WO2006094336A1

WO2006094336A1 - Generation of diploid reprogrammed cells through nuclear inactivation

Info

Publication number: WO2006094336A1
Application number: PCT/AU2006/000285
Authority: WO
Inventors: Paul Verma; Daniele Pralong
Original assignee: Australian Stem Cell Centre Limited
Priority date: 2005-03-07
Filing date: 2006-03-07
Publication date: 2006-09-14

Abstract

The present invention provides a method for producing reprogrammed diploid or effectively diploid adult cells. The method comprises (a) providing a first population of diploid adult cells; (b) providing a second population of pluripotent cells (c) treating the second population of pluripotent cells with: at least one nuclear arrest agent; or one cell cycle inhibitor; or a combination of at least one nuclear arrest agent and at least one cell cycle inhibitor; (d) fusing the first and second populations of cells; and (e) allowing the population of fused cells to undergo mitosis; and isolating from the population of fused cells reprogrammed diploid or effectively diploid adult cells.

Description

GENERATION OF DIPLOID REPROGRAMMED CELLS THROUGH NUCLEAR

INACTIVATION

FIELD OF THE INVENTION

[0001] The present invention relates to methods of generating reprogrammed diploid or effectively diploid cells from heterokaryons generated by fusion of 2 or more cells.

BACKGROUND OF THE INVENTION

[0002] Pluripotent cells are cells which have the ability to self renew and which, under specific conditions, have the potential to differentiate to become derivative cells of all three germ lineages. Pluripotent cells exist in many forms and come from a variety of sources including sources which are embryonic, adult and foetal. Pluripotent cells can be isolated from primates or species other than primates. International patent application WO97/32033 and U.S. Pat. No. 5,453,357 describe pluripotent cells including cells from species other than rodents, and primate pluripotent cells have been described in International patent applications WO98/43679 and WO96/22362 and in U.S. Pat. No. 5,843,780.

[0003] One type of pluripotent cell is the human embryonic stem cell (hESC). hESCs are derived from the inner cell mass of a blastocyst-stage human embryo, and are capable of undergoing an unlimited number of cell divisions without differentiating while maintaining a stable, diploid complement of chromosomes. hESCs are pluripotent and, under various culture regimes, can give rise to any of the differentiated cell types derived from the three primary germ layers of the embryo (the endoderm, mesoderm and ectoderm). The path of differentiation from a hESC to a fully-differentiated cell involves a series of steps resulting in a series of cell intermediates. As the differentiation process advances, it leads to a progressive diminution of the differentiation potential of each resulting cell.

[0004] At present the sole mechanism for deriving new lines of hESCs is using excess human embryos which have been created using Assisted Reproductive Technologies. This process of derivation is described in U.S. Patent Number 6,200,806. It is subject to ethical limitations since human embryos are destroyed and also practical limitations as derivations are both difficult and limited by the number of available embryos.

[0005] Currently there is a need for new hESC lines, for a number of reasons. First, there appears to be contamination of many existing lines due to the fact that they have been exposed to non-human feeder cells - which will pose clinical and regulatory problems in terms of using the cell lines for therapeutics. Second, different lines appear to behave differently under controlled conditions - leading to the need for further experimentation with greater numbers of lines. Third, the impact on genetic variation cannot be documented, as the existing cells do not represent a cross section of different populations and polymorphisms. Finally, there are limited cell lines which have the genotypes and phenotypes for particular diseases — which limits applications for toxicology testing, study of diseases and the like.

[0006] In addition, creation of ESC lines from certain non-human species in which there are currently no ESC lines or very limited numbers of ESC lines is desired for multiple potential agriculture and human therapeutic use. For example, bovine ESC lines do not currently exist, despite the numerous attempts to create such lines by scientists around the world. The creation of such lines would allow optimization of techniques for producing animals with desired traits and potentially improving the quality of bovine-based agricultural products.

[0007] Cell reprogramming is a process that alters or reverses the differentiation status of a partially differentiated cell or terminally differentiated cell. It includes alteration or reversion to a multipotent or pluripotent state, or transdifferentiation into a different cell type. Using a pluripotent cell with a functional nucleus results in a cell which is polyploid (ie. >2N), typically a hybrid tetraploid (4N). Such cells have limited therapeutic application due to their ploidy and furthermore, are not autologous with the adult cell donor due to the ES cell contribution. They also are problematic in terms of use for screening assays and drug development due to potential impacts of their ploidy status — such as not truly exhibiting the characteristics of the therapeutic indication. They are also problematic in agricultural applications as cloned offspring must be diploid to be viable.

[0008] Given the need for new ESC lines, and the practical and ethical implications associated with destroying human embryos, there is a desire in the art to devise new reprogramming methodologies for creating improved ESC lines.

SUMMARY OF THE INVENTION

[0009] The present invention describes methods for creating new pluripotent stem cell lines and lines created using such methods. Central to the invention is a method for producing such a pluripotent line, comprising fusion of a adult, preferably somatic, diploid cell with an existing pluripotent cell which has been treated to prevent the pluripotent nucleus from replicating during cell division of the somatic cell. The existing pluripotent cell used for the fusion may be of the same species as the diploid adult cell (e.g., for the production of new hESC lines) or derived from a different species (e.g., for the production of a bovine embryonic stem cells).

[0010] Enucleation of pluripotent cells prior to their fusion with an adult cell does not cause reprogramming of the adult cell - see Do and Scholer Stem Cells 2004:22:941-949. In contrast, the present invention provides reprogrammed diploid or effectively diploid adult cells without requiring the enucleation of pluripotent cells - effectively overcoming the difficulties of techniques described in the prior art. It also provides a mechanism for deriving new lines of pluripotent cells without the ethical or practical implications of destroying additional human embryos.

[0011] In a first aspect of the present invention, there is provided a method for producing reprogrammed diploid or effectively diploid adult cells, the method comprising:

(a) providing a first population of diploid adult cells;

(b) providing a second population of pluripotent cells

(c) treating the second population of pluripotent cells with:

(i) at least one nuclear arrest agent; or

(ii) one cell cycle inhibitor; or

(iii) a combination of at least one nuclear arrest agent and at least one cell cycle inhibitor;

(d) fusing the first and second populations of cells;

(e) allowing the population of fused cells to undergo mitosis;

(f) isolating from the population of fused cells reprogrammed diploid or effectively diploid adult cells. BRIEF DESCRIPTION OF THE FIGURES

[0012] Figure 1 shows a flow diagram of the summary of the methods of the invention.

[0013] Figure 2 shows a flow diagram of the method according to one embodiment of the invention.

[0014] Figure 3 shows a flow diagram of the method according to one embodiment of the invention.

[0015] Figure 4 shows a flow diagram of the method according to one embodiment of the invention.

[0016] Figure 5 shows the dose-dependent effect of mitomycin C on the proliferation of mouse embryonic stem cells.

[0017] Figure 6 shows the effect of mitomycin C on the proliferation of mouse embryonic stem cells.

[0018] Figure 7 shows the effect of mitomycin C on the differentiation of Oct4-GFΫ mouse embryonic stem cells.

[0019] Figures 8 and 9 show ploidy of adult cells from neomycin-resistant colonies.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides a method of producing reprogrammed diploid or effectively diploid adult cells which are suitable for use as cell lines for human disease models. The reprogrammed cells will also have therapeutic, agricultural and/or drug testing applications.

[0021] In a first embodiment of the present invention, there is provided a method for producing reprogrammed diploid or effectively diploid adult cells, the method comprising:

(a) providing a first population of diploid adult cells;

(b) providing a second population of pluripotent cells

(c) treating the second population of pluripotent cells with: (i) at least one nuclear arrest agent; or

(ii) one cell cycle inhibitor; or

(d) fusing the first and second populations of cells;

(e) allowing the population of fused cells to undergo mitosis;

(f) isolating from the population of fused cells reprogrammed diploid or

(g) effectively diploid adult cells.

[0022] In a preferred embodiment the first population of diploid adult cells is treated with chromatin remodelling agents or reprogramming factors.

[0023] In a further preferred embodiment the first population of diploid adult cells and/or second population of pluripotent cells is treated with at least one cytoskeletal inhibitor before or after step (d).

[0024] In one embodiment the pluripotent cells are 2N, whilst in an alternate embodiment the pluripotent cells are 4N. In a still further embodiment the pluripotent cells are greater than 4N.

[0025] In a second aspect there is established from the reprogrammed diploid or effectively diploid adult cells a cell line, whereby the cell line maintains its pluripotent characteristics.

[0026] In another preferred embodiment, both the diploid adult cells and the pluripotent cells are from the same species in origin (e.g., human.). This would, for example, allow the creation of cell lines that exhibit the same characteristics as human embryonic stem cells.

[0027] As noted above, there is currently a desire in the art to create pluripotent cell lines with the desired genotypes and phenotypes of particular species. For example, bovine ESC lines do not currently exist, despite the numerous attempts to create such lines from scientists around the world. The creation of such lines would allow production of animals with desired traits and optimization of techniques for improving the quality of bovine- based agricultural products. In fourth specific embodiment, the diploid adult cell is from a different species (e.g., bovine) than the pluripotent cell (e.g. murine.)

[0028] Potential uses of therapeutic pluripotent cells (particularly human embryonic stem cells and human adult stem cells which are derived from cord blood and other sources) are varied; defined therapeutic opportunities exist in the renal, cardiac, haematology and respiratory fields, among others. One of the challenges involved in the use of pluripotent cells for a number of these applications involves overcoming any immune responses generated by the introduction of allogeneic cells into the body. Reprogramming provides an approach for generating autologous pluripotent cells, which can then be differentiated along the desired differentiation pathway. Use of autologous cells in cell therapy offers a major advantage over allogeneic (non-autologous) cells, which are likely to be subject to immunological rejection. Autologous cells are unlikely to elicit significant immunological responses.

[0029] Thus, in a fifth embodiment of the present invention there is provided a reprogrammed pluripotent cell line for use in the treatment of a disease or condition in a subject. It is preferred that in this embodiment of the invention that ESC are immunologically compatible with the subject to be treated.

[0030] Another potential use of pluripotent cells includes the use of such cells to develop high throughput screening assays which could be used for the discovery of drugs and their development and validation. To this end, it would be useful to have pluripotent cells which have the genotype which leads to a particular therapeutic indication - such as cystic fibrosis. At present there is only a limited ability to create pluripotent cells with genotypes which lead to particular therapeutic indications — due to a lack of embryos available with such genotypes which can be used for embryonic stem cell derivation. Creating cell lines by reprogramming adult cells with the desired genotype would allay this issue.

[0031] In a still further preferred embodiment of the present invention there is provided a cell line according to the present invention for use in drug discovery, drug assessment or drug development.

[0032] A feature of this invention is the ability to reprogram adult stem cells possessing a specific genotype to become pluripotent cells with that genotype, for the use in discovery of drugs, their development and validation. It has been established that individuals with different genotypes may have subtly or radically different reactions to particular medications - due to their genetic differences. This has led to the concept of personalised medicine which is essentially the concept of proposing medication for a patient based upon prior knowledge of how they are likely to react to such medication - due to their genotype. The ability to undertake toxicity testing and screening activities using pluripotent cell lines with known genetic differences will allow those skilled in the art to develop profiles of the activities of compounds based on genetic differences which will allow more informed prescriptions of these compounds.

[0033] A cell line according to the present invention may be used in toxicity testing.

[0034] The ability to develop pluripotent cell lines with a known genotype will allow those skilled in the art to study the differentiation and development of those cells with reference to the genotype. For example, it will be possible to compare the development and differentiation of cells with a genotype which leads to a known phenotype with the development and differentiation of cells which have a different genotype and a different known phenotype. Also, the ability to develop pluripotent cell lines from patients presenting a known phenotype of unknown genotypic origin will provide the ability to study the differentiation and development of those cells with reference to acquisition of this phenotype during early development. This will provide unprecedented tools to unravel the causes of poorly understood conditions with suspected developmental origins.

[0035] As such, a cell line according to the present invention may be used for study of human development and human disease.

[0036] The availability of pluripotent cells and their differentiated progeny from domestic animals has widespread applications in animal production and farm management. For example, it has been proposed that by using pluripotent cells as donors for somatic cell nuclear transfer (SCNT), the efficiency of cloning outcomes and the viability of clones produced would be greatly increased, when compared to traditional methods of using differentiated cells as donors. This allows the multiplication of genetically valuable livestock which would have advantages in terms of the uniformity of meat products and animal management. A separate feature of this invention is the ability to create such cell lines for use in animal production and farm management.

[0037] In another embodiment of the present invention the cell line may be used in agricultural and farm management processes. [0038] As noted above, pluripotent cells provide an excellent mechanism for studying development and disease. Accordingly the cell line may be used for study of animal development and animal diseases.

[0039] Gene therapy is a technology that has been proposed as a potential avenue for the treatment of human or animal disease. In short, gene therapy is used where disease is caused by a genetic mutation or by an addition or deletion of genetic material. The traditional approach to gene therapy is to "repair" the genetic defect by inserting a normal copy of the gene into a non-specific location in the genome to replace the non-functional gene or circumvent the defect by transfer of a functional copy of the defective gene (which may or may not integrate into the host genome; in other words is episomally retained in the target cells). The normal copy of the gene is "inserted" by infecting the target cells of the patient with a retrovirus, adenovirus, adeno-associated virus, herpes simplex virus or the like. There have been problems with this practise including side effects in clinical trials which have presented symptoms analogous to leukaemia. The present invention offers a new avenue for exploring the use of gene therapy as it allows the production of autologous pluripotent cells. These cells have unique applicability for gene therapy techniques due to their immuno-compatibility and their ability to differentiate into the tissue types which are affected by the disease. Essentially, genetic manipulation of the pluripotent cell can occur in vitro - and an abnormal gene could be repaired through homologous recombination, or selective reverse mutation or other appropriate techniques known by those skilled in the art. Expression of the gene could also be modified by genetically altering the promoter region or the like. The pluripotent cell with the altered genome would be then either, partially differentiated and introduced or transplanted to the tissue or organ of the patient that is affected by the disease or alternatively provided to the patient in pluripotent form. Use of these cells could also be prophylactic.

[0040] In a still further embodiment the cell line of the present invention may be genetically modified for use in gene therapy applications in humans or other animals.

[0041] Agents which may be used in the present invention include:

1. DNA cross-linkers/cleavers

[0042] The action of these drugs results in the cross-linking of complementary DNA strands, which in turn prevents DNA replication. DNA strand breakage also occurs. This category comprises drugs such as mitomycin C, bleomycin, phleomycin, actinomycin D cyclophosphamide and chlorambucil (this list is non-exhaustive). Other agents such as cisplastin induce intra-strand crosslinks.

2. DNA intercalators

[0043] These agents complex with DNA and topoisomerases I or II. An example is etoposide, which enhance double and single-strand cleavage and prevent DNA re-ligation. Other examples include psorolins and inactine. Etoposide has also been reported to cause nuclear expulsion. Another example in this category is camptothecin, which binds irreversibly to the DNA-topoisomerase I complex, inhibiting the reassociation of DNA after cleavage by topoisomerase I and traps the enzyme in a covalent linkage with DNA. This offers a potential advantage compared to the effects of mitomycin C which are partly reversible.

3. DNA synthesis inhibitors

[0044] These agents are base analogs which incorporate into DNA and thereby results in further inhibition of DNA synthesis. This category comprises drugs such as (β-D- Arabinofuranosyl)cytosine (Ara-C). It is often reported that these drugs do not affect transcription.

5. DNA polymerase inhibitors

[0045] These agents act directly on the enzyme responsible for DNA replication. A representative drug is aphidicolin. DNA itself is not altered; thus the presence of cytotoxic DNA degradation product in the cell is avoided.

6. Irradiation.

[0046] DNA damage can be caused by radiations such as X-rays, α-particles or UVs. Using these treatments avoids the presence of residual agents in fused cells.

[0047] It is preferred that the nuclear arrest agent or cell cycle inhibitor is selected from the group consisting of mitomycin C, bleomycin, phleomycin, actinomycin D cyclophosphamide, chlorambucil, cytochalasin B, aphidicholin, nocodazole, cisplastin, etoposide, psorolin, actine, (β-D-Arabinofuranosyl)cytosine (Ara-C), aphidicolin, X-rays, gamma-irradiation, α-particles and UV irradiation. [0048] In a preferred embodiment of the invention the nuclear arrest agent is mitomycin C or gamma-irradiation.

[0049] In a preferred embodiment of the invention the cell cycle arrest agent is cytochalasin B, aphidicholin or nocodazole.

[0050] It is preferred that the cytoskeletal inhibitor is cytochalasin B or cytochalasin D. In another preferred embodiment the cytoskeletal inhibitor is cytochalasin E or dihydrocytochalasin B.

[0051] The adult diploid cells produced according to the invention are reprogrammed to exhibit characteristics of multipotency or pluripotency. This makes them particularly amenable for the generation of cell lines for treatment of disease (autologous or allogeneic therapeutic use); for drug discovery or development assays; for toxicity testing; for agricultural purposes; for study of human disease and study of animal disease.

[0052] The adult cells may be of any suitable type. For example, the cells may be derived from any organ, bone marrow, skin, muscle, adipocyte or neural tissue. The cells may also be derived from foetal tissue. For therapeutic or drug testing applications, preferably the adult cell is a human cell. However the adult cells may be derived from any vertebrate including murine, human, bovine, ovine, porcine, caprine, equine and chicken.

[0053] It will be understood by those skilled in the art that the adult cells of the invention may extend to karyoplasts. A karyoplast is a nucleus of an adult cell surrounded by a plasma membrane. It includes nuclei surrounded by a thin layer of cytoplasm. Methods of deriving karyoplasts are well known to those skilled in the art. Karyoplasts can also include denuded nuclei with little or no surrounding cytoplasm and no plasma membrane. A karyoplast may also include a non-viable cell with no intact membrane; (see for example US Patent Application 20030046722).

[0054] Karyoplasts may be obtained from adult cells by micromanipulation, density gradient centrifugation or centrifugal enucleation in the presence of the cytoskeletal disrupting agent, such as cytochalasin B or cytochalasin D. These procedures allow isolation of karyoplasts with minimal cytoplasmic component. Karyoplasts with no cytoplasm can be generated by lysis, sonication or density gradient centrifugation of cells in the absence of cytoskeletal disrupting agents. They can also be generated by piezo pulse or mechanical disruption of the plasma membrane. [0055] The term pluripotent as used herein refers to cells which are not committed to differentiate only towards one adult phenotype and which can self renew. The pluripotent cells may be derived from cells selected from a variety of sources including embryonic sources, fetal sources and adult sources, including from blood which is isolated from the umbilical cord. The pluripotent cells may be modified such that they are diploid or tetraploid or polyploid. It is possible that having more than one nuclei in the pluripotent cell will increase the efficiency of the generation of the diploid (or effectively diploid) reprogrammed cells due to facilitating the expulsion of nuclei from the heterokaryon. In addition, polyploid pluripotent cells appear to have the benefit of gene dosage effects to induce efficient reprogramming of the somatic nucleus.

[0056] Pluripotent cells include embryonic stem (ES) cells, primordial genu (PG) cells, embryonic carcinoma (EC) cells and embryonic germ (EG) cells or derivatives or mixtures thereof. The pluripotent cells may also be selected or enriched from the group consisting of mesenchymal, neural stem cells, cord blood stem or hematopoietic stem cells. The pluripotent cells may be human or may be derived from any vertebrate including murine, human, bovine, ovine, porcine, caprine, equine and chicken. Preferably the pluripotent cells will come from the same species as the adult cells. The cells may be isolated by methods known to those skilled in the art.

[0057] The step of placing the first and second populations in intimate contact according to step (d) or (c) may take any suitable form. Preferably, the cell contact comprises subjecting the cells to a fusion step.

[0058] Fusion of cells may be achieved for example by electrical pulse or by exposure to polyethylene glycol (PEG) or exposure to Sendai virus. A combination of electrofusion and PEG treatment may also be used. The cells can be treated to induce adhesion prior to fusion e.g., by treatment with phyto-hemagglutin.

[0059] The cell fusion step leads to the production of large, multinucleate, aneuploid, euploid or polyploid cells that contain an increased pool of cytoplasm. Such fused cells may contain two or more diploid (unfused) nuclei, or polyploid (at least 4N) chromosomes.

[0060] More preferably, during the cell fusion step, the adult cell nucleus and the nucleus of the pluripotent cell are maintained as separate nuclei by maintaining the cells at low temperature, by utilising a cell cycle arrester, such as aphidocolin, or a cytoskeletal inhibitor (e.g., cytochalasin B, cytochalasin D) or combinations thereof. [0061] By permitting the generation of the reprogrammed adult cell from the fused cell, it is meant that the fused cell may be further manipulated to yield a reprogrammed diploid adult cell or that the fused cell may undergo cellular division resulting in a daughter cell which is a reprogrammed diploid adult cell.

[0062] Preferably, the method according to the first aspect comprises the further step of separating the reprogrammed diploid or effectively diploid cells from other cells.

[0063] The separation step may comprise ensuring that the source of adult cells has a marker which will be expressed when the cell (or a daughter cell) has become reprogrammed. For example, the adult cell may be genetically modified to express Green Fluorescent Protein (GFP) under the control of the Oct4 promoter - so that when the cell is reprogrammed GFP will be produced. This (or another appropriate) selectable marketer will enable a person who is skilled in the art to undertake the isolation of the reprogrammed cells. Preferably, the selectable marker is a marker under the control of a promoter which will only be activated in pluripotent cells. The selected cells may be separated by fluorescent activated cell sorting (FACS).

[0064] In another embodiment of the invention, the adult cells may be pre-treated with chromatin remodelling agents or reprogramming factors. Preferably, the chromatin remodelling agents are selected from the group consisting of substances which promote demethylation of nucleic acids (e.g., 5-Aza-2'-deoxycytidine), deacetylation of histone proteins (e.g., Trichostatin A), histone exchange (e.g., nucleoplasm^) or chromatin condensation (e.g., mitotic extracts; G2/M cyclin). Alternatively adult cells from dinucleotide methyl transferase knock down animals where nucleic acid methylation cannot take place may be used.

[0065] When the hybrid cell undergoes cell division, one of two outcomes can be expected. In one outcome, the nucleus of the pluripotent cell pre-treated to induce nuclear arrest, remains bound by its nuclear membrane and is segregated with one of the resulting daughter cells. In another outcome, the nucleus of the pre-treated pluripotent cell undergoes chromosome condensation at the same time as the replicating adult cell nucleus. As a result the nuclear membrane of the pre-treated cell breaks down and this may result in the release of some chromosomes, or fragments thereof, into the cytoplasm. In this way the cell becomes effectively diploid in that it will have a nuclear component derived from the adult/somatic cell comprising 2N (which will be transcribed in the usual manner) but will potentially also have additional whole chromosomes or chromosomal fragments, from the pluripotent cell, contained in the cytoplasm - which would not be transcribed. As such the cell is effectively diploid - despite having additional chromosomes or chromosomal fragments contained within the cytoplasm.

[0066] The reprogrammed diploid or effectively diploid adult cells derived from the cell hybrid may be expanded to generate a cell line. The cell line is preferably stable in culture in vitro.

[0067] For in vitro applications such as for drug testing, marker genes may be used to identify reprogrammed diploid or effectively diploid adult cells. Suitable markers would be familiar to those skilled in the art.

[0068] The cells may be maintained in any suitable cell culture medium in the presence of factor(s) that promote maintenance of a pluripotent state. For pluripotent cells, factors include a gpl30 agonist such as the cytokine leukaemia inhibitory factor (LIF). Oncostatin M, CNTF, CTl or IL-6 with the soluble IL-6 receptor, and IL-11 and other gpl30 agonists at equivalent levels may also be used.

[0069] Preferably, the cells may be cultured in the presence of suitable factor(s) under conditions suitable for their proliferation and maintenance in vitro. This includes the use of serum including foetal calf serum and bovine serum, or the medium may be serum free. Other growth enhancing components such as insulin, transferrin and sodium selenite may be added to improve growth of the cells. As would be readily apparent to those skilled in the art, the growth enhancing components will be dependent upon the cell types cultured, other growth factors present, attachment factors and amounts of serum present.

[0070] The cells may be cultured for a time sufficient to establish the cells in culture ie. a time when the cells equilibrate in the culture medium. Preferably, the cells are cultured for approximately 2 to 14, preferably 3 to 10 days.

[0071] The cell culture medium may be any cell culture medium appropriate to sustain the cells. The cell culture medium will, of course, vary according to the source of the cells - that is the species from which they originated. For example, for mouse cells the culture medium is preferably DMEM containing high glucose, supplemented with 15% FBS, 1% Glutamax, O.lmM beta mercaptoethanol, Penicillin/Streptomycin, Non essential amino acids and 1000IU LIF. The cells are preferably cultured at 37°C in 5% CO_2. For other species the culture media will vary - as described by those skilled in the art. [0072] In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following non-limiting examples.

EXAMPLE 1

[0073] An overview of the invention is described in Figure 1. Pluripotent cells (for example ES cells or other cells as described above) are pre-treated with at least one nuclear arrest agent (for example, mitomycin C, gamma irradiation) or at least one cell cycle inhibitor (for example cytochalasin B, aphidicholine or nocodazole) or a combination of at least one nuclear arrest agent and one cell cycle inhibitor. Nuclear arrest agents preferably prevent replication of chromosomes but not translation of transcripts (mRNAs) which already exist within the cell. The pluripotent cell is then fused with an adult cell (for example cell derived from skin or other cell as described above) or karyoplast of an adult cell to produce a heterokaryon. The adult cell may or may not be pre-treated with chromatin remodelling agents such as substances which promote demethylation of nucleic acids (e.g., 5-Aza-2'-deoxycytidine), deacetylation of histone proteins (e.g., Trichostatin A (TSA)), histone exchange (e.g., nucleoplasms) or chromatin condensation (e.g., mitotic extracts; G2/M cyclin; nocodazole). A heterokaryon is a cell comprising both nuclei derived from the pluripotent cell and the adult cell which are bound by common cytoplasm. Three approaches are proposed for generating a diploid or effectively diploid reprogrammed adult cell from the heterokaryon:

(a) spontaneous enucleation of nucleus originating from pluripotent cell (Figure 2);

(b) replication induced elimination of the nucleus from the pluripotent cell (where this nucleus will segregate only with the parental or daughter cell) (Figure 3);

(c) effectively diploid cell line model of disease (where the nuclear membrane of the nucleus originating from the pluripotent cell breaks down and the chromosomes are released into the cytoplasm)(Figure 4).

[0074] Figure 2 outlines the model for the spontaneous enucleation of the nucleus originating from the pluripotent cell (in the form of a karyoplast.) A heterokaryon is derived by the fusion of a pre-treated (as above) pluripotent cell and an adult cell. The heterokaryon is treated with cytochalasin B which is a cyto-skeletal inhibitor. In addition, treatment of cells with certain concentrations of cytochalasin B can result in nuclear expulsion thus resulting in the spontaneous generation of diploid cells comprising either the nucleus derived from the adult cell or the nucleus derived from the pluripotent cell. Cells are later sorted using traditional marker systems to isolate the desired population of reprogrammed cells with the nucleus originating from the adult cell.

[0075] Figure 3 outlines the model for replication induced karyoplast/nucleus elimination. In this model, the pluripotent cell (e.g., embryonic stem cell) is treated with a nuclear arrest agent such as mitomycin C which cross links DNA strands thereby preventing DNA replication and transcription and accordingly results in a nucleus which is non functional. Fusion of the pre-treated pluripotent cell with an adult cell results in a heterokaryon with only one replicating nucleus, the adult one. When this nucleus undergoes replication one of two outcomes can be expected with the MMC -treated nucleus either remaining bound by its nuclear membrane (Figure 3) or the nucleus possibly undergoing chromosomal condensation and nuclear breakdown along with the adult nucleus (as described in Figure 4). In the former instance, when the adult cell undergoes cytokinesis (ie. a process of cell division resulting in two separate daughter cells each with its own nucleus), the entire "inactive" nucleus (originating from the pluripotent cell) will be segregated with one of the resulting daughter cells. This results in one daughter adult cell that retains the nucleus from the pluripotent cell and the other daughter cell which is a reprogrammed adult diploid cell.

[0076] Figure 4 outlines the model for creating an 'effectively' diploid cell line as a model of disease. In the latter instance mentioned above, the nucleus from the pluripotent cells is induced to undergo chromosomal condensation by the replicating adult nucleus. As a result, the nuclear membrane will break down, which may result in the release of some chromosomes originating from the pluripotent cell into the surrounding cytoplasm.

However, these chromosomes remain inactive due their treatment with mitomycin C which is irreversible. When the nuclear membranes re-form, some of the chromosomes originating from the pluripotent cell may remain in the cytoplasm and following cytokinesis, may be located in the diploid daughter cell or in the daughter cell that contains the inactive pluripotent cell nucleus. These cells may have limited use for therapy due to the presence of occasional chromosomes originating from the pluripotent cell, however as the chromosomes from the pluripotent cell are inactive, these cells are "effectively" diploid adult cells which can be used for drug testing and studies of disease models. EXAMPLE 2

[0077] Nuclear inactivation of embryonic stem cells (ESCs) using a nuclear arrest agent prior to fusion to somatic cells results in the reprogramming of the somatic genome and loss of the inactivated ESC genome during cell division in the fused cell. The nuclear arrest agent used in the following experiments is mitomycin C (MMC).

Material and Methods

Effect of MMC on the growth and differentiation of mouse embryonic stem cells

[0078] Mouse tetraploid ESCs (Tl cells) were plated at 500,000 cells/well in 6 well tissue cultures trays. Tl cells were used due to their well-documented reprogramming ability (Upton et al., 4th Annual ISSCR Meeting 2006). The following day cells were treated for 4 h with doses of MMC between 0.1 and 30 μg/ml. At the end of treatment cells were washed 6 X with 4 ml PBS, and cultured for a further 2 h. Cells from each group were then trypsinised, counted, and distributed to 4 wells of 6 well tissue culture trays. Cells were observed for changes in morphology, and viable, attached cells were counted 1, 2, or 4 days after MMC treatment. Oct4-GFP mouse diploid ESCs were also treated with 10 μg/ml of MMC and observed after 24 hrs. These cells contain a randomly inserted transgene where the expression of GFP in under the control of regulatory elements of the pluripotent factor Oct4, and permit a rapid assessment of Oct4 expression.

Fusion of MMC treated Tl cells to Oct4-neo mouse embryonic fibroblasts

[0079] Diploid mouse embryonic fibroblasts were used as a source of somatic cells. Oct4- neo MEFs were chosen, in which a neomycin resistance cassette is placed under the control of the pluripotent factor Oct4. Restoring Oct4 activity is an essential reprogramming event. These cells allow the monitoring of Oct4 activity through neomycin selection. Tl cells were plated at 500O00 cells/well in 4 well tissue cultures trays. The following day cells were treated for 4 h with 1 μM mitomycin C, and washed 3 X with 4 ml PBS. 1x10 ⁶ Oct4-neo MEFs were spun into each well, and fused using polyethylene glycol (50%, pH 7.5). Two hours after fusion cells were trypsinised and replated in 10 cm dishes. Selection of neomycin-resistant colonies, ploidy screening and cryopreservation

[0080] Starting 48 h after fusion cultures were treated for 6 days with neomycin at 500 μg/ml. Resistant colonies were picked-up individually and further cultured in the presence of neomycin. DNA content (ploidy) of cells was measured using propidium iodide (PI) flow cytometry on permeabilised cells. Cells from cultures containing near diploid cells were restained with the vital stain Hoechst 33342, and diploid cells FACS sorted and propagated clonally. Propagated clones were re-screened for ploidy. Cells were cryopreserved at each critical characterization step.

Results and Discussion

Effect of MMC on the growth and differentiation of mouse embryonic stem cells: determining the appropriate concentration of MMC

[0081] A dose of 30 μM/ml MMC (10 μg/ml) is commonly used for the preparation of inactivated MEF feeder-layers for the culture of ESCs {Robertson, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. IRL Press, Oxford and Washington D.C. 1987:71-112). Treating Tl cells with 30 μM/ml MMC resulted in rapid cell loss on the day of treatment (Figure 5, insert; 72% down from control) and massive cell death 24 h after treatment (Figure 5, insert; 97% down from control). Also, treating Oct4-GFP ESCs with only 10 μM MMC resulted in striking changes in colony morphology (Figure 6) together with loss of GFP expression (Figure 7), indicating differentiation. Figure 6 shows the effect of MMC on the proliferation of Tl cells: 1. cells at day 1 before counting of viable attached cells (A: control and B: 30 μM MMC). These cells are non-attached and floating. In 2. cells are at day 2 (A: control and B: 1 μM MMC) cells are attached and mostly consist of undifferentiated single cell colonies. The cells at day 2 are growing on a background of MEFs.

[0082] MMC has been used in the context of elucidating DNA repair mechanisms in mouse ESCs. It appeared that mouse ESCs are highly sensitive to MMC (Moynahan et ah, Cancer Research 2001:61:4842-4850), with a LD50 of 0.5 μM. The challenge in these experiments was to find a treatment which did not cause cell death or differentiation and yet blocked cell division for a significant amount of time (48h or more, in order to allow early reprogramming event to take place). Figure 5 indicates that between 0.1 and 3 μM MMC caused a dose-dependent effect on the proliferation of Tl cells. The effects of lower doses were reversible, whereas inhibition caused by 1 μM persisted over at least 2 days (Figures 5 and 6). On the basis of these experiments, a concentration of 1 μM MMC was chosen for inactivation of Tl cells prior to fusion to MEFs.

Fusion of mitomycin C treated cell to Oct4-neo mouse embryonic fibroblasts

[0083] In two separate experiments Oct4-neo MEFs were fused with Tl cells pretreated with 1 μM MMC or with control Tl cells. A first, preliminary experiment resulted in the obtention of 23 neomycin-resistant colonies for controls and 37 neomycin-resistant colonies for MMC pretreated cells (Table 1). Colonies resulting from MMC-pretreated cells grew slowly compared to controls. Colonies were picked-up, expanded and frozen down. Ploidy was assessed for 14 of the MMC-pretreated colonies. The majority of these colonies displayed a profile showing the presence of ~ 6N cells compared to the Tl 4N control and 2N MEFs, as expected for the fusion products of 4N and 2N cells (Figure 8, MMC 1). However, 5 colonies showed a relatively high proportion of cells with an apparent ploidy of about 2N, indicating potential chromosome loss in a sub-population of cells (Figure 8, MMC 2).

[0084] A second experiment resulted in the obtention of 30 neomycin-resistant colonies for controls and 16 neomycin-resistant colonies for MMC pretreated cells. 28 and 13 colonies in each group were picked-up and expanded. Ploidy was assessed for 5 of the control colonies and 7 of the MMC-pretreated colonies (Table 1). Both groups displayed a profile showing the presence of ~ 6N cells compared to the Tl 4N control (Figure 9). As in the first experiment, 4 MMC colonies displayed a relatively high proportion of cells with a ploidy of ~ 2N (Figure 9, e.g. MMC 12). Importantly, this population was absent from Tl 4N controls and all control fusion cultures tested, and therefore resulted specifically from the MMC pre-treatment.

Table i

Experiment 1

Experiment 2

EXAMPLE 3

2n vs 4n ESCs for reprogramming somatic donor cells

[0085] Aim: To compare the rate of reactivation of an Oct4-GFP transgene in somatic cells when fused with either tetraploid or diploid ESCs.

[0086] Materials and methods.

[0087] Cell lines used

[0088] mEFs used in these experiments were isolated from MTK-neo 2 +/+ females which had been mated to OG2 +/+ males. mEFs were isolated at day 13.5. [0089] Diploid ESCs were D3 ESCs which contained a hygromycin resistance cassette. Tetraploid ESCs were the result of a fusion of two diploid D3 ESC lines containing hygromycin and puromycin resistance cassettes respectively.

[0090] Fusion and measurement oftransgene reactivation

[0091] Tetraploid or diploid ESCs were plated a 500, 000 cells per well on Nunc Tissue culture treated 4 well plates. The following day, 1,000,000 OG2/MTKneo mEFs were plated on top of ESCs and plates were sun at 1200RPM for 10 minutes. Medium was aspirated from 2 wells, and 500μl PEG (50%, pH 7.5, 37°C) was added to 1 well and 500μl PBS was added to the other. Plate was incubated on bench top for exactly 2 minutes, then most of PEG aspirated and 200μl PBS- was added gently to both wells and swilled. A further 2 lots of 200μl PBS- was added and swilled before wells were aspirated and washed twice with 500μl PBS-. Medium was then replaced. Cells were returned to incubator and left to recover for 4hrs. Cells were then dissociated with trypsin and re- plated on 3x60mm plates per well.

[0092] Over the following 2 days, plates were examined using epifluorescence and the number of GFP positive colonies were counted.

[0093] Results and Discussion

[0094] Reactivation of Oct4-GFP transgene

[0095] On average, 16 GFP-positive colonies were observed for diploid ESC fusions, whereas 67 GFP positive colonies were observed for 4n ESC fusions. These results suggest a 4 times greater transgene reactivation efficiency for 4n over 2n ESCs. Reactivation of the Oct4-GFP transgene is seen as an indicator of reprogramming of the somatic genome. These results suggest that tetraploid ESCs are better at reprogramming somatic cells than diploid ESCs.

[0096] The experiments described above in Examples 1 and 2 show that nuclear inactivation of ESCs does result in a change in DNA content in reprogrammed cells obtained by fusion of these cells to somatic cells. Strategies proposed to isolate the low ploidy population of cells thereby generated are as follows.

[0097] Due to the possible rapid exit of pluripotent genetic material from the ~2N population, the somatic genome may be insufficiently reprogrammed in these cells. Culturing the cells in the presence of neomycin, which selects for the expression of endogenous Oct4, could cause the loss of cells where this expression is weak. This could be remedied by taking cells off neomycin selection as soon as cells of a lower ploidy are isolated. Alternatively, Oct4-neo MEFs could be generated from animals which possess a doxycyclin-inducible Oct4 element (e.g., Hochedlinger et al, Cell 2005: 121 :465-477)DP. Here, insufficient expression of Oct4 in these cells could be increased by doxycyclin treatment.

[0098] Residual amounts of MMC from the pretreated ESCs could destabilize the somatic genome. Resistance to MMC has been associated to the multidrug-resistance gene MDR-I (e.g., Hayes et al., BJU International 2001:87:245-250). MDR-I gene expression could be induced once pluripotent and somatic cells have been fused to facilitate MMC elimination and prevent damage to the reprogrammed genome.

[0099] Irradiation, as opposed to drug treatment, could be used to inactivate the pluripotent genome, thus avoiding the presence of residual agents in fused cells.

[0100] Using Oct4-GFP MEFs, rather than Oct4-Neo MEFs, would allow a rapid isolation of reprogrammed cells soon after fusion using FACS. Cell ploidy could be analyzed at the same time. This would represent a significant gain of time compared to antibiotic selection and expansion, and may increase the likelihood of isolating and stabilizing early cell populations from these experiments.

[0101] Isolated diploid adult cells could then be analysed for contribution of genome of pluripotent or somatic origin using microsatellite DNA polymorphism analysis.

[0102] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0103] All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application. [0104] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

1. A method for producing reprogrammed diploid or effectively diploid adult cells, the method comprising:

(a) providing a first population of diploid adult cells;

(b) providing a second population of pluripotent cells

(c) treating the second population of pluripotent cells with:

(i) at least one nuclear arrest agent; or

(ii) one cell cycle inhibitor; or

(d) fusing the first and second populations of cells;

(e) allowing the population of fused cells to undergo mitosis;

isolating from the population of fused cells reprogrammed diploid or effectively diploid adult cells.

2. A method according to claim 1 wherein the first population of diploid cells is treated with one or more chromatin remodelling agents or reprogramming factors.

3. A method according to claim 1 or claim 2 wherein the nuclear arrest agent or cell cycle inhibitor is selected from the group consisting of mitomycin C, bleomycin, phleomycin, actinomycin D cyclophosphamide, chlorambucil, cytochalasin B, aphidicholin, nocodazole, cisplastin, etoposide, psorolin, actine,

(β-D-Arabinofuranosyl)cytosine (Ara-C), aphidicolin, X-rays, gamma-irradiation, α-particles and UV irradiation.

4. A method according to any one of claims 1 to 3 wherein the nuclear arrest agent is mitomycin C or gamma-irradiation.

5. A method according to any one of claims 1 to 3 wherein the cell cycle inhibitor is selected from the group consisting of cytochalasin B, aphidicholin and nocodazole.

6. A method according to any one of claims 1 to 5 wherein the first population of diploid cells and/or second population of pluripotent cells is treated with at least one cytoskeletal inhibitor before or after step (d).

7. A method according to claim 6 wherein the cytoskeletal inhibitor is cytochalasin B or cytochalasin D.

8. A method according to claim 6 wherein the cytoskeletal inhibitor is cytochalasin E or dihydrocytochalasin B.

9. A method according to any one of claims 1 to 8 wherein both the diploid adult cells and the pluripotent cells are from the same species in origin.

10. A method according to claim 9 wherein the diploid adult cells and the pluripotent cells are selected from the group consisting of human, murine, bovine, ovine, porcine, caprine, equine and chicken cells.

11. A method according to any one of claims 1 to 10 wherein the diploid adult cells and the pluripotent cells are human cells.

12. A method according to any one of claims 1 to 10 wherein the diploid adult cells are from a different species than the pluripotent cells.

13. A method according to any one of claims 1 to 12 wherein the pluripotent cells are

2N.

14. A method according to any one of claims 1 to 12 wherein the pluripotent cells are 4N.

15. A method according to any one of claims 1 to 12 wherein the pluripotent cells are greater than 4N.

16. A cell line established from the reprogrammed diploid or effectively diploid adult cells obtained according to the method of any one of claims 1 to 15, wherein the cell line maintains it pluripotent characteristics.

17. A cell line according to claim 16 for use in the treatment of a disease or a condition in a subject.

18. A cell line according to claim 17 wherein the pluripotent diploid or effectively diploid adult cells are immunologically compatible with the subject to be treated.

19. A cell line according to claim 16 for use in drug discovery, drug assessment or drug development.

20. A cell line according to claim 16 for use in toxicity testing.

21. A cell line according to claim 16 for study in human development and human disease.

22. A cell line according to claim 16 for use agricultural and farm management practices.

23. A cell line according to claim 16 for study of animal development and animal diseases.

24. A cell line according to claim 16 which cell line is genetically modified for use in gene therapy applications in humans.

25. A cell line according to claim 16 which cell line is genetically modified for use in gene therapy applications in animals.