence of a combination of CRA(s) comprising, consisting or consisting essentially of a KLF4 inducer (eg. 15d-PGJ2); an NADPH oxidase inhibitor (eg. Sinomenine); an HDAC1 inhibitor (eg. valproic acid); a GPR modulator (eg. GPR30 Agonist), a P53 inhibitor (eg. Pifithrin-a, Cyclic (PFT)) and a demethylating agent (eg. 5-aza-2'-deoxycytidine (5-AZA)); and optionally incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days. The exposure in the presence of the chemicals may be for about 1 , 2 or 3 or more days at a time. The exposure in the absence of the chemicals may be for about 1 , 2 or 3, 4,, 5 or 6 or more days at a time.

(Protocol 4) In one embodiment, the method comprises contacting the cell with a CRA selected from the grou p consisting of a DNA methyltransferase inhibitor (eg. RG108); a histone deacetylase inhibitor (eg. Scriptaid); a KLF4 inducer (eg. 1 5d-PGJ2), a retinoic acid receptor agonist (eg. TTNPB); a HDAC1 inhibitor (eg. valproic acid); and a protein tyrosine phosphatase PTP inhibitor (eg. Sodium Orthovanadate (SOV)); a demethylating agent (eg. 5-aza-2'- deoxycytidine (5-AZA)); an inhibitor of Rho-associated protein kinase (eg. Y-27632); a histone methyltransferase inhibitor (eg. BIX 01294) and a voltage gated sodium channel stabiliser (eg. 5,5-Diphenylhydantoin sodium salt (Phenytoin); an NADPH oxidase inhibitor (eg. Sinomenine); a GPR modulator (eg. GPR30 Agonist); and a P53 inhibitor (eg. Pifithrin-a, Cyclic (PFT)).

(Protocol 4) Suitably, the method comprises contacting the cell with a combination of CRA(s) comprising, consisting or consisting essentially of a DNA methyltransferase inhibitor (eg. RG108); a histone deacetylase inhibitor (eg. Scriptaid); a KLF4 inducer (eg. 15d-PGJ2), a retinoic acid receptor agonist (eg. TTNPB); a HDAC1 inhibitor (eg. valproic acid); and a protein tyrosine phosphatase PTP inhibitor (eg. Sodium Orthovanadate (SOV)); a combination of CRA(s) comprising, consisting or consisting essentially of a demethylating agent (eg. 5-aza-2'- deoxycytidine (5-AZA)); a histone deacetylase inhibitor (eg. Scriptaid); a KLF4 inducer (eg. 15d-PGJ2), an inhibitor of Rho-associated protein kinase (eg. Y-27632); a histone methyltransferase inhibitor (eg. BIX 01294) and a voltage gated sodium channel stabiliser (eg. 5,5-Diphenylhydantoin sodium salt (Phenytoin); and a combination of CRA(s) comprising, consisting or consisting essentially of a KLF4 inducer (eg. 15d-PGJ2), an NADPH oxidase inhibitor (eg. Sinomenine); a HDAC1 inhibitor (eg. valproic acid); a GPR modulator (eg. GPR30 Agonist); a P53 inhibitor (eg. Pifithrin-a, Cyclic (PFT)); and a demethylating agent (eg. 5-aza-2'- deoxycytidine (5-AZA)).

(Protocol 4) More suitably, the method comprises contacting the cell with a combination of CRA(s) comprising, consisting or consisting essentially of a DNA methyltransferase inhibitor (eg. RG108); a histone deacetylase inhibitor (eg. Scriptaid); a KLF4 inducer (eg. 15d-PGJ2), a retinoic acid receptor agonist (eg. TTNPB); a HDAC1 inhibitor (eg. valproic acid); and a protein tyrosine phosphatase PTP inhibitor (eg. Sodium Orthovanadate (SOV)); incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days; contacting the cell with a CRA selected from the group consisting of a combination of CRA(s) comprising, consisting or consisting essentially of a demethylating agent (eg. 5-aza-2'-deoxycytidine (5- AZA)); a histone deacetylase inhibitor (eg. Scriptaid); a KLF4 inducer (eg. 15d-PGJ2), an inhibitor of Rho-associated protein kinase (eg. Y-27632); a histone methyltransferase inhibitor (eg. BIX 01294) and a voltage gated sodium channel stabiliser (eg. 5,5-Diphenylhydantoin sodium salt (Phenytoin); incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days; contacting the cell with a CRA selected from the group consisting of a KLF4 inducer (eg. 15d-PGJ2), an NADPH oxidase inhibitor (eg. Sinomenine); a HDAC1 inhibitor (eg. valproic acid); a GPR modulator (eg. GPR30 A gonist); a P53 inhibitor (eg. Pifithrin-a, Cyclic (PFT)); and a demethylating agent (eg. 5-aza-2'-deoxycytidine (5-AZA)); and optionally incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days. The exposure in the presence of the chemicals may be for about 1 , 2 or 3 or more days at a time. The exposure in the absence of the chemicals may be for about 1 , 2 or 3 or more days at a time.

(Protocol 5) In one embodiment, the method comprises contacting the cell with a CRA selected from the group consisting of a KLF4 inducer (eg. 15d-PGJ2), an NADPH oxidase inhibitor (eg. Sinomenine) ; a HDAC1 inhibitor (eg. valproic acid); a GPR modulator (eg. GPR30 Agonist); a P53 inhibitor (eg. Pifithrin-a, Cyclic (PFT)); and a demethylating agent (eg. 5-aza-2'- deoxycytidine (5-AZA)); a histone deacetylate inhibitor (eg. HDAC Inhibitor III (M344)); a PPAR gamma agonist (eg. Ciglitazone);a retinoic acid receptor agonist (eg. TTNPB); a caspase-3 Inhibitor (eg. caspase-3 Inhibitor VII); a xanthine oxidase inhibitor (eg. 3,5,7,8- tetrazabicyclo[4.3.0] nona-3,5,9-trien-2-one (allopurinol)); and a H2 histamine receptor antagonist (eg . Ranitidine).

(Protocol 5) Suitably, the method comprises contacting the cell with a combination of CRA(s) comprising, consisting or consisting essentially of a KLF4 inducer (eg. 15d-PGJ2), an NADPH oxidase inhibitor (eg. Sinomenine); a HDAC1 inhibitor (eg. valproic acid); a GPR modulator (eg. GPR30 Agonist); a P53 inhibitor (eg. Pifithrin-a, Cyclic (PFT)); and a demethylating agent (eg. 5-aza-2'-deoxycytidine (5-AZA)); and a combination of CRA(s) comprising, consisting or consisting essentially of a histone deacetylate inhibitor (eg. HDAC Inhibitor III (M344)); a PPAR gamma agonist (eg. Ciglitazone);a retinoic acid receptor agonist (eg. TTNPB); a caspase-3 Inhibitor (eg. caspase-3 Inhibitor VII); a xanthine oxidase inhibitor (eg. allopurinol); and a H2 histamine receptor antagonist (eg. Ranitidine).

(Protocol 5) More suitably, the method comprises contacting the cell with a combination of CRA(s) comprising, consisting or consisting essentially of a KLF4 inducer (eg. 15d-PGJ2), an NADPH oxidase inhibitor (eg. Sinomenine); a HDAC1 inhibitor (eg. valproic acid); a GPR modulator (eg. GPR30 Agonist); a P53 inhibitor (eg. Pifithrin-a, Cyclic (PFT)); and a demethylating agent (eg. 5-aza-2'-deoxycytidine (5-AZA)); incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days; contacting the cell with a CRA selected from the group consisting of a histone deacetylate inhibitor (eg. HDAC Inhibitor III (M344)); a PPAR gamma agonist (eg. Ciglitazone);a retinoic acid receptor agonist (eg. TTNPB); a caspase-3 Inhibitor (eg. caspase-3 Inhibitor VII); a xanthine oxidase inhibitor (eg. allopurinol); and a H2 histamine receptor antagonist (eg. Ranitidine); incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days; and contacting the cell with a CRA selected from the group consisting of a KLF4 inducer (eg. 15d-PGJ2), an NADPH oxidase inhibitor (eg. Sinomenine); a HDAC1 inhibitor (eg. valproic acid); a GPR modulator (eg. GPR30 Agonist); a P53 inhibitor (eg. Pifithrin-a, Cyclic (PFT)); and a demethylating agent (eg. 5-aza-2'- deoxycytidine (5-AZA)); and optionally incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days. The exposure in the presence of the chemicals may be for about 1 , 2 or 3 or more days at a time. The exposure in the absence of the chemicals may be for about 1 , 2 or 3 or more days at a time.

(Protocol 6) In one embodiment, the method comprises contacting the cell with a CRA selected from the group consisting of a demethylating agent (eg. 5-aza-2'-deoxycytidine (5-AZA)); a KLF4 inducer (eg. 15d-PGJ2); an NADPH oxidase inhibitor (eg. Sinomenine); and a HDAC1 inhibitor (eg. valproic acid).

(Protocol 6) Suitably, the method comprises contacting the cell with a combination of CRA(s) comprising, consisting or consisting essentially of a demethylating agent (eg. 5-aza-2'- deoxycytidine (5-AZA)); a KLF4 inducer (eg. 15d-PGJ2); an NADPH oxidase inhibitor (eg. Sinomenine) ; and a HDAC1 inhibitor (eg. valproic acid); incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days; and optionally repeating each of the steps two or more times - such as three, four or five or more times.

(Protocol 6) More suitably, the method comprises contacting the cell with a combination of CRA(s) comprising, consisting or consisting essentially of a demethylating agent (eg. 5-aza-2'- deoxycytidine (5-AZA)); a KLF4 inducer (eg. 15d-PGJ2); an NADPH oxidase inhibitor (eg. Sinomenine) ; and a HDAC1 inhibitor (eg. valproic acid); incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days; contacting the cell with a combination of CRA(s) comprising, consisting or consisting essentially of a demethylating agent (eg. 5-aza-2'-deoxycytidine (5-AZA)); a KLF4 inducer (eg. 15d-PGJ2); an NADPH oxidase inhibitor (eg. Sinomenine); and a HDAC1 inhibitor (eg. valproic acid); incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days; contacting the cell with a combination of CRA(s) comprising, consisting or consisting essentially of a demethylating agent (eg. 5-aza-2'-deoxycytidine (5-AZA)); a KLF4 inducer (eg. 15d-PGJ2); an NADPH oxidase inhibitor (eg. Sinomenine); and a HDAC1 inhibitor (eg. valproic acid); and optionally incubating the cell in the absence of one or more CRA(s) (eg. all CRA(s)) for one or more days. The exposure in the presence of the chemicals may be for about 1 , 2 or 3 or more days. The exposure in the absence of the chemicals may be for about 1 , 2 or 3 or more days.

For some embodiments, the efficiency of reprogramming a cell into an CIMP cell is at least about 0.2% - such as at least about 0.3%, 0.4%, 0.5%, 0.7%, 0.9%, 1 %, 1 .2%, 1 .4%, 1.6%, 1.8% or 1 .9%.

For some embodiments, the efficiency of reprogramming a cell into an CIMP cell is at least about 2% - such as at least about 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40%,

Com binations of CRA(s)

A further aspect relates to combinations of CRA(s) that can be used to reprogram a cell into a CIMP cell. The combination may comprise, consist or consist essentially of 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more CRA(s). The combinations may be used in kits. (Protocol 1 -day1 -4) & (Protocol 1 -day7-10) & (Protocol 4-day1 -3) The combination may comprise, consist or consist essentially of a DNA methyltransferase inhibitor; a histone deacetylase inhibitor; a KLF4 inducer, a retinoic acid receptor agonist; an HDAC1 inhibitor; and a protein tyrosine phosphatase PTP inhibitor. Thus, for example, the combination may comprise, consist or consist essentially of RG108, Scriptaid, 15d-PGJ2, TTNPB, valproic acid and sodium orthovanadate.

(Protocol 1 -day14-17) The combination may comprise, consist or consist essentially of a histone deacetylase inhibitor; a KLF4 inducer, a MEK/ERK inhibitor; a protein tyrosine phosphatase PTP inhibitor; a H2 histamine receptor antagonist and Pregnant mare serum Gonadotropin (PMSG). Thus, for example, the combination may comprise, consist or consist essentially of trichostatin A, 15d-PGJ2, PD98059, sodium orthovanadate , ranitidine and PMSG. (Protocol 2-day1 -3) The combination may comprise, consist or consist essentially of an NADPH oxidase inhibitor; a MEK/ERK inhibitor; a histone methyltransferase inhibitor; a protein tyrosine phosphatase PTP inhibitor; and a calcium channel agonist. Thus, for example, the combination may comprise, consist or consist essentially of sinomenine, PD98059, U0126, BIX 01294, sodium orthovanadate and bay K8644.

(Protocol 2-day7-10) & (Protocol 4 day 7-10) The combination may comprise, consist or consist essentially of a demethylating agent; a histone deacetylase inhibitor; a KLF4 inducer, an inhibitor of Rho-associated protein kinase; a histone methyltransferase inhibitor and a voltage gated sodium channel stabiliser. Thus, for example, the combination may comprise, consist or consist essentially of 5-aza-2'-deoxycytidine, Scriptaid), 15d-PGJ2, Y-27632, BIX 01294) and 5,5-Diphenylhydantoin sodium salt (Phenytoin).

(Protocol 2-day14-17) & (Protocol 3-day14-17) & (Protocol 4-day14-17) & (Protocol 5-day1 -3) & (Protocol 5-day 14-17) The combination may comprise, consist or consist essentially of a KLF4 inducer, an NADPH oxidase inhibitor, an HDAC1 inhibitor; a GPR modulator, a P53 inhibitor and a demethylating agent. Thus, for example, the combination may comprise, consist or consist essentially of 15d-PGJ2, sinomenine), valproic acid, GPR30Ag+, cyclic pifithrin-a and 5-aza-2'- deoxycytidine.

(Protocol 3-day 1 -3) The combination may comprise, consist or consist essentially of a DNA methyltransferase inhibitor, a deacetylase inhibitor, a PPAR gamma agonist, an NADPH oxidase inhibitor, a PKC activator and an a1 -adrenoceptor antagonist. Thus, for example, the combination may comprise, consist or consist essentially of RG108; sodium butyrate, ciglitazone, sinomenine, phorbol-12-myristate-13-acetate and verapamil hydrochloride.

(Protocol 5-day7-10) The combination may comprise, consist or consist essentially of a histone deacetylate inhibitor, a PPAR gamma agonist, a retinoic acid receptor agonist, a caspase-3 Inhibitor, a xanthine oxidase inhibitor; and a H2 histamine receptor antagonist. Thus, for example, the combination may comprise, consist or consist essentially of HDAC Inhibitor III (M344)), ciglitazone, TTNPB (Arotinoid acid, 4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)-1 -propenyl]benzoic acid), caspase-3 Inhibitor VII (2-(4-Methyl-8-(morpholin-4- ylsulfonyl)-1 ,3-dioxo-1 ,3-dihydro-2H-pyrrolo[3,4-c]quinolin-2-yl)ethyl acetate) allopurinol and Ranitidine.

(Protocol 6) The combination may comprise, consist or consist essentially of a demethylating agent, a KLF4 inducer, an NADPH oxidase inhibitor and a HDAC1 inhibitor. Thus, for example, the combination may comprise, consist or consist essentially of 5-aza-2'-deoxycytidine, 15d- PGJ2, sinomenine and valproic acid.

A combination described herein may be used together with one or more further combinations in order to reprogram a cell. Accordingly, groups of combinations of CRA(s) may be used.

(Protocol 1 ) By way of example, the combination comprising, consisting or consisting essentially of a DNA methyltransferase inhibitor; a histone deacetylase inhibitor; a KLF4 inducer, a retinoic acid receptor agonist; an HDAC1 inhibitor; and a protein tyrosine phosphatase PTP inhibitor may be used together with a combination comprising, consisting or consisting essentially of a histone deacetylase inhibitor; a KLF4 inducer, a MEK/ERK inhibitor; a protein tyrosine phosphatase PTP inhibitor; a H2 histamine receptor antagonist and Pregnant mare serum Gonadotropin (PMSG).

(Protocol 2) By way of further example, the combination comprising, consisting or consisting essentially of an NADPH oxidase inhibitor; a MEK/ERK inhibitor; a histone methyltransferase inhibitor; a protein tyrosine phosphatase PTP inhibitor; and a calcium channel agonist may be use together with (i) the combination comprising, consisting or consisting essentially of a demethylating agent; a histone deacetylase inhibitor; a KLF4 inducer, an inhibitor of Rho- associated protein kinase; a histone methyltransferase inhibitor and a voltage gated sodium channel stabiliser; and (ii) the combination comprising, consisting or consisting essentially of a KLF4 inducer, an NADPH oxidase inhibitor, an HDAC1 inhibitor; a GPR modulator, a P53 inhibitor and a demethylating agent.

(Protocol 3) By way of further example, the combination comprising, consisting or consisting essentially of a DNA methyltransferase inhibitor, a deacetylase inhibitor, a PPAR gamma agonist, an NADPH oxidase inhibitor, a PKC activator and an a 1 -adrenoceptor antagonist may be used together with a combination comprising, consisting or consisting essentially of a KLF4 inducer, an NADPH oxidase inhibitor, an HDAC1 inhibitor; a GPR modulator, a P53 inhibitor and a demethylating agent.

(Protocol 4) By way of further example, the combination comprising, consisting or consisting essentially of a DNA methyltransferase inhibitor; a histone deacetylase inhibitor; a KLF4 inducer, a retinoic acid receptor agonist; an HDAC1 inhibitor; and a protein tyrosine phosphatase PTP inhibitor may be used together with (i) a combination comprising, consisting or consisting essentially of a demethylating agent; a histone deacetylase inhibitor; a KLF4 inducer, an inhibitor of Rho-associated protein kinase; a histone methyltransferase inhibitor and a voltage gated sodium channel stabiliser; and (ii) a combination comprising, consisting or consisting essentially of a KLF4 inducer, an NADPH oxidase inhibitor, an HDAC1 inhibitor; a GPR modulator, a P53 inhibitor and a demethylating agent.

(Protocol 5) By way of further example, the combination comprising, consisting or consisting essentially of a KLF4 inducer, an NADPH oxidase inhibitor, an HDAC1 inhibitor; a GPR modulator, a P53 inhibitor and a demethylating agent may be used together with a combination comprising, consisting or consisting essentially of a histone deacetylate inhibitor, a PPAR gamma agonist, a retinoic acid receptor agonist, a caspase-3 Inhibitor, a xanthine oxidase inhibitor; and a H2 histamine receptor antagonist.

The combination may comprise, consist or consist essentially of f olic acid and a histone deacetylase inhibitor.

The combination may comprise, consist or consist essentially of a steroid hormone - such as glucocorticoid, a demethylating agent, and a histone deacetylase inhibitor.

The combination may comprise, consist or consist essentially of an inhibitor of Rho-associated protein kinases, a demethylating agent, a histone deacetylase inhibitor, a GPR modulator and a PPAR gamma agonist.

The combination may comprise, consist or consist essentially of a demethylating agent and a histone deacetylase inhibitor.

The combination may comprise, consist or consist essentially of a steroid hormone - such as glucocorticoid - and folic acid.

The combination may comprise, consist or consist essentially of a Wnt activator, a DNA methyltransferase inhibitor, a histone deacetylase inhibitor, a STAT3/PI3K activator and a PPAR gamma agonist.

The combination may comprise, consist or consist essentially of a NADPH oxidase inhibitor, a DNA methyltransferase inhibitor, a demethylating agent, and a histone deacetylase inhibitor. The combination may comprise, consist or consist essentially of an inhibitor of Rho-associated protein kinases, a demethylating agent, a histone deacetylase inhibitor, and a protein tyrosine phosphatase PTP inhibitor.

The combination may comprise, consist or consist essentially of a DNA methyltransferase inhibitor, a demethylating agent, a histone deacetylase inhibitor.

The combination may comprise, consist or consist essentially of ascorbic acid; a PKC activator and a PPAR gamma agonist. The combination may comprise, consist or consist essentially of a demethylating agent and a histone deacetylase inhibitor.

The combination may comprise, consist or consist essentially of a wnt agonist; a MEK/ERK inhibitor; a STAT3/PI3K activator and a histone methyltransferase inhibitor.

The combination may comprise, consist or consist essentially of a DNA methyltransferase inhibitor, a demethylating agent, a histone deacetylase inhibitor, an ATP dependent chromatin remodelling agent, a steroid hormone - such as glucocorticoid, a KLF4 inducer, an AMPK activator; and a MEK/ERK inhibitor.

The combination may comprise, consist or consist essentially of a GPR modulator and a HDAC1 inhibitor.

The combination may comprise, consist or consist essentially of a demethylating agent, a histone deacetylase inhibitor, a KLF4 inducer, an inhibitor of Rho-associated protein kinase, a histone methyltransferase inhibitor and a voltage gated sodium channel stabiliser.

The combination may comprise, consist or consist essentially of a DNA methyltransferase inhibitor, a histone deacetylase inhibitor, a KLF4 inducer, a retinoic acid receptor agonist, a HDAC1 inhibitor and a protein tyrosine phosphatase PTP inhibitor.

The combination may comprise, consist or consist essentially of a retinoic acid receptor agonist, a KLF4 inducer, a MEK/ERK inhibitor, a protein tyrosine phosphatase PTP inhibitor, a H2 histamine receptor antagonist and PMSG.

The combination may comprise, consist or consist essentially of a demethylating agent, a deacetylase inhibitor, a retinoic acid receptor agonist, a wnt agonist, a H2 histamine receptor antagonist and GOL.

The combination may comprise, consist or consist essentially of a histone deacetylase inhibitor, a PPAR gamma agonist, a retinoic acid receptor agonist, a caspase-3 Inhibitor, a xanthine oxidase inhibitor, and a H2 histamine receptor antagonist.

The combination may comprise, consist or consist essentially of a deacetylase inhibitor, a PPAR gamma agonist, a histone H3 demethylase inhibitor, a MEK/ERK inhibitor, a caspase-3 Inhibitor and GDL.

The combination may comprise, consist or consist essentially of a KLF4 inducer, a NADPH oxidase inhibitor, a HDAC1 inhibitor, a GPR modulator, a p53 inhibitor, and a demethylating agent.

In one embodiment, the CRA comprises, consists or consists essentially of a chemical that is or is derived from a histone deacetylase inhibitor, preferably, the histone deacetylase inhibitor is or is derived from a carboxylic acid - such as butyric acid (CH₃CH2CH2-COOH) or a salt thereof - such as sodium butyrate. In one embodiment, the CRA comprises, consists or consists essentially of chemicals that are or are derived from a PI3K inducer and an HDAC1 enzyme inhibitor. Suitably the PI3K inducer is an agonist of a G-protein-coupled receptor for oestrogen - such as the GPR30 Agonist. Suitably the HDAC1 enzyme inhibitor is an analogue of valeric acid analogue of valeric acid - such as valproic acid or a salt thereof.

The combination may be a mixture of two or more of the combinations described herein.

In a further aspect, there is provided a method of reprogramming a cell into a CIMP cell comprising the steps of: (i) providing a cell; (ii) incubating said cell in the presence of a combination of two or more CRA(s); and/or a repetition of one or more of the same or different CRA(s); and (iii) reprogramming the cell, optionally, wherein said cell that is being reprogrammed is incubated in the absence of at least one (eg. all) CRA(s) for a period of time. Suitably, said cell that is being reprogrammed is incubated in the absence of at least one CRA after step (ii).

Suitably, said method comprises: (i) providing a cell; (ii) incubating said cell in the presence of: a combination of two or more CRA(s); and/or a repetition of one or more of the same or different CRA(s); (iii) incubating said cell in the absence of at least one (eg. all) CRA; (iv) optionally repeating either step (ii) or step (ii) and (iii) one or more times; and (v) reprogramming the cell. Suitably, said cell is incubated in the presence of two or more of the same or different combinations of CRA(s) at the same or a different time and/or said cell is incubated in the presence of two or more repetitions of the same or different CRA(s).

There is also provided a method of reprogramming a cell into an CIMP cell comprising the steps of: (i) providing a cell; (ii) contacting said cell with one or more CRA(s) that remodel chromatin and initiate CIMP cell formation; (iii) contacting said cell with one or more CRA(s) that induce CIMP cell formation; (iv) contacting said cell with one or more CRA(s) that stabilise and expand said CIMP cell; and (v) obtaining a CIMP cell, wherein the CRA(s) specified in step (ii), (ii) or (iv) is a combination of two or more CRA(s); or a repetition of one or more of the same or different CRA(s).

There is also provided a method for identifying one or CRA(s) for reprogramming a cell into an CIMP cell comprising the steps of: (i) providing a cell; (ii) incubating said cell in the presence of: a combination of two or more chemicals; and/or a repetition of one or more of the same or different chemicals; and (iii) determining if said cell has been reprogrammed in to a CIMP cell, optionally, wherein said cell is incubated in the absence of the chemicals for a period of time; and wherein the presence of a CIMP cell is indicative that the one or more chemicals are able to reprogram the cell.

Specialisation of CIMP cells The CIMP cells described herein may undergo specialisation. By way of example, the CIMP cells may undergo further specialisation into one or more semi-differentiated or differentiated cells. Suitably, the CIMP cell undergoes further specialisation such that it is a more lineage restricted cell than the CIMP cell. Protocols and kits for specialising (eg. differentiating) cells are available in the art. By way of example, a cell of the ectoderm lineage may be reprogrammed into a CIMP cell and then further differentiated into, for example, cells of the hematopoietic lineage - such as megakaryocytes, plasma cells, and/or macrophages/monocytes. By way of further example, a cell of the mesoderm lineage may be reprogrammed into a CIMP cell and then further differentiated into, for example, adipocytes/lipocytes, bone cells - such as osteocytes, or a neural cell. By way of further example, a cell of the endoderm lineage may be reprogrammed into a CIMP cell and then further reprogrammed into, for example, epithelial cells of the pharynx, including the eustachian tube, the tonsils, the thyroid gland, parathyroid glands, and thymus gland; or cells of the larynx, trachea, lungs, urinary bladder or urethra.

A further aspect relates to a method of reprogramming a cell of the ectoderm lineage via a CIMP cell comprising the steps of: (i) providing a cell of the ectoderm lineage; (ii) contacting said cell with one or more of the CRA(s) (eg. combinations of CRA(s)) described herein; (iii) obtaining a CIMP cell; (iv) differentiating the CIMP cell in to a desired cell type - such as a cell of the hematopoietic lineage - such as a megakaryocyte, a plasma cell, a macrophage and/or a monocyte, wherein the cell to be reprogrammed has not been genetically manipu lated and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

A further aspect relates to a method of reprogramming a cell of the mesoderm lineage via a CIMP cell comprising the steps of: (i) providing a cell of the mesoderm lineage; (ii) contacting said cell with one or more of the CRA(s) (eg. combinations of CRA(s)) described herein; (iii) obtaining a CIMP cell; (iv) differentiating the CIMP cell in to a desired cell type - such as an adipocyte/lipocyte, a bone cell (eg. an osteocyte), and/or a neural cell, wherein said cell of the mesoderm lineage not been genetically manipulated and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency . A further aspect relates to a method of reprogramming a cell of the endoderm lineage via a CIMP cell comprising the steps of: (i) providing a cell of the endoderm lineage; (ii) contacting said cell with one or more of the CRA(s) (eg. combinations of CRA(s)) described herein; (iii) obtaining a CIMP cell; (iv) differentiating the CIMP cell in to a desired cell type, wherein said cell of the endoderm lineage has not been genetically manipulated and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency. Generating a transgenic non-human anim al

Another embodiment relates to a method of generating a transgenic non-human animal comprising the steps of: (i) introducing the CIMP cell into a non-human blastocyst; (ii) transferring the blastocyst into the uterus of a female non-human animal; and (iii) allowing the blastocyst to develop into an embryo. The method for generating a transgenic non-human animal may be carried out using methods for generating transgenic non-human animals using embryonic stem cells but replacing the embryonic stem cells with CIMP cells. Such methods have been described in the art in, for example, Hogan, B., R. Beddington, et al. (1994), "Manipulating the Mouse Embryo: A Laboratory Manual", Cold Spring Harbour Press. Briefly, said method may comprise introducing the CIMP cell into a non-human preim plantation embryo - such as a morula or a blastocyst. The chimaeric embryo may then be transferred into the uterus of a pseudopregnant non-human female where it develops into an embryo that is eventually born.

Generating a transgenic non-human animal line from CIMP cells is based on the pluripotency of the cells (/e., their ability, once injected into a host developing embryo - such as a blastocyst or morula - to participate in embryogenesis and contribute to the germ cells of the resulting animal). As outlined above, the blastocysts containing the injected CIMP cells are allowed to develop in the uterus of a pseudopregnant non-human female and is born as a chimera. The resultant transgenic non-human animal is chimeric for cells originating from CIMP cells and may be back crossed to wild-type non-human animals and screened for animals carrying only the genetic content of a CIMP cell so as to identify transgenic animals homozygous for the combination of DNA segments. The transgenic non-human animal may be a transgenic mouse, rat, hamster, dog, monkey, rabbit, pig, sheep or cow. In a further aspect, a transgenic non- human animal obtained or obtainable by the methods described herein is provided . Methods for identifyi ng a CIMP cell

A further aspect relates to methods for identifying one or CRA(s) for reprogramming a cell into a CIMP cell.

In one embodiment, the one or more CRA(s) may be identified using the methods based on split-split and split-pool culture as described in WO2004/031369. As described therein, the cell culture techniques can be based on a dynamic process involving serial culture steps performed in a defined sequence to achieve a desired effect.

Accordingly, a further aspect relate to a method for identifying one or CRA(s) for reprogramming a cell into a CIMP cell comprising the steps of: (a) providing a first set of groups of cell units each comprising one or more cells, and exposing said groups to one or more chemicals, preferably small molecules; (b) subdividing one or more of said groups to create a further set of groups of cell units; (c) exposing said further groups to one or more further chemicals, preferably small molecules; (d) optionally, repeating steps (b) - (c) iteratively as required; and (e) assessing the effect on a given cell unit of the one or more chemicals, preferably small molecules to which it has been exposed, wherein the presence of a CIMP cell is indicative that the one or more chemicals, preferably small molecules is a CRA able to reprogram the cell into a CIMP cell. Optionally, the cell units may be incubated in the absence of one or more (eg. all) CRA(s) following at least one (eg. each) exposure to the chemicals, preferably small molecules for a period of time - such as 1 , 2, 3, 4, 5, or 6 more days.

A further aspect relates to a method for identifying one or more CRA(s) for reprogramming a cell into a CIMP cell comprising the steps of: a) providing a first set of groups of cell units each comprising one or more cells, and exposing said groups to one or more chemicals, preferably small molecules; (b) pooling two or more of said groups to form at least one second pool; (c) subdividing the second pool to create a further set of groups of cell units; (d) exposing said further groups to one or more (further) chemicals, preferably small molecules; (e) optionally, repeating steps (b) - (d) iteratively as required; and (f) assessing the effect on a given cell unit of the one or more chemicals, preferably small molecules to which it has been exposed, wherein the presence of a CIMP cell is indicative that the one or more chemicals, preferably small molecules are CRA(s) able to reprogram a cell into a CIMP cell. Optionally, the cell units may be incubated in the absence of one or more (eg. all) chemicals, preferably small molecules following at least one (eg. each) exposure to the chemicals, preferably small molecules for a period of time - such as 1 , 2, 3, 4, 5, or 6 more days.

A further aspect relates to a method for identifying one or CRA(s) for reprogramming a cell into a CIMP cell comprising the steps of: a) providing a first set of groups of cell units each comprising one or more cells, and exposing said groups to one or more chemicals, preferably small molecules (b) pooling two or more of said groups to form at least one second pool; (c) subdividing the second pool to create a further set of groups of cell units; (d) exposing said further groups to one or more (further) chemicals, preferably small molecules; and (e) optionally, repeating steps (b) - (d) iteratively as required, wherein the presence of a CIMP cell is indicative that the one or more chemicals, preferably small molecules are CRA(s) able to reprogram a cell into a CIMP cell. Optionally, the cell units may be incubated in the absence of one or more (eg. all) chemicals, preferably small molecules following at least one (eg. each) exposure to the CRA(s) for a period of time - such as 1 , 2, 3, 4, 5, or 6 more days.

A further aspect relates to a method for identifying one or more CRA(s) for reprogramming a cell into a CIMP cell comprising the steps of: a) providing a first set of groups of cell units each comprising one or more cells, and exposing said groups to one or more chemicals, preferably small molecules; (b) pooling two or more of said groups to form at least one second pool; (c) subdividing the second pool to create a further set of groups of cell units; (d) exposing said further groups to one or more (further) chemicals, preferably small molecules; (e) optionally, repeating steps (b) - (d) iteratively as required; and (f) assessing the effect on a given cell unit of the chemicals, preferably small molecules to which it has been exposed, wherein the presence of a CIMP cell is indicative that the one or more chemicals, preferably small molecules are CRA(s) able to reprogram a cell into a CIMP cell. Optionally, the cell units may be incubated in the absence of one or more (eg. all) CRA(s) following at least one (eg. each) exposure to the CRA(s) for a period of time - such as 1 , 2, 3, 4, 5, or 6 more days.

Suitably, the groups of cell units are reprogrammed in a culture medium. Suitably, the method comprises the first step of providing the cell to be reprogrammed in a culture medium in the absence of one or more chemicals, preferably small molecules. Suitably, the cell to be reprogrammed has not been genetically manipulated and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency . Suitably, the groups of cell units are rescued in a maintenance medium.

As described herein the chemicals that are used may be single chemicals and/or a combination of chemicals which may be the same or different.

In one embodiment, the cell units are labelled and the label(s) reflect(s) the CRA(s) to which the cell unit has been exposed.

In one embodiment, the label is spatially encoded.

In one embodiment, the label is selected from the group consisting of an oligonucleotide, a peptide, a fluorescent compound , a secondary amine, a halocarbon, a mixture of stable isotopes, a bar code, a bead or a tag - such as an optical tag or a radiofrequency encoding tag. The label may be a group of labels, each added at a specific culturing step; or a label added at the beginning or the experiment which is modified according to, or tracked during, the culturing steps to which the cell unit is exposed; or simply a positional reference, which allows the culturing steps used to be deduced. A label may also be a device that reports or records the location or the identity of a cell unit at any one time, or assigns a unique identifier to the cell unit. Examples of labels are molecules of unique sequence, structure or mass; or fluorescent molecules or objects such as beads; or radiofrequency and other transponders; or objects with unique markings or shapes.

Tags

Tags may be conjugated to a microcarrier, such as a cell-associated microcarrier. Subsequent detection and identification provides for an unambiguous record of the chronology and identity of the cell culture conditions to which the cell unit has been exposed. Various molecular or macromolecular tags may be used in combination with the microcarriers so long as they can be detected. The tags typically comprise uniquely shaped or objects modified with markings and/or coloured and/or fluorescent compounds. In one embodiment the tags which are used to label cell units have one or more (preferably all) of the following qualities: i. They are small in size relative to the microcarrier they are labelling and/or smaller than the mean pore size of a porous microcarrier;

ii. They are capable of forming a complex with the microcarrier such that binding persists throughout the experiment and so unbound tags can be separated from the complex without affecting the labelled cell units;

iii. They are separable from cell units with which they have formed a complex under conditions which do not perturb the unique qualities of the tags;

iv. They are made of one or more inert substances which do not substantially affect the biology of the cell unit and which in turn is not affected by the cell units or their biology;

v. They are obtainable in large numbers and moreover in many related but distinct variants which are easily distinguishable using an appropriate technique; and/or

vi. They are distinguished by a method which is convenient, highly reliable and which can be automated.

In one embodiment, the tag is a microsphere-such as a fluorescent and/or coloured microsphere. More than 2000 different microspheres made by emulsion or suspension polymerization, precipitation etc. and comprised of polystyrene, other polymers, copolymers, terpolymers and/or silica etc. are available in a variety of sizes, densities, colours etc. A common type of microsphere is the Polystyrene (PS) and styrene/divinylbenzene copolymer (S/DVB) microsphere. Other polymers include polymethylmethacrylate (PMMA), polyvinyltoluene (PVT), styrene/butadiene (S/B) copolymer, styrene/vinyltoluene (S/VT) copolymer. Many of these microspheres can be functionalised, for instance by carboxyl groups as in the CML microspheres, or by amino functionalized or nitrogen-containing compounds , like primary, secondary, tertiary, and quaternary aliphatic amines, aromatic amines, and pyridines or sulfoxides which offer alternative coupl ing reactions to the COOH beads.

Suitably, the microsphere is a hydrophilic microsphere. More suitably the microsphere is a polystyrene microsphere. Most suitably, the microsphere is a surface-modified microsphere such as a carboxylate modified (CML) microsphere.

In one embodiment, one or more CML microspheres are complexed together with one or more microcarriers with positive charge. CML microspheres have a highly charged surface layer of carboxyl groups derived from a copolymerisation process. The surface is somewhat porous and relatively hydrophilic, but retains overall hydrophobic properties. The charge density of these particles ranges from about 10-125 A² per carboxyl group, and they are stable to high concentrations of electrolytes (up to 1 M univalent salt). The CML latex will adsorb proteins and other biomolecules, but much less strongly than hydrophobic microspheres.

In some embodiments, conjugates of microspheres and proteins, e.g. streptavidin are prepared. For example, conjugates with CML microspheres may be prepared as follows. CML microspheres may be activated using a water soluble carbodiimide reagent that makes the carboxyl groups reactive with primary amines on the proteins to be coupled. A 50 mM reaction buffer at pH 6.0 is prepared. Sodium acetate or 2-[N-morpholino]ethanesulfonic acid (MES) are suitable buffers. The protein is dissolved in the reaction buffer at a concentration of 10 mg/mL. A 1 % (w/v) suspension of microspheres is prepared in the reaction buffer. One volume protein solution to ten volumes microsphere suspension is prepared and the mixture allowed to incubate, at room temperature for 20 minutes. A solution of 10 mg/mL (52 μMοΙ/mL) of 1 -ethyl- 3-(3-dimethylaminopropyl)carbodiimide (EDAC) in deionized water is prepared and used immediately. A calculated amount of EDAC solution to the microsphere suspension is added and the pH of the reaction mixture adjusted to 6.5±0.2 with 0.1 N NaOH. The mixture is incubated on a rocker or mixing wheel for 2 hours at room temperature. Unbou nd protein is removed and stored in storage buffer.

Advantageously, CML and other microspheres can be obtained in various formats - such as various colors (e.g. blue, red, green, yellow, black), various fluorophores (e.g. Pacific Blue (blue), Alexa Fluor^® (blue), Fluorescein (green), Fluorescein (red), Firefli™ (red) Fluorescein (green) , Fluorescein (red) or Fluorescein and Rhodamine (red green) and various sizes (e.g. 5.4 μm (1 .14 x 10¹⁰ beads/gram), and 7.6 pm (4.10 x 10⁹ beads/gram)). CML and other microspheres may be prepared such that they are loaded with one or more visible dyes and/or fluorophores.

In one embodiment, tags, such as microspheres, are not coated with proteins.

Advantageously, CML microspheres not coated with proteins (e.g. streptavidin) adhere extremely tightly to microcarriers,. By varying various parameters in the fabrication process, commercial microsphere providers, such as Bangs Laboratories, can manufacture bead sets which can be distinguished based on differing sizes (e.g. bead sets of 4.4 pm and 5.5 pm diameter). Beads within each size group can be furthermore distinguished from each other based on differing fluorescence intensity owing to differential loading with a single fluorescent dye. It is possible to use many different dyes with different absorption or emission characteristics, which can be attached to the microcarriers described herein. Accordingly, tag diversity may result from varying tag size and/or fluorophore loading (i.e. fluor intensity) and/or fluorophore identity/combination. In particular, tag diversity may result from the type of fluorophore they carry (e.g. beads can be loaded with either UV2 or Starfire Red); size (e.g. for each fluorophore there are 5 different bead sizes: 1 .87, 4.41 , 5.78, 5.37 and 9.77 microns) and/or the quantity of fluorophore they carry (5 different intensities of each dye are available) . Other fl uorophores , such as TRITC, may be used. Filters can then be used to detect the at least 4 different dyes on any given bead-such as the TRITC filter (ex 540/25; dm 565; ba 605/55) for TRITC visualization from Nikon; the DAPI filter (ex 340-380; dm 400; ba 435-485) for UV2 visualization from Nikon; the GFP-B filter (ex 460-500; dm 505; ba 510-560) for FITC visualization from Nikon and the Cy5 filter set (cat no 41008 from Chroma Technology) for Strarfire Red visualization.

Microspheres can be dyed internally or externally, with visible or fluorescent dyes. Internal dyeing occurs when the dye is integrated into the microsphere mass, typically by soaking the microsphere in a solution containing a dye or fluorophore. External modification occurs when a dye is conjugated to the surface of the microsphere, for instance modification of a CML microsphere with an isothiocyanate derivative as described herein. Accordingly, in some embodiments, the microsphere may be dyed internally or externally, with visible or fluorescent dyes. It is furthermore possible to use 'quantum dots' to obtain a very high number of different fluorescent labels which can be read conveniently.

In one embodiment, flow cytometry may be used to determine the identity of one or more microspheres. Microspheres designed for use in flow cytometry readout are available in the art. In certain embodiments, quantum dots are preferable due to the fact they do not fade (photo- bleach) when exposed to light. For instance the fluorophore FITC is known to photo-bleach and cell units treated with tags containing FITC are ideally handled in the dark and are difficult to analyse reliably. Quantum dots may be incorporated into microspheres at the time of polymerizing the polystyrene resulting in even loading of tags. Quantum dots are available in many colours and they can be excited at the same wavelength so allowing visualization of multiple colours without filters, by using a colour CCD camera. Further background information on Quantum dots is available from U.S. Pat. No. 6,322,901 , U.S. Pat. No. 6,576,291 , US2003/0017264, U.S. Pat. No. 6,423,551 , U.S. Pat. No. 6,251 ,303, U.S. Pat. No. 6,319,426 U.S. Pat. No. 6,426,513, U.S. Pat. No. 6,444,143, US2002/0045045 , U.S. Pat. No. 5,990,479, U.S. Pat. No. 6,207,392, U.S. Pat. No. 6,251 ,303, U.S. Pat. No. 6,319,426, U.S. Pat. No. 6,426,513 and U.S. Pat. No. 6,444,143.

Advantageously, the tags are protected against degradation by the components of the cell culture, for example by chemical or other modification or by encapsulation. Encapsulation of tags can take place in many different media, for example in beads as already described herein- such as those from Bangs Laboratories Inc. (Fishers Ind., USA), and encapsulation may be used to standardise tag dosage in addition to providing components for tag amplification and/or detection (for example by providing PCR primers for use with a DNA tag). Detection of tags can be accomplished by a variety of methods familiar to those skilled in the art. Methods include mass spectrometry, nuclear magnetic resonance, sequencing, hybridisation, antigen detection, electrophoresis, spectroscopy, microscopy, image analysis, fluorescence detection, efc. In some embodiments, since the tags typically contain a colour or a fluorophore then flow cytometry, microscopy, spectroscopy, image analysis and/or fluorescence detection may be used. The tags do not necessarily have to be distinguished by their chemical or molecular structure in the first instance. Multiple variations of the non-chemical tagging strategy can be devised to determine the identity of a given cell unit in a mixture or of deducing the identity of the different cell units that comprise a mixture. For instance optical or visual methods of tagging have been described where different shaped objects, graphically encoded objects or different colours denote the identity of a sample (for example see 1998, Guiles et al, Angew. Chem. Intl Ed Engl, vol. 37, p926; Luminex Corp, Austin Tex., USA; BD Biosciences; Memobead Technologies, Ghent, Belgium). Suitably, the tag may be a charged tag (e.g. a negatively charged tag). Suitably, the tag may be complexed with a microcarrier - such as a porous microcarrier. Typically, the microcarrier has a net charge. Typically, the microcarrier and tag have opposite charges. The microcarrier may comprise, consist or consist essentially of protein, cellulose, polyethylene, polystyrol, glass collagen, collagen-gylcose-aminoglycan, gelatine, or derivatives thereof. The microcarrier may be selected from the group consisting of a Cultispher-G microcarrier, a Cultispher-GL microcarrier, a Cultispher-S microcarrier, an Informatrix microcarrier, a Microsphere microcarrier, a Siran microcarrier, a FibraCel® Disks microcarrier, a Cytoline microcarrier (e.g. a Cytoline 1 microcarrier or a Cytoline 2 microcarrier), a Cytodex microcarrier (e.g. a Cytodex 1 , Cytodex 2 or Cytodex 3 microcarrier), a Cytopore microcarrier (e.g. a Cytopore 1 microcarrier or a Cytopore 2 microcarrier), a Biosilon microcarrier, a Bioglass microcarrier, a FACT III microcarrier, a Collagen C microcarrier, a Hillex II microcarrier, a ProNectin F microcarrier, a Plastice microcarrier, a Plastic Plus microcarrier, a Nunc 2D MicroHex™ microcarrier, a Glass microcarrier (Sigma Aldrich), a DE 52/53 microcarrier or combinations or derivatives thereof.

Suitably, the charged tag is a sphere - such as a microsphere , that is about <20 pm or less in diameter.

The microsphere may be a carboxylate modified (CML) microsphere. In a further embodiment, the tag is a rod-shaped particle. Suitably, the rod-shaped tag is a nanowire. The nanowire may comprise, consist or consist essentially of various metals - such as gold or silver. The nanowire may consist of different length segments made from various metals such as silver and/or gold. Suitably, the nanowire is about 1 pm or less in diameter and/or is about 10 pm or less in length. The nanowire may be a nanowire as described in Science vol. 294, p. 137-141 (2001 ). Accordingly, there is also described a complex comprising a microcarrier and a nanowire. Briefly, nanowires are multimetal microrods intrinsically encoded with submicrometer stripes. Complex patterns can be generated by sequential electrochemical deposition of metal ions onto templates with uniformly sized pores. Advantageously, the nanowires are small enough to be used as tags that may be added after each split. This is more convenient as it is necessary to read tags only in the positive microcarriers. Parameters for the rod-shaped particle-such as the nanowire-include but are not limited to size, optical properties and/or metal composition. In one embodiment, the optical properties are selected from the group consisting of: light reflectivity- such as light reflectivity of a particular wavelength, colour, the fluorescence emission wavelength(s) and the fluorescence emission intensity. In some embodiments, the rod-shaped particle-such as the nanowire is externally dyed. Microcarriers that can be used together with the rod -shaped tag are described herein.

Rod shaped tags and charge-neutral porous microcarriers may be used. Without wishing to be bound by an particular theory it is believed that smaller tags penetrate the pores of the microcarriers better and become jammed (presumably due to size asymmetry). Accordingly, the binding of nanowires is better than, for example, the binding of microsphere tags and results in a high level permanent tagging. In another embodiment, one or more polystyrene microbeads are complexed together with one or more Cultispher-G microcarriers. For some embodiments, the tag is not a DNA tag. In some embodiments, the tag is an externally dyed tag.

In a further embodiment, the tags use radio waves to transmit information, as in RFID tags. RFID generally employs transponde rs (RF tags), antennae and readers. An RF tag is a small electronic circuit, usually encased in glass or plastic, which in its simplest form provides access to a unique identification code that may be 'read', without contact or line of sight, by suitable electronics. Tags may also store information generated by the user, again without contact or line of sight. A 'reader' is an electronic unit that transfers information to and from one or more tags (it should be noted that the term reader is used interchangeably to mean both a read only and read/write unit). The size and features of a reader may vary considerably, and it may operate in isolation, or be connected to a remote computer system . An antenna is used to transmit information from a reader to a tag, and to receive information sent by an RF tag. The size and format of an antenna will reflect the specific application, and may range from a small circular coil to large planar structures. An RFID system may operate in isolation, or be connected to a remote computer for more comprehensive interpretation and manipulation of identification and associated data derived from a tag. One RFID strategy is described in Nicolaou et al (1995, Angew Chem IntI Ed Engl, vol. 34, p. 2289) and comprises: (i) a porous enclosure containing a synthesis substrate and the semiconductor tag; (ii) the solid phase synthesis resin; (iii) a glass- encased Single or Multiple Addressable Radiofrequency Tag semiconductor unit capable of receiving, storing and emitting radiofrequency signals. A sim ilar device could be adapted to growing and following cell units simply by replacing the solid phase synthesis resin with tissue culture microcarriers or suitable cell units. More variations of this can be envisaged including but not limited to (coated or uncoated) RF tags on which cells are grown directly, or RF tags implanted into cell units or organisms.

Compositions In a further aspect, there is provided a composition comprising a CIMP cell and one or more constituents that maintain the viability of the cell. The constituents may be cell medium constituents and/or constituents facilitating administration to a patient.

The composition may be used in a variety of experimental and therapeutic scenarios. Since the CIMP cells are free of transgenic expression elements and contain an unmodified set of endogenous genes, they are expected to be beneficial in gene therapy, regenerative medicine, cell therapy and/or drug screening and the like.

Gene therapy, which is based on introducing therapeutic DNA constructs for correcting a genetic defect into somatic cells by ex vivo or in vivo techniques is one of the most important applications of gene transfer. Suitable vectors and methods for in vitro or in vivo gene therapy are described in the literature and are known to the person skilled in the art (Davis PB, Cooper MJ., AAPS J. (2007), 19;9(1 ):E1 1 -7; Li S, Ma Z., Curr Gene Ther. (2001 ), 1 (2):201 -26). Cells obtained from a patient could, for example, be genetically corrected by methods known in the art and described above and subsequently be reprogrammed into CIMP cells. The CIMP cells may have the pheno- and/or genotype of ES cells.

Regenerative medicine may be used to potentially cure any disease that results from malfunctioning, damaged or failing tissue by either regenerating the damaged tissues in vivo or by growing the tissues and organs in vitro and subsequently implanting them into the patient. The CIMP cells being capable of differentiating into virtually any tissue (ectoderm, mesoderm, endoderm cells) can be used in any aspect of regenerative medicine and hence drastically reduce the need for ES cells.

The CIMP cells and/or their differentiated progeny may also be used to identify drug targets and test potential therapeutics hence reducing the need for ES cells and in vivo studies. Experimental setups and methods to identify and/or assess effects of a potential drug including, for example, target-site and -specificity, toxicity, bioavailability, are well-known to the person skilled in the art.

Further, the CIMP cells and/or their differentiated progeny may be used to study the prevention and treatment of birth defects or study cell differentiation.

The CIMP cells and/or their differentiated progeny may be utilized for repairing or regenerating a tissue or cell lineage in a subject. The method includes obtaining the reprogrammed cell as described herein and administering the cell to a subject (eg., a subject having a myocardial infarction, congestive heart failure, stroke, ischemia, peripheral vascular disease, alcoholic liver disease, cirrhosis, Parkinson's disease, Alzheimer's disease, diabetes, cancer, arthritis, wound healing, immunodeficiency, aplastic anemia, anemia, and genetic disorders) and similar diseases, where an increase or replacement of a particular cell type/tissue or cellular de- differentiation is desirable. In one embodiment, the subject has damage to the tissue or organ , and the administering provides a dose of cells sufficient to increase a biological function of the tissue or organ or to increase the number of cell present in the tissue or organ. In another embodiment, the subject has a disease, disorder, or condition, and wherein the administering provides a dose of cells sufficient to ameliorate or stabilize the disease, disorder, or condition. In yet another embodiment, the subject has a deficiency of a particular cell type, such as a circulating blood cell type and wherein the administering restores such circulating blood cells. The CIMP cells and/or their differentiated progeny may also be used for drug discovery on rare cell types that are prepared by the methods described herein. Thus drug targets and potential therapeutics can be tested on the rare cell types prepared by the methods described herein thereby reducing the need for ES cells and in vivo studies and providing a plentiful supply of such cells.

The CIMP cells and/or their differentiated progeny may also be used as disease models by isolating a cell from a subject with a certain disease and reprogramming the cell into a cell with that disease. Thus, the mechanisms underlying the disease process of the cell can be investigated. The composition may be a pharmaceutical composition.

Pharmaceutical com positions

In a further aspect, there is provided a pharmaceutical composition comprising one or more CRA(s) or combinations thereof admixed with a pharmaceutically acceptable carrier, diluent, excipient or adju vant and/or combinations thereof.

In a further aspect, there is provided a process of preparing a pharmaceutical composition comprising admixing one or more CRA(s) or combinations thereof with a pharmaceutically acceptable diluent, carrier, excipient or adjuvant and/or combinations thereof.

In a further aspect, there is provided a method of preventing and/or treating a disease comprising administering the pharmaceutical composition to a subject.

The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).

Preservatives, stabilisers, dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and s uspending agents may be also used. There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, the pharmaceutical composition may be formulated to be administered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form , for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be administered by a number of routes. If the agent is to be administered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.

Where appropriate, the pharmaceutical compositions may be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or the pharmaceutical compositions can be injected parenterally, for example, intravenously, intramuscularly or subcutaneously. For parenteral administration, the com positions may be best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

The one or more CRA(s) or combinations t hereof may be administered in the form of a pharmaceutically acceptable or active salt. Pharmaceutically-acceptable salts are well known to those skilled in the art, and for example, include those mentioned by Berge et al, in J.Pharm.Sci., 66, 1-19 (1977).

The routes for administration (delivery) may include, but are not limited to, one or more of oral (e.g. as a tablet, capsule, or as an ingestable solution), topical, mucosal (e.g. as a nasal spray or aerosol for inhalation), nasal, parenteral (e.g. by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral) , transdermal, rectal, buccal, vaginal, epidural, sublingual.

Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compou nd, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The pharmaceutical composition comprising one or more CRA(s) or combinations thereof admixed with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant and/or combinations thereof may be administered simultaneously, separately or sequentially. In particular, the CRA(s) that as part of a combination of CRA(s) may be administered simultaneously, separately or sequentially. Kits

The one or more CRA(s) or combinations thereof for use in the methods described herein are suited for the preparation of kits. Such a kit may comprise containers, each with one or more of the CRA(s) or combinations thereof (typically in concentrated form) that are described herein. A set of instructions will also typically be included. Where the kit comprises combinations of CRA(s) then the individual components of the combination may be provided in individual form, with appropriate instructions for mixing same, or combinations thereof that are ready mixed.

Derivative

Derivatives of the CRA(s) are also described herein. A derivative may be a chemically modified CRA - such as a replacement of a hydrogen by a halo group, an alkyl group, an acyl group or an amino group and the like. The modification may increase or decrease one or more hydrogen bonding interactions, charge interactions, hydrophobic interactions, Van Der Waals interactions or dipole interactions.

General techniques

The present invention employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and cell culture, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1 -3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N. Y ); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing : Essential Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach , Irl Press; and, D. M. J. Lilley and J. E. Dahlberg , 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press.

Exam ples Example 1 Materials and methods

Cell culture

Mouse dermal fibroblasts (MDF) were purchased from CHEMICON (cat no. DF-BALBC). MDF cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (see Table 6) supplemented with 10% fetal calf serum, 2 mM glutamine, 1 % pen/strep on uncoated 10 cm tissue culture dishes at 37°C, 5% C02.

C17.2 cells are an immortalized, multipotent neural stem cell (NSC) line derived from neonatal mouse cerebellum (Snyder et al. Cell 68, 33-51 , 1992). The cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 5% horse serum, 2 mM glutamine, 1 % pen/strep on uncoated 10 cm tissue culture dishes at 37°C, 5% C02.

Combinatorial Cell c ulture

The process of combinatorial cell culture is explained in WO2004/031369. Differentiated cells were seeded on the surface of specialised microscopic beads (microcarriers). These cell units were randomly split into a number of sets and cultured separately under different conditions for a given time. The cell units in each group were subsequently washed, then pooled and mixed thoroughly, and once again split into random sets which were cultured under further different conditions. This split-pool procedure was repeated for three cycles.

Cell units subjected to this iterative split-pool protocol systematically sample all possible combinations of conditions in a predetermined experimental matrix. The different cell culture protocols tested in an experiment where beads are split T times into N numbers of sets is equal to N^T (the "complexity" of the experiment). It is thus feasible to sample millions of combinations of cell culture conditions in a single experiment.

Owing to the large nu mber of cell culture protocols tested, there is a probability that one or more cell units will have been exposed to conditions that promote the reprogramming of cells into CIMP cells. These cell units can be identified by pluripotent-specific markers - such as Alkaline phosphatase, SSEA1 , Oct4 and/or Nanog expression.

CombiCult using chemical compounds

The cell culture conditions used in Combinatorial Cell Culture comprised bioactive chemical compounds with activities such as epigenetic modifi cation activities and effects on intracellular signal transduction. The set of chemicals with epigenetic modu lation activity included chromatin modifying agents - such as DNA methyltransferase inhibitors, histone deacetylase inhibitors, histone methyltransferase inhibitors and ATP dependent chromatin remodelling agents. The set of chemicals with effects on intracellular signal transduction included chemicals known to modulate pathways that intersect with the signal transduction mechanism of Nanog, Klf4, PI3K, MEK/ERK, telomerase; GPR, Wnt, RAR, Calcium signalling, anti-appoptotic compounds, and other intracellular effectors.

In order to classify the chemicals into functionally relevant groups, we presumed three likely stages of cellular de-differentiation: i) chromatin remodelling and CIMP priming, ii) CIMP induction and iii) CIMP stabilization and expansion.

For each of these processes, a unique set of chemicals were selected and formulated in culture media either alone or in combinations. These media were tested in 1000 -15,000 combinations over 10 or 21 days using Combinatorial Cell Culture.

Screening of cell units to identify CIMP cells

Alkaline phosphatase (ALP) staining was used for the initial screening of cell units subjected to CombiCult. This was performed on 500-800 bead aliquots using Millipore ALP Detection Kit (SCR004) or Vector Red substrate kit (Vector Labs) according to manufacturer's instructions. If a sample proved positive for ALP, then its corresponding non-stained master sample were cultured further in order to expand and characterise the cells.

CIMP propagation and expansion

Cell units from experiments in which ALP expression had been detected were maintained in serum-free clonal iSTEM media (SCS-SF-ES-01 , Stem cell sciences), or in ESGRO (SF001 - 500, Chemicon) without feeder cells. iSTEM medium is said to eliminate differentiation signals to sustain mouse ES cell self-renewal and maintain stem cells in their ground state via blocking exogenous signals (to pERK) rather than providing exogenous signals to STAT3 via LIF. ESGRO is a completely defined (serum-free) medium containing BMP4 and LIF that has been shown to support mouse ES cells under feeder-free and serum-free conditions (Ying et a/., 2003, Cell 1 15:281 -292). This enabled selective expansion of CIMP cells in cell units bearing such cells. Optionally, bead cultures were trypsinized, cells detached from the beads were further propagated as monolayer cultures in gelatine coated dishes.

Detection of pluripotency markers in reprogrammed cells

To detect the expression of pluripotency markers, cells were cultured in mES media in 48-well cell culture plates either on the beads on which they appeared, or replated on tissue culture plastic. In any case cells were fixed with 4% PFA and permeabilized with 0.5% Triton X-100, then stained with primary antibodies against SSEA1 (Millipore), Oct4 (Abeam or Millipore), and Nanog (Abeam) followed by staining with suitable secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen). Nuclei were counterstained with Hoechst dye or DAPI (Invitrogen). Cells were imaged using a Nikon Eclipse TE 2000-S inverted fluorescence microscope equipped with Nikon DS-F1 1 camera.

Differentiation of CI MP cells

In order to demonstrate the pluripotency of CIMP cells, the developmental potential of neuroectoderm-derived NSC-CIMP cells into mesoderm lineages was tested, and developmental potential of mesoderm-derived MDF-CIMP cells into neuroectoderm lineages. Ectodermal NSC-derived CIMP cells were efficiently differentiated into several mesodermal cell types, including haematopoietic, osteogenic and adipogenic cell types.

MDF derived CIMP cells were efficiently differentiated into several other mesodermal cell types, including osteogenic and adipogenic cell types, as well as into neuronal and astroglia cell types of neuroectoderm. The differentiation protocols used are described below. a) Haematopoietic differentiation

A two-step protocol based on semisolid methylcellulose-based media was used (Keller ef a/, Mol Cell 8/0/ 13: 473, 1993).

In the first step, cells were prepared in a semi-solid medium to promote differentiation into embroid bodies (EBs).

Semi-solid medium consist of Iscove's MDM (IMDM) 1 % methylcellulose (MC) 15% FBS, plus 200mM L-Glutamine, MTG (150uM) , m SCF (40ng/ml).

Cell suspensions (3000 cells/ml) prepared in semi-solid medium were plated in non-adherent petri dishes (e.g. Sterilin 25 compartment, adding 2.5ml per well).

From day 7, the culture media was topped up every 3-5 days with dilute methylcellulose medium (0.5% MC plus 15% FBS) containing hematopoietic growth factors (m SCF, 160 ng/ml; m IL3, 10ug/ml, m LI6, 10 ug/ml; human EPO 3U/ml). for up to 20 days.

In the second step, the haematopoietic colony forming assay was carried out using Methocult

TM M3434 media (Stemcell Technologies Inc.) in accordance with the manufacturer's recommendations.

Briefly, EBs were harvested and disrupted into single cells. Cells from the disrupted EBs were first prepared in IMDM 2% FBS (10⁶ cells/ml), this was then mixed with 10 volume of replating medium consisting of 1 % methylcellulose, 15% FBS, 1 % bovine serum albumin, 100uM 2- mercaptoethanol , 2 mM L-glutamine, rm SCF (50 ng/ml), rm IL3 (10 ng/ml), rh IL6 (10 ng/ml) and rh erythropoietin (3 U/ml). These mixtures were plated in petri dishes (e.g. Sterilin 25 compartment, adding 2.5ml per well). After 2-3 weeks, the number of colonies was counted. The morphology of the colonies was observed using an inverted light microscope. Colony forming units were extracted by pipetting and further characterised by Wright's-Giesma staining (VWR, West Sussex, UK). Briefly, cell preparations from CFU were smeared and fixed on glass slides, stained with Wright-Giemsa (WG) solution, mounted with DePeX mounting medium (VWR, West Sussex, UK) and then observed using a light microscope. b) Macrophages and phagoctyosis functional assay from CIMP cells

To differentiate CIMP cells into macrophages, cell microcarrier beads were first washed in DMEM and then transferred into Stemline II medium (Sigma) containing mSCF (40 ng/ml). This was cultured for 4 days. Samples were then tranfered into Stemline II plus mSCF (40 ng/ml), mTPO (40 ng/ml) hBMP2 (5ng/ml), hTGFbl (5ng/ml) for 2 days. On day 7, samples were then transferred into Stemline II plus mlL3 (30 ng/ml), hlL6 (20 ng/ml), mTPO (20 ng/ml) for an additional 7 days. Media was changed every two days. All the cytokines were purchsed from R and D Systems. To assess phagocytosis, the bead suspensions were transferred into a 48-well culture plates, incubated with E. coli BioParticles® tetramethylrhodamine conjugates (Invitrogen, E2862) according to manufactures protocol. Cells were then rinsed with PBS, treated with trypan blue to quench the cell-surface attached particles, washed and analyzed on fluorescence microscope. c) Osteogenic differentiation

CIMP cells were plated at 10,000 cells per well in a 48-well plate and allowed to adhere for 24 hours. Osteogenic differentiation was stimulated by adding differentiation medium consisting of alpha-MEM supplemented with 10% fetal bovine serum, 50 uM ascorbic acid-2-phosphate (Sigma-Aldrich), 100 nM dexamethasone (Sigma-Aldrich), 10 tti M β-glycerophosphate ((Sigma- Aldrich) and 50 ng/ml rhBMP-2 ( R & D Systems) and 1 % pen/strep (Invitrogen). Medium was changed every 2-3 days for a total of 21 days in culture. Differentiated cells were detected by Alizarin red staining. d) Adipogenic differen tiation

CIMP cells were cultured in differentiation media from a commercially available kit (R& D systems) comprising hydrocortisone, isobutylmethylxanthine (IBMX), and indomethacin in alpha-MEM with 10% FBS. Medium was changed every 2-3 days for a total of 21 days in culture. Differentiated cells were detected by oil red O staining of lipid vacuoles in mature adipocytes. e) Neurogenic differentiation

CIMP cells derived from MDF were plated in NDiff™ N2B27 medium (Stem Cell Sciences, cat no. SCS-SF-NB-02) medium onto gelatin coated multi-well (6-well) tissue culture plates at 10,000 cells/cm². Medium was changed every 2 days and neuronal differentiation was monitored by cellular morphology and staining for neuronal markers from day 10 to day 16. For glial cell differentiation 1 % FCS was added in N2B27 media from day 10. Differentiated cells were detected by immunocyochemistry. Briefly, cells were fixed with 4% PFA and permeabilized with 0.5% Triton X-100. The cells were, then stained with primary antibodies against beta- tubulin III (Sigma) and/or GFAP (Sigma), followed by staining with suitable secondary antibod ies conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen) . Nuclei were counterstained with Hoechst dye or DAPI (Invitrogen). Cells were imaged using a Nikon Eclipse TE 2000-S inverted fluorescence microscope equipped with Nikon DS-F1 1 camera.

Example 2

The lineage-committed mouse neural stem cell line (NSC) was cultured using a bead-based cell culture system and exposed to combinations of CRA chemicals using CombiCult.

Three CombiCult experiments were performed according to the matrices shown in Tables 1 -3. In the first experiment (see Table 1 for details of media), NSC cells seeded on 80,000 gelatine beads were initially split (split 1 ) into 13 media containing cocktails of CRA(s) and cultured therein for three days, then transferred to mES medium for a further three days. On day 7, cell units were washed in 10 volumes of DMEM then pooled and s plit (split 2) into 10 different media containing cocktails of CRA(s) and cultured therein for three days, then transferred to mES medium for a further four days. On day 14, cell units were washed in 10 volumes of DMEM then pooled and split (split 3) into 12 different media containing cocktails of CRA(s) and cultured therein for three days, then transferred to mES medium for a further four days. This protocol thus tested (13 x 10 x 12 =) 1560 combinations of cell culture med ia containing CRA(s). On day 21 aliquots of cell units were tested for expression of ALP.

Results: ALP+ beads were detected in the following 4 conditions (out of 12); split 3 conditions 3, 5, 6 and 7, while others were negative for ALP activity. The percentage of ALP+ beads were in the range of 0.3 to 0.7%.

In the second experiment (see Table 2 for details of media), NSC cells seeded on 170,000 gelatin beads were initially split (split 1 ) into 20 different media containing cocktails of CRA(s) and cultured therein for one day, then transferred to mES medium for a further two days. On day 4, cell units were washed in 10 volumes of DMEM and then pooled and split (split 2) into 15 different media containing cocktails of CRA(s) and cultured therein for one day, then transferred to mES medium for a further two days. On day 7, cell units were washed in 10 volume of DMEM then pooled and split (split 3) into 19 different media containing cocktails of CRA(s) and cultured therein for one day, then transferred to mES medium for a further two days. This protocol thus tested (20 x 15 x 19 =) 5,700 combinations of cell culture media containing CRA(s). On day 10, aliquots of cell units were tested for expression of ALP.

Results: ALP+ beads were detected in the following 3 conditions (out of 19); split 3 conditions 2, 6, and 12, while others were negative for ALP activity. The percentage of ALP+ beads were in the range of 0.3 to 0.4%.

In the third experiment (see Table 3 for details of media), NSC cells seeded on 96,000 gelatin beads were initially split (split 1 ) into 24 different media containing cocktails of CRA(s) and cultured therein for one day, then transferred to mES medium for a further two days. On day 4, cell units were washed in 10 volume of DMEM then pooled and split (split 2) into 24 different cocktails of CRA(s) and cultured therein for one day, then transferred to mES medium for a further two days. On day 7, cell units were washed in 10 volume of DMEM then pooled and split (split 3) into 24 different media containing cocktails of CRA(s) and cultured therein for one day, then transferred to mES medium for a further two days. This protocol thus tested (24 x 24 x 24 =) 13,824 combinations of cell culture media contain ing CRA(s). On day 1 0, aliquots of cell units were tested for expression of ALP.

Results: ALP+ beads were detected in the following 2 conditions (out of 24); split 3 conditions 7 and 20, while others were negative for ALP activity. The percentage of ALP+ beads was 0.7%.

An alkaline phosphatase screen was used for the detection of CIMP cells in aliquots of cell units from these three experiments. Cell units from experiments that produced ALP positive cells were further cultured in serum-free clonal media iSTEM (SCS) or in ESGRO (Millipore) in the absence of feeder cells to enable selective expansion of CIMP cells.

CIMP cells were further passaged on day 28 as a singe cell suspension using 0.25% trypsin/EDTA and seeded at 30,000 cells per cm² for routine culture. All cells were grown on Nunclon cell culture dishes or multiwell plates (Fisher Scientific) coated with 0.1 % (w/v) gelatin (Millipore).

Cell units were characterised by detecting the expression of pluripotency markers using immunocytochemistry and histochemical staining for ALP. NSC-derived CIMP cells stained positively for ALP, SSEA1 , Oct4 and Nanog (Figures 1 and 2). Immunostaining of these pluripotency markers was comparable to that of mES (data not shown).

In order to demonstrate that NSC-derived CIMP cells have an enlarged developmental capacity they were differentiated into cells of the haematopoietic lineage - such as megakaryocytes, plasma cells, and macrophages/monocytes - and were characterised by CD34 immunostaining (Figure 3B) followed by morphological assessment and Wright-Giesma staining (Figure 3A and 3C) and phagocytosis functional assay (Figure 4).

Furthermore, when exposed to differentiation conditions known to direct the appearance of adipogenic and osteogenic cells from mesenchymal stem cells, NSC-derived CIMP cells were able to differentiate towards these lineages as determined by positive staining using Alizarin red and Oil red O, respectively (see Figure 5). When NSC cells were exposed to the same protocols as a control, there was no evidence of the appearance of osteocytes or adipocytes.

Example 3

Lineage-committed NSC or MDF cells were cultured in a bead-based cell culture system and exposed to combinations of media containing CRA(s) using CombiCult (see Table 4 for details of media).

NSC cells seeded on 101 ,250 cellulose beads were initially split (split 1 ) into 15 different media containing cocktails of CRA(s) spiked with unique tags and cultured therein for three days, then transferred to mES medium for a further three days. On day 7, cell units were washed in 10 volumes of DMEM then pooled and split (split 2) into 15 different media containing cocktails of CRA(s) spiked with unique tags and cultured therein for three days, then transferred to mES medium for a further four days. On day 14, cell units were washed in 10 volumes of DMEM, then pooled and split (split 3) into 15 different media containing cocktails of CRA(s) spiked with unique tags and cultured therein for three days, then transferred to mES medium for a further four days. This protocol thus tested (15 x 15 x 15 =) 3,375 combinations of cell culture media containing CRA(s). On day 21 , aliquots of cell units were tested for expression of ALP.

Results: ALP+ beads were detected in the following 7 conditions (out of 15); split 3 conditions 1 , 5, 6, 7, 8, 9 and 13, while others were negative for ALP activity. The percentage of ALP+ beads was in the range of 0.2 to 1 .6%.

Tags from cell units bearing CIMP cells were analysed to deduce the cell culture protocols that gave rise to these cells. Some of these protocols are listed in Table 5.

MDF or NSC cells cultured according to the protocols listed in Table 5 gave rise to CIMP cells characterised by positive histochemical staining for alkaline phosphatase (Figure 2B and Figure 6C) and/or antibody staining for SSEA1 , Oct4 and Nanog protein expression (Figure 2C and Figure 7). The estimated reprogramming efficiency was in the region of 2 - 40 % for NSC cells and 1 - 30 % for MDF cells. This was based on ALP positive staining area after replating of all cells as monolayer cultures and/or Oct4 immunostaining analysis.

Protocol 1 (NSC CIMP cells 10%, MDF CIMP cells 4%); Protocol 2 (NSC CIMP cells 5%, MDF CIMP cells 5%); Protocol 3 (NSC CIMP cells 2%, MDF CIMP cells 1 %); Protocol 4 (NSC CIMP cells 40%, MDF CIMP cells 25%); Protocol 5 (NSC CIMP cells 5%, MDF CIMP cells 30%); Protocol 6 (NSC CIMP cells 40%, MDF CIMP cells 30%).

Furthermore MDF-derived CIMP cells were able to differentiate into adipocytes, osteocytes (Figure 8), as well as into neuroectoderm lineages such as neurons and astroglia (Figure 9). NSC-derived CIMP cells were able to differentiate into several mesoderm lineages such as adipocytes and osteocytes (Figure 5), and haematopoietic lineages (Figure 4). Example 4

The common chemical classes used to obtain CI MP cells were analysed across each of the validated protocols set forth in Table 5. The results are shown in Table 7. The most common chemical classes which appeared in five out of the five validated protocols were PPAR gamma agonist(s), HDAC inhibitor(s), DNA methyltransferase inhibitor(s) and PI3K inducer(s). Thus all of the five validated protocols required the presence of these CRA(s). The next most common chemical classes were NADPH oxidase inhibitor(s), GPR modulator(s) and p53 inhibitor(s), which appeared in four out of the five protocols. Thus four out of the five validated protocols also required the presence of these CRA(s). The next most common chemical class was anti- apoptosis agent(s) which appeared in three out of the five protocols. Thus three out of the five validated protocols also required the presence of these CRA(s) The next commonest chemical class was histone methyltransferase(s) which appeared in two out of the five protocols. Thus two out of the five validated protocols also required the presence of this CRA.

Example 5

The common chemical classes that were used to obtain CIMP cells were analysed for each step of the reprogramming across the validated protocols set forth in Table 5. Classes of chemicals were identified with a cut-off value of≥3 and are listed in Table 8. The most common chemical classes required in step 1 were HDAC inhibitor(s), PPAR gamma agonist(s), DNA methyltransferase inhibitor(s), NADPH oxidase inhibitor(s) and PI3K inducer(s). The most common chemical classes required in step 2 were HDAC inhibitor(s), PPAR gamma agonist(s), DNA methyltransferase inhibitor(s), histone methyltransferase inhibitor(s) and anti-apoptosis agent(s). The most common chemical classes required in step 3 were PPAR gamma agonist(s), HDAC inhibitor(s), DNA methyltransferase inhibitor(s), NADPH oxidase inhibitor(s), GPR modulator(s) and PI3K inducer(s).

Further aspects of this invention are set forth in the following paragraphs .

1 . A method of reprogramming a cell into a chemically induced multi-lineage potential (CIMP) cell comprising the steps of: (i) contacting the cell to be reprogrammed with a medium comprising one or more chemical reprogramming agents (CRAs); and (ii) obtaining a CIMP cell, wherein the cell to be reprogrammed has not been genetically manipulated and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

2. The method according to paragraph 1 , wherein the medium is a culture medium comprising one or more CRA(s). 3. The method according to paragraph 1 or paragraph 2, wherein expression of one or more pluripotency genes is induced in the CIMP cell and one or more proteins are expressed by the one or more pluripotency genes in the CIMP cell following contact with the one more CRA(s).

4. The method according to any of the preceding paragraphs, wherein said cell to be reprogrammed is contacted with one or more CRA(s) simultaneously, separately or sequentially.

5. The method according to any of the preceding paragraphs , wherein said cell to be reprogrammed is contacted with one or more of the same CRA(s) separately or sequentially.

6. The method according to any of the preceding paragraphs , wherein said cell to be reprogrammed is contacted with one or more different CRA(s) (eg. a combination of different CRA(s)) simultaneously, separately or sequentially.

7. The method according to any of the preceding paragraphs , wherein said cell to be reprogrammed is incubated in the absence of one or more CRA(s) for a period of time.

8. The method according to paragraph 7, wherein the medium is the same medium as used in step (i) with the proviso that all of the CRA(s) are absent therefrom.

9. The method according to any of the preceding paragraphs , wherein said cell to be reprogrammed is simultaneously, separately or sequentially contacted with one or CRA(s) for one or more days and then incubated in the absence of said one or more CRA(s) for one or more days, and optionally repeated one or more times.

10. The method according to paragraph 8, wherein the method is repeated using the same or different CRA(s).

1 1 . The method according to any of the preceding paragraphs , wherein the CRA is selected from the group consisting of a chromatin modifying agent that primes CIMP cell formation ; a CRA that induces CIMP cell formation and a CRA that stabilises and expands a CIMP cell or a combination of one or more thereof.

12. The method according to any of the preceding paragraphs, wherein after the reprogramming the medium is replaced with maintenance medium.

13. The method according to any of the preceding paragraphs , wherein said cell is reprogrammed by: (i) contacting said cell with one or more CRA(s) that remodels chromatin and primes CIMP cell formation; (ii) contacting said cell with one or more CRA(s) that induce CIMP cell formation; and (iii) contacting said cell with one or more CRA(s) that stabilise and expand said CIMP cell.

14. The method according to any of the preceding paragraphs , wherein after the reprogramming, said CIMP cell is expanded.

15. The method according to paragraph 14, wherein said CIMP cell is differentiated into a more lineage restricted cell. 16. The method according to any of the preceding paragraphs, wherein said CIMP cell is differentiated into a cell that is different to the cell from which the CIMP cell was derived.

17. The method according to any of paragraphs 14 to 16, wherein said expanded CIMP cell is a population of homogenous cells.

18. The method according to any of the preceding paragraphs, wherein the CRA is selected from the group consisting of: a DNA methyltransferase inhibitor; a histone deacetylase inhibitor; a KLF4 inducer, a retinoic acid receptor agonist; an HDAC1 inhibitor; and a protein tyrosine phosphatase PTP inhibitor, a MEK/ERK inhibitor, a protein tyrosine phosphatase PTP inhibitor; a H2 histamine receptor antagonist, pregnant mare serum Gonadotropin (PMSG), an NADPH oxidase inhibitor, a histone methyltransferase inhibitor, a calcium channel agonist, a demethylating agent, an inhibitor of Rho-associated protein kinase, a voltage gated sodium channel stabiliser, a GPR modulator, a P53 inhibitor, a PPAR gamma agonist, a caspase-3 Inhibitor, a xanthine oxidase inhibitor or a combination of two or more thereof.

19. A method of reprogramming a cell into a CIMP cell comprising the steps of: (i) providing a cell; (ii) contacting said cell with one or more CRA(s) that remodel chromatin and prime CIMP cell formation; (iii) contacting said cell with one or more CRA(s) that induce CIMP cell formation;

(iv) contacting said cell with one or more CRA(s) that stabilise and expand said CIMP cell; and

(v) obtaining a CIMP cell.

20. A CIMP cell obtained or obtainable by the method of any of paragraphs 1 to 19.

21 . A CIMP cell.

22. A composition comprising one or more of the CIMP cells according to paragraph 20 or 21 and one or more constituents that maintain the viability of said cell.

23. A method of reprogramming a cell via a CIMP cell comprising the steps of: (i) contacting the cell to be reprogrammed with a medium comprising one or more CRA(s); (ii) obtaining a CIMP cell; and (iii) differentiating the CIMP cell into a more lineage restricted cell, wherein the cell to be reprogrammed has not been genetically manipulated and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

24. A method for identifying one or CRA(s) for reprogramming a cell into a CIMP cell comprising the steps of: (i) contacting the cell to be reprogrammed with a medium comprising one or more chemicals; and (ii) determining if said cell has been reprogrammed in to a CIMP cell, wherein the presence of a CIMP cell is indicative that the one or more chemicals are CRA(s) able to reprogram the cell into a CIMP cell, wherein the cell to has not been genetically manipulated and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

25. A method of reprogramming a cell into a desired cell type comprising the steps of: i) obtaining a cell; ii) reprogramming the cell into a CIMP cell according to the method of any of paragraphs 1 to 19; and iii) culturing the CIMP cell to differentiate the cell into a desired cell type.

26. A method of treating a (non-human) subject comprising: i) obtaining a cell from a (non- human) subject; ii) reprogramming the cell into a CIMP cell according to the method of any of paragraphs 1 to 19; iii) culturing the CIMP cell to differentiate the cell into a desired cell type suitable for treating a condition; and iv) introducing the cell into the subject, thereby treating the condition.

27. A combination comprising, consisting or consisting essentially of: (i) a DNA methyltransferase inhibitor; a histone deacetylase inhibitor; a KLF4 inducer, a retinoic acid receptor agonist; an HDAC 1 inhibitor; and a protein tyrosine phosphatase PTP inhibitor; or (ii) a histone deacetylase inhibitor; a KLF4 inducer, a MEK/ERK inhibitor; a protein tyrosine phosphatase PTP inhibitor; a H2 histamine receptor antagonist and Pregnant mare serum Gonadotropin (PMSG); or (iii) an NADPH oxidase inhibitor; a MEK/ERK inhibitor; a histone methyltransferase inhibitor; a protein tyrosine phosphatase PTP inhibitor; and a calcium channel agonist.; or (iv) a demethylating agent; a histone deacetylase inhibitor; a KLF4 inducer, an inhibitor of Rho-associated protein kinase; a histone methyltransferase inhibitor and a voltage gated sodium channel stabiliser; or (v) a KLF4 inducer, an NADPH oxidase inhibitor, an HDAC1 inhibitor; a GPR modulator, a P53 inhibitor and a demethylating agent; or (vi) a DNA methyltransferase inhibitor, a deacetylase inhibitor, a PPAR gamma agonist, an NADPH oxidase inhibitor, a PKC activator and an a1 -adrenoceptor antagonist; or (vii) a histone deacetylate inhibitor, a PPAR gamma agonist, a retinoic acid receptor agonist, a caspase-3 Inhibitor, a xanthine oxidase inhibitor; and a H2 histamine receptor antagonist; or (viii) a demethylating agent, a KLF4 inducer, an NADPH oxidase inhibitor and a HDAC1 inhibitor.

28. A pharmaceutical composition comprising one or more of the combinations according to paragraph 27 admixed with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

29. A method for generating a transgenic non-human animal comprising the steps of: (i) providing a CIMP cell according to paragraph 20 or paragraph 21 or a composition according to paragraph 28; (ii) introducing the cell into a non-human blastocyst; (iii) transferring the blastocyst into the uterus of a female non-human animal; and (iv) allowing the blastocyst to develop into an embryo.

30. A transgenic non-human animal obtained or obtainable by the method of paragraph 29.

31 . A CIMP cell according to paragraph 20 or 21 or the composition of paragraph 28 for use in the treatment of disease.

32. A CIMP cell according to paragraph 20 or paragraph 21 or the composition of paragraph 28 for the treatment of disease. 33. Use of a CIMP cell according to paragraph 20 or paragraph 21 in the manufacture of a composition for the treatment of a disease that results from malfunctioning, damaged or failing tissue.

34. A method of treating a disease in a subject comprising administering the pharmaceutical composition according to paragraph 28 to said subject.

35. A combination according to paragraph 27 or a pharmaceutical composition according to paragraph 28 for treating a disease.

36. Use of a combination according to paragraph 27 or a pharmaceutical composition according to paragraph 28 in the manufacture of a composition for the treatment of a disease that results from malfunctioning, damaged or failing tissue.

37. A method for reprogramming a cell into a CIMP cell comprising the use of one or more of the combinations according to paragraph 27.

38. The method according to paragraph 37, wherein the cell to be reprogrammed is or is derived from the ectoderm, endoderm or mesoderm lineage.

39. Use of one or more of the combinations according to paragraph 27 for reprogramming a cell.

40. A kit for reprogramming a cell into a CIMP cell comprising one or more of the combinations according to paragraph 27 and optionally a set of instructions for performing same.

The CIMP cell may be reffered to as a "chemically induced pluripotent stem cell" (ChlPS cell). The CRA(s) may be referred to as a chemical de-differentiating agent(s) (CDDA(s)) .

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in cellular and molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS 1 . A method of reprogramming a cell via a chemically induced multi-lineage potential (CIMP) cell comprising the steps of:

(i) contacting the cell to be reprog rammed with a medium comprising one or more, preferably two or more, chemical reprogramming agents (CRAs);

(ii) obtaining a CIMP cell in which the expression of one or more pluripotency genes is induced therein and one or more pluripotency proteins are expressed by the one or more induced pluripotency genes following contact with the one more CRA(s) such that the cell has gained potency and the capacity to differentiate into an increased number of cell types as compared to the cell of step (i); and

(iii) differentiating the CIMP cell into a more lineage restricted cell,

preferably, wherein the cell to be reprogrammed has not been genetically manipulated to induce pluripotency and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

2. The method according to claim 1 , wherein the one or more pluripotency proteins that are expressed in step (ii) comprise, consist or consist essentially of pluripotency proteins selected from the group consisting of alkaline phosphatase (ALP), SSEA1 , Oct4 or Nanog or a combination of two or more thereof, preferably, wherein the one or more pluripotency proteins that are expressed in step (ii) comprise, consist or consist essentially of alkaline phosphatase (ALP), SSEA1 , Oct4 and Nanog .

3. The method according to claim 1 or claim 2, wherein the CRA(s) are small molecules.

4. The method according to any of claims 1 to 3, wherein the CIMP cell is phenotypically at an earlier developmental stage than the cell in step (i) and is morphologically different to the cell in step (i).

5. The method according to any of the preceding claims, wherein the CRA(s) is selected from the group consisting of: a DNA methyltransferase inhibitor(s); a histone deacetylase inhibitor(s); a KLF4 inducer(s), a retinoic acid receptor agonist(s); an HDAC1 inhibitor(s); a protein tyrosine phosphatase PTP inhibitor(s), a MEK/ERK inhibitor(s), a protein tyrosine phosphatase PTP inhibitor(s); a H2 histamine receptor antagonist(s), pregnant mare serum Gonadotropin(s) (PMSG), an NADPH oxidase inhibitor(s) , a histone methyltransferase inhibitor(s), a calcium channel agonist(s), a demethylating agent(s), an inhibitor of Rho- associated protein kinase(s), a voltage gated sodium channel stabiliser(s), a GPR modulator(s), a P53 inhibitor(s), a Peroxisome Proliferator-Activated Receptor (PPAR) gamma agonist(s), a caspase-3 Inhibitor(s) and a xanthine oxidase inhibitor(s) or a combination of two or more thereof.

6. The method according to any of claims 1 to 4, wherein the CRA(s) is selected from the group consisting of: a PPAR gamma agonist(s), a histone deacetylase inhibitor(s), a DNA methyltransferase inhibitor(s), and a P13K inducer(s) or a combination of two or more thereof or a combination thereof; preferably, wherein the CRA(s) are selected from a combination of: a PPAR gamma agonist(s), a histone deacetylase inhibitor(s), a DNA methyltransferase inhibitor(s), a P13K inducer(s), an NADPH oxidase inhibitor(s), a GPR modulator(s) and a p53 inhibitor(s) or a combination of two or more thereof or a combination thereof; preferably, wherein the CRA(s) are selected from: a PPAR gamma agonist(s), a histone deacetylase inhibitor(s), a DNA methyltransferase inhibitor(s), a P13K inducer(s), an NADPH oxidase inhibitor(s), a GPR modulator(s), a p53 inhibitor(s) and an anti-apoptosis agent(s) or a combination of two or more thereof or a combination thereof; preferably wherein the CRA(s) are selected from: a PPAR gamma agonist(s), a histone deacetylase inhibitor(s), a DNA methyltransferase inhibitor(s), a P13K inducer(s), an NADPH oxidase inhibitor(s), a GPR modulator(s), a p53 inhibitor(s), an anti-apoptosis agent(s) and a histone methyltransferase inhibitor(s) or a combination of two or more thereof or a combination thereof.

7. The method according to any of the preceding claims, wherein said cell to be reprogrammed is contacted with the CRA(s) simultaneously, separately or sequentially.

8. The method according to any of the preceding claims, wherein said cell to be reprogrammed is incubated in the absence of one or more of the CRA(s) for a period of time.

9. The method according to any of the preceding claims, wherein said cell to be reprogrammed is contacted with the following combinations of CRA(s):

(i) an HDAC inhibitor, a PPAR gamma agonist, a DNA methyltransferase inhibitor, a NADPH oxidase inhibitor and a P13K inducer;

(ii) an HDAC inhibitor, a PPAR gamma agonist, a DNA methyltransferase inhibitor, a histone methyltransferase inhibitor and an anti-apoptosis agent; and

(iii) an HDAC inhibitor, a PPAR gamma agonist, a DNA methyltransferase inhibitor, a NADPH oxidase inhibitor, a GPR modulator and a p53 inhibitor.

10. The method according to any of the preceding claims, wherein said CIMP cell is differentiated in step (iii) into a cell that is different to the cell of step (i) and/or step (ii), preferably, wherein the CIMP cell is differentiated in step (iii) into a cell of a different germ layer to the cell of step (i) and/or step (ii).

11 . The method according to any of the preceding claims, wherein after the reprogramming, said CIMP cell is expanded, preferably, wherein said expanded CIMP cell is a population of homogenous cells.

12. A cell obtained or obtainable by the method of any of claims 1 to 1 1 .

13. A method of reprogramming a cell into a chemically induced multi-lineage potential (CIMP) cell comprising the steps of:

(i) contacting the cell to be reprogrammed with a medium comprising one or more, preferably, two or more, CRA(s); and

(ii) obtaining a CIMP cell in which the expression of one or more pluripotency genes is induced therein and one or more pluripotency proteins are expressed by the one or more induced pluripotency genes following contact with the CRA(s) such that the cell has gained potency and the capacity to differentiate into an increased number of cell types as compared to the cell of step (i),

preferably, wherein the cell to be reprogrammed has not been genetically manipulated and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

14. A CIMP cell obtained or obtainable by the method of claim 13.

15. A CIMP cell, wherein said cell is more potent than the cell from which it was derived following contact with one or more, preferably two or more, CRA(s) and wherein the expression of one or more pluripotency genes is induced in the CIMP cells and one or more proteins are expressed by the one or more induced pluripotency genes such that the cell has gained potency and the capacity to differentiate into an increased number of cell types as compared to the cell from which it was derived, wherein said CIMP cell has the capacity to differentiate into a more lineage restricted cell and pre ferably, wherein the cell to be reprogrammed has not been genetically manipulated to induce pluripotency and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

16. A method for identifying one or more, preferably two or more, CRA(s) for reprogramming a cell into a CIMP cell comprising the steps of: (i) contacting the cell to be reprog rammed with a medium comprising one or more, chemicals, preferably small molecules; and

(ii) determining if the expression of one or more pluripotency genes is induced therein and one or more proteins are expressed by the one or more induced pluripotency genes following contact with the chemicals such that the cell has gained potency and the capacity to differentiate into an increased number of cell types as compared to the cell of step (i);

(iii) determining if said cell of step (i) has been reprogrammed in to a CIMP cell, wherein the presence of a CIMP cell is indicative that the chemicals are CRA(s) able to reprogram the cell into a CIMP cell,

preferably, wherein the cell to has not been genetically manipulated and/or contacted with one or more exogenous reprogramming proteins or nucleic acids encoding same to induce pluripotency.

17. A combination comprising, consisting or consisting essentially of:

(i) a DNA methyltransferase inhibitor; a histone deacetylase inhibitor; a KLF4 inducer, a retinoic acid receptor agonist; an HDAC1 inhibitor; and a protein tyrosine phosphatase PTP inhibitor; or

(ii) a histone deacetylase inhibitor; a KLF4 inducer, a MEK/ERK inhibitor; a protein tyrosine phosphatase PTP inhibitor; a H2 histamine receptor antagonist and Pregnant mare serum Gonadotropin (PMSG); or

(iii) an NADPH oxidase inhibitor; a MEK/ERK inhibitor; a histone methyltransferase inhibitor; a protein tyrosine phosphatase PTP inhibitor; and a calcium channel agonist; or

(iv) a demethylating agent; a histone deacetylase inhibitor; a KLF4 inducer, an inhibitor of Rho-associated protein kinase; a histone methyltransferase inhibitor and a voltage gated sodium channel stabiliser; or

(v) a KLF4 inducer, an NADPH oxidase inhibitor, an HDAC1 inhibitor; a GPR modulator, a P53 inhibitor and a demethylating agent; or

(vi) a DNA methyltransferase inhibitor, a deacetylase inhibitor, a PPAR gamma agonist, an NADPH oxidase inhibitor, a PKC activator and an a1 -adrenoceptor antagonist; or

(vii) a histone deacetylate inhibitor, a PPAR gamma agonist, a retinoic acid receptor agonist, a caspase-3 Inhibitor, a xanthine oxidase inhibitor; and a H2 histamine receptor antagonist; or

(viii) a demethylating agent, a KLF4 inducer, an NADPH oxidase inhibitor and a HDAC1 inhibitor; or

(ix) a PPAR gamma agonist, a histone deacetylase inhibitor, a DNA methyltransferase inhibitor, or a P13K inducer; or (x) a PPAR gamma agonist, a histone deacetylase inhibitor, a DNA methyltransferase inhibitor, a P13K inducer, an NADPH oxidase inhibitor, a GPR modulator and a p53 inhibitor; or

(xi) a PPAR gamma agonist, a histone deacetylase inhibitor, a DNA methyltransferase inhibitor, or a P13K inducer, an NADPH oxidase inhibitor, a GPR modulator, a p53 inhibitor and an anti-apoptosis agent; or

(xii) a PPAR gamma agonist, a histone deacetylase inhibitor, a DNA methyltransferase inhibitor, a P13K inducer, an NADPH oxidase inhibitor, a GPR modulator, a p53 inhibitor, an anti-apoptosis agent and a histone methyltransferase inhibitor; or

(xiii) an HDAC inhibitor, a PPAR gamma agonist, a DNA methyltransferase inhibitor, a NADPH oxidase inhibitor and a P13K inducer; or

(xiv) an HDAC inhibitor, a PPAR gamma agonist, a DNA methyltransferase inhibitor, a histone methyltransferase inhibitor and an anti-apoptosis agent; or

(xv) an HDAC inhibitor, a PPAR gamma agonist, a DNA methyltransferase inhibitor, a NADPH oxidase inhibitor, a GPR modulator and a p53 inhibitor.

18. A pharmaceutical composition comprising one or more of the combinations according to claim 17 admixed with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.

19. A method for generating a transgenic non-human animal comprising the steps of:

(i) providing a CIMP cell according to claim 14 or claim 15 or a composition according to claim 18;

(ii) introducing the cell into a non-human blastocyst;

(iii) transferring the blastocyst into the uterus of a female non-human animal; and

(iv) allowing the blastocyst to develop into an embryo.

20. A transgenic non-human animal obtained or obtainable by the method of claim 19.

21 . The cell according to claim 12, the CIMP cell according to claim 14 or claim 15 or a cell derived or derivable from said cell or said CIMP cell or the composition of claim 18 for use in the treatment of disease; or for the treatment of disease.

22. Use of the cell according to claim 12, the CIMP cell according to claim 14 or claim 15 or a cell derived or derivable from said cell or said CIMP cell or the composition of claim 18 in the manufacture of a composition for the treatment of a disease that results from malfunctioning, damaged or failing tissue.

23. A method of treating a disease in a subject comprising administering the pharmaceutical composition according to claim 18 to said subject.

24. One or more combinations according to claim 17 or the pharmaceutical composition according to claim 18 for treating a disease.

25. Use of one or more combinations according to claim 17 or the pharmaceutical composition according to claim 18 in the manufacture of a composition for the treatment of a disease that results from malfunctioning, damaged or failing tissue.

26. A method for reprogramming a cell into a CIMP cell comprising the use of one or more of the combinations according to claim 17, preferably, wherein the cell to be reprogrammed is or is derived from the ectoderm, endoderm or mesoderm lineage.

27. Use of one or more of the combinations according to claim 17 for reprogramming a cell.

28. A kit for reprogramming a cell into a CIMP cell comprising one or more of the combinations according to claim 17 and optionally a set of instructions for performing same.

29. A method, cell, combination, composition , transgenic non-human animal, use or kit substantially as described herein and with reference to the accompanying drawings.